Research Advances in Quorum Sensing InhibitionResearch Advances in Quorum Sensing Inhibition: I...
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Research Advances in Quorum Sensing Inhibition Book of abstracts 2015 SANTIAGO DE COMPOSTELA I International Symposium on Quorum Sensing Inhibition and Satellite Meeting on Novel Anti-Fouling Strategies
Transcript of Research Advances in Quorum Sensing InhibitionResearch Advances in Quorum Sensing Inhibition: I...
Research Advances in Quorum Sensing Inhibition
Supported by:
Book of abstracts
2015
SANTIAGO DE COMPOSTELA
I International Symposium on Quorum Sensing Inhibition
and Satellite Meeting on Novel Anti-Fouling Strategies
Research Advances in Quorum Sensing Inhibition
I International Symposium on Quorum Sensing Inhibition
and Satellite Meeting on Novel Anti-Fouling
Strategies
Book of abstracts
supported by:
EDITED BY ANA OTERO
MANUEL ROMERO MARIA ISABEL REYERO
2015
SANTIAGO DE COMPOSTELA
Research Advances in Quorum Sensing Inhibition
I International Symposium on Quorum Sensing Inhibition
and Satellite Meeting on Novel Anti-Fouling
Strategies
Book of abstracts
supported by:
EDITED BY ANA OTERO
MANUEL ROMERO MARIA ISABEL REYERO
2015
SANTIAGO DE COMPOSTELA
Research Advances in Quorum Sensing Inhibition: I International Symposium on Quorum Sensing Inhibition and Satellite Meeting on Novel Anti-Fouling Strategies, Book of Abstracts/ Edited by Ana Otero, Manuel Romero and Maria Isabel Reyero.
Front cover illustration: The marine alga Delisea pulchra avoids bacterial fouling by secreting furanones that mimic the quorum sensing signals N-acyl homoserine lactones (AHLs), blocking signal reception and avoiding the formation of bacterial biofilm. The picture was published in 1999 and shows the “halo of silence” generated when D. pulchra is embedded in an agar plate with the quorum sensing, pigment producing bacterium Chromobacterium violaceum and N-oxohexanoyl-L-homoserine lactone (Manefiel et al., 1999. Microbiology 145(2):283-291). The picture has been reprinted with the permission of the authors.
© Ana Otero, 2015
Edited by
Ana Otero
Printed by
Campus na nube
Servicio de reprografía, edición e impresión dixital da USC
Dep.Legal: C 852 2015
ISBN 978 84 606 8286 8
PREFACE
In the last few decades, there have been a number of key scientific milestones which have drastically changed our understanding of the microbial world. These include the discovery, in the 1960s and 1970s, of the existence of bacterial intercellular signals responsible for driving pneumococcal competence, the expression of luminescence in marine vibrios, and fruiting body formation in myxobacteria. Unknown to scientists then, this constituted the tip of the iceberg of a plethora of intricate cell-cell signalling mechanisms developed by bacteria to coordinate group behaviour, later named as Quorum Sensing (QS). These were the first steps which changed the concept of bacteria working as single entities to a behaviour closer to that of multicellular organisms. The burst on the understanding of the different mechanisms governing QS-mediated bacterial responses, during the early 1990’s, revealed a central role for QS in the control of bacterial pathogenicity. This raised the possibility of exploiting QS systems as a novel target in antimicrobial warfare. In the late 1990’s and early 2000’s two important discoveries supported this idea. The first one was when the red seaweed Delisea pulchra was shown to have developed a strategy to prevent bacteria from colonising its surfaces through the production of furanones which mimics the QS N-acylhomoserine lactone signals. These furanones effectively interfered with the QS systems of colonizing bacteria blocking signal reception and hence algal colonisation (Givskov et al., 1996). We have chosen a picture of this phenomenon as the cover for this book. The second discovery was the identification of the AiiA lactonase from Bacillus as the first bacterial enzyme inhibiting QS systems through the degradation of QS signal molecules (Dong et al., 2000). More than 15 years have passed since these discoveries and, during this time, the amount of knowledge on the intricate mechanisms driving QS-mediated signalling in bacteria and the possible strategies to interfere with them has been steadily increasing. As evidenced by the contributions included in this book of abstracts, most efforts have been so far focused on the use of quorum sensing inhibition (QSI) to fight pathogenic bacteria. Still, other applications of QSI such as antifouling, are gaining momentum and will have a special focus on this symposium through a satellite meeting on ‘Novel antifouling strategies’. This is the reason behind the sponsoring of this meeting by the EU-OCEAN funded project Byefouling.
This international symposium was conceived with the purpose of gathering our current knowledge on QSI and evaluate the possibilities of seeing this field applied to sectors ranging from the clinic to the environment in the coming years.
Ana Otero
Miguel Cámara
References Givskov M, de Nys R, Manefield M, Gram L, Maximilien R, Eberl L, Molin S, Steinberg PD, Kjelleberg S. 1996. Eukaryotic interference with homorserine lactone-mediated prokaryotic signalling. J Bacteriol 178:6618-6622.
Dong YH, Xu JL, Li XZ, Zhang LH. 2000. AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora. PNAS 97:3526-3531.
Research Advances in Quorum Sensing Inhibition: I International Symposium on Quorum Sensing Inhibition and Satellite Meeting on Novel Anti-Fouling Strategies, Book of Abstracts/ Edited by Ana Otero, Manuel Romero and Maria Isabel Reyero.
Front cover illustration: The marine alga Delisea pulchra avoids bacterial fouling by secreting furanones that mimic the quorum sensing signals N-acyl homoserine lactones (AHLs), blocking signal reception and avoiding the formation of bacterial biofilm. The picture was published in 1999 and shows the “halo of silence” generated when D. pulchra is embedded in an agar plate with the quorum sensing, pigment producing bacterium Chromobacterium violaceum and N-oxohexanoyl-L-homoserine lactone (Manefiel et al., 1999. Microbiology 145(2):283-291). The picture has been reprinted with the permission of the authors.
© Ana Otero, 2015
Edited by
Ana Otero
Printed by
Campus na nube
Servicio de reprografía, edición e impresión dixital da USC
Dep.Legal: C 852 2015
ISBN 978 84 606 8286 8
PREFACE
In the last few decades, there have been a number of key scientific milestones which have drastically changed our understanding of the microbial world. These include the discovery, in the 1960s and 1970s, of the existence of bacterial intercellular signals responsible for driving pneumococcal competence, the expression of luminescence in marine vibrios, and fruiting body formation in myxobacteria. Unknown to scientists then, this constituted the tip of the iceberg of a plethora of intricate cell-cell signalling mechanisms developed by bacteria to coordinate group behaviour, later named as Quorum Sensing (QS). These were the first steps which changed the concept of bacteria working as single entities to a behaviour closer to that of multicellular organisms. The burst on the understanding of the different mechanisms governing QS-mediated bacterial responses, during the early 1990’s, revealed a central role for QS in the control of bacterial pathogenicity. This raised the possibility of exploiting QS systems as a novel target in antimicrobial warfare. In the late 1990’s and early 2000’s two important discoveries supported this idea. The first one was when the red seaweed Delisea pulchra was shown to have developed a strategy to prevent bacteria from colonising its surfaces through the production of furanones which mimics the QS N-acylhomoserine lactone signals. These furanones effectively interfered with the QS systems of colonizing bacteria blocking signal reception and hence algal colonisation (Givskov et al., 1996). We have chosen a picture of this phenomenon as the cover for this book. The second discovery was the identification of the AiiA lactonase from Bacillus as the first bacterial enzyme inhibiting QS systems through the degradation of QS signal molecules (Dong et al., 2000). More than 15 years have passed since these discoveries and, during this time, the amount of knowledge on the intricate mechanisms driving QS-mediated signalling in bacteria and the possible strategies to interfere with them has been steadily increasing. As evidenced by the contributions included in this book of abstracts, most efforts have been so far focused on the use of quorum sensing inhibition (QSI) to fight pathogenic bacteria. Still, other applications of QSI such as antifouling, are gaining momentum and will have a special focus on this symposium through a satellite meeting on ‘Novel antifouling strategies’. This is the reason behind the sponsoring of this meeting by the EU-OCEAN funded project Byefouling.
This international symposium was conceived with the purpose of gathering our current knowledge on QSI and evaluate the possibilities of seeing this field applied to sectors ranging from the clinic to the environment in the coming years.
Ana Otero
Miguel Cámara
References Givskov M, de Nys R, Manefield M, Gram L, Maximilien R, Eberl L, Molin S, Steinberg PD, Kjelleberg S. 1996. Eukaryotic interference with homorserine lactone-mediated prokaryotic signalling. J Bacteriol 178:6618-6622.
Dong YH, Xu JL, Li XZ, Zhang LH. 2000. AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora. PNAS 97:3526-3531.
The Symposium is supported by the EU funded project BYEFOULING and the Universidade de Santiago de Compostela (USC, Spain), with the collaboration of the European Society for Marine Biotechnology.
SCIENTIFIC COMMITTEE: Dr. Ana Otero, Universidade de Santiago de Compostela (USC), Spain.
Prof. Miguel Cámara, University of Nottingham, UK.
Dr. Yves Dessaux, Institute for integrative biology of the cell. CEA-CNRS-Université Paris-
Sud,France.
Dr. Tom Defoirdt, Ghent University, Belgium.
Dr. Karen Tait, Plymouth Marine Laboratory, UK.
Dr. Inmaculada Llamas, Universidad de Granada, Spain.
SATELLITE MEETING SCIENTIFIC AND ORGANISING COMMITTE Dr. Ana Otero, n er a e e ant a o e o o te a a n
Prof. Yehuda Benayhu, Tel Aviv University, Israel
Dr. Joao Tedim, Smallmatek, Portugal
Peter Van Aken, Lonza Group Ltd., Switzerland
Dr. Claire Hellio. BioDIMar(UBO/IUEM/LEMAR Bioprospection platform) Université de Bretagne Occidentale, France
LOCAL ORGANIZING COMMITTEE: Dr. Manuel Romero, Universidade de Santiago de Compostela (USC), Spain.
Celia Mayer, Universidade de Santiago de Compostela (USC), Spain.
Isabel Freire, Universidade de Santiago de Compostela (USC), Spain.
Andrea Muras, Universidade de Santiago de Compostela (USC), Spain.
Hugo a e Cunha, Universidade de Santiago de Compostela (USC), Spain.
orena a n er a e e ant a o e o o te a a n
Isabel Reyero, Universidade de Santiago de Compostela (USC), Spain.
QSI, Santiago de Compostela 2015
INDEX
KEYNOTE LECTURES
Williams P. The art of antibacterial aarfare – Deception through interference with quorum sensing………………………………………………………………………………..…………….. 13
Rodríguez Iglesias P. Regulatory aspects of chemical substances under REACH and Biocidal Products Regulation……………………………………………………………………... 14
INVITED LECTURES
Cámara M. Silencing quorum sensing in Pseudomonas aeruginosa…............................. 17
Defoirdt T. Quorum sensing inhibition to control bacterial disease: aquaculture as an example...................................................................................................................... 18
Dessaux Y., Grandclément, C., Faure D. Quorum quenching: actors, biological roles and applications…………………………………………………………………………………….…… 19
Tait K. Havenhand, J., Cámara, M., Williams, P. AHLs and macrofouling wihin the marine environment…………………………………………………………………………….…………... 20
Federle MJ. Inhibiting Rgg pheromone receptors to disrupt quorum sensing in Gram-positive bacteria……………………………………………………………………………..…….. 21
ORAL COMUNICATIONS
Streit WR. Metagenomes as potent resources for the identification of novel QQ genes with anti-biofilm activities………………………………………………..……………………….. 25
Torres M., Uroz, S., Quesada, E., Llamas, I. Screening of the quorum quenching activity in a metagenomic library from a hypersaline-soil sample taken in Rambla Salada (Murcia, Spain)........................................................................................................................ 26
Bertini EV, Leguina CV., Castellanos de Figueroa LI, Nieto-Peñalver CG. Inactivation of QS molecules by endophytic yeasts from sugarcane………………………………………... 27
Mayer C, Romero M, Muras A, Otero A. Aii20J, a novel thermostable lactonase from Tenacibaculum sp.strain 20J can quench AHL-mediated acid resistance in E.coli……….. 28
Garge S, Nerurkar A. Quorum quenching N-acyl homoserine lactonase from soil isolate Lysinibacillus sp. Gs50………………………………………………………………….............. 29
Rueda NJ, Suarez, MO, Romero N, Correa E, Ordúz S, Flórez A, Flórez AM. Genetic improvement of aiiA gene encoding N-acyl homoserine lactonase from Bacillusthuringiensis by Error-Prone PCR………………………………………………………………... 30
The Symposium is supported by the EU funded project BYEFOULING and the Universidade de Santiago de Compostela (USC, Spain), with the collaboration of the European Society for Marine Biotechnology.
SCIENTIFIC COMMITTEE: Dr. Ana Otero, Universidade de Santiago de Compostela (USC), Spain.
Prof. Miguel Cámara, University of Nottingham, UK.
Dr. Yves Dessaux, Institute for integrative biology of the cell. CEA-CNRS-Université Paris-
Sud,France.
Dr. Tom Defoirdt, Ghent University, Belgium.
Dr. Karen Tait, Plymouth Marine Laboratory, UK.
Dr. Inmaculada Llamas, Universidad de Granada, Spain.
SATELLITE MEETING SCIENTIFIC AND ORGANISING COMMITTE Dr. Ana Otero, n er a e e ant a o e o o te a a n
Prof. Yehuda Benayhu, Tel Aviv University, Israel
Dr. Joao Tedim, Smallmatek, Portugal
Peter Van Aken, Lonza Group Ltd., Switzerland
Dr. Claire Hellio. BioDIMar(UBO/IUEM/LEMAR Bioprospection platform) Université de Bretagne Occidentale, France
LOCAL ORGANIZING COMMITTEE: Dr. Manuel Romero, Universidade de Santiago de Compostela (USC), Spain.
Celia Mayer, Universidade de Santiago de Compostela (USC), Spain.
Isabel Freire, Universidade de Santiago de Compostela (USC), Spain.
Andrea Muras, Universidade de Santiago de Compostela (USC), Spain.
Hugo a e Cunha, Universidade de Santiago de Compostela (USC), Spain.
orena a n er a e e ant a o e o o te a a n
Isabel Reyero, Universidade de Santiago de Compostela (USC), Spain.
QSI, Santiago de Compostela 2015
INDEX
KEYNOTE LECTURES
Williams P. The art of antibacterial aarfare – Deception through interference with quorum sensing………………………………………………………………………………..…………….. 13
Rodríguez Iglesias P. Regulatory aspects of chemical substances under REACH and Biocidal Products Regulation……………………………………………………………………... 14
INVITED LECTURES
Cámara M. Silencing quorum sensing in Pseudomonas aeruginosa…............................. 17
Defoirdt T. Quorum sensing inhibition to control bacterial disease: aquaculture as an example...................................................................................................................... 18
Dessaux Y., Grandclément, C., Faure D. Quorum quenching: actors, biological roles and applications…………………………………………………………………………………….…… 19
Tait K. Havenhand, J., Cámara, M., Williams, P. AHLs and macrofouling wihin the marine environment…………………………………………………………………………….…………... 20
Federle MJ. Inhibiting Rgg pheromone receptors to disrupt quorum sensing in Gram-positive bacteria……………………………………………………………………………..…….. 21
ORAL COMUNICATIONS
Streit WR. Metagenomes as potent resources for the identification of novel QQ genes with anti-biofilm activities………………………………………………..……………………….. 25
Torres M., Uroz, S., Quesada, E., Llamas, I. Screening of the quorum quenching activity in a metagenomic library from a hypersaline-soil sample taken in Rambla Salada (Murcia, Spain)........................................................................................................................ 26
Bertini EV, Leguina CV., Castellanos de Figueroa LI, Nieto-Peñalver CG. Inactivation of QS molecules by endophytic yeasts from sugarcane………………………………………... 27
Mayer C, Romero M, Muras A, Otero A. Aii20J, a novel thermostable lactonase from Tenacibaculum sp.strain 20J can quench AHL-mediated acid resistance in E.coli……….. 28
Garge S, Nerurkar A. Quorum quenching N-acyl homoserine lactonase from soil isolate Lysinibacillus sp. Gs50………………………………………………………………….............. 29
Rueda NJ, Suarez, MO, Romero N, Correa E, Ordúz S, Flórez A, Flórez AM. Genetic improvement of aiiA gene encoding N-acyl homoserine lactonase from Bacillusthuringiensis by Error-Prone PCR………………………………………………………………... 30
QSI, Santiago de Compostela 2015
Müller C, Birmes FS, Rückert C, Kalinowski J, Fetzner S. Bacterial degradation of the Pseudomonas quinolone signal PQS and identification of new quorum quenching enzymes…………………………………………………………………………………………….. 31
De la Fuente-Nuñez C, Reffuveille F, Brackman G, Coenye T, Hancock REW. Peptides that eradicate multidrug-resistant biofilms and protect against lethal Pseudomonas aeruginosa infections……………………………………………………………………………… 32
Palliyil S, Downham C, Broadbent I, Charlton K, Porter A. Protective effect of quorum quenching monoclonal antibodies in lethal Pseudomonas infection……………………….. 33
Ivanova K, Fernandes MM, Tzanov T. Sonochemical coatings of acylase nanoparticles inhibit Pseudomonas aeruginosa biofilm formation and virulence factors production………………………………………………………………………………………..…. 34
Utari PD, van Merkerk R, Quax WJ. Utilizing AHL acylase to disrupt biofilm formation of hospital-acquired pathogen Acinetobacter nosocomialis M2………………………………... 35
Bhargava N, Sharma P, Capalash N. Unchartering quorum quenching potential in G.glabra for attenuation of quorum sensing mediated virulence of A.baumannii…………………………………………………………………………………………… 36
Slachmuylders L, Brackman G, Coenye T. Insight into the effect of the quorum sensing inhibitor baicalin hydrate on Burkholderia cepacia complex biofilm susceptibility………………………………………………………………………………………… 37
Cavaleiro E., Duarte AS, Esteves AC, Correia A, Whitcombe MJ, Piletska EV, Piletsky SA, Chianella I. Methacrylate-based polymers to inhibiotic bacterial quorum sensing……………………………………………………………………………………………… 38
Pande GSJ, Natrah FMI, Kumar U, Bossier P, Defoirdt T. AHL-degrading bacteria isolated from microalgae protect prawn larvae from Vibrio campbellii infection……………………....................................................................................... 39
Yang Q, Aamdal-Scheie A, Benneche T, Defoirdt T. Novel quorum sensing-disrupting thiophenones with a promising potential to treat vibriosis in aquaculture……………………. 40
Ascenso OS, Rui F, Miguel AS, Marques JC, Xavier KB, Ventura MR. Study and modulation of inter-species quorum sensing by AI-2 analogues……………….…………….. 41
Park H, Lee K, Shin H-K, Holzapfel W. Inhibition of autoinducer-2 leads to gut microbiota modulation……………………………………………………………………………………….…. 42
Lee K, Park H, Shin H-K, Holzapfel W. Autoinducer-2 quorum sensing influences on bacterial growth under specific stress conditions………………………………………………. 43
Talagas A, Perchat S, Lereclus D, Nessler, S. Structural analysis of NprR, a bifunctional quorum sensor of the Bacillus cereus group………………………………..…………………... 44
Brackman G, Van den Driessche F, Coenye T. How does the quorum sensing inhibitor hamamelitanning increase Staphylococcus aureus biofilm susceptibility?....................... 45
QSI, Santiago de Compostela 2015
Murugan K, Sekar K, Al-Sohaibani S. Anticaries, antibiofilm and quorum sensing inhibitory activity of stigmasterol-3-D-glucoside isolated from Mimusops elegi L………………………............................................................................................................ 46
Jaiyen Y, Rui F, Steele V, Murray E, Chan W, Williams P. Discovery of inhibitors of the Staphylococcus…………………………………………………………………………………….. 47
García-Contreras R, Castañeda P, Maeda T, Wood TK. Is spreading of resistance against quorum quenchers possible?................................................................................... 48
POSTER COMUNICATIONS
Zhang Y, Brackman G, Coenye T. Interfering with biofilm formation of bacteria involved in chronic wound infections by enzymatic quorum quenching……………………................... 51
Birmes FS, Fetzner S. Mycobacterium abscessus, an emerging pathogen in cystic fibrosis patients, degrades the Pseudomonas quinolone signal……………......................... 52
Müller C, Birmes FS, Fetzner S. Rhodococcus erythropolis BG43, an isolate able to degrade HHQ, PQS and related alkylhydroxyquinolines……………………………….……… 53
Cocotl-Yañez M, Dafhnis-Calas F, Krasnogor N, Cámara M, Heeb S. Engineered quorum quenching bacterial coatings and skins against Pseudomonas aeruginosa infections……………………………………………………………………………………………. 54
Porzio E, Andrenacci D, Manco G. Quorum quenching activity of archaeal lactonases against Pseudomonas aeruginosa in a Drosophila infection model…………….................... 55
Torres M, Quesada E, Llamas I. Potential biotechnological applications of two strains of Alteromonas with high quorum-quenching activity………..................................................... 56
Freire I, Nascimento P, Reyero I, Muras A, Mayer C, Milhazes-Cunha H, Romero M, Otero A. Use of the quorum quenching strain Tenacibaculum sp. 20J to improve survival in mollusc larvae cultures…………………………………………………………………………. 57
Mayer C, Romero M, Muras A, Rumbo S, Gato E, Tomas M, Otero A. Effect of the quorum quenching lactonase Aii20J on biofilm formation by different strains and mutants of Acinetobacter baumannii………………………………………………………………….….… 58
Rey D, Mayer C, Muras A., Romero M, Otero A. Identification and cloning of enzymes responsible for quorum quenching activity in the marine bacterium Maribacterulvicola…..………………………………………………………………………………………….. 59
Singh BN. Disabling Pseudomonas aeruginosa quorum sensing by bio-silver nanoparticles……………………………………………………………………………………….. 60
Bodelón G, Montes-García V, López-Puente V, Rodal-Cedeira S, Costas C, Pérez-Juste J, Pastoriza-Santos I, Liz-Marzán LM. Non-invasive plasmonic detection and imaging of quorum sensing in biofilms of Pseudomonas aeruginosa……………………………….…….. 61
Costas C, López-Puente V, Bodelón G, González-Bello C, Pérez-Juste J, Pastoriza-Santos I, Liz-Marzán LM. Analysis of the interactions between Pseudomonas aeruginosa LasR and quorum sensing modulators by SERS…………………………............................. 62
QSI, Santiago de Compostela 2015
Müller C, Birmes FS, Rückert C, Kalinowski J, Fetzner S. Bacterial degradation of the Pseudomonas quinolone signal PQS and identification of new quorum quenching enzymes…………………………………………………………………………………………….. 31
De la Fuente-Nuñez C, Reffuveille F, Brackman G, Coenye T, Hancock REW. Peptides that eradicate multidrug-resistant biofilms and protect against lethal Pseudomonas aeruginosa infections……………………………………………………………………………… 32
Palliyil S, Downham C, Broadbent I, Charlton K, Porter A. Protective effect of quorum quenching monoclonal antibodies in lethal Pseudomonas infection……………………….. 33
Ivanova K, Fernandes MM, Tzanov T. Sonochemical coatings of acylase nanoparticles inhibit Pseudomonas aeruginosa biofilm formation and virulence factors production………………………………………………………………………………………..…. 34
Utari PD, van Merkerk R, Quax WJ. Utilizing AHL acylase to disrupt biofilm formation of hospital-acquired pathogen Acinetobacter nosocomialis M2………………………………... 35
Bhargava N, Sharma P, Capalash N. Unchartering quorum quenching potential in G.glabra for attenuation of quorum sensing mediated virulence of A.baumannii…………………………………………………………………………………………… 36
Slachmuylders L, Brackman G, Coenye T. Insight into the effect of the quorum sensing inhibitor baicalin hydrate on Burkholderia cepacia complex biofilm susceptibility………………………………………………………………………………………… 37
Cavaleiro E., Duarte AS, Esteves AC, Correia A, Whitcombe MJ, Piletska EV, Piletsky SA, Chianella I. Methacrylate-based polymers to inhibiotic bacterial quorum sensing……………………………………………………………………………………………… 38
Pande GSJ, Natrah FMI, Kumar U, Bossier P, Defoirdt T. AHL-degrading bacteria isolated from microalgae protect prawn larvae from Vibrio campbellii infection……………………....................................................................................... 39
Yang Q, Aamdal-Scheie A, Benneche T, Defoirdt T. Novel quorum sensing-disrupting thiophenones with a promising potential to treat vibriosis in aquaculture……………………. 40
Ascenso OS, Rui F, Miguel AS, Marques JC, Xavier KB, Ventura MR. Study and modulation of inter-species quorum sensing by AI-2 analogues……………….…………….. 41
Park H, Lee K, Shin H-K, Holzapfel W. Inhibition of autoinducer-2 leads to gut microbiota modulation……………………………………………………………………………………….…. 42
Lee K, Park H, Shin H-K, Holzapfel W. Autoinducer-2 quorum sensing influences on bacterial growth under specific stress conditions………………………………………………. 43
Talagas A, Perchat S, Lereclus D, Nessler, S. Structural analysis of NprR, a bifunctional quorum sensor of the Bacillus cereus group………………………………..…………………... 44
Brackman G, Van den Driessche F, Coenye T. How does the quorum sensing inhibitor hamamelitanning increase Staphylococcus aureus biofilm susceptibility?....................... 45
QSI, Santiago de Compostela 2015
Murugan K, Sekar K, Al-Sohaibani S. Anticaries, antibiofilm and quorum sensing inhibitory activity of stigmasterol-3-D-glucoside isolated from Mimusops elegi L………………………............................................................................................................ 46
Jaiyen Y, Rui F, Steele V, Murray E, Chan W, Williams P. Discovery of inhibitors of the Staphylococcus…………………………………………………………………………………….. 47
García-Contreras R, Castañeda P, Maeda T, Wood TK. Is spreading of resistance against quorum quenchers possible?................................................................................... 48
POSTER COMUNICATIONS
Zhang Y, Brackman G, Coenye T. Interfering with biofilm formation of bacteria involved in chronic wound infections by enzymatic quorum quenching……………………................... 51
Birmes FS, Fetzner S. Mycobacterium abscessus, an emerging pathogen in cystic fibrosis patients, degrades the Pseudomonas quinolone signal……………......................... 52
Müller C, Birmes FS, Fetzner S. Rhodococcus erythropolis BG43, an isolate able to degrade HHQ, PQS and related alkylhydroxyquinolines……………………………….……… 53
Cocotl-Yañez M, Dafhnis-Calas F, Krasnogor N, Cámara M, Heeb S. Engineered quorum quenching bacterial coatings and skins against Pseudomonas aeruginosa infections……………………………………………………………………………………………. 54
Porzio E, Andrenacci D, Manco G. Quorum quenching activity of archaeal lactonases against Pseudomonas aeruginosa in a Drosophila infection model…………….................... 55
Torres M, Quesada E, Llamas I. Potential biotechnological applications of two strains of Alteromonas with high quorum-quenching activity………..................................................... 56
Freire I, Nascimento P, Reyero I, Muras A, Mayer C, Milhazes-Cunha H, Romero M, Otero A. Use of the quorum quenching strain Tenacibaculum sp. 20J to improve survival in mollusc larvae cultures…………………………………………………………………………. 57
Mayer C, Romero M, Muras A, Rumbo S, Gato E, Tomas M, Otero A. Effect of the quorum quenching lactonase Aii20J on biofilm formation by different strains and mutants of Acinetobacter baumannii………………………………………………………………….….… 58
Rey D, Mayer C, Muras A., Romero M, Otero A. Identification and cloning of enzymes responsible for quorum quenching activity in the marine bacterium Maribacterulvicola…..………………………………………………………………………………………….. 59
Singh BN. Disabling Pseudomonas aeruginosa quorum sensing by bio-silver nanoparticles……………………………………………………………………………………….. 60
Bodelón G, Montes-García V, López-Puente V, Rodal-Cedeira S, Costas C, Pérez-Juste J, Pastoriza-Santos I, Liz-Marzán LM. Non-invasive plasmonic detection and imaging of quorum sensing in biofilms of Pseudomonas aeruginosa……………………………….…….. 61
Costas C, López-Puente V, Bodelón G, González-Bello C, Pérez-Juste J, Pastoriza-Santos I, Liz-Marzán LM. Analysis of the interactions between Pseudomonas aeruginosa LasR and quorum sensing modulators by SERS…………………………............................. 62
QSI, Santiago de Compostela 2015
Huedo P, Yero D, Martínez-Servat S, Daura X, Gibert I. New insights into the QS regulatory network of Stenotrophomonas maltophilia…………………………....................... 63
Morohoshi T, Shiogai S, Ochiai S, Azuma T, Ishizuka M, Ikeda T. Inhibition of biofilm formation of Gram-negative bacteria using quorum-quenching compounds……….............. 64
Lu C, Maurer C, Kirsch B, Steinbach A, Weiβ S, Pawar S, Hartmann RW. Quinolone-based PqsR antagonists provide in vivo proof-of-concept for PQS-targeting anti-virulence strategy………………………………………………………………………………...................... 65
Thomann A, Martins AG, Brengel C, Weidel E, Plaza A, Börger C, Empting M, Hartmann RW. Biological evaluation of an in vivo-potent dual target PQS-quorum sensing inhibitor that hinders biofilm formation……………………………………………………………..…….... 66
Lafayette I, Dubern J-F,Do QT, Lazenby J, Halliday N, Heeb S, Williams P, Cámara M. Discovery of novel small-molecules for the inhibition of Pseudomonas quinolone signalling............................................................................................................................... 67
Zender M, Kirsch B, Maurer CK, Empting M, Hartmann RW. Development of novel antagonists of PqsR, an important regulator of Pseudomonas aeruginosa virulence……......................................................................................................................... 68
a a , Kumar V, Kumar Singh P, Dohare S, Narvi SS, Agarwal, V. Inhibition of quorum sensing controlled virulence phenotypes in Pseudomonas aeruginosa PAO1 by using eucalyptus oil…………………………………………………………….…………………………. 69
Pérez-López M, Soto-Hernández M, García-Contreras R, Martínez-Vázquez M, San Miguel-Chávez R, Castillo-Juárez I. Regulatory effect of fatty acids from seeds (Amaranthus hypochondriacus L., Salvia hispánica L.and Helianthus annuus L.) in the quorum sensing systems of Chromobacterium violaceum……………………………….…… 70
Muñoz-Casares N, Soto-Hernández M, García-Contreras R, Martínez-Vázquez M, Aguilar-Rodríguez S, Montes de Oca Mejía M, Castillo-Juárez I. Phytochemical study and evaluation of quorum sensing system inhibition of barks Ceiba spp…………….................. 71
Busetti A, Flynn P, Graham, WG, Gilmore B.F. Quorum sensing inhibition and attenuation of virulence by atmospheric pressure non thermal plasma……......................... 72
Tait K, Ransome E, Munn C. Bacterial communication and climate change: consequences for the coral ecosystem………………………………………..…………..….… 73
Sato R, Yamaguchi T, Someya N, Ikeda T, Morohoshi T. Analysis of N-acylhomoserine lactone-degrading mechanisms in the coagulase-negative staphylococci……………….….. 74
Pérez-Pascual D, Metton C, Besset C, Monnet V, Gardan R. SHP-RovS cell-cell communication mechanism, a target for quorum quenching?............................................... 75
Endo EH, Nakamura TU, Nakamura CV, Días Filho BP. Anti-biofilm effect of Rosmarinus officinalis against MRSA Staphylococcus aureus…………………………………………….… 76
Ryu Eun-Ju, Sim J, Ko Y-K, Jung K-S, Choi B-K. Application of quorum sensing inhibitors prevent biofilm formation of periodontophathogens…………………….................. 77
QSI, Santiago de Compostela 2015
Muras A, Mayer C, Romero M, Ferrer M, Mira A., Otero, A. Interference with quorum sensing signals to prevent biofilm formation by Streptococcus mutans………………..…..... 78
SATELLITE METING ON NOVEL ANTI-FOULING STRATEGIES
INVITED LECTURES
Burguess JG. Towards commercialization of a novel marine nuclease for biofilm removal and prevention……………………………………………………………………………………… 81
Hellio C. Biomimetics approaches for the development of new antifouling strategies……………………………………………………………………………………………. 82
ORAL COMUNICATIONS
Tedim J, Simon C. The FP7-Ocean EU project BYEFOULING: an interdisciplinary approach to novel anti-fouling strategies……………………………………………………….. 85
Stübing D, van Haare J. Synergistic fouling control technologies – project overview and first results………………………………………………………………………………………….. 86
Blas E, Barros R, Guedella E. New biocoating for corrosion inhibition in metal surfaces……………………………………………………………………………………………... 87
Piller C, Hoch-Gunter E, Weis M, Larroze S,Teixeira T,Goldenberg L, Antipov A, Benayahu Y. Effects of novel anti-fouling paints on aquatic species of different trophic levels………………………………………………………………………………………………… 88
Rodríguez-Ezpeleta N, Menchaca I, Zorita I, Alonso L, Franco J. Monitoring biofouling on hard substrata through DNA based approaches…………………….………………..……. 89
Wittig L, Grunwald I, Wunder A, Isaksson D. Bio-based and bio-active biofouling control strategies……………………………………………………………………………………….…… 90
Martín-Rodríguez AJ, Álvarez-Méndez SJ, Martín VS, Fernández JJ. Novel quorum sensing disruptors and their antifouling implications………………………………………..…. 91
Louzao I, Sui C, Winzer K, Fernandez-Trillo F, Alexander C. Polymer mediated bacterial clustering: cell-adhesive properties of cationic homo- and copolymers………………….….. 92
Matteucci G, Esposito M, Fiesoletti F, James V, Napolano L, Pascale C, Rossini P, Testoni A. Test on the environmental impacts of two commercial antifouling coating systems……………………………………………………………………………………………… 93
QSI, Santiago de Compostela 2015
Huedo P, Yero D, Martínez-Servat S, Daura X, Gibert I. New insights into the QS regulatory network of Stenotrophomonas maltophilia…………………………....................... 63
Morohoshi T, Shiogai S, Ochiai S, Azuma T, Ishizuka M, Ikeda T. Inhibition of biofilm formation of Gram-negative bacteria using quorum-quenching compounds……….............. 64
Lu C, Maurer C, Kirsch B, Steinbach A, Weiβ S, Pawar S, Hartmann RW. Quinolone-based PqsR antagonists provide in vivo proof-of-concept for PQS-targeting anti-virulence strategy………………………………………………………………………………...................... 65
Thomann A, Martins AG, Brengel C, Weidel E, Plaza A, Börger C, Empting M, Hartmann RW. Biological evaluation of an in vivo-potent dual target PQS-quorum sensing inhibitor that hinders biofilm formation……………………………………………………………..…….... 66
Lafayette I, Dubern J-F,Do QT, Lazenby J, Halliday N, Heeb S, Williams P, Cámara M. Discovery of novel small-molecules for the inhibition of Pseudomonas quinolone signalling............................................................................................................................... 67
Zender M, Kirsch B, Maurer CK, Empting M, Hartmann RW. Development of novel antagonists of PqsR, an important regulator of Pseudomonas aeruginosa virulence……......................................................................................................................... 68
a a , Kumar V, Kumar Singh P, Dohare S, Narvi SS, Agarwal, V. Inhibition of quorum sensing controlled virulence phenotypes in Pseudomonas aeruginosa PAO1 by using eucalyptus oil…………………………………………………………….…………………………. 69
Pérez-López M, Soto-Hernández M, García-Contreras R, Martínez-Vázquez M, San Miguel-Chávez R, Castillo-Juárez I. Regulatory effect of fatty acids from seeds (Amaranthus hypochondriacus L., Salvia hispánica L.and Helianthus annuus L.) in the quorum sensing systems of Chromobacterium violaceum……………………………….…… 70
Muñoz-Casares N, Soto-Hernández M, García-Contreras R, Martínez-Vázquez M, Aguilar-Rodríguez S, Montes de Oca Mejía M, Castillo-Juárez I. Phytochemical study and evaluation of quorum sensing system inhibition of barks Ceiba spp…………….................. 71
Busetti A, Flynn P, Graham, WG, Gilmore B.F. Quorum sensing inhibition and attenuation of virulence by atmospheric pressure non thermal plasma……......................... 72
Tait K, Ransome E, Munn C. Bacterial communication and climate change: consequences for the coral ecosystem………………………………………..…………..….… 73
Sato R, Yamaguchi T, Someya N, Ikeda T, Morohoshi T. Analysis of N-acylhomoserine lactone-degrading mechanisms in the coagulase-negative staphylococci……………….….. 74
Pérez-Pascual D, Metton C, Besset C, Monnet V, Gardan R. SHP-RovS cell-cell communication mechanism, a target for quorum quenching?............................................... 75
Endo EH, Nakamura TU, Nakamura CV, Días Filho BP. Anti-biofilm effect of Rosmarinus officinalis against MRSA Staphylococcus aureus…………………………………………….… 76
Ryu Eun-Ju, Sim J, Ko Y-K, Jung K-S, Choi B-K. Application of quorum sensing inhibitors prevent biofilm formation of periodontophathogens…………………….................. 77
QSI, Santiago de Compostela 2015
Muras A, Mayer C, Romero M, Ferrer M, Mira A., Otero, A. Interference with quorum sensing signals to prevent biofilm formation by Streptococcus mutans………………..…..... 78
SATELLITE METING ON NOVEL ANTI-FOULING STRATEGIES
INVITED LECTURES
Burguess JG. Towards commercialization of a novel marine nuclease for biofilm removal and prevention……………………………………………………………………………………… 81
Hellio C. Biomimetics approaches for the development of new antifouling strategies……………………………………………………………………………………………. 82
ORAL COMUNICATIONS
Tedim J, Simon C. The FP7-Ocean EU project BYEFOULING: an interdisciplinary approach to novel anti-fouling strategies……………………………………………………….. 85
Stübing D, van Haare J. Synergistic fouling control technologies – project overview and first results………………………………………………………………………………………….. 86
Blas E, Barros R, Guedella E. New biocoating for corrosion inhibition in metal surfaces……………………………………………………………………………………………... 87
Piller C, Hoch-Gunter E, Weis M, Larroze S,Teixeira T,Goldenberg L, Antipov A, Benayahu Y. Effects of novel anti-fouling paints on aquatic species of different trophic levels………………………………………………………………………………………………… 88
Rodríguez-Ezpeleta N, Menchaca I, Zorita I, Alonso L, Franco J. Monitoring biofouling on hard substrata through DNA based approaches…………………….………………..……. 89
Wittig L, Grunwald I, Wunder A, Isaksson D. Bio-based and bio-active biofouling control strategies……………………………………………………………………………………….…… 90
Martín-Rodríguez AJ, Álvarez-Méndez SJ, Martín VS, Fernández JJ. Novel quorum sensing disruptors and their antifouling implications………………………………………..…. 91
Louzao I, Sui C, Winzer K, Fernandez-Trillo F, Alexander C. Polymer mediated bacterial clustering: cell-adhesive properties of cationic homo- and copolymers………………….….. 92
Matteucci G, Esposito M, Fiesoletti F, James V, Napolano L, Pascale C, Rossini P, Testoni A. Test on the environmental impacts of two commercial antifouling coating systems……………………………………………………………………………………………… 93
QSI, Santiago de Compostela 2015
POSTER COMUNICATIONS
Arregui L, Soler A, Liébana R, Santos A, Marquina D, Muñagorri F, Serrano S. A tool for controlling biofouling on membrane bioreactor (MBR) wastewater systems based on the selection of quorum quenching bacteria………………………………………..……………….. 97
Fernández-Hermida X. Hidroboya£: The “Sample & Hold” platform to overcome the fouling problem…………………………………………………………………………….…….…. 98
Hoch-Gutner E, Piller C, Avelelas F, Martins R, Malheiro E, Maia F, Tedim J, Benayahu Y. Novel anti-fouling nanomaterials: effects on macrofoulers from the Eastern Mediterranean and the northern Gulf of Aqaba (Red Sea)………………………................... 99
Freire I, Muras A, Reyero I, Otero A. Inhibitory activity in extracts of the marine microalga Isochrysis aff. galbana clon T-ISO against different marine microalgae……………….……. 100
Muras A, Freire I, Mayer C, Otero A. Effect of Bacillus licheniformis CECT20T on biofilm formation by marine bacteria…………………………............................................................. 101
Muras A, Mayer C, Freire I, Otero A. Monitoring bacterial biofilm development with xCELLigence® technology……………………………………………………….…….…………. 102
Author index...……………………………………….……………………………………………. 103
Notes………………………………………………………………………………………………... 111
QSI, Santiago de Compostela 2015
Keynote lectures
QSI, Santiago de Compostela 2015
POSTER COMUNICATIONS
Arregui L, Soler A, Liébana R, Santos A, Marquina D, Muñagorri F, Serrano S. A tool for controlling biofouling on membrane bioreactor (MBR) wastewater systems based on the selection of quorum quenching bacteria………………………………………..……………….. 97
Fernández-Hermida X. Hidroboya£: The “Sample & Hold” platform to overcome the fouling problem…………………………………………………………………………….…….…. 98
Hoch-Gutner E, Piller C, Avelelas F, Martins R, Malheiro E, Maia F, Tedim J, Benayahu Y. Novel anti-fouling nanomaterials: effects on macrofoulers from the Eastern Mediterranean and the northern Gulf of Aqaba (Red Sea)………………………................... 99
Freire I, Muras A, Reyero I, Otero A. Inhibitory activity in extracts of the marine microalga Isochrysis aff. galbana clon T-ISO against different marine microalgae……………….……. 100
Muras A, Freire I, Mayer C, Otero A. Effect of Bacillus licheniformis CECT20T on biofilm formation by marine bacteria…………………………............................................................. 101
Muras A, Mayer C, Freire I, Otero A. Monitoring bacterial biofilm development with xCELLigence® technology……………………………………………………….…….…………. 102
Author index...……………………………………….……………………………………………. 103
Notes………………………………………………………………………………………………... 111
QSI, Santiago de Compostela 2015
Keynote lectures
ISQSI, Santiago de Compostela 2015 Keynote Lectures
1
The art of antibacterial warfare - Deception through interference with quorum sensing
Paul Williams
Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham, University Park, Nottingham, U.K. E-mail: [email protected]
Keywords: Quorum sensing inhibitors, Staphylococcus aureus, agr system, autoinducing peptides
Abstract The ability of unicellular micro-organisms to synchronize their behaviour at the population level through quorum sensing (QS) is now recognized as a common adaptive response to environmental challenges. QS enables bacterial populations to mount co-operative responses that improve access to nutrients or particular environmental niches, promote collective defence against other competitor prokaryotes or higher organisms and facilitate survival through differentiation into morphological forms better able to combat environmental threats. Through QS, bacterial populations can modify their nature and dynamics, and act as a community accomplishing tasks that individual bacterial cells would find impossible to achieve. Although we now appreciate that QS signal molecules are chemically diverse, their basic mechanism of action is conserved in that the structure of the QS signal molecule contains information that is directed by a ‘‘sender’’ cell/organism to a ‘‘receiver’’ cell/organism. Since QS often controls virulence and biofilm development, it is an attractive target for anti-infective agents particularly given the global health threat posed by multi-antibiotic resistant bacteria and the lack of new antibiotics entering the clinic. Other potential advantages of targeting QS include expansion of the repertoire of bacterial targets, preserving the host microflora and reducing the pressures that drive selection for resistance.
The common architecture of QS systems provides multiple molecular targets for agents that interfere with QS-mediated cell-to-cell communication, namely (i) the biosynthesis of the signal molecule by the ‘‘sender’’ cell, (ii) the functionality and availability of the signal itself, and (iii) the reception/decoding of the message contained in QS signal molecule by the ‘‘receiver’’ cell. With respect to the discovery of QS inhibitors (QSIs), an appreciation of the need to conserve steric requirements for optimal QS ligand/receptor/synthase interactions, has enabled QSIs to be readily discovered through structural modification of native QS signals. Bacterial strains originally developed as biosensors for QS signal molecules have proved useful as screens for QSIs from chemical libraries and natural products. Recent progress in exploiting our current knowledge of QS systems as targets for anti-infective agents will be explored for bacterial pathogens in general but in particular for Staphylococcus aureus.
ISQSI, Santiago de Compostela 2015 Keynote Lectures
1
The art of antibacterial warfare - Deception through interference with quorum sensing
Paul Williams
Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham, University Park, Nottingham, U.K. E-mail: [email protected]
Keywords: Quorum sensing inhibitors, Staphylococcus aureus, agr system, autoinducing peptides
Abstract The ability of unicellular micro-organisms to synchronize their behaviour at the population level through quorum sensing (QS) is now recognized as a common adaptive response to environmental challenges. QS enables bacterial populations to mount co-operative responses that improve access to nutrients or particular environmental niches, promote collective defence against other competitor prokaryotes or higher organisms and facilitate survival through differentiation into morphological forms better able to combat environmental threats. Through QS, bacterial populations can modify their nature and dynamics, and act as a community accomplishing tasks that individual bacterial cells would find impossible to achieve. Although we now appreciate that QS signal molecules are chemically diverse, their basic mechanism of action is conserved in that the structure of the QS signal molecule contains information that is directed by a ‘‘sender’’ cell/organism to a ‘‘receiver’’ cell/organism. Since QS often controls virulence and biofilm development, it is an attractive target for anti-infective agents particularly given the global health threat posed by multi-antibiotic resistant bacteria and the lack of new antibiotics entering the clinic. Other potential advantages of targeting QS include expansion of the repertoire of bacterial targets, preserving the host microflora and reducing the pressures that drive selection for resistance.
The common architecture of QS systems provides multiple molecular targets for agents that interfere with QS-mediated cell-to-cell communication, namely (i) the biosynthesis of the signal molecule by the ‘‘sender’’ cell, (ii) the functionality and availability of the signal itself, and (iii) the reception/decoding of the message contained in QS signal molecule by the ‘‘receiver’’ cell. With respect to the discovery of QS inhibitors (QSIs), an appreciation of the need to conserve steric requirements for optimal QS ligand/receptor/synthase interactions, has enabled QSIs to be readily discovered through structural modification of native QS signals. Bacterial strains originally developed as biosensors for QS signal molecules have proved useful as screens for QSIs from chemical libraries and natural products. Recent progress in exploiting our current knowledge of QS systems as targets for anti-infective agents will be explored for bacterial pathogens in general but in particular for Staphylococcus aureus.
ISQSI, Santiago de Compostela 2015 Keynote Lectures
14
Regulatory aspects of chemical substances under REACH and Biocidal Products Regulation
Pilar Rodríguez Iglesias
European Chemicals Agency (ECHA). P.O. Box 400, FI-00121, Helsinki, Finland. E-mail: [email protected]
Keywords: REACH, chemicals, biocides, substance, ECHA
Abstract REACH is a regulation of the European Union (Regulation (EC) 1907/2006), adopted to improve the protection of human health and the environment from the risks that can be posed by chemicals, while enhancing the competitiveness of the EU chemicals industry. It also promotes alternative methods for the hazard assessment of substances in order to reduce the number of tests on animals.
In principle, REACH applies to all chemical substances (i.e. those used in industrial processes but also in our day-to-day lives). REACH places the burden of proof on companies. To comply with the regulation, companies must identify and manage the risks linked to the substances they manufacture and market in the EU. They have to demonstrate to ECHA how the substance can be safely used, and they must communicate the risk management measures to the users.
If the risks cannot be managed, authorities can restrict the use of substances in different ways. In the long run, the most hazardous substances should be substituted with less dangerous ones.
REACH stands for Registration, Evaluation, Authorisation and Restriction of Chemicals. It entered into force on 1 June 2007.
The Biocidal Products Regulation (BPR, Regulation (EU) 528/2012) concerns the placing on the market and use of biocidal products, which are used to protect humans, animals, materials or articles against harmful organisms like pests or bacteria, by the action of the active substances contained in the biocidal product. This regulation aims to improve the functioning of the biocidal products market in the EU, while ensuring a high level of protection for humans and the environment.
All biocidal products require an authorisation before they can be placed on the market, and the active substances contained in that biocidal product must be previously approved. There are, however, certain exceptions to this principle.
The BPR aims to harmonise the market at Union level, simplify the approval of active substances and authorisation of biocidal products, and introduce timelines for Member State evaluations, opinion-forming and decision-making. It also promotes the reduction of animal testing by introducing mandatory data sharing obligations and encouraging the use of alternative testing methods.
The regulation is applicable since 1 September 2013, with a transitional period for certain provisions.
ISQSI, Santiago de Compostela 2015
15
Invited lectures
ISQSI, Santiago de Compostela 2015 Keynote Lectures
14
Regulatory aspects of chemical substances under REACH and Biocidal Products Regulation
Pilar Rodríguez Iglesias
European Chemicals Agency (ECHA). P.O. Box 400, FI-00121, Helsinki, Finland. E-mail: [email protected]
Keywords: REACH, chemicals, biocides, substance, ECHA
Abstract REACH is a regulation of the European Union (Regulation (EC) 1907/2006), adopted to improve the protection of human health and the environment from the risks that can be posed by chemicals, while enhancing the competitiveness of the EU chemicals industry. It also promotes alternative methods for the hazard assessment of substances in order to reduce the number of tests on animals.
In principle, REACH applies to all chemical substances (i.e. those used in industrial processes but also in our day-to-day lives). REACH places the burden of proof on companies. To comply with the regulation, companies must identify and manage the risks linked to the substances they manufacture and market in the EU. They have to demonstrate to ECHA how the substance can be safely used, and they must communicate the risk management measures to the users.
If the risks cannot be managed, authorities can restrict the use of substances in different ways. In the long run, the most hazardous substances should be substituted with less dangerous ones.
REACH stands for Registration, Evaluation, Authorisation and Restriction of Chemicals. It entered into force on 1 June 2007.
The Biocidal Products Regulation (BPR, Regulation (EU) 528/2012) concerns the placing on the market and use of biocidal products, which are used to protect humans, animals, materials or articles against harmful organisms like pests or bacteria, by the action of the active substances contained in the biocidal product. This regulation aims to improve the functioning of the biocidal products market in the EU, while ensuring a high level of protection for humans and the environment.
All biocidal products require an authorisation before they can be placed on the market, and the active substances contained in that biocidal product must be previously approved. There are, however, certain exceptions to this principle.
The BPR aims to harmonise the market at Union level, simplify the approval of active substances and authorisation of biocidal products, and introduce timelines for Member State evaluations, opinion-forming and decision-making. It also promotes the reduction of animal testing by introducing mandatory data sharing obligations and encouraging the use of alternative testing methods.
The regulation is applicable since 1 September 2013, with a transitional period for certain provisions.
ISQSI, Santiago de Compostela 2015
15
Invited lectures
ISQSI, Santiago de Compostela 2015 Invited lectures
17
Silencing quorum sensing in Pseudomonas aeruginosa
Miguel Cámara
Centre of Biomolecular Sciences, School of Life Sciences, University of Nottingham, Nottingham NG7 2RD, United Kingdom. E-mail: [email protected]
Keywords: Pseudomonas aeruginosa, PQS, alkylquinolones, biofilms, antimicrobials, peptide nucleic acids
Abstract Pseudomonas aeruginosa is a major opportunistic pathogen in cystic fibrosis, wound and nosocomial infections due to its high levels of intrinsic and acquired resistance to antibiotics, presenting a serious problem to the public health systems. This organism can adapt to many different environments using highly intricate regulatory networks. Quorum sensing (QS) plays a central role in this adaptation processes and hence is key for the success of this organism during infection regulating the production of many virulence traits. P. aeruginosa has several quorum sensing systems including the LasR/I and RhlR/I systems and their cognate signal molecules, N-(3-oxododecanoyl)-L-homoserine lactone and N-butanoyl-L-homoserine lactone respectively. P. aeruginosa also produces 2-alkyl-4-quinolones (AQ) signal molecules as QS molecules. The major P. aeruginosa AQ signal molecules are 2-heptyl-4-quinolone (HHQ) and 2-heptyl-3-hydroxy-4-quinolone (PQS). These QS systems play an important role in biofilm formation and their resistance to antibiotics. We have shown they are active in vivo during human infection, especially in the CF lung, and can interfere with host responses to infection.
For the above reasons, QS in P. aeruginosa represents a target for the development of novel antibacterials which can attenuate bacterial virulence so that the pathogen fails to adapt to the host environment. Interference with either the synthesis or the transduction of a QS signal is core to this attenuation. We are currently investigating several ways to inhibit QS in P. aeruginosa. These include: (i) searching for molecules which can antagonise the interaction of PQS/HHQ with their cognate regulatory protein PqsR and (ii) using antisense PNAs (Peptide Nucleic Acids) which prevent the translation of pqsA, the first gene of the pqsA-E operon involved in the biosynthesis of AQ and their response. Using these two approaches we have identified QS inhibitors which can attenuate the virulence of P. aeruginosa and most importantly sensitise biofilms to antibiotics. Details of these studies and their potential impact in the clinic will be present.
ISQSI, Santiago de Compostela 2015 Invited lectures
17
Silencing quorum sensing in Pseudomonas aeruginosa
Miguel Cámara
Centre of Biomolecular Sciences, School of Life Sciences, University of Nottingham, Nottingham NG7 2RD, United Kingdom. E-mail: [email protected]
Keywords: Pseudomonas aeruginosa, PQS, alkylquinolones, biofilms, antimicrobials, peptide nucleic acids
Abstract Pseudomonas aeruginosa is a major opportunistic pathogen in cystic fibrosis, wound and nosocomial infections due to its high levels of intrinsic and acquired resistance to antibiotics, presenting a serious problem to the public health systems. This organism can adapt to many different environments using highly intricate regulatory networks. Quorum sensing (QS) plays a central role in this adaptation processes and hence is key for the success of this organism during infection regulating the production of many virulence traits. P. aeruginosa has several quorum sensing systems including the LasR/I and RhlR/I systems and their cognate signal molecules, N-(3-oxododecanoyl)-L-homoserine lactone and N-butanoyl-L-homoserine lactone respectively. P. aeruginosa also produces 2-alkyl-4-quinolones (AQ) signal molecules as QS molecules. The major P. aeruginosa AQ signal molecules are 2-heptyl-4-quinolone (HHQ) and 2-heptyl-3-hydroxy-4-quinolone (PQS). These QS systems play an important role in biofilm formation and their resistance to antibiotics. We have shown they are active in vivo during human infection, especially in the CF lung, and can interfere with host responses to infection.
For the above reasons, QS in P. aeruginosa represents a target for the development of novel antibacterials which can attenuate bacterial virulence so that the pathogen fails to adapt to the host environment. Interference with either the synthesis or the transduction of a QS signal is core to this attenuation. We are currently investigating several ways to inhibit QS in P. aeruginosa. These include: (i) searching for molecules which can antagonise the interaction of PQS/HHQ with their cognate regulatory protein PqsR and (ii) using antisense PNAs (Peptide Nucleic Acids) which prevent the translation of pqsA, the first gene of the pqsA-E operon involved in the biosynthesis of AQ and their response. Using these two approaches we have identified QS inhibitors which can attenuate the virulence of P. aeruginosa and most importantly sensitise biofilms to antibiotics. Details of these studies and their potential impact in the clinic will be present.
ISQSI, Santiago de Compostela 2015 Invited lectures
18
Quorum sensing inhibition to control bacterial disease: aquaculture as an example
Tom Defoirdt
Laboratory of Aquaculture & Artemia Reference Center, Ghent University. Rozier 44, 9000 Gent, Belgium. E-mail: [email protected]
Keywords: Gnotobiotic models, quorum sensing inhibitors, AHL, AI-2, CAI-1, indole
Abstract Due to large-scale use of antibiotics, many pathogenic bacteria have acquired (multiple) resistance, and therefore, alternative treatments to control disease are needed. More than ten years ago, we proposed inhibition of quorum sensing, bacterial cell-to-cell communication, as a novel strategy to control infections caused by antibiotic-resistant bacteria in aquaculture.
In this presentation, I will discuss our current knowledge on the impact of quorum sensing and quorum sensing disruption on the virulence of aquaculture pathogens towards different host organisms. We found that the three-channel quorum sensing system of Vibrio harveyi regulates the expression of different virulence factors and the virulence of the bacterium towards gnotibiotic brine shrimp (Artemia franciscana) and giant river prawn (Macrobrachium rosenbergii) larvae. Remarkably, the impact of the three signal molecules on virulence of Vibrioharveyi was different for the two hosts, with acylhomoserine lactones (AHLs) playing no role in virulence towards brine shrimp and cholerae autoinducer-1 (CAI-1) playing no role in virulence towards river prawn. In contrast to what we observed in Vibrio harveyi, the three channel quorum sensing system of Vibrio anguillarum was found to play no role in virulence towards gnotobiotic sea bass (Dicentrarchus labrax) larvae. However, we found that another signalling molecule, indole, played a major role in virulence of this pathogen. Finally, we found that AHL quorum sensing controls the virulence of Aeromonas species towards larvae of the freshwater fish species burbot (Lota lota).
The most important quorum sensing-disrupting agents reported thus far include compounds that interfere with quorum sensing signal detection and signal transduction, and signal molecule-degrading bacteria. Quorum sensing-disrupting compounds studied in our laboratory include antagonistic AHL molecules, brominated furanones, brominated thiophenones and cinnamaldehyde. All of these compounds were found to increase survival of aquaculture animals when challenged to pathogenic bacteria. Furthermore, we found that metabolites produced by some micro-algae that are frequently used in aquaculture are also able to disrupt quorum sensing in Gram-negative bacteria. Finally, we found that signal molecule-degrading bacteria isolated from aquaculture settings have a positive effect on survival of challenged aquaculture animals, e.g. giant river prawn larvae challenged to Vibrio harveyi.
In conclusion, the data we obtained thus far indicate that quorum sensing disruption is a valid alternative biocontrol strategy for aquaculture, that biocontrol agents with quorum sensing-disrupting activity can be obtained from the aquatic environment and that these agents have a beneficial effect on cultured organisms.
ISQSI, Santiago de Compostela 2015 Invited lectures
19
Quorum quenching: actors, biological roles and applications
Yves Dessaux, Catherine Grandclément, Denis Faure
Department of Microbiology, Institute for integrative biology of the cell, CEA-CNRS-Université Paris-Sud, avenue de la terrasse, 91198 Gif sur Yvette, France.
E-mail: [email protected]
Keywords: quorum sensing inhibitor, quorum quenching enzyme, screening, metagenomics, biological control, soft-rot bacteria
Abstract Numerous bacterial populations are able to monitor their own population density and regulate the expression of some genes accordingly. This regulatory process is termed quorum sensing (QS). It relies upon the production of QS signal(s) that each individual cell of a given bacterial population produces. The overall concentration of the signal(s) therefore mimics the cell density of this population. Once a threshold concentration of the signal is perceived by a receptor, the QS-regulated genes are expressed or repressed.
Quorum quenching (QQ) refers to all processes involved in the disturbance of the quorum sensing. QQ molecular actors are diverse in nature (enzymes, chemical compounds), mode of action (QS-signal cleavage, site competition…), and targets as all the main steps of the QS pathway that are synthesis, accumulation and perception of the QS signals may be affected. Usually enzymes inactivating QS signals are named QQ enzymes, while (bio)chemicals disrupting QS-pathways are called QS inhibitors (or QSIs). Rather than listing all known enzymes and QSI molecules, the presentation will focus on a limited number of cases chosen to exemplify the variety of actors and origins. In relation, the occurrence of quenching functions in uncultivable micro-organisms will be discussed.
The biological role of the QQ phenomenon remains an intriguing - and to a certain extent unanswered - question. QSIs could be described as elements of a chemical warfare that involves organisms competing for the same ecological niche. This description most often results, however, from circumstantial observations and arguments. QQ enzymes could contribute to the recycling or the clearing (riddance) of the QS signals. However, in a well-documented case that implicates a QS molecule of the N-acyl homoserine lactone class, experimental results revealed that a microbial QQ enzyme may be involved in a detoxification process rather than in a fine tuning of QS-regulated functions.
Whatever the biological roles of QQ are, QSI molecules and QQ enzymes have been used in numerous exploratory applications in medicine, agriculture, environmental sciences and technology. Several examples of such applications have been or will be presented by others in the meeting. Because food production and preservation will become a critical parameter in the next decades, this presentation will mostly focus on applied outcomes in agriculture, and especially on the development of procedures that aim at countering plant-macerating pathogens of the Pectobacterium genus.
ISQSI, Santiago de Compostela 2015 Invited lectures
18
Quorum sensing inhibition to control bacterial disease: aquaculture as an example
Tom Defoirdt
Laboratory of Aquaculture & Artemia Reference Center, Ghent University. Rozier 44, 9000 Gent, Belgium. E-mail: [email protected]
Keywords: Gnotobiotic models, quorum sensing inhibitors, AHL, AI-2, CAI-1, indole
Abstract Due to large-scale use of antibiotics, many pathogenic bacteria have acquired (multiple) resistance, and therefore, alternative treatments to control disease are needed. More than ten years ago, we proposed inhibition of quorum sensing, bacterial cell-to-cell communication, as a novel strategy to control infections caused by antibiotic-resistant bacteria in aquaculture.
In this presentation, I will discuss our current knowledge on the impact of quorum sensing and quorum sensing disruption on the virulence of aquaculture pathogens towards different host organisms. We found that the three-channel quorum sensing system of Vibrio harveyi regulates the expression of different virulence factors and the virulence of the bacterium towards gnotibiotic brine shrimp (Artemia franciscana) and giant river prawn (Macrobrachium rosenbergii) larvae. Remarkably, the impact of the three signal molecules on virulence of Vibrioharveyi was different for the two hosts, with acylhomoserine lactones (AHLs) playing no role in virulence towards brine shrimp and cholerae autoinducer-1 (CAI-1) playing no role in virulence towards river prawn. In contrast to what we observed in Vibrio harveyi, the three channel quorum sensing system of Vibrio anguillarum was found to play no role in virulence towards gnotobiotic sea bass (Dicentrarchus labrax) larvae. However, we found that another signalling molecule, indole, played a major role in virulence of this pathogen. Finally, we found that AHL quorum sensing controls the virulence of Aeromonas species towards larvae of the freshwater fish species burbot (Lota lota).
The most important quorum sensing-disrupting agents reported thus far include compounds that interfere with quorum sensing signal detection and signal transduction, and signal molecule-degrading bacteria. Quorum sensing-disrupting compounds studied in our laboratory include antagonistic AHL molecules, brominated furanones, brominated thiophenones and cinnamaldehyde. All of these compounds were found to increase survival of aquaculture animals when challenged to pathogenic bacteria. Furthermore, we found that metabolites produced by some micro-algae that are frequently used in aquaculture are also able to disrupt quorum sensing in Gram-negative bacteria. Finally, we found that signal molecule-degrading bacteria isolated from aquaculture settings have a positive effect on survival of challenged aquaculture animals, e.g. giant river prawn larvae challenged to Vibrio harveyi.
In conclusion, the data we obtained thus far indicate that quorum sensing disruption is a valid alternative biocontrol strategy for aquaculture, that biocontrol agents with quorum sensing-disrupting activity can be obtained from the aquatic environment and that these agents have a beneficial effect on cultured organisms.
ISQSI, Santiago de Compostela 2015 Invited lectures
19
Quorum quenching: actors, biological roles and applications
Yves Dessaux, Catherine Grandclément, Denis Faure
Department of Microbiology, Institute for integrative biology of the cell, CEA-CNRS-Université Paris-Sud, avenue de la terrasse, 91198 Gif sur Yvette, France.
E-mail: [email protected]
Keywords: quorum sensing inhibitor, quorum quenching enzyme, screening, metagenomics, biological control, soft-rot bacteria
Abstract Numerous bacterial populations are able to monitor their own population density and regulate the expression of some genes accordingly. This regulatory process is termed quorum sensing (QS). It relies upon the production of QS signal(s) that each individual cell of a given bacterial population produces. The overall concentration of the signal(s) therefore mimics the cell density of this population. Once a threshold concentration of the signal is perceived by a receptor, the QS-regulated genes are expressed or repressed.
Quorum quenching (QQ) refers to all processes involved in the disturbance of the quorum sensing. QQ molecular actors are diverse in nature (enzymes, chemical compounds), mode of action (QS-signal cleavage, site competition…), and targets as all the main steps of the QS pathway that are synthesis, accumulation and perception of the QS signals may be affected. Usually enzymes inactivating QS signals are named QQ enzymes, while (bio)chemicals disrupting QS-pathways are called QS inhibitors (or QSIs). Rather than listing all known enzymes and QSI molecules, the presentation will focus on a limited number of cases chosen to exemplify the variety of actors and origins. In relation, the occurrence of quenching functions in uncultivable micro-organisms will be discussed.
The biological role of the QQ phenomenon remains an intriguing - and to a certain extent unanswered - question. QSIs could be described as elements of a chemical warfare that involves organisms competing for the same ecological niche. This description most often results, however, from circumstantial observations and arguments. QQ enzymes could contribute to the recycling or the clearing (riddance) of the QS signals. However, in a well-documented case that implicates a QS molecule of the N-acyl homoserine lactone class, experimental results revealed that a microbial QQ enzyme may be involved in a detoxification process rather than in a fine tuning of QS-regulated functions.
Whatever the biological roles of QQ are, QSI molecules and QQ enzymes have been used in numerous exploratory applications in medicine, agriculture, environmental sciences and technology. Several examples of such applications have been or will be presented by others in the meeting. Because food production and preservation will become a critical parameter in the next decades, this presentation will mostly focus on applied outcomes in agriculture, and especially on the development of procedures that aim at countering plant-macerating pathogens of the Pectobacterium genus.
ISQSI, Santiago de Compostela 2015 Invited lectures
20
AHLs and macrofouling within the marine environment Karen Tait1, Jon Havenhand2, Miguel Cámara3, Paul Williams3
1Plymouth Marine Laboratory, Plymouth, PL1 3DH, UK. 2 Department of Biological and Environmental Sciences, University of Gothenburg, Tjärnö, Sweden. 3 Centre for
Biomolecular Sciences, Nottingham University, Nottingham, NG7 2RD, UK. E-mail: [email protected]
Keywords: marine, biofouling, Ulva, acorn barnacle, settlement
Abstract Many reports have described enhanced settlement of invertebrate larvae and macro-algae on bacterial biofilms, yet few have demonstrated the nature of the cue responsible. Biofouling communities are dominated by N-acyl homoserine lactone (AHL) producing bacteria, with concentrations of ~ 600 pmol cm-2 detected in rocky shore biofilms. Previous research has shown AHLs enhance the settlement of zoospores of the green seaweed Ulva. Using cyprids of the acorn barnacle Balanus improvisus, the hypothesis that these organisms utilise AHLs as a cue in surface selection was tested. Biofilms of the bacteria Vibrio anguillarum, Aeromonas hydrophila and Sulfitobacter sp. attracted cyprids. However, when AHL production was inactivated, either by mutation of the AHL synthetic genes or by expression of an AHL-degrading gene (aiiA), the ability of the bacteria to attract was abolished. In addition, cyprids were attracted to biofilms of E. coli expressing recombinant AHL synthase genes, but not to E.coli that did not produce AHLs. Finally, synthetic AHLs resulted in increased cyprid settlement. Thus, similar to Ulva, the ecological success of a barnacle may also dependent on a bacterial signalling process. This strengthens the theory that AHLs are a universal cue for surface selection, influencing the development and functioning of biofouling communities.
ISQSI, Santiago de Compostela 2015 Invited lectures
21
Inhibiting Rgg pheromone receptors to disrupt quorum sensing in Gram-positive bacteria
Michael J. Federle
Department of Medicinal Chemistry and Pharmacognosy and the Center for Pharmaceutical Biotechnology, University of Illinois at Chicago.
E-mail: [email protected]
Abstract Gram-positive bacteria coordinate varieties of social behaviours, important to environmental, commensal, and pathogenic bacteria, through intercellular communication circuits that primarily utilize peptide signals. Rgg quorum-sensing pathways, widespread in the phylum Firmicutes, employ cytoplasmic pheromone receptors (Rgg transcription factors) that directly bind and elicit gene expression responses to imported peptide signals. In the human-restricted pathogen Streptococcus pyogenes, the Rgg2/Rgg3 regulatory circuit controls biofilm development and lysozyme resistance in response to the short hydrophobic peptides SHP2 and SHP3. We have identified specific environmental factors, likely encountered in vivo, that stimulate pheromone production. Using Rgg-SHP as a model receptor-ligand target, we sought to identify chemical compounds that specifically inhibited Rgg quorum-sensing circuits. Individual compounds from a diverse library of known drugs and drug-like molecules were screened for their ability to disrupt Rgg:FITC-SHP complexes using a fluorescence-polarization (FP) assay. The best hits were found to bind Rgg3 in vitro with sub-micromolar affinities, specifically abolished transcription of Rgg2/3-controlled genes, and prevented biofilm development in S. pyogenes without affecting bacterial growth. Furthermore, the top hit from these screens, cyclosporin A, as well as a non-immunosuppressive analog, inhibited Rgg-SHP pathways in multiple species of Streptococcus. The X-ray crystal structures of a Streptococcus Rgg2 protein alone and in complex with cyclosporin A have been solved and will provide a structural template in the design of enhanced inhibitors. Finally, we have also identified a streptococcal protease, capable of degrading SHP pheromones and inhibiting quorum-sensing responses, and whose expression is under the control of a central virulence regulator. Efforts to understand the underlying biology for which quorum sensing provides benefits to S. pyogenes, together with approaches that identify means to manipulate quorum-sensing pathways, has provided important perspective in the development of QS inhibitors and consideration for how therapeutics might be applied to avoid unintended consequences.
ISQSI, Santiago de Compostela 2015 Invited lectures
20
AHLs and macrofouling within the marine environment Karen Tait1, Jon Havenhand2, Miguel Cámara3, Paul Williams3
1Plymouth Marine Laboratory, Plymouth, PL1 3DH, UK. 2 Department of Biological and Environmental Sciences, University of Gothenburg, Tjärnö, Sweden. 3 Centre for
Biomolecular Sciences, Nottingham University, Nottingham, NG7 2RD, UK. E-mail: [email protected]
Keywords: marine, biofouling, Ulva, acorn barnacle, settlement
Abstract Many reports have described enhanced settlement of invertebrate larvae and macro-algae on bacterial biofilms, yet few have demonstrated the nature of the cue responsible. Biofouling communities are dominated by N-acyl homoserine lactone (AHL) producing bacteria, with concentrations of ~ 600 pmol cm-2 detected in rocky shore biofilms. Previous research has shown AHLs enhance the settlement of zoospores of the green seaweed Ulva. Using cyprids of the acorn barnacle Balanus improvisus, the hypothesis that these organisms utilise AHLs as a cue in surface selection was tested. Biofilms of the bacteria Vibrio anguillarum, Aeromonas hydrophila and Sulfitobacter sp. attracted cyprids. However, when AHL production was inactivated, either by mutation of the AHL synthetic genes or by expression of an AHL-degrading gene (aiiA), the ability of the bacteria to attract was abolished. In addition, cyprids were attracted to biofilms of E. coli expressing recombinant AHL synthase genes, but not to E.coli that did not produce AHLs. Finally, synthetic AHLs resulted in increased cyprid settlement. Thus, similar to Ulva, the ecological success of a barnacle may also dependent on a bacterial signalling process. This strengthens the theory that AHLs are a universal cue for surface selection, influencing the development and functioning of biofouling communities.
ISQSI, Santiago de Compostela 2015 Invited lectures
21
Inhibiting Rgg pheromone receptors to disrupt quorum sensing in Gram-positive bacteria
Michael J. Federle
Department of Medicinal Chemistry and Pharmacognosy and the Center for Pharmaceutical Biotechnology, University of Illinois at Chicago.
E-mail: [email protected]
Abstract Gram-positive bacteria coordinate varieties of social behaviours, important to environmental, commensal, and pathogenic bacteria, through intercellular communication circuits that primarily utilize peptide signals. Rgg quorum-sensing pathways, widespread in the phylum Firmicutes, employ cytoplasmic pheromone receptors (Rgg transcription factors) that directly bind and elicit gene expression responses to imported peptide signals. In the human-restricted pathogen Streptococcus pyogenes, the Rgg2/Rgg3 regulatory circuit controls biofilm development and lysozyme resistance in response to the short hydrophobic peptides SHP2 and SHP3. We have identified specific environmental factors, likely encountered in vivo, that stimulate pheromone production. Using Rgg-SHP as a model receptor-ligand target, we sought to identify chemical compounds that specifically inhibited Rgg quorum-sensing circuits. Individual compounds from a diverse library of known drugs and drug-like molecules were screened for their ability to disrupt Rgg:FITC-SHP complexes using a fluorescence-polarization (FP) assay. The best hits were found to bind Rgg3 in vitro with sub-micromolar affinities, specifically abolished transcription of Rgg2/3-controlled genes, and prevented biofilm development in S. pyogenes without affecting bacterial growth. Furthermore, the top hit from these screens, cyclosporin A, as well as a non-immunosuppressive analog, inhibited Rgg-SHP pathways in multiple species of Streptococcus. The X-ray crystal structures of a Streptococcus Rgg2 protein alone and in complex with cyclosporin A have been solved and will provide a structural template in the design of enhanced inhibitors. Finally, we have also identified a streptococcal protease, capable of degrading SHP pheromones and inhibiting quorum-sensing responses, and whose expression is under the control of a central virulence regulator. Efforts to understand the underlying biology for which quorum sensing provides benefits to S. pyogenes, together with approaches that identify means to manipulate quorum-sensing pathways, has provided important perspective in the development of QS inhibitors and consideration for how therapeutics might be applied to avoid unintended consequences.
ISQSI, Santiago de Compostela 2015
2
Oral communications
ISQSI, Santiago de Compostela 2015
2
Oral communications
ISQSI, Santiago de Compostela 2015 Oral communications – session 1. Update on Quorum quenching enzymes
25
Metagenomes as potent resources for the identification of novel QQ genes with anti-biofilm activities
Wolfgang R. Streit
Abteilung für Mikrobiologie und Biotechnologie, Biozentrum Klein Flottbek, Universität Hamburg, Ohnhorststr. 18, D-22609 Hamburg, Germany.
E-mail: [email protected]
Keywords: QQ genes, metagenomes, single cell analyses
Abstract In many Gram-negative bacteria the expression of a number of virulence factors, as well as biofilm formation, are controlled by quorum sensing (QS). N-acylhomoserine lactones (AHLs) are an important class of signalling molecules involved in bacterial QS and in many pathogenic bacteria infection and host colonization are AHL-dependent. The AHL signalling molecules are subject to inactivation by hydrolases (EC 3) (i.e. N-acylhomoserine lactonases and acyl-homoserine-lactone acylases) but also to some extent by other enzymes. Here I will report on our efforts to identify AHL- hydrolysing enzymes from single bacterial isolates and from metagenomes for the inhibition of biofilm formation (1, 2). I will summarize metagenome screening technologies and give and overview on the enzyme diversity involved in QQ activities and their impact on various potent biofilm-forming organisms (3). Finally, I will highlight results from single cell analysis with respect to the expression and regulation of QQ genes in the Gram-negative and plant-associated soil bacterium Sinorhizobium fredii.
References 1. Bijtenhoorn P, Schipper C, Hornung C, Quitschau M, Grond S, Weiland N, Streit WR.
2011. BpiB05, a novel metagenome-derived hydrolase acting on N-acylhomoserine lactones. J Biotechnol 155:86-94.
2. Bijtenhoorn P, Mayerhofer H, Müller-Dieckmann J, Utpatel C, Schipper C, Hornung C, Szesny M, Grond S, Thürmer A, Brzuszkiewicz E, Daniel R, Dierking K, Schulenburg H, Streit WR. 2011. A novel metagenomic short-chain dehydrogenase/reductase attenuates Pseudomonas aeruginosa biofilm formation and virulence on Caenorhabditis elegans. PLoS One 6:e26278.
3. Kisch JM, Utpatel C, Hilterhaus L, Streit WR, Liese A. 2014. Pseudomonas aeruginosa biofilm growth inhibition on medical plastic materials by immobilized esterases and acylase. ChemBioChem 15:1911-1919.
ISQSI, Santiago de Compostela 2015 Oral communications – session 1. Update on Quorum quenching enzymes
25
Metagenomes as potent resources for the identification of novel QQ genes with anti-biofilm activities
Wolfgang R. Streit
Abteilung für Mikrobiologie und Biotechnologie, Biozentrum Klein Flottbek, Universität Hamburg, Ohnhorststr. 18, D-22609 Hamburg, Germany.
E-mail: [email protected]
Keywords: QQ genes, metagenomes, single cell analyses
Abstract In many Gram-negative bacteria the expression of a number of virulence factors, as well as biofilm formation, are controlled by quorum sensing (QS). N-acylhomoserine lactones (AHLs) are an important class of signalling molecules involved in bacterial QS and in many pathogenic bacteria infection and host colonization are AHL-dependent. The AHL signalling molecules are subject to inactivation by hydrolases (EC 3) (i.e. N-acylhomoserine lactonases and acyl-homoserine-lactone acylases) but also to some extent by other enzymes. Here I will report on our efforts to identify AHL- hydrolysing enzymes from single bacterial isolates and from metagenomes for the inhibition of biofilm formation (1, 2). I will summarize metagenome screening technologies and give and overview on the enzyme diversity involved in QQ activities and their impact on various potent biofilm-forming organisms (3). Finally, I will highlight results from single cell analysis with respect to the expression and regulation of QQ genes in the Gram-negative and plant-associated soil bacterium Sinorhizobium fredii.
References 1. Bijtenhoorn P, Schipper C, Hornung C, Quitschau M, Grond S, Weiland N, Streit WR.
2011. BpiB05, a novel metagenome-derived hydrolase acting on N-acylhomoserine lactones. J Biotechnol 155:86-94.
2. Bijtenhoorn P, Mayerhofer H, Müller-Dieckmann J, Utpatel C, Schipper C, Hornung C, Szesny M, Grond S, Thürmer A, Brzuszkiewicz E, Daniel R, Dierking K, Schulenburg H, Streit WR. 2011. A novel metagenomic short-chain dehydrogenase/reductase attenuates Pseudomonas aeruginosa biofilm formation and virulence on Caenorhabditis elegans. PLoS One 6:e26278.
3. Kisch JM, Utpatel C, Hilterhaus L, Streit WR, Liese A. 2014. Pseudomonas aeruginosa biofilm growth inhibition on medical plastic materials by immobilized esterases and acylase. ChemBioChem 15:1911-1919.
ISQSI, Santiago de Compostela 2015 Oral communications – session 1. Update on Quorum quenching enzymes
26
Screening of the quorum quenching activity in a metagenomic library from a hypersaline-soil sample taken in Rambla Salada
(Murcia, Spain) Marta Torres1,2, Stéphane Uroz3, Emilia Quesada1,2, Inmaculada Llamas1,2
1Departament of Microbiology, Faculty of Pharmacy, University of Granada, Granada, Spain. 2Institute of Biotechnology, Center for Biomedical Research, Health-Sciences Technology Park, Armilla, Spain. 3French National Institute for Agricultural Research
(INRA), Nancy, France. E-mail: [email protected]
Keywords: quorum quenching, metagenomics, hypersaline soil
Abstract Many microorganisms use the genetic regulatory mechanism known as quorum sensing (QS) to monitor their cell density and establish a coordinated behaviour. QS depends on the production and release of signal molecules or autoinducers to the extracellular medium where they accumulate until they reach a threshold concentration and control the expression of numerous genes (1).
Many important functions of microorganisms in nature are controlled by QS, and therefore many of the competing organisms have developed different strategies to interfere with these communication systems (2). These include the enzymatic inactivation of signal molecules, referred to as quorum quenching (QQ).
In recent years the QS and QQ mechanisms of numerous bacterial species have been described. Nevertheless, there is very little data concerning these systems of regulation in hypersaline environments, and the information is even less in uncultured halophilic microorganisms. Our research group described for the first time the QS communication in halophilic bacteria of the genus Halomonas (3) for the first time and later on in 43 members of the Halomonadaceae family. But there are not many studies on the disruption of QS in hypersaline habitats.
In this communication we present the construction of a metagenomic library of 2.5x105 clones from a hypersaline soil sample taken in Rambla Salada (Murcia, Spain). The screening of the QQ activity of the clones was performed using the biosensor Chromobacterium violaceum CV026. One clone was selected that allowed the degradation of a wide range of N-acylhomoserine lactone-type signal molecules.
References 1. Fuqua WC, Winans SC, Greenberg EP. 1994. QS in bacteria: the LuxR-LuxI family of cell
density-responsive transcriptional regulators. J Bacteriol 176:269-275.
2. Uroz S, Dessaux Y, Oger P. 2009. QS and QQ: the yin and yang of bacterial communication. ChemBioChem 10:205-216.
3. Llamas I, Quesada E, Martínez-Cánovas MJ, Gronquist M, Eberhard A, González JE. 2005. QS in halophilic bacteria: detection of N-acylhomoserine lactones in the exopolysaccharide-producing species of Halomonas. Extremophiles 9:333-341.
ISQSI, Santiago de Compostela 2015 Oral communications – session 1. Update on Quorum quenching enzymes
27
Inactivation of QS molecules by endophytic yeasts from sugarcane
Elisa V. Bertini1, A. Carolina del V. Leguina1, Lucía I. Castellanos de Figueroa1,2 Carlos G. Nieto-Peñalver1,2
1Planta Piloto de Procesos Industriales Microbiológicos (PROIMI – CONICET). Av. Belgrano y Pje. Caseros (4000), San Miguel de Tucumán, Tucumán, Argentina.
2Falcultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, Tucumán, Argentina. E-mail: [email protected]
Keywords: quorum sensing, quorum quenching, yeast, lactonase, acylase, sugarcane
Abstract Quorum sensing (QS) enables the microorganisms to coordinate the gene expression in response to bacterial population density. QS involves the production of small signal molecules called autoinducers, being N-acyl homoserine lactones (AHLs) one of the most studied in Gram-negative bacteria. Quorum quenching (QQ) is a process in which the QS systems are disrupted through the inhibition of the signal production, its enzymatic inactivation or the interference of the receptor. Lactonases, acylases and oxidoreductases that inactivate AHLs have been isolated from both Prokaryotes and Eukaryotes. QQ is considered as a potential way in which microorganisms interfere with the QS signalization of others that share the same ecological niche. The function in nature of endophytic yeasts has remained largely obscure. In particular, the QQ properties of these fungal microorganisms have not been studied. The overall objective of this work was to characterize the ecological function of the QQ activities of endophytic yeasts isolated from sugarcane (Saccharum officinarum) in the interactions with both pathogenic and beneficial bacteria. Three different genera of yeasts isolated from leaves, apoplastic and internode tissues of the LCP 85-384 variety of sugarcane, the most utilized in the Northwest of Argentina, showed AHL-inactivating activities. Among them, Rhodotorula sp. 7Apo1 was selected for a further characterization. This strain presented both lactonase and acylase-like enzymatic activities against both short- and long-chain AHLs. Cultivation of Rhodotorula sp. 7Apo1 in media supplemented with different combinations of QS molecules, including substituted and unsubstituted AHLs, showed that the presence of one molecule does not interfere with the inactivation of the other. In cocultures with the phytopathogen P. syringae pv. syringae, the yeast Rhodotorula sp. 7Apo1 could inactivate the AHLs produced. An endophytic bacterium synthesizing at least two AHLs was isolated from sugarcane and identified as Agrobacterium sp. 6N2. The yeast Rhodotorula sp. 7Apo1 could also partially inactivate the Agrobacterium sp. 6N2 QS molecules. These results suggest that QQ activity from endophytic yeasts could play a remarkable role modulating the bacterial QS systems of microorganisms colonizing the same niche.
Acknowledgement This work was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, PIP0257).
ISQSI, Santiago de Compostela 2015 Oral communications – session 1. Update on Quorum quenching enzymes
26
Screening of the quorum quenching activity in a metagenomic library from a hypersaline-soil sample taken in Rambla Salada
(Murcia, Spain) Marta Torres1,2, Stéphane Uroz3, Emilia Quesada1,2, Inmaculada Llamas1,2
1Departament of Microbiology, Faculty of Pharmacy, University of Granada, Granada, Spain. 2Institute of Biotechnology, Center for Biomedical Research, Health-Sciences Technology Park, Armilla, Spain. 3French National Institute for Agricultural Research
(INRA), Nancy, France. E-mail: [email protected]
Keywords: quorum quenching, metagenomics, hypersaline soil
Abstract Many microorganisms use the genetic regulatory mechanism known as quorum sensing (QS) to monitor their cell density and establish a coordinated behaviour. QS depends on the production and release of signal molecules or autoinducers to the extracellular medium where they accumulate until they reach a threshold concentration and control the expression of numerous genes (1).
Many important functions of microorganisms in nature are controlled by QS, and therefore many of the competing organisms have developed different strategies to interfere with these communication systems (2). These include the enzymatic inactivation of signal molecules, referred to as quorum quenching (QQ).
In recent years the QS and QQ mechanisms of numerous bacterial species have been described. Nevertheless, there is very little data concerning these systems of regulation in hypersaline environments, and the information is even less in uncultured halophilic microorganisms. Our research group described for the first time the QS communication in halophilic bacteria of the genus Halomonas (3) for the first time and later on in 43 members of the Halomonadaceae family. But there are not many studies on the disruption of QS in hypersaline habitats.
In this communication we present the construction of a metagenomic library of 2.5x105 clones from a hypersaline soil sample taken in Rambla Salada (Murcia, Spain). The screening of the QQ activity of the clones was performed using the biosensor Chromobacterium violaceum CV026. One clone was selected that allowed the degradation of a wide range of N-acylhomoserine lactone-type signal molecules.
References 1. Fuqua WC, Winans SC, Greenberg EP. 1994. QS in bacteria: the LuxR-LuxI family of cell
density-responsive transcriptional regulators. J Bacteriol 176:269-275.
2. Uroz S, Dessaux Y, Oger P. 2009. QS and QQ: the yin and yang of bacterial communication. ChemBioChem 10:205-216.
3. Llamas I, Quesada E, Martínez-Cánovas MJ, Gronquist M, Eberhard A, González JE. 2005. QS in halophilic bacteria: detection of N-acylhomoserine lactones in the exopolysaccharide-producing species of Halomonas. Extremophiles 9:333-341.
ISQSI, Santiago de Compostela 2015 Oral communications – session 1. Update on Quorum quenching enzymes
27
Inactivation of QS molecules by endophytic yeasts from sugarcane
Elisa V. Bertini1, A. Carolina del V. Leguina1, Lucía I. Castellanos de Figueroa1,2 Carlos G. Nieto-Peñalver1,2
1Planta Piloto de Procesos Industriales Microbiológicos (PROIMI – CONICET). Av. Belgrano y Pje. Caseros (4000), San Miguel de Tucumán, Tucumán, Argentina.
2Falcultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, Tucumán, Argentina. E-mail: [email protected]
Keywords: quorum sensing, quorum quenching, yeast, lactonase, acylase, sugarcane
Abstract Quorum sensing (QS) enables the microorganisms to coordinate the gene expression in response to bacterial population density. QS involves the production of small signal molecules called autoinducers, being N-acyl homoserine lactones (AHLs) one of the most studied in Gram-negative bacteria. Quorum quenching (QQ) is a process in which the QS systems are disrupted through the inhibition of the signal production, its enzymatic inactivation or the interference of the receptor. Lactonases, acylases and oxidoreductases that inactivate AHLs have been isolated from both Prokaryotes and Eukaryotes. QQ is considered as a potential way in which microorganisms interfere with the QS signalization of others that share the same ecological niche. The function in nature of endophytic yeasts has remained largely obscure. In particular, the QQ properties of these fungal microorganisms have not been studied. The overall objective of this work was to characterize the ecological function of the QQ activities of endophytic yeasts isolated from sugarcane (Saccharum officinarum) in the interactions with both pathogenic and beneficial bacteria. Three different genera of yeasts isolated from leaves, apoplastic and internode tissues of the LCP 85-384 variety of sugarcane, the most utilized in the Northwest of Argentina, showed AHL-inactivating activities. Among them, Rhodotorula sp. 7Apo1 was selected for a further characterization. This strain presented both lactonase and acylase-like enzymatic activities against both short- and long-chain AHLs. Cultivation of Rhodotorula sp. 7Apo1 in media supplemented with different combinations of QS molecules, including substituted and unsubstituted AHLs, showed that the presence of one molecule does not interfere with the inactivation of the other. In cocultures with the phytopathogen P. syringae pv. syringae, the yeast Rhodotorula sp. 7Apo1 could inactivate the AHLs produced. An endophytic bacterium synthesizing at least two AHLs was isolated from sugarcane and identified as Agrobacterium sp. 6N2. The yeast Rhodotorula sp. 7Apo1 could also partially inactivate the Agrobacterium sp. 6N2 QS molecules. These results suggest that QQ activity from endophytic yeasts could play a remarkable role modulating the bacterial QS systems of microorganisms colonizing the same niche.
Acknowledgement This work was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, PIP0257).
ISQSI, Santiago de Compostela 2015 Oral communications – session 1. Update on Quorum quenching enzymes
28
Aii20J, a novel thermo-stable lactonase from Tenacibaculum sp. strain 20J can quench AHL-mediated acid resistance in
E.coli
Celia Mayer1, Manuel Romero1,2 ,Andrea Muras1, Ana Otero1
1Department of Microbiology and Parasitology, Faculty of Biology-CIBUS, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain.
2 Preset address: School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, NG7 2RD Nottingham. UK. E-mail: [email protected]
Keywords: Quorum sensing, quorum quenching, lactonases, acid resistence, Escherichia coli
Abstract
Numerous human, animal and plant pathogenic bacteria coordinate important biological functions, including the expression of virulence factors, through a cell-density-dependent gene regulation system known as quorum sensing (QS). Since in the absence of QS the pathogenic capacity of the bacteria is highly impaired, the inhibition of quorum-sensing, also known as Quorum Quenching (QQ) constitutes a promising strategy for the control of bacterial pathogens. Recently, the marine bacterium Tenacibaculum sp. strain 20J was identified, presenting a very wide spectrum activity against N-acyl homoserine lactones (AHLs), the most common QS signals in Gram-negative pathogens.
The gene coding the enzyme responsible of the wide spectrum QQ activity from the marine bacterium Tenacibaculum sp. strain 20J was identified, cloned and overexpressed. A functional screening in a fosmid genomic library was performed to obtain the DNA sequence of the aii20J gene. A putative lactonase gene was identified by pyrosequencing the 40 kb insert in the only positive clone obtained in the library. The sequence codifies a novel metallo-β-lactamase of 286 amino acidic residues with less than 31% identity to AiiA-type AHL lactonases. The gene is present in several other strains of the genus Tenacibaculum. The purified enzyme has a broad substrate spectrum activity, degrading all the AHLs tested, with or without oxo- substitutions, with a certain preference against long-chain AHLs.
Besides being much more active and having a wider spectrum activity than the lactonase AiiA from Bacillus, Aii20J is highly thermo-resistant, and is not affected by protease K and D-chymotrypsin, being active at a wide range of pH, which makes it suitable as feed additive. Metal ions improve the hydrolytic activity of Aii20J due to its metallo-enzyme condition. In addition, purified enzyme does not interfere with the action of any of the β-lactam antibiotics and β-lactamases inhibitors tested.
Although E. coli does not produce AHLs, E. coli cells respond to the AHLs released by other bacterial species, activating, among other genes, the glutamate-dependent acid resistance system, which contribute to the survival of pathogenic E. coli in different acidic environments as specific foods and in the gastrointestinal tract. The addition of Aii20J to E. coli K-12, in which the acid-resistance system had been activated by exogenous addition of AHLs, resulted in a significant reduction of survival (50%) after exposure to acid environment. The strong and non-specific QQ activity of Aii20J, with clear advantages in comparison with the enzymes from Bacillus species and other known AHL-lactonases, confirm this enzyme as novel promising candidate anti-pathogenic agent with diverse applications.
ISQSI, Santiago de Compostela 2015 Oral communications – session 1. Update on Quorum quenching enzymes
29
Quorum quenching N-acyl homoserine lactonase from soil isolate Lysinibacillus sp. Gs50
Sneha Garge, Anuradha Nerurkar
Department of Microbiology and Biotechnology Centre, Faculty of Science, The Maharaja Sayajirao University of Baroda,Vadodara, India.
E-mail: [email protected]
Keywords: Quorum sensing, Quorum quenching, AHL lactonase, Lysinibacillus sp.
Abstract Quorum sensing is an example of cell-to-cell communication in bacteria and depends on the production, secretion and response to small, diffusible signal molecules called autoinducers which are Acyl homoserine Lactones (AHL) in Gram-negative bacteria. Lysinibacillus sp. Gs50 isolated form soil was found to be the best among 97 isolates screened for their AHL degradation ability but not able to utilise it as sole carbon source for growth. AHL-degrading enzymes such as lactonases have been widely characterized in Bacillus species but have not been yet reported in Lysinibacillus sp. Isolate Lysinibacillus sp.Gs50 treated AHL could be restored under acidic conditions indicated the presence of putative AHL lactonase in it. The isolate was able to degrade AHL of various acyl chain lengths. Furthermore, the enzyme was found to be localised into the membrane of the bacteria. To identify the gene responsible for AHL degradation, primers were designed against hypothetical protein Bsph_3377 [Lysinibacillus sphaericus C3-41] (NCBI Reference Sequence: YP_001698999.1) for the putative AHL lactonase. Using these primers gene was amplified and cloned into expression vector pET22b(+) which resulted in the expression of 35kDa size protein. The soft rot attenuation studies on potato by recombinant E.coli BL21 expressing the AHL lactonse against the plant pathogen Pectobacterium carotovorum carotovorum BR1 (PccBR1, laboratory isolate) resulted in significant decrease in maceration caused by PccBR1. Mass spectrum analysis to ascertain the mechanism of the enzyme are being conducted. Thus, this is the first report of the quorum quenching N-acyl homoserine lactonase from Lysinibacillus sp. and its application to quorum quenching as biocontrol mechanism against plant pathogen Pectobacterium carotovorum carotovorum BR1, the causal agent of soft rot.
ISQSI, Santiago de Compostela 2015 Oral communications – session 1. Update on Quorum quenching enzymes
28
Aii20J, a novel thermo-stable lactonase from Tenacibaculum sp. strain 20J can quench AHL-mediated acid resistance in
E.coli
Celia Mayer1, Manuel Romero1,2 ,Andrea Muras1, Ana Otero1
1Department of Microbiology and Parasitology, Faculty of Biology-CIBUS, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain.
2 Preset address: School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, NG7 2RD Nottingham. UK. E-mail: [email protected]
Keywords: Quorum sensing, quorum quenching, lactonases, acid resistence, Escherichia coli
Abstract
Numerous human, animal and plant pathogenic bacteria coordinate important biological functions, including the expression of virulence factors, through a cell-density-dependent gene regulation system known as quorum sensing (QS). Since in the absence of QS the pathogenic capacity of the bacteria is highly impaired, the inhibition of quorum-sensing, also known as Quorum Quenching (QQ) constitutes a promising strategy for the control of bacterial pathogens. Recently, the marine bacterium Tenacibaculum sp. strain 20J was identified, presenting a very wide spectrum activity against N-acyl homoserine lactones (AHLs), the most common QS signals in Gram-negative pathogens.
The gene coding the enzyme responsible of the wide spectrum QQ activity from the marine bacterium Tenacibaculum sp. strain 20J was identified, cloned and overexpressed. A functional screening in a fosmid genomic library was performed to obtain the DNA sequence of the aii20J gene. A putative lactonase gene was identified by pyrosequencing the 40 kb insert in the only positive clone obtained in the library. The sequence codifies a novel metallo-β-lactamase of 286 amino acidic residues with less than 31% identity to AiiA-type AHL lactonases. The gene is present in several other strains of the genus Tenacibaculum. The purified enzyme has a broad substrate spectrum activity, degrading all the AHLs tested, with or without oxo- substitutions, with a certain preference against long-chain AHLs.
Besides being much more active and having a wider spectrum activity than the lactonase AiiA from Bacillus, Aii20J is highly thermo-resistant, and is not affected by protease K and D-chymotrypsin, being active at a wide range of pH, which makes it suitable as feed additive. Metal ions improve the hydrolytic activity of Aii20J due to its metallo-enzyme condition. In addition, purified enzyme does not interfere with the action of any of the β-lactam antibiotics and β-lactamases inhibitors tested.
Although E. coli does not produce AHLs, E. coli cells respond to the AHLs released by other bacterial species, activating, among other genes, the glutamate-dependent acid resistance system, which contribute to the survival of pathogenic E. coli in different acidic environments as specific foods and in the gastrointestinal tract. The addition of Aii20J to E. coli K-12, in which the acid-resistance system had been activated by exogenous addition of AHLs, resulted in a significant reduction of survival (50%) after exposure to acid environment. The strong and non-specific QQ activity of Aii20J, with clear advantages in comparison with the enzymes from Bacillus species and other known AHL-lactonases, confirm this enzyme as novel promising candidate anti-pathogenic agent with diverse applications.
ISQSI, Santiago de Compostela 2015 Oral communications – session 1. Update on Quorum quenching enzymes
29
Quorum quenching N-acyl homoserine lactonase from soil isolate Lysinibacillus sp. Gs50
Sneha Garge, Anuradha Nerurkar
Department of Microbiology and Biotechnology Centre, Faculty of Science, The Maharaja Sayajirao University of Baroda,Vadodara, India.
E-mail: [email protected]
Keywords: Quorum sensing, Quorum quenching, AHL lactonase, Lysinibacillus sp.
Abstract Quorum sensing is an example of cell-to-cell communication in bacteria and depends on the production, secretion and response to small, diffusible signal molecules called autoinducers which are Acyl homoserine Lactones (AHL) in Gram-negative bacteria. Lysinibacillus sp. Gs50 isolated form soil was found to be the best among 97 isolates screened for their AHL degradation ability but not able to utilise it as sole carbon source for growth. AHL-degrading enzymes such as lactonases have been widely characterized in Bacillus species but have not been yet reported in Lysinibacillus sp. Isolate Lysinibacillus sp.Gs50 treated AHL could be restored under acidic conditions indicated the presence of putative AHL lactonase in it. The isolate was able to degrade AHL of various acyl chain lengths. Furthermore, the enzyme was found to be localised into the membrane of the bacteria. To identify the gene responsible for AHL degradation, primers were designed against hypothetical protein Bsph_3377 [Lysinibacillus sphaericus C3-41] (NCBI Reference Sequence: YP_001698999.1) for the putative AHL lactonase. Using these primers gene was amplified and cloned into expression vector pET22b(+) which resulted in the expression of 35kDa size protein. The soft rot attenuation studies on potato by recombinant E.coli BL21 expressing the AHL lactonse against the plant pathogen Pectobacterium carotovorum carotovorum BR1 (PccBR1, laboratory isolate) resulted in significant decrease in maceration caused by PccBR1. Mass spectrum analysis to ascertain the mechanism of the enzyme are being conducted. Thus, this is the first report of the quorum quenching N-acyl homoserine lactonase from Lysinibacillus sp. and its application to quorum quenching as biocontrol mechanism against plant pathogen Pectobacterium carotovorum carotovorum BR1, the causal agent of soft rot.
ISQSI, Santiago de Compostela 2015 Oral communications – session 1. Update on Quorum quenching enzymes
0
Genetic improvement of aiiA gene encoding N-acyl homoserine lactonase from Bacillus thuringiensis by Error-Prone PCR
N.J. Rueda1,2, M.O. Suarez1,2, N. Romero1, E. Correa3, S. Ordúz3, A. Flórez 1, A.M. Flórez A.M1
1Universidad de Santander, UDES; 2 Universidad Industrial de Santander, UIS; 3. Universidad Nacional sede Medellín. E-mail: [email protected]
Keywords: Quorum Sensing Inhibition, Lactonase, Bacillus thuringiensis, Error Prone PCR
Abstract Quorum Sensing Inhibition enzymes are a wide range of proteins with the capability of disrupting the communication signals within a microbial community and therefore, represents a promising strategy to fight bacterial infections. However, their activity is limited by recognition of the broad range of N- acyl homoserine lactones (AHLs). One way to reduce their spectrum and increase their biochemical properties is by directed evolution, which mimics the process of natural selection, to evolve proteins in order to increase their biotechnological potential. In this work, we used Error Prone-PCR to generate a library using aiiA gene from Bacillus thuringiensis strain 147-11516. From this library, 10 mutants were selected according to their activity with biosensors and four, 851, 852, 944 and 947 were selected. From these, mutant 852 showed activity towards C6-HSL, C8-HSL, C6-Oxo- HSL and a reduced but detectable activity towards C12-Oxo-HSL when it was evaluated with CV026 and NTL4 strains. The activity of mutant 852 with C8-HSL was 0.9 µmol/min-1/mg-1 followed by C6-oxo-HSL (0.8 µmol/min-1/mg-1) in the first 10 min of the reaction, when 10% of the substrate was consumed. In order to quantify the activity, C8-HSL was used to characterize the mutant by HPLC-MS. The catalytic activity was 100% with C8-HSL at 0.25 mM as optimal concentration of the substrate. However the catalytic activity was also detected between 0.25 mM and 25 mM, measured at pH 7 during 60 minutes at room temperature. The thermal stability towards C8-HSL was determined from 20°C to 70°C and its hydrolytic activity was detected in a pH range between 4 and 8. We found that this mutant exhibited 2.3 times more affinity to C8-HSL (Km= 8.8) than wild type (Km= 3.81). Sequencing of mutant 852 showed a mutated region 240KRVVECSRNIY250 and an insertion S251 at the C-terminal. In vitro essays have demonstrated that this mutant was capable to attenuate soft rot disease in Solanum tuberosum and Solanum quitoense produced by Pectobacterium carotovorum and crown gall formation by Agrobacterium tumefasciens in Daucus carota. According to these results, this mutant is promising for the inhibition of Quorum Sensing Systems in pathogens in which C8-HSL is the major communication molecule such as Burkholderia species.
ISQSI, Santiago de Compostela 2015 Oral communications – session 2.QSI and Gram negative pathogens. I. QSI in Pseudomonas
1
_
Bacterial degradation of the Pseudomonas quinolone signal PQS and identification of new quorum quenching enzymes
Christine Müller1, Franziska S. Birmes1, Christian Rückert2, Jörn Kalinowski2, Susanne Fetzner1
1Institute of Molecular Microbiology and Biotechnology, University of Münster, Corrensstrasse 3, D-48149 Münster, Germany; 2Center for Biotechnology (CeBiTec), Bielefeld University, D-33615 Bielefeld, Germany. E-mail: [email protected]
Keywords: Pseudomonas aeruginosa, alkylquinolone, HHQ, PQS, Rhodococcus erythropolis, PQS-cleaving dioxygenase
Abstract The opportunistic pathogen Pseudomonas aeruginosa besides N-acylhomoserine lactones (AHLs) employs the alkylquinolones PQS (Pseudomonas quinolone signal, 2-heptyl-3-hydroxy-4(1H)-quinolone) and HHQ (2-heptyl-4(1H)-quinolone) as quorum sensing signal molecules. Alkylquinolone signalling is involved in the control of virulence factor production, and it influences biofilm maturation (1). Whereas numerous enzymes acting on AHLs have been described, enzymes that specifically inactivate alkylquinolone signals have not been identified so far (2).
Rhodococcus erythropolis BG43 is able to degrade the signal molecules PQS and HHQ to anthranilate (3). Two candidate gene clusters possibly coding for the degradative enzymes were identified in the genome of strain BG43. Each cluster comprises genes coding for a putative hydrolase, a monooxygenase, and a ring-cleaving dioxygenase. Transcription of the genes is upregulated up to 10-fold by PQS. Heterologous gene expression studies and in vitro enzyme assays indicated (i) an NADH-dependent hydroxylation of HHQ to PQS by one of the monooxygenases, (ii) cleavage of PQS to N-octanoylanthranilate by both dioxygenases, and (iii) subsequent release of anthranilate mediated by both hydrolases. Most interestingly, genes that may confer the ability to convert HHQ and PQS are conserved in Mycobacterium fortuitum and among many strains of M. abscessus. Cell-free extracts of M. abscessus (DSM 44196) and M.fortuitum (DSM 46621) indeed catalysed HHQ and PQS conversion.
Heterologous expression of a gene from R. erythropolis BG43 coding for a PQS-cleaving enzyme in P. aeruginosa PAO1 significantly quenched the production of the virulence factors pyocyanin and rhamnolipids and reduced the synthesis of the siderophore pyoverdine. Thus, the toolbox of quorum quenching enzymes is expanded by new PQS dioxygenases.
References
1. Heeb S, Fletcher MP, Chhabra SR, Diggle SP, Williams P, Cámara M. 2011. Quinolones: from antibiotics to autoinducers. FEMS Microbiol Lett 35:247 274.
2. Fetzner S. 2014. Quorum quenching enzymes. J Biotechnol, doi: 10.1016/j.jbiotec.2014.09.001.
3. Müller C, Birmes FS, Niewerth H, Fetzner S. 2014. Conversion of the Pseudomonas aeruginosa quinolone signal and related alkylhydroxyquinolines by Rhodococcus sp. strain BG43. Appl Environ Microbiol 80:7266 7274.
ISQSI, Santiago de Compostela 2015 Oral communications – session 1. Update on Quorum quenching enzymes
0
Genetic improvement of aiiA gene encoding N-acyl homoserine lactonase from Bacillus thuringiensis by Error-Prone PCR
N.J. Rueda1,2, M.O. Suarez1,2, N. Romero1, E. Correa3, S. Ordúz3, A. Flórez 1, A.M. Flórez A.M1
1Universidad de Santander, UDES; 2 Universidad Industrial de Santander, UIS; 3. Universidad Nacional sede Medellín. E-mail: [email protected]
Keywords: Quorum Sensing Inhibition, Lactonase, Bacillus thuringiensis, Error Prone PCR
Abstract Quorum Sensing Inhibition enzymes are a wide range of proteins with the capability of disrupting the communication signals within a microbial community and therefore, represents a promising strategy to fight bacterial infections. However, their activity is limited by recognition of the broad range of N- acyl homoserine lactones (AHLs). One way to reduce their spectrum and increase their biochemical properties is by directed evolution, which mimics the process of natural selection, to evolve proteins in order to increase their biotechnological potential. In this work, we used Error Prone-PCR to generate a library using aiiA gene from Bacillus thuringiensis strain 147-11516. From this library, 10 mutants were selected according to their activity with biosensors and four, 851, 852, 944 and 947 were selected. From these, mutant 852 showed activity towards C6-HSL, C8-HSL, C6-Oxo- HSL and a reduced but detectable activity towards C12-Oxo-HSL when it was evaluated with CV026 and NTL4 strains. The activity of mutant 852 with C8-HSL was 0.9 µmol/min-1/mg-1 followed by C6-oxo-HSL (0.8 µmol/min-1/mg-1) in the first 10 min of the reaction, when 10% of the substrate was consumed. In order to quantify the activity, C8-HSL was used to characterize the mutant by HPLC-MS. The catalytic activity was 100% with C8-HSL at 0.25 mM as optimal concentration of the substrate. However the catalytic activity was also detected between 0.25 mM and 25 mM, measured at pH 7 during 60 minutes at room temperature. The thermal stability towards C8-HSL was determined from 20°C to 70°C and its hydrolytic activity was detected in a pH range between 4 and 8. We found that this mutant exhibited 2.3 times more affinity to C8-HSL (Km= 8.8) than wild type (Km= 3.81). Sequencing of mutant 852 showed a mutated region 240KRVVECSRNIY250 and an insertion S251 at the C-terminal. In vitro essays have demonstrated that this mutant was capable to attenuate soft rot disease in Solanum tuberosum and Solanum quitoense produced by Pectobacterium carotovorum and crown gall formation by Agrobacterium tumefasciens in Daucus carota. According to these results, this mutant is promising for the inhibition of Quorum Sensing Systems in pathogens in which C8-HSL is the major communication molecule such as Burkholderia species.
ISQSI, Santiago de Compostela 2015 Oral communications – session 2.QSI and Gram negative pathogens. I. QSI in Pseudomonas
1
_
Bacterial degradation of the Pseudomonas quinolone signal PQS and identification of new quorum quenching enzymes
Christine Müller1, Franziska S. Birmes1, Christian Rückert2, Jörn Kalinowski2, Susanne Fetzner1
1Institute of Molecular Microbiology and Biotechnology, University of Münster, Corrensstrasse 3, D-48149 Münster, Germany; 2Center for Biotechnology (CeBiTec), Bielefeld University, D-33615 Bielefeld, Germany. E-mail: [email protected]
Keywords: Pseudomonas aeruginosa, alkylquinolone, HHQ, PQS, Rhodococcus erythropolis, PQS-cleaving dioxygenase
Abstract The opportunistic pathogen Pseudomonas aeruginosa besides N-acylhomoserine lactones (AHLs) employs the alkylquinolones PQS (Pseudomonas quinolone signal, 2-heptyl-3-hydroxy-4(1H)-quinolone) and HHQ (2-heptyl-4(1H)-quinolone) as quorum sensing signal molecules. Alkylquinolone signalling is involved in the control of virulence factor production, and it influences biofilm maturation (1). Whereas numerous enzymes acting on AHLs have been described, enzymes that specifically inactivate alkylquinolone signals have not been identified so far (2).
Rhodococcus erythropolis BG43 is able to degrade the signal molecules PQS and HHQ to anthranilate (3). Two candidate gene clusters possibly coding for the degradative enzymes were identified in the genome of strain BG43. Each cluster comprises genes coding for a putative hydrolase, a monooxygenase, and a ring-cleaving dioxygenase. Transcription of the genes is upregulated up to 10-fold by PQS. Heterologous gene expression studies and in vitro enzyme assays indicated (i) an NADH-dependent hydroxylation of HHQ to PQS by one of the monooxygenases, (ii) cleavage of PQS to N-octanoylanthranilate by both dioxygenases, and (iii) subsequent release of anthranilate mediated by both hydrolases. Most interestingly, genes that may confer the ability to convert HHQ and PQS are conserved in Mycobacterium fortuitum and among many strains of M. abscessus. Cell-free extracts of M. abscessus (DSM 44196) and M.fortuitum (DSM 46621) indeed catalysed HHQ and PQS conversion.
Heterologous expression of a gene from R. erythropolis BG43 coding for a PQS-cleaving enzyme in P. aeruginosa PAO1 significantly quenched the production of the virulence factors pyocyanin and rhamnolipids and reduced the synthesis of the siderophore pyoverdine. Thus, the toolbox of quorum quenching enzymes is expanded by new PQS dioxygenases.
References
1. Heeb S, Fletcher MP, Chhabra SR, Diggle SP, Williams P, Cámara M. 2011. Quinolones: from antibiotics to autoinducers. FEMS Microbiol Lett 35:247 274.
2. Fetzner S. 2014. Quorum quenching enzymes. J Biotechnol, doi: 10.1016/j.jbiotec.2014.09.001.
3. Müller C, Birmes FS, Niewerth H, Fetzner S. 2014. Conversion of the Pseudomonas aeruginosa quinolone signal and related alkylhydroxyquinolines by Rhodococcus sp. strain BG43. Appl Environ Microbiol 80:7266 7274.
ISQSI, Santiago de Compostela 2015 Oral communications – session 2.QSI and Gram negative pathogens. I. QSI in Pseudomonas
2
Peptides that eradicate multidrug-resistant biofilms and protect against lethal Pseudomonas aeruginosa infections
César de la Fuente-Núñez1, Fany Reffuveille1, Gilles Brackman2, Tom Coenye2, Robert E. W. Hancock1
1Department of Microbiology and Immunology, Centre for Microbial Diseases and Immunity Research, 232-2259 Lower Mall Research Station, University of British
Columbia, Vancouver, BC, V6T 1Z4, Canada. 2Laboratory of Pharmaceutical Microbiology, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium
Keywords: Anti-biofilm, peptides, (p)ppGpp, synergy, antibiotics, animal models
Abstract Bacteria form multicellular communities known as biofilms that are estimated to cause at least 65% of all infections in humans and demonstrate a 10 to 1000-fold increase in adaptive resistance to conventional antibiotics. Currently, there are no available drugs that specifically target bacterial biofilms. Here, we identified synthetic L-, D- and retro-inverso peptides with potent anti-biofilm activity that worked by blocking the widespread bacterial second messenger (p)ppGpp, a signal involved in biofilm development. These peptides completely prevented biofilm formation and led to the eradication of mature biofilms in strains of both Gram-negative and Gram-positive bacterial pathogens including Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, methicillin resistant Staphylococcus aureus, Salmonella Typhimurium and Burkholderia cenocepacia. In addition, low levels of the peptide led to biofilm dispersal, while higher doses triggered cell death in biofilms. Moreover, these peptides showed strong synergy with conventional antibiotics, reducing the antibiotic concentrations required for complete biofilm inhibition by up to 64-fold. The most potent peptides were D-peptides, which protected invertebrates from lethal Pseudomonas aeruginosa infections. These peptides represent a novel approach against biofilm-related drug resistance.
ISQSI, Santiago de Compostela 2015 Oral communications – session 2.QSI and Gram negative pathogens. I. QSI in Pseudomonas
Protective effect of quorum quenching monoclonal antibodies in lethal Pseudomonas infection
Soumya Palliyil1, Christina Downham1, Ian Broadbent2, Keith Charlton3, Andrew J. Porter1
1Scottish Biologics Facility, University of Aberdeen, Foresterhill, Aberdeen, United Kingdom; 2Department of Management, Aberdeen Business School, Robert Gordon University, Aberdeen, United Kingdom; 3Scotia Biologics Ltd., Foresterhill, Aberdeen,
United Kingdom. E-mail: [email protected]
Keywords: P. aeruginosa, quorum sensing, homoserine lactones, monoclonal antibodies, phage display, immunotherapy
Abstract The quorum sensing molecule homoserine lactone (HSL) is considered as a difficult target to raise antibodies against, owing to its minimal antigenic properties. Using sheep immunisation and recombinant antibody technology, we have successfully developed a panel of sheep-mouse chimeric monoclonal antibodies (MAbs) which recognised HSL compounds with high sensitivity (nanomolar range) and cross-reactivity. In this study, immunomodulation of HSL molecules by MAbs was used as a novel approach to prevent P. aeruginosa infections and as tools to detect HSLs in bodily fluids as a possible first clue to an undiagnosed Gram-negative infection.
Sheep was immunised using a mixture of different HSL compounds to generate an immune response. Lead MAb clones generated from immunised sheep phage display library were tested in elastase and nematode slow killing assays to evaluate their quorum quenching activities. P.aeruginosa cultures were grown in human urine for 3 days and the culture supernatant along with HSL MAbs was used in a competition ELISA format to detect the presence of HSL compounds. For survival studies in mice, lead MAbs at 10 mg/kg or comparator drugs gentamicin and ciprofloxacin at 10 mg/kg or vehicle-only control were co-administered with P. aeruginosa PA058 inocula by intranasal route. A second treatment dose was administered intravenously 4 h post infection. Bacterial burden in the lung tissue was quantified 24 h post infection and survival was observed up to 7 days post infection.
The ability of lead MAbs to bind specifically to quorum sensing molecules has been demonstrated by a series of in vitro immunoassays and bioassays. In nematode slow killing assay, the survival rates of Caenorhabditis elegans treated with HSL-2 and HSL-4 MAbs over a period of 96 h increased from 15% to 53% and 60%, respectively. In a nonneutropenic lung model of mice infected with P. aeruginosa PA058, HSL-2 and HSL-4 MAb monotherapy demonstrated significant efficacy, prolonging survival by 83% and 67%, respectively. HSL mAbs retained functional recognition of its antigen in the presence of urine with very little reduction in sensitivity observed (IC50 value 4 nM in PBS vs 10 nM in urine).
Antibodies are an attractive method for controlling bacterial virulence by block quorum sensing signalling as these ‘antipathogenic’ drugs are less likely to develop resistance in bacteria compared to conventional antibiotics. Our HSL MAbs showed exceptional sensitivity for P. aeruginosa autoinducer compounds in biochemical assays. An immunoassay-based diagnostic system exploiting the high sensitivity of HSL MAbs could be developed to detect the presence of specific markers of infection (homoserine lactones) in bodily fluids such as urine. This improvement in MAb sensitivity is also believed to have had a corresponding positive impact on therapeutic efficacy in vivo, apparently blocking bacterial infection.
ISQSI, Santiago de Compostela 2015 Oral communications – session 2.QSI and Gram negative pathogens. I. QSI in Pseudomonas
2
Peptides that eradicate multidrug-resistant biofilms and protect against lethal Pseudomonas aeruginosa infections
César de la Fuente-Núñez1, Fany Reffuveille1, Gilles Brackman2, Tom Coenye2, Robert E. W. Hancock1
1Department of Microbiology and Immunology, Centre for Microbial Diseases and Immunity Research, 232-2259 Lower Mall Research Station, University of British
Columbia, Vancouver, BC, V6T 1Z4, Canada. 2Laboratory of Pharmaceutical Microbiology, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium
Keywords: Anti-biofilm, peptides, (p)ppGpp, synergy, antibiotics, animal models
Abstract Bacteria form multicellular communities known as biofilms that are estimated to cause at least 65% of all infections in humans and demonstrate a 10 to 1000-fold increase in adaptive resistance to conventional antibiotics. Currently, there are no available drugs that specifically target bacterial biofilms. Here, we identified synthetic L-, D- and retro-inverso peptides with potent anti-biofilm activity that worked by blocking the widespread bacterial second messenger (p)ppGpp, a signal involved in biofilm development. These peptides completely prevented biofilm formation and led to the eradication of mature biofilms in strains of both Gram-negative and Gram-positive bacterial pathogens including Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, methicillin resistant Staphylococcus aureus, Salmonella Typhimurium and Burkholderia cenocepacia. In addition, low levels of the peptide led to biofilm dispersal, while higher doses triggered cell death in biofilms. Moreover, these peptides showed strong synergy with conventional antibiotics, reducing the antibiotic concentrations required for complete biofilm inhibition by up to 64-fold. The most potent peptides were D-peptides, which protected invertebrates from lethal Pseudomonas aeruginosa infections. These peptides represent a novel approach against biofilm-related drug resistance.
ISQSI, Santiago de Compostela 2015 Oral communications – session 2.QSI and Gram negative pathogens. I. QSI in Pseudomonas
Protective effect of quorum quenching monoclonal antibodies in lethal Pseudomonas infection
Soumya Palliyil1, Christina Downham1, Ian Broadbent2, Keith Charlton3, Andrew J. Porter1
1Scottish Biologics Facility, University of Aberdeen, Foresterhill, Aberdeen, United Kingdom; 2Department of Management, Aberdeen Business School, Robert Gordon University, Aberdeen, United Kingdom; 3Scotia Biologics Ltd., Foresterhill, Aberdeen,
United Kingdom. E-mail: [email protected]
Keywords: P. aeruginosa, quorum sensing, homoserine lactones, monoclonal antibodies, phage display, immunotherapy
Abstract The quorum sensing molecule homoserine lactone (HSL) is considered as a difficult target to raise antibodies against, owing to its minimal antigenic properties. Using sheep immunisation and recombinant antibody technology, we have successfully developed a panel of sheep-mouse chimeric monoclonal antibodies (MAbs) which recognised HSL compounds with high sensitivity (nanomolar range) and cross-reactivity. In this study, immunomodulation of HSL molecules by MAbs was used as a novel approach to prevent P. aeruginosa infections and as tools to detect HSLs in bodily fluids as a possible first clue to an undiagnosed Gram-negative infection.
Sheep was immunised using a mixture of different HSL compounds to generate an immune response. Lead MAb clones generated from immunised sheep phage display library were tested in elastase and nematode slow killing assays to evaluate their quorum quenching activities. P.aeruginosa cultures were grown in human urine for 3 days and the culture supernatant along with HSL MAbs was used in a competition ELISA format to detect the presence of HSL compounds. For survival studies in mice, lead MAbs at 10 mg/kg or comparator drugs gentamicin and ciprofloxacin at 10 mg/kg or vehicle-only control were co-administered with P. aeruginosa PA058 inocula by intranasal route. A second treatment dose was administered intravenously 4 h post infection. Bacterial burden in the lung tissue was quantified 24 h post infection and survival was observed up to 7 days post infection.
The ability of lead MAbs to bind specifically to quorum sensing molecules has been demonstrated by a series of in vitro immunoassays and bioassays. In nematode slow killing assay, the survival rates of Caenorhabditis elegans treated with HSL-2 and HSL-4 MAbs over a period of 96 h increased from 15% to 53% and 60%, respectively. In a nonneutropenic lung model of mice infected with P. aeruginosa PA058, HSL-2 and HSL-4 MAb monotherapy demonstrated significant efficacy, prolonging survival by 83% and 67%, respectively. HSL mAbs retained functional recognition of its antigen in the presence of urine with very little reduction in sensitivity observed (IC50 value 4 nM in PBS vs 10 nM in urine).
Antibodies are an attractive method for controlling bacterial virulence by block quorum sensing signalling as these ‘antipathogenic’ drugs are less likely to develop resistance in bacteria compared to conventional antibiotics. Our HSL MAbs showed exceptional sensitivity for P. aeruginosa autoinducer compounds in biochemical assays. An immunoassay-based diagnostic system exploiting the high sensitivity of HSL MAbs could be developed to detect the presence of specific markers of infection (homoserine lactones) in bodily fluids such as urine. This improvement in MAb sensitivity is also believed to have had a corresponding positive impact on therapeutic efficacy in vivo, apparently blocking bacterial infection.
ISQSI, Santiago de Compostela 2015 Oral communications – session 2.QSI and Gram negative pathogens. I. QSI in Pseudomonas
4
Sonochemical coatings of acylase nanoparticles inhibit Pseudomonas aeruginosa biofilm formation and virulence
factors production Kristina Ivanova, Margarida M. Fernandes, Tzanko Tzanov
Group of Molecular and Industrial Biotechnology, Department of Chemical Engineering, Universitat Politècnica de Catalunya, Rambla Sant Nebridi 22, 08222, Terrassa, Spain.
E-mail: [email protected]
Keywords: quorum sensing inhibition, biofilms, catheters, acylase nanoparticles, antibiotic resistance
Abstract Many medically-relevant bacteria use quorum sensing (QS) signals to communicate and regulate the expression of various behaviours such as virulence factors production and biofilm formation. Bacterial cells within the biofilms established on medical devices or living tissues are more resistant to conventional antibiotics and host defenses mechanisms than their free counterparts. Currently there is an urgent need to develop novel strategies effective against drug resistant bacterial species in both planktonic and biofilm forms. Disruption of bacterial communication process through the enzymatic degradation of extracellular QS signals is an attractive alternative though to exert less selective pressure on bacterial population, consequently reducing the risk of resistance development. In this study, anti-QS nanoparticles (NPs) of acylase were in situ generated and immobilized on silicone catheters using high-intensity ultrasound. The NPs coated catheters were characterized by Fourier transform infrared analysis and scanning electron microscopy. The acylase NP coatings efficiently inhibited QS-regulated violacein pigment production in Chromobacterium violaceum and virulence factors secretion (e.g. pyocyanin, elastase and alkaline protease) of Pseudomonas aeruginosa. Moreover, when hybrid antibacterial NPs comprising acylase and gentamicin or penicillin at subinhibitory concentrations were simultaneously generated and deposited on the catheters, the developed coatings synergistically reduced the pathogenic Pseudomonas aeruginosa biofilm formation in both static and dynamic conditions. The biocompatibility analysis after 7 days of incubation with human cell fibroblasts (BJ-5ta cell line) did not show significant decrease of cells viability, suggesting the potential of the sonochemically coated silicone materials to control QS regulated biofilm occurrence and virulence factors expression.
ISQSI, Santiago de Compostela 2015 Oral communications – session 2.QSI and Gram negative pathogens. II. Other Gram negative pathogens
5
Utilizing AHL acylase to disrupt biofilm formation of hospital-acquired pathogen Acinetobacter nosocomialis M2
Putri Dwi Utari, Ronald van Merkerk, Wim J. Quax
Department of Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan, Groningen, The Netherlands. E-mail: [email protected]
Keywords: Acinetobacter nosocomialis M2, biofilm, AHL acylase, PvdQ, PvdQL 146W,Fβ24Y, QqaR
Abstract Biofilm formation is one of the most important virulence determinants in numerous pathogens, including Acinetobacter, bacteria that associated with nosocomial infection and multidrug resistant properties. Since quorum sensing regulated biofilm formation in this genus, utilization of quorum sensing inhibitors has been suggested as a potential antibiofilm strategy. One particular species, namely Acinetobacter nosocomialis strain M2 (previously known as Acinetobacter baumannii) produces wide range of signalling molecules, with 3-OH-C12-HSL as the primary AHL. The purpose of this study was to examine the efficacy of quorum quenching acylase enzymes in reducing biofilm formation of A. nosocomialis M2. Three acylases with different preference of AHLs substrate were used, namely PvdQ (prefer long chain AHLs, such as C10-C14 HSL), PvdQLα146W,Fβ24Y (engineered PvdQ, active towards C8) and QqaR (active towards C8-C14 HSL). Biofilm of A. nosocomialis M2 was grown in 96-well plate with or without addition of single acylase. The production of biofilm was measured with crystal violet staining method. Compare to control, up to 50% reduction was observed in the biofilm treated with AHL acylase. Interestingly, PvdQL 146W,Fβ24Y and QqaR shown to be the most effective in biofilm disruption.
Acknowledgement We are grateful to Dr. Paul Rather (Department of Microbiology and Immunology, Emory University School of Medicine, USA) for providing Acinetobacter nosocomialis M2. This study is funded by University of Groningen and Indonesian Directorate General of Higher Education.
ISQSI, Santiago de Compostela 2015 Oral communications – session 2.QSI and Gram negative pathogens. I. QSI in Pseudomonas
4
Sonochemical coatings of acylase nanoparticles inhibit Pseudomonas aeruginosa biofilm formation and virulence
factors production Kristina Ivanova, Margarida M. Fernandes, Tzanko Tzanov
Group of Molecular and Industrial Biotechnology, Department of Chemical Engineering, Universitat Politècnica de Catalunya, Rambla Sant Nebridi 22, 08222, Terrassa, Spain.
E-mail: [email protected]
Keywords: quorum sensing inhibition, biofilms, catheters, acylase nanoparticles, antibiotic resistance
Abstract Many medically-relevant bacteria use quorum sensing (QS) signals to communicate and regulate the expression of various behaviours such as virulence factors production and biofilm formation. Bacterial cells within the biofilms established on medical devices or living tissues are more resistant to conventional antibiotics and host defenses mechanisms than their free counterparts. Currently there is an urgent need to develop novel strategies effective against drug resistant bacterial species in both planktonic and biofilm forms. Disruption of bacterial communication process through the enzymatic degradation of extracellular QS signals is an attractive alternative though to exert less selective pressure on bacterial population, consequently reducing the risk of resistance development. In this study, anti-QS nanoparticles (NPs) of acylase were in situ generated and immobilized on silicone catheters using high-intensity ultrasound. The NPs coated catheters were characterized by Fourier transform infrared analysis and scanning electron microscopy. The acylase NP coatings efficiently inhibited QS-regulated violacein pigment production in Chromobacterium violaceum and virulence factors secretion (e.g. pyocyanin, elastase and alkaline protease) of Pseudomonas aeruginosa. Moreover, when hybrid antibacterial NPs comprising acylase and gentamicin or penicillin at subinhibitory concentrations were simultaneously generated and deposited on the catheters, the developed coatings synergistically reduced the pathogenic Pseudomonas aeruginosa biofilm formation in both static and dynamic conditions. The biocompatibility analysis after 7 days of incubation with human cell fibroblasts (BJ-5ta cell line) did not show significant decrease of cells viability, suggesting the potential of the sonochemically coated silicone materials to control QS regulated biofilm occurrence and virulence factors expression.
ISQSI, Santiago de Compostela 2015 Oral communications – session 2.QSI and Gram negative pathogens. II. Other Gram negative pathogens
5
Utilizing AHL acylase to disrupt biofilm formation of hospital-acquired pathogen Acinetobacter nosocomialis M2
Putri Dwi Utari, Ronald van Merkerk, Wim J. Quax
Department of Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan, Groningen, The Netherlands. E-mail: [email protected]
Keywords: Acinetobacter nosocomialis M2, biofilm, AHL acylase, PvdQ, PvdQL 146W,Fβ24Y, QqaR
Abstract Biofilm formation is one of the most important virulence determinants in numerous pathogens, including Acinetobacter, bacteria that associated with nosocomial infection and multidrug resistant properties. Since quorum sensing regulated biofilm formation in this genus, utilization of quorum sensing inhibitors has been suggested as a potential antibiofilm strategy. One particular species, namely Acinetobacter nosocomialis strain M2 (previously known as Acinetobacter baumannii) produces wide range of signalling molecules, with 3-OH-C12-HSL as the primary AHL. The purpose of this study was to examine the efficacy of quorum quenching acylase enzymes in reducing biofilm formation of A. nosocomialis M2. Three acylases with different preference of AHLs substrate were used, namely PvdQ (prefer long chain AHLs, such as C10-C14 HSL), PvdQLα146W,Fβ24Y (engineered PvdQ, active towards C8) and QqaR (active towards C8-C14 HSL). Biofilm of A. nosocomialis M2 was grown in 96-well plate with or without addition of single acylase. The production of biofilm was measured with crystal violet staining method. Compare to control, up to 50% reduction was observed in the biofilm treated with AHL acylase. Interestingly, PvdQL 146W,Fβ24Y and QqaR shown to be the most effective in biofilm disruption.
Acknowledgement We are grateful to Dr. Paul Rather (Department of Microbiology and Immunology, Emory University School of Medicine, USA) for providing Acinetobacter nosocomialis M2. This study is funded by University of Groningen and Indonesian Directorate General of Higher Education.
ISQSI, Santiago de Compostela 2015 Oral communications – session 2.QSI and Gram negative pathogens. II. Other Gram negative pathogens
6
Unchartering quorum quenching potential in Glycyrrhiza glabra for attenuation of quorum sensing mediated virulence of A.
baumannii Nidhi Bhargava1, Prince Sharma2, Neena Capalash1
1Department of Biotechnology, Panjab University, Chandigarh 160014, India.2Department of Microbiology, Panjab University, Chandigarh 160014, India.
E-mail: [email protected]
Keywords: Acinetobacter baumannii, Glycyrrhiza glabra, quorum sensing inhibition, biofilm, motility, superoxide dismutase, catalase
Abstract Increasing antibiotic resistance in emerging nosocomial pathogen, A. baumannii has necessitated the development of alternative therapies that can control virulence with least risk of resistance development in the pathogen. Plants are rich sources of diverse medicinal compounds with novel bioactivities.
This study aimed at investigation of quorum quenching potential in G. glabra. Bioactivity guided fractionation identified the flavonoid rich ethyl acetate extract as potentially active against quorum sensing signal (3-OH-C12-HSL) of A. baumannii. Quorum quenching activity in ethyl acetate extract of G. glabra was found at concentration ranging from 0.5 - 2.0 mg/ml, which did not affect the cell survival. Significant reduction in production of quorum sensing mediated biofilm formation and other virulence factors of A. baumannii such as motility and production of antioxidant enzymes viz-a-viz catalase and superoxide dismutase (SOD) was observed in presence of the extract. Quorum quenching activity in the extract was also found effective on P.aeruginosa as the viable cell counts of both A. baumannii and P. aeruginosa which show stable co-existence, significantly decreased on treatment of dual biofilm with the extract.
This is the first report on quorum quenching activity of G. glabra against A. baumannii which down regulated the expression of abaI with concomitant decrease in 3-OH-C12-HSL production.
ISQSI, Santiago de Compostela 2015 Oral communications – session 2.QSI and Gram negative pathogens. II. Other Gram negative pathogens
7
Insight into the effect of the quorum sensing inhibitor baicalin hydrate on Burkholderia cepacia complex biofilm susceptibility
Lisa Slachmuylders, Gilles Brackman, Tom Coenye
Laboratory of Pharmaceutical Microbiology, Ghent University, Ottergemse steenweg 460, Ghent, Belgium. E-mail: [email protected]
Keywords: Burkholderia cepacia complex, Biofilms, Antibiotics, Quorum sensing inhibition, Baicalin hydrate
Abstract Burkholderia cepacia complex (Bcc) strains are opportunistic pathogens, often causing a rapid decline in lung function in cystic fibrosis (CF) patients. Bcc strains are highly resistant to many antibacterial agents and have the ability to form biofilms. Biofilms are communities of cells embedded in a self-produced, highly hydrated matrix, and can also develop on biotic surfaces such as epithelial cells. Biofilm formation is associated with an increased tolerance to antibiotics, compared to planktonic cells. A possible alternative target to combat biofilm-related chronic infections, is the bacterial quorum sensing (QS) system. We have previously shown that the QS inhibitor (QSI) baicalin hydrate (BH), a flavonoid targeting the acyl homoserine lactone QS system in Bcc increases the susceptibility of Bcc biofilms towards tobramycin. However, several questions remained regarding this activity.
In this study, we first elucidated the mode of action of BH on the molecular level by applying RNA sequencing. Several genes were observed to be differentially regulated after treatment with BH. The observed results were investigated in more detail using appropriate phenotypic assays. Secondly, we evaluated the effect of different flavonoids and BH-analogues on QS and on Bcc biofilm susceptibility. As such, we identified the structural elements critical for their activity. Finally, we determined the spectrum of activity by investigating whether these flavonoids could increase the susceptibility of different Bcc strains against different antimicrobial agents with different modes of action.
In conclusion, by conducting this study, we not only defined how BH can be applied but also gained more insights into why BH affects biofilm susceptibility.
ISQSI, Santiago de Compostela 2015 Oral communications – session 2.QSI and Gram negative pathogens. II. Other Gram negative pathogens
6
Unchartering quorum quenching potential in Glycyrrhiza glabra for attenuation of quorum sensing mediated virulence of A.
baumannii Nidhi Bhargava1, Prince Sharma2, Neena Capalash1
1Department of Biotechnology, Panjab University, Chandigarh 160014, India.2Department of Microbiology, Panjab University, Chandigarh 160014, India.
E-mail: [email protected]
Keywords: Acinetobacter baumannii, Glycyrrhiza glabra, quorum sensing inhibition, biofilm, motility, superoxide dismutase, catalase
Abstract Increasing antibiotic resistance in emerging nosocomial pathogen, A. baumannii has necessitated the development of alternative therapies that can control virulence with least risk of resistance development in the pathogen. Plants are rich sources of diverse medicinal compounds with novel bioactivities.
This study aimed at investigation of quorum quenching potential in G. glabra. Bioactivity guided fractionation identified the flavonoid rich ethyl acetate extract as potentially active against quorum sensing signal (3-OH-C12-HSL) of A. baumannii. Quorum quenching activity in ethyl acetate extract of G. glabra was found at concentration ranging from 0.5 - 2.0 mg/ml, which did not affect the cell survival. Significant reduction in production of quorum sensing mediated biofilm formation and other virulence factors of A. baumannii such as motility and production of antioxidant enzymes viz-a-viz catalase and superoxide dismutase (SOD) was observed in presence of the extract. Quorum quenching activity in the extract was also found effective on P.aeruginosa as the viable cell counts of both A. baumannii and P. aeruginosa which show stable co-existence, significantly decreased on treatment of dual biofilm with the extract.
This is the first report on quorum quenching activity of G. glabra against A. baumannii which down regulated the expression of abaI with concomitant decrease in 3-OH-C12-HSL production.
ISQSI, Santiago de Compostela 2015 Oral communications – session 2.QSI and Gram negative pathogens. II. Other Gram negative pathogens
7
Insight into the effect of the quorum sensing inhibitor baicalin hydrate on Burkholderia cepacia complex biofilm susceptibility
Lisa Slachmuylders, Gilles Brackman, Tom Coenye
Laboratory of Pharmaceutical Microbiology, Ghent University, Ottergemse steenweg 460, Ghent, Belgium. E-mail: [email protected]
Keywords: Burkholderia cepacia complex, Biofilms, Antibiotics, Quorum sensing inhibition, Baicalin hydrate
Abstract Burkholderia cepacia complex (Bcc) strains are opportunistic pathogens, often causing a rapid decline in lung function in cystic fibrosis (CF) patients. Bcc strains are highly resistant to many antibacterial agents and have the ability to form biofilms. Biofilms are communities of cells embedded in a self-produced, highly hydrated matrix, and can also develop on biotic surfaces such as epithelial cells. Biofilm formation is associated with an increased tolerance to antibiotics, compared to planktonic cells. A possible alternative target to combat biofilm-related chronic infections, is the bacterial quorum sensing (QS) system. We have previously shown that the QS inhibitor (QSI) baicalin hydrate (BH), a flavonoid targeting the acyl homoserine lactone QS system in Bcc increases the susceptibility of Bcc biofilms towards tobramycin. However, several questions remained regarding this activity.
In this study, we first elucidated the mode of action of BH on the molecular level by applying RNA sequencing. Several genes were observed to be differentially regulated after treatment with BH. The observed results were investigated in more detail using appropriate phenotypic assays. Secondly, we evaluated the effect of different flavonoids and BH-analogues on QS and on Bcc biofilm susceptibility. As such, we identified the structural elements critical for their activity. Finally, we determined the spectrum of activity by investigating whether these flavonoids could increase the susceptibility of different Bcc strains against different antimicrobial agents with different modes of action.
In conclusion, by conducting this study, we not only defined how BH can be applied but also gained more insights into why BH affects biofilm susceptibility.
ISQSI, Santiago de Compostela 2015 Oral communications – session 2.QSI and Gram negative pathogens. II. Other Gram negative pathogens
8
Methacrylate-based polymers to inhibit bacterial quorum sensing
Eliana Cavaleiro1, Ana Sofia Duarte1, Ana Cristina Esteves1, António Correia1, Michael J.Whitcombe2 Elena V. Piletska2, Sergey A. Piletsky2, Iva Chianella3
1Department of Biology & CESAM, University of Aveiro, Aveiro, Portugal, 2Department of Chemistry, University of Leicester, Leicester, UK, 3 Centre for Biomedical
Engineering, SATM, Cranfield University, Cranfield, Beds, UK. E-mail: [email protected]
Keywords: Linear polymers, Quorum Sensing, Aeromonas hydrophila, Vibrio fischeri
Abstract Quorum sensing (QS) is a phenomenal system of cell-to-cell communication in bacteria. The bacterial gene expression is coordinate in response to environment fluctuations in cell-population density trough QS. Bacterial phenotypes such as pathogenesis, antibiotic production, bioluminescence, conjugation, sporulation, swarming, biofilm formation and the expression of several virulence factors are correlated with QS. In Gram-negative bacteria, the QS mechanism makes use of N-acyl homoserine lactones (AHLs) as signal molecules.
The aim of this work was the development of linear polymers able to adsorb AHLs which are involved in QS communication. Once the AHLs are confiscated from the environment, QS is disrupted and the virulence may be attenuated.
We have developed linear polymer using methyl methacrylate (MMA) as backbone monomer and as functional monomers the methacrylic acid and itaconic acid. The efficiency of these polymers was evaluated in Vibrio fischeri and Aeromonas hydrophila, QS-controlled phenotypes. Aeromonas hydrophila is considered as an emergent pathogen and is known to use the QS communication to synchronize its behaviour.
The results showed that V. fischeri and A. hydrophila QS-phenotypes were significantly affected by the linear polymers. Itaconic-acid linear polymers showed to be more effective than methacrylic acid one. The attenuation of QS was reverted by the extra-addition of AHLs. No cytotoxicity associated to the polymers was detected on a mammalian cell line.
Acknowledgement FCT financed Eliana Cavaleiro (SFRH/BD/47502/2008) and Ana Sofia Duarte (SFRH/BPD/46290/2008).
References: 1. Cavaleiro E, Duarte AS, Esteves AC, Correia A, Whitcombe MJ, Piletska EV, Piletsky
SA, Chianella I. 2015. Novel Linear Polymers Able to inhibit bacterial Quorum Sensing. Macromolecular Bioscience DOI: 10.1002/mabi.201400447
ISQSI, Santiago de Compostela 2015 Oral communications – session 2.QSI and Gram negative pathogens. II. Other Gram negative pathogens
9
AHL-degrading bacteria isolated from microalgae protect prawn larvae from Vibrio campbellii infection
Pande GSJ1, FMI Natrah2, U. Kumar1, P. Bossier1,T. Defoirdt1
1Lab of Aquaculture and Artemia Reference Center, Ghent University, Rozier 44, 9000 Ghent, Belgium, 2Laboratory of Marine Biotechnology, Universiti Putra Malaysia,
Serdang, Selangor, Malaysia. E-mail: [email protected]
Keywords: aquaculture, quorum quenching, quorum sensing
Abstract Biodegradation of quorum sensing (QS) molecules has been proposed as a method of anti-infective strategy to combat bacterial diseases in aquaculture. One of the most important aquaculture pathogens, Vibrio campbellii, contains a three-channel quorum sensing system with three different types of signal molecules (HAI-1, AI-2 and CAI-1, respectively) feeding a shared signal transduction cascade (1). HAI-1 is an AHL (N-acylated-L-homoserine lactone), the most common class of autoinducer used by Gram-negative bacteria. It has been reported that the HAI-1 and the AI-2 mediated channels of the Vibrio campbellii quorum sensing system are essential for full virulence to giant river prawn larvae (2).
In this study, we isolated AHL-degrading bacteria from microalgal cultures, Tetraselmis suecica and Chaetoceros muelleri, and tested their influence on algal growth and on survival of prawn larvae challenged with V. campbellii. We isolated 2 strains, Pseudomonas sp. NFMI-T and Bacillus sp. NFMI-C from Tetraselmis suecica and Chaetocheros muelleri, respectively. An N-hexanoyl-L-homoserine lactone (HHL) degradation assay showed that both isolates were able to degrade HHL. Both strains showed HHL degradation rates that were similar to those of Bacillus sp. strains isolated from the intestinal tract of shrimp and fish (3). Furthermore, we found that only a coculture of both isolates had an effect on the growth of axenic Tetraselmis suecica, whereas the mixture of both isolates resulted in a decreased chlorophyll fluorescence of axenic Chaetoceros muelleri. The isolates were still detected after 2 and 3 weeks in conventionalized microalgae culture, indicating that they can persist in open algal cultures. We also found that Bacillus sp. NFMI-C, but not Pseudomonas sp. NFMI-T, can inhibit quorum sensing-regulated luminescence of V. campbellii. Finally, addition of Bacillus sp. NFMI-C to the rearing water resulted in a significantly improved survival of larvae of the giant river prawn Macrobrachium rosenbergii when challenged with V. campbellii without any effect on larval growth. These results showed that the AHL-degrading bacteria isolated from microalgae could be useful as novel disease control strains for aquaculture.
References 1. Ruwandeepika HAD, Jayaweera TSP, Bhowmick PP, Karunasagar I, Bossier P,
Defoirdt T. 2012. Pathogenesis, virulence factors and virulence regulation of vibrios belonging to the Harveyi clade. Rev Aquacult 4:59-74. doi: 10.1111/j.1753-5131.2012.01061.x.
2. Pande GSJ, Natrah FMI, Sorgeloos P, Bossier P, Defoirdt T. 2013. The Vibrio harveyi quorum sensing signals have a different impact on virulence of the bacterium towards different hosts. Vet Microbiol 167:540-545. doi: 10.1016/j.vetmic.2013.08.021.
3. Defoirdt T, Thanh LD, Van Delsen B, De Schryver P, Sorgeloos P, Boon N, Bossier P. 2011. N-acylhomoserine lactone degrading Bacillus strains isolated from aquaculture animals. Aquacult 311:258-260. doi:10.1016/j.aquaculture.2010.11.046.
ISQSI, Santiago de Compostela 2015 Oral communications – session 2.QSI and Gram negative pathogens. II. Other Gram negative pathogens
8
Methacrylate-based polymers to inhibit bacterial quorum sensing
Eliana Cavaleiro1, Ana Sofia Duarte1, Ana Cristina Esteves1, António Correia1, Michael J.Whitcombe2 Elena V. Piletska2, Sergey A. Piletsky2, Iva Chianella3
1Department of Biology & CESAM, University of Aveiro, Aveiro, Portugal, 2Department of Chemistry, University of Leicester, Leicester, UK, 3 Centre for Biomedical
Engineering, SATM, Cranfield University, Cranfield, Beds, UK. E-mail: [email protected]
Keywords: Linear polymers, Quorum Sensing, Aeromonas hydrophila, Vibrio fischeri
Abstract Quorum sensing (QS) is a phenomenal system of cell-to-cell communication in bacteria. The bacterial gene expression is coordinate in response to environment fluctuations in cell-population density trough QS. Bacterial phenotypes such as pathogenesis, antibiotic production, bioluminescence, conjugation, sporulation, swarming, biofilm formation and the expression of several virulence factors are correlated with QS. In Gram-negative bacteria, the QS mechanism makes use of N-acyl homoserine lactones (AHLs) as signal molecules.
The aim of this work was the development of linear polymers able to adsorb AHLs which are involved in QS communication. Once the AHLs are confiscated from the environment, QS is disrupted and the virulence may be attenuated.
We have developed linear polymer using methyl methacrylate (MMA) as backbone monomer and as functional monomers the methacrylic acid and itaconic acid. The efficiency of these polymers was evaluated in Vibrio fischeri and Aeromonas hydrophila, QS-controlled phenotypes. Aeromonas hydrophila is considered as an emergent pathogen and is known to use the QS communication to synchronize its behaviour.
The results showed that V. fischeri and A. hydrophila QS-phenotypes were significantly affected by the linear polymers. Itaconic-acid linear polymers showed to be more effective than methacrylic acid one. The attenuation of QS was reverted by the extra-addition of AHLs. No cytotoxicity associated to the polymers was detected on a mammalian cell line.
Acknowledgement FCT financed Eliana Cavaleiro (SFRH/BD/47502/2008) and Ana Sofia Duarte (SFRH/BPD/46290/2008).
References: 1. Cavaleiro E, Duarte AS, Esteves AC, Correia A, Whitcombe MJ, Piletska EV, Piletsky
SA, Chianella I. 2015. Novel Linear Polymers Able to inhibit bacterial Quorum Sensing. Macromolecular Bioscience DOI: 10.1002/mabi.201400447
ISQSI, Santiago de Compostela 2015 Oral communications – session 2.QSI and Gram negative pathogens. II. Other Gram negative pathogens
9
AHL-degrading bacteria isolated from microalgae protect prawn larvae from Vibrio campbellii infection
Pande GSJ1, FMI Natrah2, U. Kumar1, P. Bossier1,T. Defoirdt1
1Lab of Aquaculture and Artemia Reference Center, Ghent University, Rozier 44, 9000 Ghent, Belgium, 2Laboratory of Marine Biotechnology, Universiti Putra Malaysia,
Serdang, Selangor, Malaysia. E-mail: [email protected]
Keywords: aquaculture, quorum quenching, quorum sensing
Abstract Biodegradation of quorum sensing (QS) molecules has been proposed as a method of anti-infective strategy to combat bacterial diseases in aquaculture. One of the most important aquaculture pathogens, Vibrio campbellii, contains a three-channel quorum sensing system with three different types of signal molecules (HAI-1, AI-2 and CAI-1, respectively) feeding a shared signal transduction cascade (1). HAI-1 is an AHL (N-acylated-L-homoserine lactone), the most common class of autoinducer used by Gram-negative bacteria. It has been reported that the HAI-1 and the AI-2 mediated channels of the Vibrio campbellii quorum sensing system are essential for full virulence to giant river prawn larvae (2).
In this study, we isolated AHL-degrading bacteria from microalgal cultures, Tetraselmis suecica and Chaetoceros muelleri, and tested their influence on algal growth and on survival of prawn larvae challenged with V. campbellii. We isolated 2 strains, Pseudomonas sp. NFMI-T and Bacillus sp. NFMI-C from Tetraselmis suecica and Chaetocheros muelleri, respectively. An N-hexanoyl-L-homoserine lactone (HHL) degradation assay showed that both isolates were able to degrade HHL. Both strains showed HHL degradation rates that were similar to those of Bacillus sp. strains isolated from the intestinal tract of shrimp and fish (3). Furthermore, we found that only a coculture of both isolates had an effect on the growth of axenic Tetraselmis suecica, whereas the mixture of both isolates resulted in a decreased chlorophyll fluorescence of axenic Chaetoceros muelleri. The isolates were still detected after 2 and 3 weeks in conventionalized microalgae culture, indicating that they can persist in open algal cultures. We also found that Bacillus sp. NFMI-C, but not Pseudomonas sp. NFMI-T, can inhibit quorum sensing-regulated luminescence of V. campbellii. Finally, addition of Bacillus sp. NFMI-C to the rearing water resulted in a significantly improved survival of larvae of the giant river prawn Macrobrachium rosenbergii when challenged with V. campbellii without any effect on larval growth. These results showed that the AHL-degrading bacteria isolated from microalgae could be useful as novel disease control strains for aquaculture.
References 1. Ruwandeepika HAD, Jayaweera TSP, Bhowmick PP, Karunasagar I, Bossier P,
Defoirdt T. 2012. Pathogenesis, virulence factors and virulence regulation of vibrios belonging to the Harveyi clade. Rev Aquacult 4:59-74. doi: 10.1111/j.1753-5131.2012.01061.x.
2. Pande GSJ, Natrah FMI, Sorgeloos P, Bossier P, Defoirdt T. 2013. The Vibrio harveyi quorum sensing signals have a different impact on virulence of the bacterium towards different hosts. Vet Microbiol 167:540-545. doi: 10.1016/j.vetmic.2013.08.021.
3. Defoirdt T, Thanh LD, Van Delsen B, De Schryver P, Sorgeloos P, Boon N, Bossier P. 2011. N-acylhomoserine lactone degrading Bacillus strains isolated from aquaculture animals. Aquacult 311:258-260. doi:10.1016/j.aquaculture.2010.11.046.
ISQSI, Santiago de Compostela 2015 Oral communications – session 2.QSI and Gram negative pathogens. II. Other Gram negative pathogens
40
Novel quorum sensing-disrupting thiophenones with a promising potential to treat vibriosis in aquaculture
Qian Yang1, Anne Aamdal-Scheie2, Tore Benneche3,Tom Defoirdt1
1Laboratory of Aquaculture & Artemia Reference Center, Ghent University, Belgium;2Department of Oral Microbiology, University of Oslo, Norway. 3Department of
Chemistry, University of Oslo, Norway. E-mail: [email protected]
Keywords: Vibrio harveyi, thiophenones, quorum sensing-disruption, bioluminescence, brine shrimp larvae, challenge test
Abstract The frequent and indiscriminate use of antibiotics in aquaculture has forced bacteria to develop resistance, and disease caused by antibiotic resistant pathogens is becoming a serious problem (1). Recent investigations have revealed that quorum sensing inhibition could be a promising alternative strategy to control disease caused by these bacteria. Brominated furanones are one of the most intensively studied classes of quorum sensing inhibitors (2). However, they are highly toxic to higher organisms. Brominated thiophenones, the sulphur analogues of brominated furanones, were recently reported to be more effective and less toxic than their furanone counterparts (3).
In this study, we determined the quorum sensing-disrupting activity, protective effect and toxicity of 20 novel brominated thiophenone compounds using our standardised gnotobiotic V. harveyi-brine shrimp model system. Results showed that most of the compounds could inhibit quorum sensing-regulated bioluminescence in V. harveyi, with the most active compounds inhibiting quorum sensing at a concentration of 0.25 µM. In vivo challenge tests with gnotobiotic brine shrimp larvae indicated that thiophenones protected the brine shrimp larvae from pathogenic V.harveyi at concentrations similar to those needed to block quorum sensing-regulated bioluminescence in vitro. Seven compounds offered a complete protection to brine shrimp larvae (no significant difference in survival when compared to unchallenged larvae). The in vivo challenge test also revealed that there was a clear relation between the structure of the thiophenones and their activity. Most of the thiophenones showed high toxicity to brine shrimp larvae at a concentration of 10 µM, and the length and position of the side-chain significantly affected the toxicity of thiophenones. Four thiophenones (TF125, TF307, TF346 and TF347) were considered to be highly promising in the treatment Vibrio disease, since they protected the brine shrimp larvae from the pathogen at 0.25 µM, whereas no severe toxicity was observed at 10 µM (the highest concentration tested).
References 1. Defoirdt T, Sorgeloos P, Bossier P. 2011. Alternatives to antibiotics for the control of
bacterial disease in aquaculture. Curr Opin Microbiol 14:251–258.
2. Manefield M, Rasmussen TB, Henzter M, Andersen JB, Steinberg P, Kjelleberg S, Givskov M. 2002. Halogenated furanones inhibit quorum sensing through accelerated LuxR turnover. Microbiology 148:1119–1127.
3. Defoirdt T, Benneche T, Brackman G, Coenye T, Sorgeloos P, Scheie AA. 2012. A quorum sensing-disrupting brominated thiophenone with a promising therapeutic potential to treat luminescent vibriosis. PLoS ONE 7:e41788.
ISQSI, Santiago de Compostela 2015 Oral communications – session 3.Interfering with AI 2
41
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Study and modulation of inter-species quorum sensing by AI-2 analogues
Osvaldo S. Ascenso,1 Fábio Rui,1,2 Ana Sofia Miguel,1 João C. Marques2, Karina B. Xavier,1,2 M. Rita Ventura1
1Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal, 2Instituto Gulbenkian de Ciência, 2780-156 Oeiras,
Portugal.
Keywords: AI-2, DPD, quorum sensing, AI-2 analogues, chemical synthesis
Abstract Autoinducer-2 (AI-2) is a signalling molecule for bacterial inter-species communication. Examples of quorum sensing regulated behaviours are biofilm formation, virulence-factor expression, antibiotic production and bioluminescence. Ultimately, the understanding of the molecular mechanisms that bacteria use to regulate their behaviours can lead to the development of new therapies to control bacterial infections, and also to develop biotechnological applications for the control of industrial scale production of beneficial bacterial products, such as antibiotics or recombinant proteins.
A synthesis of (S)-4,5-dihydroxypentane-2,3-dione (DPD, 1, Fig. 1), the precursor of AI-2, has been developed starting from methyl glycolate (1). Using the same synthetic strategy and starting from methyl (S)- and (R)-lactates, four new analogues have been prepared and tested (Fig. 1) (2).The new analogues had one more asymmetric center and the configuration of the new substituent exerted an important influence in its biological activity. Other analogues have been synthesised and tested for their quorum sensing activity, leading to useful structure/activity conclusions.
Studies towards the preparation of new DPD fluorescent markers, using different linkers, will also be presented. These new chemical tools are being used to identify novel AI-2-receptor proteins.
Figure 1: DPD and analogues synthesis.
References 1. Ascenso OS, Marques JC, Santos AR, Xavier KX, Ventura MR, Maycock CD. 2011.An
efficient synthesis of the precursor of AI-2, the signaling molecule for inter-species quorum sensing. Bioorg Med Chem 19:1236-1241.
2. Rui F, Marques JC, Miller ST, Maycock CD, Xavier KB, Ventura MR. 2012. Stereochemical diversity of AI-2 analogs modulates quorum sensing in Vibrio harveyi and Escherichia coli. Bioorg Med Chem 20:249-256.
ISQSI, Santiago de Compostela 2015 Oral communications – session 2.QSI and Gram negative pathogens. II. Other Gram negative pathogens
40
Novel quorum sensing-disrupting thiophenones with a promising potential to treat vibriosis in aquaculture
Qian Yang1, Anne Aamdal-Scheie2, Tore Benneche3,Tom Defoirdt1
1Laboratory of Aquaculture & Artemia Reference Center, Ghent University, Belgium;2Department of Oral Microbiology, University of Oslo, Norway. 3Department of
Chemistry, University of Oslo, Norway. E-mail: [email protected]
Keywords: Vibrio harveyi, thiophenones, quorum sensing-disruption, bioluminescence, brine shrimp larvae, challenge test
Abstract The frequent and indiscriminate use of antibiotics in aquaculture has forced bacteria to develop resistance, and disease caused by antibiotic resistant pathogens is becoming a serious problem (1). Recent investigations have revealed that quorum sensing inhibition could be a promising alternative strategy to control disease caused by these bacteria. Brominated furanones are one of the most intensively studied classes of quorum sensing inhibitors (2). However, they are highly toxic to higher organisms. Brominated thiophenones, the sulphur analogues of brominated furanones, were recently reported to be more effective and less toxic than their furanone counterparts (3).
In this study, we determined the quorum sensing-disrupting activity, protective effect and toxicity of 20 novel brominated thiophenone compounds using our standardised gnotobiotic V. harveyi-brine shrimp model system. Results showed that most of the compounds could inhibit quorum sensing-regulated bioluminescence in V. harveyi, with the most active compounds inhibiting quorum sensing at a concentration of 0.25 µM. In vivo challenge tests with gnotobiotic brine shrimp larvae indicated that thiophenones protected the brine shrimp larvae from pathogenic V.harveyi at concentrations similar to those needed to block quorum sensing-regulated bioluminescence in vitro. Seven compounds offered a complete protection to brine shrimp larvae (no significant difference in survival when compared to unchallenged larvae). The in vivo challenge test also revealed that there was a clear relation between the structure of the thiophenones and their activity. Most of the thiophenones showed high toxicity to brine shrimp larvae at a concentration of 10 µM, and the length and position of the side-chain significantly affected the toxicity of thiophenones. Four thiophenones (TF125, TF307, TF346 and TF347) were considered to be highly promising in the treatment Vibrio disease, since they protected the brine shrimp larvae from the pathogen at 0.25 µM, whereas no severe toxicity was observed at 10 µM (the highest concentration tested).
References 1. Defoirdt T, Sorgeloos P, Bossier P. 2011. Alternatives to antibiotics for the control of
bacterial disease in aquaculture. Curr Opin Microbiol 14:251–258.
2. Manefield M, Rasmussen TB, Henzter M, Andersen JB, Steinberg P, Kjelleberg S, Givskov M. 2002. Halogenated furanones inhibit quorum sensing through accelerated LuxR turnover. Microbiology 148:1119–1127.
3. Defoirdt T, Benneche T, Brackman G, Coenye T, Sorgeloos P, Scheie AA. 2012. A quorum sensing-disrupting brominated thiophenone with a promising therapeutic potential to treat luminescent vibriosis. PLoS ONE 7:e41788.
ISQSI, Santiago de Compostela 2015 Oral communications – session 3.Interfering with AI 2
41
_
Study and modulation of inter-species quorum sensing by AI-2 analogues
Osvaldo S. Ascenso,1 Fábio Rui,1,2 Ana Sofia Miguel,1 João C. Marques2, Karina B. Xavier,1,2 M. Rita Ventura1
1Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal, 2Instituto Gulbenkian de Ciência, 2780-156 Oeiras,
Portugal.
Keywords: AI-2, DPD, quorum sensing, AI-2 analogues, chemical synthesis
Abstract Autoinducer-2 (AI-2) is a signalling molecule for bacterial inter-species communication. Examples of quorum sensing regulated behaviours are biofilm formation, virulence-factor expression, antibiotic production and bioluminescence. Ultimately, the understanding of the molecular mechanisms that bacteria use to regulate their behaviours can lead to the development of new therapies to control bacterial infections, and also to develop biotechnological applications for the control of industrial scale production of beneficial bacterial products, such as antibiotics or recombinant proteins.
A synthesis of (S)-4,5-dihydroxypentane-2,3-dione (DPD, 1, Fig. 1), the precursor of AI-2, has been developed starting from methyl glycolate (1). Using the same synthetic strategy and starting from methyl (S)- and (R)-lactates, four new analogues have been prepared and tested (Fig. 1) (2).The new analogues had one more asymmetric center and the configuration of the new substituent exerted an important influence in its biological activity. Other analogues have been synthesised and tested for their quorum sensing activity, leading to useful structure/activity conclusions.
Studies towards the preparation of new DPD fluorescent markers, using different linkers, will also be presented. These new chemical tools are being used to identify novel AI-2-receptor proteins.
Figure 1: DPD and analogues synthesis.
References 1. Ascenso OS, Marques JC, Santos AR, Xavier KX, Ventura MR, Maycock CD. 2011.An
efficient synthesis of the precursor of AI-2, the signaling molecule for inter-species quorum sensing. Bioorg Med Chem 19:1236-1241.
2. Rui F, Marques JC, Miller ST, Maycock CD, Xavier KB, Ventura MR. 2012. Stereochemical diversity of AI-2 analogs modulates quorum sensing in Vibrio harveyi and Escherichia coli. Bioorg Med Chem 20:249-256.
ISQSI, Santiago de Compostela 2015 Oral communications – session 3.Interfering with AI 2
42
Inhibition of autoinducer-2 leads to gut microbiota modulation Hyunjoon Park1, Kyuyeon Lee1, Heuyn-Kil Shin2, Wilhelm Holzapfel1
1Department of Advanced Green Energy and Environment, and 2Department of Life Science, Handong Global University, Pohang 791-708, South Korea.
E-mail: [email protected]
Keywords: Quorum sensing, Autoinducer-2, AI-2 inhibition, Gut microbiota
Abstract Autoinducer-2 (AI-2) signalling is a universal communication system found in a wide variety of Gram-positive and Gram-negative bacteria. In theory, quorum systems do not affect microbial growth directly. However, these signalling systems have been reported to affect the expression of genes associated with corresponding adaptation to the environment. Recent studies suggest that the AI-2 signal system is even related to specific stress response. The human gastrointestinal ecosystem involves complex environmental stresses and bacterial interaction is well known. Consequently, we assumed that a change in the AI-2 universal quorum signalling status may cause significant modulation of the gut microbiota composition.
We incubated AI-2 signal molecules and their inhibitor with fresh human faecal samples dose dependently at 37°C for 8-16 hours. The DNA was then extracted to analyse changes in the bacterial community using qRT-PCR and next generation sequencing. The group treated with t purified AI-2 and its inhibitor molecules showed partial change of bacterial composition in the microbiota. Interestingly, AI-2 inhibitor treatment significantly modulated the bacterial community as compared to the control. Based on these results, we suggest that the targeting range of the AI-2 signal interaction are not limited only to viability-unrelated gene expression, and that AI-2 quorum sensing may be closely linked to bacterial interaction in the human gut ecosystem.
Acknowledgement This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2014R1A1A2059602), and supported by Global Ph.D. Fellowship Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2014H1A2A1020810).
ISQSI, Santiago de Compostela 2015 Oral communications – session 3.Interfering with AI 2
4
Autoinducer-2 quorum-sensing influences on bacterial growth under specific stress conditions
Kyuyeon Lee1, Hyunjoon Park1, Heuyn-Kil Shin2, Wilhelm Holzapfel1
1Advanced Green Energy and Environment institute. 2Department of Life Science, Handong Global University, Pohang 791-708, South Korea.
E-mail: [email protected]
Keywords: Quorum-Sensing, EHEC, luxS, Gastro-intestinal tract (GIT)
Abstract Bacteria use small autoinducer molecules to communicate both at intra-species and inter-species level by quorum sensing. One such cell density dependent signal system is the luxS-mediated universal quorum sensing using autoinducer-2 (AI-2) (1). Virulence of several pathogens is determined by an AI-2 system and is related to colonization and infection of the host. From this concept, numerous papers have suggested that AI-2 inhibition is an important strategy towards designing of a new antimicrobial agent.
Recent studies indicate that the AI-2 system is also involved in stress response (2). We observed the growth of enterohaemorrhagic Escherichia coli O157:H7 (EHEC) and its luxS-deficient strain under specific stress conditions, and we found significant differences in their growth rate in 0.6M NaCl. Interestingly, a luxS-deficient EHEC strain showed strong resistance to osmotic stress conditions compared to the wild-type strain. Transcriptional analysis showed that genes associated with extreme osmotic pressure were up-regulated in the wild-type strain, but in the luxS-deficient strain the genes were inactivated. Moreover, the same pattern was observed under gastro-intestinal tract (GIT) conditions.
These results suggest that AI-2 quorum sensing has a direct influence on bacterial growth under the specific conditions, especially in the gastro-intestinal tract. We confirmed that inhibition of AI-2 signal production improves the stress resistance of EHEC. Therefore, the environmental factors of target pathogens should be considered to provide available AI-2 quorum quenching strategy.
Acknowledgement This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF-2013R1A1A4A01012585).
References 1. Guo M, Gamby S, Zheng Y, Sintim HO. 2013. Small molecules inhibitors of AI-2 signaling
in bacteria: state-of-art and future perspectives for anti-quorum sensing agents. Int J Mol Sci 14:17694-17728.
2. Van Kessel JC, Julia C, Rutherford ST, Cong JP, Quinodoz S, Healy J, Bassler BL. 2015. Quorum sensing regulates the osmotic stress response in Vibrio harveyi. J. Bacteriol. 197:73-80.
ISQSI, Santiago de Compostela 2015 Oral communications – session 3.Interfering with AI 2
42
Inhibition of autoinducer-2 leads to gut microbiota modulation Hyunjoon Park1, Kyuyeon Lee1, Heuyn-Kil Shin2, Wilhelm Holzapfel1
1Department of Advanced Green Energy and Environment, and 2Department of Life Science, Handong Global University, Pohang 791-708, South Korea.
E-mail: [email protected]
Keywords: Quorum sensing, Autoinducer-2, AI-2 inhibition, Gut microbiota
Abstract Autoinducer-2 (AI-2) signalling is a universal communication system found in a wide variety of Gram-positive and Gram-negative bacteria. In theory, quorum systems do not affect microbial growth directly. However, these signalling systems have been reported to affect the expression of genes associated with corresponding adaptation to the environment. Recent studies suggest that the AI-2 signal system is even related to specific stress response. The human gastrointestinal ecosystem involves complex environmental stresses and bacterial interaction is well known. Consequently, we assumed that a change in the AI-2 universal quorum signalling status may cause significant modulation of the gut microbiota composition.
We incubated AI-2 signal molecules and their inhibitor with fresh human faecal samples dose dependently at 37°C for 8-16 hours. The DNA was then extracted to analyse changes in the bacterial community using qRT-PCR and next generation sequencing. The group treated with t purified AI-2 and its inhibitor molecules showed partial change of bacterial composition in the microbiota. Interestingly, AI-2 inhibitor treatment significantly modulated the bacterial community as compared to the control. Based on these results, we suggest that the targeting range of the AI-2 signal interaction are not limited only to viability-unrelated gene expression, and that AI-2 quorum sensing may be closely linked to bacterial interaction in the human gut ecosystem.
Acknowledgement This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2014R1A1A2059602), and supported by Global Ph.D. Fellowship Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2014H1A2A1020810).
ISQSI, Santiago de Compostela 2015 Oral communications – session 3.Interfering with AI 2
4
Autoinducer-2 quorum-sensing influences on bacterial growth under specific stress conditions
Kyuyeon Lee1, Hyunjoon Park1, Heuyn-Kil Shin2, Wilhelm Holzapfel1
1Advanced Green Energy and Environment institute. 2Department of Life Science, Handong Global University, Pohang 791-708, South Korea.
E-mail: [email protected]
Keywords: Quorum-Sensing, EHEC, luxS, Gastro-intestinal tract (GIT)
Abstract Bacteria use small autoinducer molecules to communicate both at intra-species and inter-species level by quorum sensing. One such cell density dependent signal system is the luxS-mediated universal quorum sensing using autoinducer-2 (AI-2) (1). Virulence of several pathogens is determined by an AI-2 system and is related to colonization and infection of the host. From this concept, numerous papers have suggested that AI-2 inhibition is an important strategy towards designing of a new antimicrobial agent.
Recent studies indicate that the AI-2 system is also involved in stress response (2). We observed the growth of enterohaemorrhagic Escherichia coli O157:H7 (EHEC) and its luxS-deficient strain under specific stress conditions, and we found significant differences in their growth rate in 0.6M NaCl. Interestingly, a luxS-deficient EHEC strain showed strong resistance to osmotic stress conditions compared to the wild-type strain. Transcriptional analysis showed that genes associated with extreme osmotic pressure were up-regulated in the wild-type strain, but in the luxS-deficient strain the genes were inactivated. Moreover, the same pattern was observed under gastro-intestinal tract (GIT) conditions.
These results suggest that AI-2 quorum sensing has a direct influence on bacterial growth under the specific conditions, especially in the gastro-intestinal tract. We confirmed that inhibition of AI-2 signal production improves the stress resistance of EHEC. Therefore, the environmental factors of target pathogens should be considered to provide available AI-2 quorum quenching strategy.
Acknowledgement This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF-2013R1A1A4A01012585).
References 1. Guo M, Gamby S, Zheng Y, Sintim HO. 2013. Small molecules inhibitors of AI-2 signaling
in bacteria: state-of-art and future perspectives for anti-quorum sensing agents. Int J Mol Sci 14:17694-17728.
2. Van Kessel JC, Julia C, Rutherford ST, Cong JP, Quinodoz S, Healy J, Bassler BL. 2015. Quorum sensing regulates the osmotic stress response in Vibrio harveyi. J. Bacteriol. 197:73-80.
ISQSI, Santiago de Compostela 2015 Oral communications – session 4.QSI and Gram positive pathogens
44
Structural analysis of NprR, a bifunctional quorum sensor of the Bacillus cereus group
Antoine Talagas1, Stéphane Perchat2, Didier Lereclus2, Sylvie Nessler1
1 Institute for Integrative Biology of the Cell (I2BC), Department of Biochemistry, Biophysics and Structural Biology (B3S), University Paris Sud, 91405 Orsay cedex,
France; 2 INRA, UMR1319 Micalis-AgroParisTech, 78352 Jouy-en-Josas cedex, France. E-mail: [email protected]
Keywords: regulation, peptide binding, functional switch, conformational change, molecular mechanism, crystal structure
Abstract In sporulating bacteria, the differentiation process is tuned by communication systems involving signalling peptides and regulators of the RNPP family. The defining members of this family (Rap, NprR, PrgX and PlcR) are involved in important bacterial processes such as sporulation and virulence and are considered as interesting targets for the development of new antibacterial agents. RNPP proteins are characterized by a TPR-type peptide-binding domain. Except for the Rap proteins, they also contain an N-terminal HTH-type DNA-binding domain and display a transcription factor activity.
At the onset of sporulation, the quorum sensor NprR interacts with its signalling peptide NprX and activates the expression of genes required for the necrotrophic lifestyle of B. thuringiensis (1). The NprR-NprX complex adopts a tetrameric structure allowing the specific binding of the protein on the DNA target sites (2), thus activating the transcription of the corresponding genes (3). In sharp contrast, in the absence of NprX, and independently of its transcriptional activator function, NprR negatively affects the sporulation process. A structural and mutational analysis of NprR allowed us to demonstrate that NprR prevents expression of Spo0A-regulated genes by acting on Spo0F. Direct interaction between NprR and Spo0F was demonstrated. The close structural relationship between NprR and the Rap proteins suggests that NprR has both a transcriptional regulator activity and a Rap-like function.
In conclusion, NprR is a dual-function protein regulated through a quorum-sensing system. We propose a molecular mechanism for the structural and functional switch induced by peptide binding.
References 1. Dubois T, Perchat S, Verplaetse E, Gominet M, Lemy C, Aumont-Nicaise M, Grenha R,
Nessler S, Lereclus D. 2013. Activity of the Bacillus thuringiensis NprR-NprX cell-cell communication system is co-ordinated to the physiological stage through a complex transcriptional regulation. Mol Microbiol. 88:48-63.
2. Zouhir S, Perchat S, Nicaise M, Perez J, Guimaraes B, Lereclus D, Nessler S. 2013. Peptide-binding dependent conformational changes regulate the transcriptional activity of the quorum-sens or NprR. Nucleic Acids Res 41:7920-7933.
3. Perchat S, Dubois T, Zouhir S, Gominet M, Poncet S, Lemy C, Aumont-Nicaise M, Deutscher J, Gohar M, Nessler S, Lereclus D. 2011. A cell-cell communication system regulates protease production during sporulation in bacteria of the Bacillus cereus group. Mol Microbiol 82:619-33.
ISQSI, Santiago de Compostela 2015 Oral communications – session 4.QSI and Gram positive pathogens
45
How does the quorum sensing inhibitor hamamelitannin increase Staphylococcus aureus biofilm susceptibility?
G. Brackman1, F. Van den Driessche1, T. Coenye1
1Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium. E-mail: [email protected]
Keywords: Biofilm, Quorum Sensing, hamamelitannin, Staphylococcus aureus
Abstract Biofilm-associated infections caused by Staphylococcus aureus are often very difficult to treat and novel targets are needed to combat these infections. We have previously shown that the quorum sensing (QS) modulator 2',5-di-O-galloyl-D-hamamelose (hamamelitannin, HAM) increases the susceptibility of S. aureus biofilms towards vancomycin (VAN) in vitro as well as invivo. However, the mechanism of action of HAM at the molecular level has not yet been elucidated.
Two parallel strategies were followed in order to gain insights in the way HAM affects QS. First, we evaluated the effect of HAM on the susceptibility of biofilms of S. aureus strains with mutations in the various QS systems (e.g. agrBCDA, traP, luxS) or in genes involved in biofilm formation and virulence (e.g. icaA, sarA, codY). Secondly, using Illumina sequencing we identified genes that were differentially expressed in untreated biofilms and biofilms treated with VAN alone or with the combination VAN+HAM. Results obtained with both strategies were further investigated using the appropriate tools.
No loss in HAM activity was observed for most of the mutants. In contrast, HAM did not affect biofilm susceptibility of S. aureus strains with mutations in agrA or traP gene. This suggests that these genes are involved in mediating the activity of HAM. Using RNA sequencing, we identified a large number of genes that were differentially expressed after treatment. Treatment with HAM (alone or in combination with VAN) resulted in a differential regulation of genes involved in biosynthesis of lysine, D-alanine, glutamine consuming pathways, and eDNA production and autolysis. In addition, the upregulation of genes encoding virulence factors (e.g. tst, hla, yent1, hlgB) during VAN treatment was not observed when VAN was combined with HAM.
HAM reduces the upregulation of peptidoglycan biosynthesis normally observed after treatment with VAN. In addition, less eDNA is present in the matrix of HAM treated biofilms. This possibly leads to the increased susceptibility of S. aureus biofilm cells towards VAN. Our results further indicate that combination therapy could positively affect morbidity since the upregulation of virulence factors observed for VAN treatment are not observed when VAN is combined with HAM.
ISQSI, Santiago de Compostela 2015 Oral communications – session 4.QSI and Gram positive pathogens
44
Structural analysis of NprR, a bifunctional quorum sensor of the Bacillus cereus group
Antoine Talagas1, Stéphane Perchat2, Didier Lereclus2, Sylvie Nessler1
1 Institute for Integrative Biology of the Cell (I2BC), Department of Biochemistry, Biophysics and Structural Biology (B3S), University Paris Sud, 91405 Orsay cedex,
France; 2 INRA, UMR1319 Micalis-AgroParisTech, 78352 Jouy-en-Josas cedex, France. E-mail: [email protected]
Keywords: regulation, peptide binding, functional switch, conformational change, molecular mechanism, crystal structure
Abstract In sporulating bacteria, the differentiation process is tuned by communication systems involving signalling peptides and regulators of the RNPP family. The defining members of this family (Rap, NprR, PrgX and PlcR) are involved in important bacterial processes such as sporulation and virulence and are considered as interesting targets for the development of new antibacterial agents. RNPP proteins are characterized by a TPR-type peptide-binding domain. Except for the Rap proteins, they also contain an N-terminal HTH-type DNA-binding domain and display a transcription factor activity.
At the onset of sporulation, the quorum sensor NprR interacts with its signalling peptide NprX and activates the expression of genes required for the necrotrophic lifestyle of B. thuringiensis (1). The NprR-NprX complex adopts a tetrameric structure allowing the specific binding of the protein on the DNA target sites (2), thus activating the transcription of the corresponding genes (3). In sharp contrast, in the absence of NprX, and independently of its transcriptional activator function, NprR negatively affects the sporulation process. A structural and mutational analysis of NprR allowed us to demonstrate that NprR prevents expression of Spo0A-regulated genes by acting on Spo0F. Direct interaction between NprR and Spo0F was demonstrated. The close structural relationship between NprR and the Rap proteins suggests that NprR has both a transcriptional regulator activity and a Rap-like function.
In conclusion, NprR is a dual-function protein regulated through a quorum-sensing system. We propose a molecular mechanism for the structural and functional switch induced by peptide binding.
References 1. Dubois T, Perchat S, Verplaetse E, Gominet M, Lemy C, Aumont-Nicaise M, Grenha R,
Nessler S, Lereclus D. 2013. Activity of the Bacillus thuringiensis NprR-NprX cell-cell communication system is co-ordinated to the physiological stage through a complex transcriptional regulation. Mol Microbiol. 88:48-63.
2. Zouhir S, Perchat S, Nicaise M, Perez J, Guimaraes B, Lereclus D, Nessler S. 2013. Peptide-binding dependent conformational changes regulate the transcriptional activity of the quorum-sens or NprR. Nucleic Acids Res 41:7920-7933.
3. Perchat S, Dubois T, Zouhir S, Gominet M, Poncet S, Lemy C, Aumont-Nicaise M, Deutscher J, Gohar M, Nessler S, Lereclus D. 2011. A cell-cell communication system regulates protease production during sporulation in bacteria of the Bacillus cereus group. Mol Microbiol 82:619-33.
ISQSI, Santiago de Compostela 2015 Oral communications – session 4.QSI and Gram positive pathogens
45
How does the quorum sensing inhibitor hamamelitannin increase Staphylococcus aureus biofilm susceptibility?
G. Brackman1, F. Van den Driessche1, T. Coenye1
1Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium. E-mail: [email protected]
Keywords: Biofilm, Quorum Sensing, hamamelitannin, Staphylococcus aureus
Abstract Biofilm-associated infections caused by Staphylococcus aureus are often very difficult to treat and novel targets are needed to combat these infections. We have previously shown that the quorum sensing (QS) modulator 2',5-di-O-galloyl-D-hamamelose (hamamelitannin, HAM) increases the susceptibility of S. aureus biofilms towards vancomycin (VAN) in vitro as well as invivo. However, the mechanism of action of HAM at the molecular level has not yet been elucidated.
Two parallel strategies were followed in order to gain insights in the way HAM affects QS. First, we evaluated the effect of HAM on the susceptibility of biofilms of S. aureus strains with mutations in the various QS systems (e.g. agrBCDA, traP, luxS) or in genes involved in biofilm formation and virulence (e.g. icaA, sarA, codY). Secondly, using Illumina sequencing we identified genes that were differentially expressed in untreated biofilms and biofilms treated with VAN alone or with the combination VAN+HAM. Results obtained with both strategies were further investigated using the appropriate tools.
No loss in HAM activity was observed for most of the mutants. In contrast, HAM did not affect biofilm susceptibility of S. aureus strains with mutations in agrA or traP gene. This suggests that these genes are involved in mediating the activity of HAM. Using RNA sequencing, we identified a large number of genes that were differentially expressed after treatment. Treatment with HAM (alone or in combination with VAN) resulted in a differential regulation of genes involved in biosynthesis of lysine, D-alanine, glutamine consuming pathways, and eDNA production and autolysis. In addition, the upregulation of genes encoding virulence factors (e.g. tst, hla, yent1, hlgB) during VAN treatment was not observed when VAN was combined with HAM.
HAM reduces the upregulation of peptidoglycan biosynthesis normally observed after treatment with VAN. In addition, less eDNA is present in the matrix of HAM treated biofilms. This possibly leads to the increased susceptibility of S. aureus biofilm cells towards VAN. Our results further indicate that combination therapy could positively affect morbidity since the upregulation of virulence factors observed for VAN treatment are not observed when VAN is combined with HAM.
ISQSI, Santiago de Compostela 2015 Oral communications – session 4.QSI and Gram positive pathogens
46
Anticaries, antibiofilm and quorum sensing inhibitory activity of stigmasterol-3-D-glucoside isolated from Mimusops elengi L.
Kasi Murugan1, Kuppusamy Sekar2,Saleh Al-Sohaibani1
1Department of Botany & Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia. 2Research and development center, Bharathiar University,
Coimbatore, India. E-mail: [email protected]
Keywords: GC-MS, IR, NMR, molecular docking, Stigmasterol-3-D-Glucoside
Abstract
Dental caries is now a day recognized as a pending public health problem. They are difficult to cure due the primary etiological agent’s ability of forming a highly resistant and organized biofilm. The present study aims at the isolation, characterization and evaluation of the antibiofilm and quorum sensing inhibition potential of bioactive compound from ethnomedicinal natural toothbrush plant Mimusops elengi L.
The stems of oral hygiene maintaining natural toothbrush plant M. elengi used by Malayali tribals of Kolli hills, India were collected and their bioactive compounds were successively extracted with hexane, chloroform, ethyl acetate together with methanol solvents employing solvent extraction. They were screened for their anticaries activity against the cariogenic biofilm forming Streptococcus mutans, Lactobacillus casei and Staphylococcus aureus bacterial isolates. The bioactive compound contributing antibiofilm and quorum sensing inhibitory activity was isolated from the highly active ethyl acetate extract using bioactivity guided fractionation. The isolated and purified compound was identified using physical, GC-MS and spectroscopic investigation (UV, IR, 1H NMR, 13C NMR, 2D NMR). The isolated compound’s concentration-dependent inhibition and interference with the exopolysaccharide synthesis, biofilm formation as well as the quorum sensing (QS) were determined using biofilm specific and molecular docking studies.
M. elengi bioactive compound isolated and identified as Stigmasterol-3-D-Glucoside at 25 µg/ml inhibit 80-94% of biofilm formation and the epi-fluorescence microscopy studies revealed it’s invitro biofilm and bacterial adherence inhibition. The molecular docking study code of information showed high binding energy interaction with Streptococcus mutans antigen I/II (3QE5) and Gram-positive bacteria Rap phosphatase quorum sensing receptors (4I1A).
M. elengi compound stigmasterol-3-D-glucoside exhibits dose-dependent in vitro anticaries and biofilm inhibitory activity due to its ability to suppress the growth of cariogenic isolates and possible interfere with QS.
Further studies on M. elengi stigmasterol-3-D-glucoside would lead to novel antimicrobial QS targeting antibiofilm agent development.
ISQSI, Santiago de Compostela 2015 Oral communications – session 4.QSI and Gram positive pathogens
47
Discovery of inhibitors of the Staphylococcus aureus global regulator, agr
Yanin Jaiyen1, Fabio Rui2, Victoria Steele1, Ewan Murray1, Weng Chan2, Paul Williams1
1School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, 2School of Pharmacy, University of Nottingham,
University Park, Nottingham NG7 2RD.
Keywords: Staphylococcus aureus, agr inhibitor, anti-virulence factors
Abstract Staphylococcus aureus is one of the most important pathogenic bacteria. Developing new drug therapies is an urgent issue as many clinical cases report drug resistance against almost every antibiotic currently available. S. aureus infection is enabled by a highly co-ordinated change in the bacterial genome expression that is ultimately controlled by a few key regulators such as the S. aureus quorum sensing (QS) system agr. Here we have screened a small library of related novel compounds for their ability to reduce S. aureus exotoxin production in a variety of S. aureus strains, including the highly virulent MRSA USA300. Data indicate that one of our compounds (FR44) reduced the production of exotoxins alpha-haemolysin and TSST but also increased the expression of protein A circumstantially implying that FR44 may be targeting the S. aureus agr system. Significantly, this compound did not inhibit bacterial growth at the concentrations that inhibited toxin production therefore there would be less selective pressure for mutants resistant to this novel compound to arise. In conclusion, our data imply that FR44 targets and inhibits the S. aureus QS system agr and we are currently working to decipher the mechanism by which it achieves this.
ISQSI, Santiago de Compostela 2015 Oral communications – session 4.QSI and Gram positive pathogens
46
Anticaries, antibiofilm and quorum sensing inhibitory activity of stigmasterol-3-D-glucoside isolated from Mimusops elengi L.
Kasi Murugan1, Kuppusamy Sekar2,Saleh Al-Sohaibani1
1Department of Botany & Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia. 2Research and development center, Bharathiar University,
Coimbatore, India. E-mail: [email protected]
Keywords: GC-MS, IR, NMR, molecular docking, Stigmasterol-3-D-Glucoside
Abstract
Dental caries is now a day recognized as a pending public health problem. They are difficult to cure due the primary etiological agent’s ability of forming a highly resistant and organized biofilm. The present study aims at the isolation, characterization and evaluation of the antibiofilm and quorum sensing inhibition potential of bioactive compound from ethnomedicinal natural toothbrush plant Mimusops elengi L.
The stems of oral hygiene maintaining natural toothbrush plant M. elengi used by Malayali tribals of Kolli hills, India were collected and their bioactive compounds were successively extracted with hexane, chloroform, ethyl acetate together with methanol solvents employing solvent extraction. They were screened for their anticaries activity against the cariogenic biofilm forming Streptococcus mutans, Lactobacillus casei and Staphylococcus aureus bacterial isolates. The bioactive compound contributing antibiofilm and quorum sensing inhibitory activity was isolated from the highly active ethyl acetate extract using bioactivity guided fractionation. The isolated and purified compound was identified using physical, GC-MS and spectroscopic investigation (UV, IR, 1H NMR, 13C NMR, 2D NMR). The isolated compound’s concentration-dependent inhibition and interference with the exopolysaccharide synthesis, biofilm formation as well as the quorum sensing (QS) were determined using biofilm specific and molecular docking studies.
M. elengi bioactive compound isolated and identified as Stigmasterol-3-D-Glucoside at 25 µg/ml inhibit 80-94% of biofilm formation and the epi-fluorescence microscopy studies revealed it’s invitro biofilm and bacterial adherence inhibition. The molecular docking study code of information showed high binding energy interaction with Streptococcus mutans antigen I/II (3QE5) and Gram-positive bacteria Rap phosphatase quorum sensing receptors (4I1A).
M. elengi compound stigmasterol-3-D-glucoside exhibits dose-dependent in vitro anticaries and biofilm inhibitory activity due to its ability to suppress the growth of cariogenic isolates and possible interfere with QS.
Further studies on M. elengi stigmasterol-3-D-glucoside would lead to novel antimicrobial QS targeting antibiofilm agent development.
ISQSI, Santiago de Compostela 2015 Oral communications – session 4.QSI and Gram positive pathogens
47
Discovery of inhibitors of the Staphylococcus aureus global regulator, agr
Yanin Jaiyen1, Fabio Rui2, Victoria Steele1, Ewan Murray1, Weng Chan2, Paul Williams1
1School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, 2School of Pharmacy, University of Nottingham,
University Park, Nottingham NG7 2RD.
Keywords: Staphylococcus aureus, agr inhibitor, anti-virulence factors
Abstract Staphylococcus aureus is one of the most important pathogenic bacteria. Developing new drug therapies is an urgent issue as many clinical cases report drug resistance against almost every antibiotic currently available. S. aureus infection is enabled by a highly co-ordinated change in the bacterial genome expression that is ultimately controlled by a few key regulators such as the S. aureus quorum sensing (QS) system agr. Here we have screened a small library of related novel compounds for their ability to reduce S. aureus exotoxin production in a variety of S. aureus strains, including the highly virulent MRSA USA300. Data indicate that one of our compounds (FR44) reduced the production of exotoxins alpha-haemolysin and TSST but also increased the expression of protein A circumstantially implying that FR44 may be targeting the S. aureus agr system. Significantly, this compound did not inhibit bacterial growth at the concentrations that inhibited toxin production therefore there would be less selective pressure for mutants resistant to this novel compound to arise. In conclusion, our data imply that FR44 targets and inhibits the S. aureus QS system agr and we are currently working to decipher the mechanism by which it achieves this.
ISQSI, Santiago de Compostela 2015 Oral communications – session 4.QSI and Gram positive pathogens
48
Is spreading of resistance against quorum quenchers possible?
Rodolfo García-Contreras1, Paulina Castañeda1, Toshinari Maeda2, Thomas K. Wood3
1 Department of Microbiology and Parasitology, Faculty of Medicine, UNAM, Mexico City, Mexico.2 Department of Biological Functions Engineering, Kyushu Institute of
Technology, Kitakyushu, Japan, 3Department of Chemical Engineeringy, Pennsylvania State University, University Park, PA, USA. E-mail: [email protected]
Keywords: Quorum quenching, Pseudomonas aeruginosa, virulence factors, private and public goods, resistance, environmental stress
Abstract Quorum quenching is a novel therapy to fight bacterial infections that, unlike conventional antibiotic treatments, is focused on reducing the damage caused by the pathogens (virulence) rather than focused on inhibiting growth. Based on this ideal, it was predicted that this approach will be either impervious or at least much less prone to the selection of resistance in bacterial populations; however, recently resistance mechanisms against well characterized quorum quenchers has been found in the laboratory as well as in clinical strains, demonstrating the rise of resistance against these kind of compounds is possible (1-3). Nevertheless, it has been argued that even if resistance mechanisms against quorum quenchers exist, this fact do not guarantee that resistance will spread. In the present work, I discuss recent insights derived from the latest experimental works developed in order to address this question. In addition, I demonstrate how environmental conditions like the stress produced by the host immune system may influence the spread of resistance (4) and eventually lead to the spread of quorum-quenching-resistant bacteria in the clinical setting.
Acknowledgement RGC is supported by the grant SEP/CONACyT-Mexico No.152794
References 1. García-Contreras R, Maeda T, Wood TK. 2013. Resistance to quorum-quenching
compounds. Appl Environ Microbiol 79:6840-6.
2. García-Contreras R, Martinez-Vazquez M, Velazquez Guadarrama N, Villegas Paneda AG, Hashimoto T, Maeda T, Quezada H, Wood TK. 2013. Resistance to the quorum-quenching compounds brominated furanone C-30 and 5-fluorouracil in Pseudomonas aeruginosa clinical isolates. Pathog Dis 68:8-11.
3. Maeda, T., García-Contreras R, Pu M, Sheng L, Garcia LR, Tomas M, Wood TK. 2012. Quorum quenching quandary: resistance to antivirulence compounds. ISME J 6:493-501.
4. García-Contreras R, Nunez-López L, Jasso-Chávez R, Kwan BW, Belmont JA, Rangel- Vega A, Maeda T, Wood TK. 2014. Quorum sensing enhancement of the stress response promotes resistance to quorum quenching and prevents social cheating. ISME J 9: 115–125.
ISQSI, Santiago de Compostela 2015
49
Poster communications
ISQSI, Santiago de Compostela 2015 Oral communications – session 4.QSI and Gram positive pathogens
48
Is spreading of resistance against quorum quenchers possible?
Rodolfo García-Contreras1, Paulina Castañeda1, Toshinari Maeda2, Thomas K. Wood3
1 Department of Microbiology and Parasitology, Faculty of Medicine, UNAM, Mexico City, Mexico.2 Department of Biological Functions Engineering, Kyushu Institute of
Technology, Kitakyushu, Japan, 3Department of Chemical Engineeringy, Pennsylvania State University, University Park, PA, USA. E-mail: [email protected]
Keywords: Quorum quenching, Pseudomonas aeruginosa, virulence factors, private and public goods, resistance, environmental stress
Abstract Quorum quenching is a novel therapy to fight bacterial infections that, unlike conventional antibiotic treatments, is focused on reducing the damage caused by the pathogens (virulence) rather than focused on inhibiting growth. Based on this ideal, it was predicted that this approach will be either impervious or at least much less prone to the selection of resistance in bacterial populations; however, recently resistance mechanisms against well characterized quorum quenchers has been found in the laboratory as well as in clinical strains, demonstrating the rise of resistance against these kind of compounds is possible (1-3). Nevertheless, it has been argued that even if resistance mechanisms against quorum quenchers exist, this fact do not guarantee that resistance will spread. In the present work, I discuss recent insights derived from the latest experimental works developed in order to address this question. In addition, I demonstrate how environmental conditions like the stress produced by the host immune system may influence the spread of resistance (4) and eventually lead to the spread of quorum-quenching-resistant bacteria in the clinical setting.
Acknowledgement RGC is supported by the grant SEP/CONACyT-Mexico No.152794
References 1. García-Contreras R, Maeda T, Wood TK. 2013. Resistance to quorum-quenching
compounds. Appl Environ Microbiol 79:6840-6.
2. García-Contreras R, Martinez-Vazquez M, Velazquez Guadarrama N, Villegas Paneda AG, Hashimoto T, Maeda T, Quezada H, Wood TK. 2013. Resistance to the quorum-quenching compounds brominated furanone C-30 and 5-fluorouracil in Pseudomonas aeruginosa clinical isolates. Pathog Dis 68:8-11.
3. Maeda, T., García-Contreras R, Pu M, Sheng L, Garcia LR, Tomas M, Wood TK. 2012. Quorum quenching quandary: resistance to antivirulence compounds. ISME J 6:493-501.
4. García-Contreras R, Nunez-López L, Jasso-Chávez R, Kwan BW, Belmont JA, Rangel- Vega A, Maeda T, Wood TK. 2014. Quorum sensing enhancement of the stress response promotes resistance to quorum quenching and prevents social cheating. ISME J 9: 115–125.
ISQSI, Santiago de Compostela 2015
49
Poster communications
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
51
Interfering with biofilm formation of bacteria involved in chronic wound infections by enzymatic quorum quenching
Yunhui Zhang, Gilles Brackman, Tom Coenye
Laboratory of Pharmaceutical Microbiology, Ghent University, Ottergemsesteenweg 460, B-9000 Gent, Belgium. E-mail: [email protected]
Keywords: Biofilm, Quorum quenching enzyme, Acinetobacter baumanii, Pseudomonas aeruginosa
Abstract Acinetobacter baumanii and Pseudomonas aeruginosa are two important Gram-negative species causing serious chronic wound infections. Biofilm formation by these species is a major reason for failure of wound treatment due to the higher antimicrobial tolerance. Quorum sensing regulates biofilm formation of these pathogens, thus interfering with quorum sensing may be an effective way to treat biofilm infection in chronic wounds. Recently, a novel marine-derived N-acyl homoserine lactonase, MomL, was identified from Muricauda olearia and was reported to reduce the virulence of P.aeruginosa. Here, we investigated the effect of MomL on single- or dual-species biofilms formed by A. baumanii and P. aeruginosa strains. We have successfully formed the single- and dual-species biofilms in both microtiter plate and a wound model system comprising artificial dermis, bovine plasma and horse blood to more closely mimic the in vivo situation. The susceptibilities of single- and dual-species biofilms to different antibiotics were compared. The purified MomL was lyophilized with sucrose and added to the mature biofilm or during the biofilm formation. The effects of different combinations of MomL and antibiotics on the susceptibility of biofilms were examined. These results provide a possibility to use MomL in combating bacteria biofilm infections.
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
51
Interfering with biofilm formation of bacteria involved in chronic wound infections by enzymatic quorum quenching
Yunhui Zhang, Gilles Brackman, Tom Coenye
Laboratory of Pharmaceutical Microbiology, Ghent University, Ottergemsesteenweg 460, B-9000 Gent, Belgium. E-mail: [email protected]
Keywords: Biofilm, Quorum quenching enzyme, Acinetobacter baumanii, Pseudomonas aeruginosa
Abstract Acinetobacter baumanii and Pseudomonas aeruginosa are two important Gram-negative species causing serious chronic wound infections. Biofilm formation by these species is a major reason for failure of wound treatment due to the higher antimicrobial tolerance. Quorum sensing regulates biofilm formation of these pathogens, thus interfering with quorum sensing may be an effective way to treat biofilm infection in chronic wounds. Recently, a novel marine-derived N-acyl homoserine lactonase, MomL, was identified from Muricauda olearia and was reported to reduce the virulence of P.aeruginosa. Here, we investigated the effect of MomL on single- or dual-species biofilms formed by A. baumanii and P. aeruginosa strains. We have successfully formed the single- and dual-species biofilms in both microtiter plate and a wound model system comprising artificial dermis, bovine plasma and horse blood to more closely mimic the in vivo situation. The susceptibilities of single- and dual-species biofilms to different antibiotics were compared. The purified MomL was lyophilized with sucrose and added to the mature biofilm or during the biofilm formation. The effects of different combinations of MomL and antibiotics on the susceptibility of biofilms were examined. These results provide a possibility to use MomL in combating bacteria biofilm infections.
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
52
Mycobacterium abscessus, an emerging pathogen in cystic fibrosis patients, degrades the Pseudomonas quinolone signal
Franziska S. Birmes, Susanne Fetzner
Institute of Molecular Microbiology and Biotechnology; University of Münster.Corrensstr. 3; 48149 Münster, Germany. E-mail: franzi.birmes@uni-
muenster.de
Keywords: Pseudomonas aeruginosa, Mycobacterium abscessus, alkylquinolones, PQS, HHQ, cystic fibrosis
Abstract The opportunistic pathogen Pseudomonas aeruginosa is one of the major causes of nosocomial infections, and colonizes the lungs of cystic fibrosis patients. It regulates its virulence via a complex quorum sensing (QS) network including N-acylhomoserine lactones and the alkylquinolones (AQs) 2-heptyl-3-hydroxy-4(1H)-quinolone (Pseudomonas quinolone signal, PQS) and 2-heptyl-4(1H)-quinolone (HHQ) as signal molecules (1).
With Rhodococcus erythropolis BG43, the first strain able to degrade both PQS and HHQ, as well as the respiratory electron transport inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), was isolated recently (2). Homologues of its genes involved in AQ degradation are conserved in many Mycobacterium abscessus strains. M. abscessus is an emerging pathogen causing pseudotuberculous lung disease in patients with cystic fibrosis (3).
Desalted cell free extracts of M. abscessus DSM 44196 are able to rapidly degrade PQS and HHQ. PQS conversion does not require cosubstrates, whereas conversion of HHQ takes place in the presence of NADH. Additionally, the cell-free extracts of M. abscessus supplemented with NADH are able to convert the antibiotic HQNO synthesized by P. aeruginosa into PQS, transiently forming an intermediate with the mass of a hydroxylated form of HQNO.
The PQS cleaving enzyme Hod from Arthrobacter sp. Rue61a, despite its low catalytic efficiency, nevertheless reduces the virulence of P. aeruginosa PAO1 when added exogenously to cultures (4).
Taken together, the findings tentatively suggest that M. abscessus strains due to their ability to inactivate the AQ signals PQS and HHQ may have the potential to interfere with P. aeruginosa QS and hence reduce synthesis of virulence factors.
Acknowledgements: We thank Prof. Kalinowski, Bielefeld University, for sequencing the genome of R. erythropolis BG43.
References 1. Heeb S, Fletcher MP, Chhabra SR, Diggle SP, Williams P, Cámara M. 2011. Quinolones:
from antibiotics to autoinducers. FEMS Microbiol Lett 35:247-274.
2. Müller C, Birmes FS, Niewerth H, Fetzner S. 2014. Conversion of the Pseudomonas aeruginosa quinolone signal and related alkylhydroxyquinolines by Rhodococcus sp. Strain BG43. Appl Environ Microbiol 80:7266-7274.
3. Razvi S, Saiman L. 2007. Nontuberculous mycobacteria in cystic fibrosis. Pediatr Infect Dis J Mar 26:263-264
4. Pustelny C, Albers A, Büldt-Karentzopoulos, Parschat K, Chhabra SR, Cámara M, Williams P, Fetzner S. 2009. Dioxygenase-mediated quenching of quinolone dependent quorum sensing in Pseudomonas aeruginosa. Chem Biol 16:1259-1267.
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
5
Rhodococcus erythropolis BG43, an isolate able to degrade HHQ, PQS and related alkylhydroxyquinolines Christine Müller, Franziska S. Birmes, Susanne Fetzner
Institute of Molecular Microbiology and Biotechnology, University of Münster, Corrensstrasse 3, D-48149 Münster, Germany. E-mail: [email protected]
Keywords: Pseudomonas aeruginosa, PQS, HHQ, Rhodococcus erythropolis, HQNO
Abstract Quorum sensing (QS) enables bacteria to communicate with each other and to regulate their gene expression in a cell-density dependent manner. The opportunistic pathogen Pseudomonas aeruginosa controls its production of virulence factors and biofilm formation via a complex QS network employing N-acylhomoserine lactones (AHLs) as well as the alkylquinolones (AQs) 2-heptyl-3-hydroxy-4(1H)-quinolone (Pseudomonas quinolone signal, PQS) and 2-heptyl-4(1H)-quinolone (HHQ) as signalling molecules (1). Since there is major interest to develop novel strategies to treat P. aeruginosa infections, interference of QS by signal degradation to reduce its virulence is a promising tool.
To detect novel AQ-converting enzymes, we obtained several isolates from enrichment cultures supplemented with HHQ or PQS. Cell suspensions of one isolate which was assigned to the species Rhodococcus erythropolis converted 20 µM PQS and HHQ within 30 and 110 minutes, respectively. HHQ conversion was accompanied by transient formation of PQS and anthranilic acid, suggesting that the first step of HHQ degradation is the hydroxylation of HHQ to PQS, which subsequently undergoes cleavage of the heterocyclic ring and further degradation to anthranilic acid. Comparison of the rates of HHQ and PQS conversion by crude cell extracts from PQS-induced and non-induced cells suggested that the expression of genes coding for the AQ-converting enzymes is inducible (2).
Remarkably, R. erythropolis BG43 was also able to convert the respiratory electron transport inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide, which is also produced by P. aeruginosa, to PQS. Moreover, strain BG43 contains a qsdA gene encoding an AHL-lactonase which might confer to R. erythropolis BG43 the potential to disrupt AHL-based QS circuits.
Isolation and characterization of the AQ degrading strain R. erythropolis BG43 open up new perspectives to interfere with QS and virulence (2).
References 1. Heeb S, Fletcher MP, Chhabra SR, Diggle SP, Williams P, Cámara M. 2011. Quinolones:
from antibiotics to autoinducers. FEMS Microbiol Lett. 35:247 274.
2. Müller C, Birmes FS, Niewerth H, Fetzner S. 2014. Conversion of the Pseudomonas aeruginosa quinolone signal and related alkylhydroxyquinolines by Rhodococcus sp. strain BG43. Appl Environ Microbiol. 80:7266 7274.
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
52
Mycobacterium abscessus, an emerging pathogen in cystic fibrosis patients, degrades the Pseudomonas quinolone signal
Franziska S. Birmes, Susanne Fetzner
Institute of Molecular Microbiology and Biotechnology; University of Münster.Corrensstr. 3; 48149 Münster, Germany. E-mail: franzi.birmes@uni-
muenster.de
Keywords: Pseudomonas aeruginosa, Mycobacterium abscessus, alkylquinolones, PQS, HHQ, cystic fibrosis
Abstract The opportunistic pathogen Pseudomonas aeruginosa is one of the major causes of nosocomial infections, and colonizes the lungs of cystic fibrosis patients. It regulates its virulence via a complex quorum sensing (QS) network including N-acylhomoserine lactones and the alkylquinolones (AQs) 2-heptyl-3-hydroxy-4(1H)-quinolone (Pseudomonas quinolone signal, PQS) and 2-heptyl-4(1H)-quinolone (HHQ) as signal molecules (1).
With Rhodococcus erythropolis BG43, the first strain able to degrade both PQS and HHQ, as well as the respiratory electron transport inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), was isolated recently (2). Homologues of its genes involved in AQ degradation are conserved in many Mycobacterium abscessus strains. M. abscessus is an emerging pathogen causing pseudotuberculous lung disease in patients with cystic fibrosis (3).
Desalted cell free extracts of M. abscessus DSM 44196 are able to rapidly degrade PQS and HHQ. PQS conversion does not require cosubstrates, whereas conversion of HHQ takes place in the presence of NADH. Additionally, the cell-free extracts of M. abscessus supplemented with NADH are able to convert the antibiotic HQNO synthesized by P. aeruginosa into PQS, transiently forming an intermediate with the mass of a hydroxylated form of HQNO.
The PQS cleaving enzyme Hod from Arthrobacter sp. Rue61a, despite its low catalytic efficiency, nevertheless reduces the virulence of P. aeruginosa PAO1 when added exogenously to cultures (4).
Taken together, the findings tentatively suggest that M. abscessus strains due to their ability to inactivate the AQ signals PQS and HHQ may have the potential to interfere with P. aeruginosa QS and hence reduce synthesis of virulence factors.
Acknowledgements: We thank Prof. Kalinowski, Bielefeld University, for sequencing the genome of R. erythropolis BG43.
References 1. Heeb S, Fletcher MP, Chhabra SR, Diggle SP, Williams P, Cámara M. 2011. Quinolones:
from antibiotics to autoinducers. FEMS Microbiol Lett 35:247-274.
2. Müller C, Birmes FS, Niewerth H, Fetzner S. 2014. Conversion of the Pseudomonas aeruginosa quinolone signal and related alkylhydroxyquinolines by Rhodococcus sp. Strain BG43. Appl Environ Microbiol 80:7266-7274.
3. Razvi S, Saiman L. 2007. Nontuberculous mycobacteria in cystic fibrosis. Pediatr Infect Dis J Mar 26:263-264
4. Pustelny C, Albers A, Büldt-Karentzopoulos, Parschat K, Chhabra SR, Cámara M, Williams P, Fetzner S. 2009. Dioxygenase-mediated quenching of quinolone dependent quorum sensing in Pseudomonas aeruginosa. Chem Biol 16:1259-1267.
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
5
Rhodococcus erythropolis BG43, an isolate able to degrade HHQ, PQS and related alkylhydroxyquinolines Christine Müller, Franziska S. Birmes, Susanne Fetzner
Institute of Molecular Microbiology and Biotechnology, University of Münster, Corrensstrasse 3, D-48149 Münster, Germany. E-mail: [email protected]
Keywords: Pseudomonas aeruginosa, PQS, HHQ, Rhodococcus erythropolis, HQNO
Abstract Quorum sensing (QS) enables bacteria to communicate with each other and to regulate their gene expression in a cell-density dependent manner. The opportunistic pathogen Pseudomonas aeruginosa controls its production of virulence factors and biofilm formation via a complex QS network employing N-acylhomoserine lactones (AHLs) as well as the alkylquinolones (AQs) 2-heptyl-3-hydroxy-4(1H)-quinolone (Pseudomonas quinolone signal, PQS) and 2-heptyl-4(1H)-quinolone (HHQ) as signalling molecules (1). Since there is major interest to develop novel strategies to treat P. aeruginosa infections, interference of QS by signal degradation to reduce its virulence is a promising tool.
To detect novel AQ-converting enzymes, we obtained several isolates from enrichment cultures supplemented with HHQ or PQS. Cell suspensions of one isolate which was assigned to the species Rhodococcus erythropolis converted 20 µM PQS and HHQ within 30 and 110 minutes, respectively. HHQ conversion was accompanied by transient formation of PQS and anthranilic acid, suggesting that the first step of HHQ degradation is the hydroxylation of HHQ to PQS, which subsequently undergoes cleavage of the heterocyclic ring and further degradation to anthranilic acid. Comparison of the rates of HHQ and PQS conversion by crude cell extracts from PQS-induced and non-induced cells suggested that the expression of genes coding for the AQ-converting enzymes is inducible (2).
Remarkably, R. erythropolis BG43 was also able to convert the respiratory electron transport inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide, which is also produced by P. aeruginosa, to PQS. Moreover, strain BG43 contains a qsdA gene encoding an AHL-lactonase which might confer to R. erythropolis BG43 the potential to disrupt AHL-based QS circuits.
Isolation and characterization of the AQ degrading strain R. erythropolis BG43 open up new perspectives to interfere with QS and virulence (2).
References 1. Heeb S, Fletcher MP, Chhabra SR, Diggle SP, Williams P, Cámara M. 2011. Quinolones:
from antibiotics to autoinducers. FEMS Microbiol Lett. 35:247 274.
2. Müller C, Birmes FS, Niewerth H, Fetzner S. 2014. Conversion of the Pseudomonas aeruginosa quinolone signal and related alkylhydroxyquinolines by Rhodococcus sp. strain BG43. Appl Environ Microbiol. 80:7266 7274.
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
54
Engineered quorum quenching bacterial coatings and skins against Pseudomonas aeruginosa infections
Miguel Cocotl-Yañez1, Felix Dafhnis-Calas1, Natalio Krasnogor2, Miguel Cámara1, Stephan Heeb1
1School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, and 2School of Computing Science, Newcastle University,
Newcastle NE1 7RU, United Kingdom.E-mail: [email protected]
Keywords: Pseudomonas aeruginosa, Biofilm, Biosensor
Abstract Infectious diseases are a major cause of death, disability, economic and ultimately social upheaval accounting for more than 12 million deaths a year worldwide. As multiple antibiotic resistances become ever more widespread, current efforts concentrate on the development of novel antimicrobial and therapeutic strategies. However, early interventions based on state–of–the–art diagnostic strategies and novel treatments that accelerate the detection and management of bacterial colonisation is paramount to substantially reduce the occurrence and impact of infections. Many bacterial pathogens rely on quorum sensing signalling molecules (QSSM) to control biofilm formation and virulence, the most studied being the N–acyl homoserine lactones (AHLs) produced by Gram–negative bacteria. Enzymes that degrade QSSM such as the AiiA lactonase from Bacillus spp, which hydrolyses the lactone bond of a wide range of AHLs including those produced by the human opportunistic pathogen Pseudomonas aeruginosa (PA), can interfere with QS mechanisms to attenuate virulence and increase the susceptibility of biofilms to currently available antibiotics. AHLs have been detected in vivo during PA infections using a number of approaches. These include the construction of Escherichia coli–based biosensors expressing LuxR-type AHL-dependent transcriptional regulators which upon interaction with exogenous AHLs induce particular reporter genes. Hence the detection of AHLs can be exploited for the diagnosis of infectious diseases and the engineering of biologically reactive coatings. Here we report the design and construction of sticky non–pathogenic E. coli biosensor communities which will form strong biofilms (to be used as coatings or artificial skins) and which will sense PA and be induced to produce the AiiA quorum–quenching lactonase. Thus, this kind of engineered coatings should be able to detect, respond and neutralise incoming PA cells by disarming them before having the opportunity cause host damage. The construction has been designed to be modular in order to facilitate its adaptation to the detection of other signal molecules, the induction of different genes in response to their detection, and the stable insertion of the resulting constructs in a variety of bacterial hosts other than E. coli.
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
55
Quorum quenching activity of archaeal lactonases against Pseudomonas aeruginosa in a Drosophila infection model
Elena Porzio1, Davide Andrenacci2, Giuseppe Manco1
1Institute of Protein Biochemistry; 2 Institute of Genetics and Biophisics; National Research Council of Italy, Via P. Castellino 111, 80131, Naples, Italy.
E-mail: [email protected]
Keywords: Pseudomonas aeruginosa, thermostable lactonases, biofilm, Phosphotriesterase-like Lactonases, Pseudomonas virulence, infections, quorum quenching
Abstract Pseudomonas aeruginosa is the leading cause of clinically relevant infections in cystic fibrosis (CF) due to resistance to antibiotics, which requires new strategies to control bacterial growth. The mechanism of quorum sensing (QS) regulates the expression of several virulence factors among which the biofilm formation, given that some bacterial strains with mutations in the QS system, are less virulent in animal models (1). The QS signals may be interrupted by enzymes such as lactonases. In humans there are three lactonases: PON1, PON2 and PON3 (2). PON1 can protect Drosophila melanogaster from Pseudomonas infections (6). Within a project founded by the Italian Cystic Fibrosis Foundation, we began studying members of the family of thermostable Phosphotriesterase-like Lactonases (PLL) to fight infection by Pseudomonas aeruginosa, as these enzymes have lactonase activity on signalling molecules used by the bacterial pathogen (4-6). Here we will report results showing that these enzymes are capable of reducing the production of virulence factors by P. aeruginosa in vitro, and in the in vivo animal model Drosophila melanogaster.
References 1. Parsek, MR, and Singh, PK. 2003. Bacterial biofilms: an emerging link to disease
pathogenesis. Annu Rev Microbiol 57:677-701.
2. Draganov DI, Teiber JF, Speelman A, Osawa Y, Sunahara R and La Du BN. 2005. Human paraoxonases (PON1 PON2, and PON3) are lactonases with overlapping and distinct substrate specificities. J Lipid Res 46:1239-1247.
3. Stoltz DA, Ozer EA, Taft PJ, Barry M, Liu L, Kiss PJ, Moninger TO, Parsek MR, Zabner J. 2008. Drosophila are protected from Pseudomonas aeruginosa lethality by transgenic expression of paraoxonase-1. J Clin Invest 118:3123-3131.
4. Merone L, Mandrich L, Rossi M, Manco G. 2005. A thermostable phosphotriesterase from the archaeon Sulfolobus solfataricus: cloning, overexpression and properties. Extremophiles 9:297-305
5. Porzio E, Merone L, Mandrich L, Rossi M, Manco G. 2007. A new phosphotriesterase from Sulfolobus acidocaldarius and its comparison with the homologue from Sulfolobus solfataricus. Biochimie 89:625-36.
6. Elias M, Dupuy J, Merone L, Mandrich L, Porzio E, Moniot S, Rochu D, Lecomte C, Rossi M, Masson P, Manco G, Chabriere E. 2008. Structural basis for natural lactonase and promiscuous phosphotriesterase activities. J Mol Biol 379:1017-28.
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
54
Engineered quorum quenching bacterial coatings and skins against Pseudomonas aeruginosa infections
Miguel Cocotl-Yañez1, Felix Dafhnis-Calas1, Natalio Krasnogor2, Miguel Cámara1, Stephan Heeb1
1School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, and 2School of Computing Science, Newcastle University,
Newcastle NE1 7RU, United Kingdom.E-mail: [email protected]
Keywords: Pseudomonas aeruginosa, Biofilm, Biosensor
Abstract Infectious diseases are a major cause of death, disability, economic and ultimately social upheaval accounting for more than 12 million deaths a year worldwide. As multiple antibiotic resistances become ever more widespread, current efforts concentrate on the development of novel antimicrobial and therapeutic strategies. However, early interventions based on state–of–the–art diagnostic strategies and novel treatments that accelerate the detection and management of bacterial colonisation is paramount to substantially reduce the occurrence and impact of infections. Many bacterial pathogens rely on quorum sensing signalling molecules (QSSM) to control biofilm formation and virulence, the most studied being the N–acyl homoserine lactones (AHLs) produced by Gram–negative bacteria. Enzymes that degrade QSSM such as the AiiA lactonase from Bacillus spp, which hydrolyses the lactone bond of a wide range of AHLs including those produced by the human opportunistic pathogen Pseudomonas aeruginosa (PA), can interfere with QS mechanisms to attenuate virulence and increase the susceptibility of biofilms to currently available antibiotics. AHLs have been detected in vivo during PA infections using a number of approaches. These include the construction of Escherichia coli–based biosensors expressing LuxR-type AHL-dependent transcriptional regulators which upon interaction with exogenous AHLs induce particular reporter genes. Hence the detection of AHLs can be exploited for the diagnosis of infectious diseases and the engineering of biologically reactive coatings. Here we report the design and construction of sticky non–pathogenic E. coli biosensor communities which will form strong biofilms (to be used as coatings or artificial skins) and which will sense PA and be induced to produce the AiiA quorum–quenching lactonase. Thus, this kind of engineered coatings should be able to detect, respond and neutralise incoming PA cells by disarming them before having the opportunity cause host damage. The construction has been designed to be modular in order to facilitate its adaptation to the detection of other signal molecules, the induction of different genes in response to their detection, and the stable insertion of the resulting constructs in a variety of bacterial hosts other than E. coli.
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
55
Quorum quenching activity of archaeal lactonases against Pseudomonas aeruginosa in a Drosophila infection model
Elena Porzio1, Davide Andrenacci2, Giuseppe Manco1
1Institute of Protein Biochemistry; 2 Institute of Genetics and Biophisics; National Research Council of Italy, Via P. Castellino 111, 80131, Naples, Italy.
E-mail: [email protected]
Keywords: Pseudomonas aeruginosa, thermostable lactonases, biofilm, Phosphotriesterase-like Lactonases, Pseudomonas virulence, infections, quorum quenching
Abstract Pseudomonas aeruginosa is the leading cause of clinically relevant infections in cystic fibrosis (CF) due to resistance to antibiotics, which requires new strategies to control bacterial growth. The mechanism of quorum sensing (QS) regulates the expression of several virulence factors among which the biofilm formation, given that some bacterial strains with mutations in the QS system, are less virulent in animal models (1). The QS signals may be interrupted by enzymes such as lactonases. In humans there are three lactonases: PON1, PON2 and PON3 (2). PON1 can protect Drosophila melanogaster from Pseudomonas infections (6). Within a project founded by the Italian Cystic Fibrosis Foundation, we began studying members of the family of thermostable Phosphotriesterase-like Lactonases (PLL) to fight infection by Pseudomonas aeruginosa, as these enzymes have lactonase activity on signalling molecules used by the bacterial pathogen (4-6). Here we will report results showing that these enzymes are capable of reducing the production of virulence factors by P. aeruginosa in vitro, and in the in vivo animal model Drosophila melanogaster.
References 1. Parsek, MR, and Singh, PK. 2003. Bacterial biofilms: an emerging link to disease
pathogenesis. Annu Rev Microbiol 57:677-701.
2. Draganov DI, Teiber JF, Speelman A, Osawa Y, Sunahara R and La Du BN. 2005. Human paraoxonases (PON1 PON2, and PON3) are lactonases with overlapping and distinct substrate specificities. J Lipid Res 46:1239-1247.
3. Stoltz DA, Ozer EA, Taft PJ, Barry M, Liu L, Kiss PJ, Moninger TO, Parsek MR, Zabner J. 2008. Drosophila are protected from Pseudomonas aeruginosa lethality by transgenic expression of paraoxonase-1. J Clin Invest 118:3123-3131.
4. Merone L, Mandrich L, Rossi M, Manco G. 2005. A thermostable phosphotriesterase from the archaeon Sulfolobus solfataricus: cloning, overexpression and properties. Extremophiles 9:297-305
5. Porzio E, Merone L, Mandrich L, Rossi M, Manco G. 2007. A new phosphotriesterase from Sulfolobus acidocaldarius and its comparison with the homologue from Sulfolobus solfataricus. Biochimie 89:625-36.
6. Elias M, Dupuy J, Merone L, Mandrich L, Porzio E, Moniot S, Rochu D, Lecomte C, Rossi M, Masson P, Manco G, Chabriere E. 2008. Structural basis for natural lactonase and promiscuous phosphotriesterase activities. J Mol Biol 379:1017-28.
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
56
Potential biotechnological applications of two strains of Alteromonas with high quorum-quenching activity
Marta Torres1,2, Emilia Quesada1,2, Inmaculada Llamas1,2
1Departament of Microbiology, Faculty of Pharmacy, University of Granada, Granada, Spain. 2Institute of Biotechnology, Center for Biomedical Research, Health-Sciences
Technology Park, Armilla, Spain. E-mail: [email protected]
Keywords: quorum quenching, aquaculture, agriculture
Abstract In order to control pathogenic bacteria without developing antibiotic-resistant strains, alternative methods need to be investigated. It is well known that the production of virulence factors by many pathogenic microorganisms, such as exoproteases in Erwinia carotovora or metalloproteases in Vibrio anguillarum, depends on the intercellular communication system called quorum sensing (QS), which involves the production and release of signal molecules or autoinducers (1). Given the link between QS and virulence, the disruption of these signalling processes has emerged as an attractive new-therapeutic strategy for the treatment of a variety of infections. One of the possibilities for interfering with QS systems is quorum quenching (QQ), which involves degrading the signalling molecules via enzymes, such as acylases and lactonases (2). Since QS is not essential for bacterial survival, QQ strategies may well diminish pathogenicity without imposing the level of selective pressure associated with antimicrobial-drugs treatments.
In this communication we present the screening of QQ activity among 450 strains isolated from a bivalve hatchery in Granada (Spain), and the selection of two strains, which degrade a wide range of N-acylhomoserine lactones. The selected strains, identified as members of the genus Alteromonas, were used to conduct co-cultivation experiments with bacterial pathogens of molluscs, fish and plants. The results obtained suggest that the two strains selected in this work may have biotechnological applications in both aquaculture and agriculture.
References 1. Croxatto A, Chalker VJ, Lauritz J, Jass J, Hardman A, Williams P, Cámara M, Milton DL.
2002. VanT, a homologue of Vibrio harveyi luxR, regulates serine, metalloprotease, pigment and biofilm formation in Vibrio anguillarum. J Bacteriol 184:1617–1629.
2. Defoirdt T, Boon N, Sorgeloos P, Verstraete W, Bossier P. 2008. Quorum sensing and quorum quenching in Vibrio harveyi: lessons learned from in vivo work. ISME J 2:19-26.
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
57
Use of the quorum quenching strain Tenacibaculum sp. 20J to improve survival in mollusc larvae cultures
Isabel Freire1, Pedro Nascimento1, Isabel Reyero1, Andrea Muras1, Celia Mayer1, Hugo Milhazes-Cunha1, Manuel Romero2, Ana Otero1
1Department of Microbiology and Parasitology, Faculty of Biology, University of Santiago de Compostela, Spain, 2Present address: Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham NG7 2RD Nottingham, UK. E-mail:
[email protected], [email protected]
Keywords: 20J, bacteria, quorum sensing, quorum quenching, larvae
Abstract One of the major challenges for the sustainable development of aquaculture is the search of alternatives to antibiotics to control bacterial infections. The problem of pathogenic bacteria is especially acute in mollusc larvae cultures, in which very high mortalities are often derived from the presence of opportunistic pathogens, belonging in many cases to the genus Vibrio. One of the most promising strategies for the control of pathogenic bacteria is the interference with their quorum sensing systems that control the expression of virulence factors. Quorum sensing inhibition or Quorum Quenching (QQ) has special interest in the field of aquaculture since many pathogenic bacteria infecting fish and invertebrates, including Vibrio spp., use this mechanism to attack their hosts in a coordinated manner. The marine isolate Tenacibaculum sp. strain 20J was selected in a previous work (1) for its wide-spectrum QQ activity against N-acyl homoserine lactones (AHLs) and was demonstrated to produce an AHL-degrading lactonase called Aii20J. In order to check the capacity of strain 20J to improve survival in mollusc larvae cultures, 103 and 105 CFUs/mL of 20J were added to cultures of clams (Venerupis pullastra and Ruditapes decussatus) and oyster (Ostrea edulis) larvae under standard conditions at laboratory scale (2.3 L). The addition of living cells of strain 20J improved survival for the 3 species tested compared to control larvae cultures (30 to 45 % survival improvement). Interestingly, the addition of 105 CFUs/mL of 20J showed better survival rates than chloramphenicol treated larvae cultures: 20 to 40 % survival improvement for V. pullastra and O. edulis respectively. In order to test if QQ activity of 20J could explain the increased survival of larvae cultures, additional experiments were carried out with mussel larvae challenged with the pathogenic strain Vibrio neptunius (145.98) and supplemented with purified Aii20J (2 µg/mL). Results showed a 3-fold increase of survival after 48 hours in Aii20J-treated challenged cultures, suggesting that the QQ activity of strain 20J may be crucial for the positive effect of the addition of living cells to larvae cultures. In conclusion, results demonstrate the feasibility of using living cells of strain 20J or its purified enzyme Aii20J to improve survival in mollusc larvae cultures.
Acknowledgements Vibrio neptunius (145.98) was kindly provided by Prof. Juan Luis Barja from the Group of Pathology in Aquaculture of the USC. This work has been supported by Fundación Barrié de la Maza and Fundación Ramón Areces (CIVP16A1814).
References 1. Romero M. Martin-Cuadrado AB. Roca-Rivada A. Cabello AM. Otero A. 2011. Quorum
quenching in cultivable bacteria from dense marine coastal microbial communities. FEMS Microbiol Ecol 75:205-217.
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
56
Potential biotechnological applications of two strains of Alteromonas with high quorum-quenching activity
Marta Torres1,2, Emilia Quesada1,2, Inmaculada Llamas1,2
1Departament of Microbiology, Faculty of Pharmacy, University of Granada, Granada, Spain. 2Institute of Biotechnology, Center for Biomedical Research, Health-Sciences
Technology Park, Armilla, Spain. E-mail: [email protected]
Keywords: quorum quenching, aquaculture, agriculture
Abstract In order to control pathogenic bacteria without developing antibiotic-resistant strains, alternative methods need to be investigated. It is well known that the production of virulence factors by many pathogenic microorganisms, such as exoproteases in Erwinia carotovora or metalloproteases in Vibrio anguillarum, depends on the intercellular communication system called quorum sensing (QS), which involves the production and release of signal molecules or autoinducers (1). Given the link between QS and virulence, the disruption of these signalling processes has emerged as an attractive new-therapeutic strategy for the treatment of a variety of infections. One of the possibilities for interfering with QS systems is quorum quenching (QQ), which involves degrading the signalling molecules via enzymes, such as acylases and lactonases (2). Since QS is not essential for bacterial survival, QQ strategies may well diminish pathogenicity without imposing the level of selective pressure associated with antimicrobial-drugs treatments.
In this communication we present the screening of QQ activity among 450 strains isolated from a bivalve hatchery in Granada (Spain), and the selection of two strains, which degrade a wide range of N-acylhomoserine lactones. The selected strains, identified as members of the genus Alteromonas, were used to conduct co-cultivation experiments with bacterial pathogens of molluscs, fish and plants. The results obtained suggest that the two strains selected in this work may have biotechnological applications in both aquaculture and agriculture.
References 1. Croxatto A, Chalker VJ, Lauritz J, Jass J, Hardman A, Williams P, Cámara M, Milton DL.
2002. VanT, a homologue of Vibrio harveyi luxR, regulates serine, metalloprotease, pigment and biofilm formation in Vibrio anguillarum. J Bacteriol 184:1617–1629.
2. Defoirdt T, Boon N, Sorgeloos P, Verstraete W, Bossier P. 2008. Quorum sensing and quorum quenching in Vibrio harveyi: lessons learned from in vivo work. ISME J 2:19-26.
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
57
Use of the quorum quenching strain Tenacibaculum sp. 20J to improve survival in mollusc larvae cultures
Isabel Freire1, Pedro Nascimento1, Isabel Reyero1, Andrea Muras1, Celia Mayer1, Hugo Milhazes-Cunha1, Manuel Romero2, Ana Otero1
1Department of Microbiology and Parasitology, Faculty of Biology, University of Santiago de Compostela, Spain, 2Present address: Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham NG7 2RD Nottingham, UK. E-mail:
[email protected], [email protected]
Keywords: 20J, bacteria, quorum sensing, quorum quenching, larvae
Abstract One of the major challenges for the sustainable development of aquaculture is the search of alternatives to antibiotics to control bacterial infections. The problem of pathogenic bacteria is especially acute in mollusc larvae cultures, in which very high mortalities are often derived from the presence of opportunistic pathogens, belonging in many cases to the genus Vibrio. One of the most promising strategies for the control of pathogenic bacteria is the interference with their quorum sensing systems that control the expression of virulence factors. Quorum sensing inhibition or Quorum Quenching (QQ) has special interest in the field of aquaculture since many pathogenic bacteria infecting fish and invertebrates, including Vibrio spp., use this mechanism to attack their hosts in a coordinated manner. The marine isolate Tenacibaculum sp. strain 20J was selected in a previous work (1) for its wide-spectrum QQ activity against N-acyl homoserine lactones (AHLs) and was demonstrated to produce an AHL-degrading lactonase called Aii20J. In order to check the capacity of strain 20J to improve survival in mollusc larvae cultures, 103 and 105 CFUs/mL of 20J were added to cultures of clams (Venerupis pullastra and Ruditapes decussatus) and oyster (Ostrea edulis) larvae under standard conditions at laboratory scale (2.3 L). The addition of living cells of strain 20J improved survival for the 3 species tested compared to control larvae cultures (30 to 45 % survival improvement). Interestingly, the addition of 105 CFUs/mL of 20J showed better survival rates than chloramphenicol treated larvae cultures: 20 to 40 % survival improvement for V. pullastra and O. edulis respectively. In order to test if QQ activity of 20J could explain the increased survival of larvae cultures, additional experiments were carried out with mussel larvae challenged with the pathogenic strain Vibrio neptunius (145.98) and supplemented with purified Aii20J (2 µg/mL). Results showed a 3-fold increase of survival after 48 hours in Aii20J-treated challenged cultures, suggesting that the QQ activity of strain 20J may be crucial for the positive effect of the addition of living cells to larvae cultures. In conclusion, results demonstrate the feasibility of using living cells of strain 20J or its purified enzyme Aii20J to improve survival in mollusc larvae cultures.
Acknowledgements Vibrio neptunius (145.98) was kindly provided by Prof. Juan Luis Barja from the Group of Pathology in Aquaculture of the USC. This work has been supported by Fundación Barrié de la Maza and Fundación Ramón Areces (CIVP16A1814).
References 1. Romero M. Martin-Cuadrado AB. Roca-Rivada A. Cabello AM. Otero A. 2011. Quorum
quenching in cultivable bacteria from dense marine coastal microbial communities. FEMS Microbiol Ecol 75:205-217.
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
58
Effect of the quorum quenching lactonase Aii20J on biofilm formation by different strains and mutants of Acinetobacter
baumannii Celia Mayer1, Manuel Romero1,2, Andrea Muras1, Soraya Rumbo3, Eva Gato3,
María Tomás3, Ana Otero1
1Departamento de Microbioloxía e Parasitoloxía, Facultade de Bioloxía-CIBUS, Universidade de Santiago de Compostela, Spain, 2Present address: Centre for
Biomolecular Sciences, School of Life Sciences, University of Nottingham NG7 2RD Nottingham, UK, 3Departamento de Microbioloxía, Complexo Hospitalario Universitario
A Coruña-INIBIC, Spain. E-mail: [email protected], [email protected]
Keywords: Acinetobacter baumannii, quorum sensing, biofilm, quorum quenching, acyl-homoserine-lactones, lactonase
Abstract Acinetobacter baumannii is an important Gram-negative nosocomial pathogen responsible for numerous healthcare-associated infections worldwide with an increasing interest due to the emergence of multidrug-resistant (MDR) strains. Among the virulence factors, one of the main causes for the persistence of A. baumannii is the biofilm formation, which has been reported to be regulated by quorum sensing (QS). A. baumannii presents a typical LuxI/LuxR-type quorum-sensing network that involves the abaI synthase and the abaR receptor and is mediated by long substituted N-acyl homoserine lactones (AHLs) such as OHC12-HSL. Therefore, the interference with QS systems, a process generally termed quorum quenching (QQ), represents an interesting anti-pathogenic strategy to control biofilm formation in this bacterium.
Additionally, several reports have demonstrated the contribution of resistance-nodulation-cell division (RND)-type efflux systems in conferring multidrug resistance in this pathogen. RND efflux pump have been related to QS regulation by the selection of AHLs in P. aeruginosa via the efflux system MexAb-OprM. In A. baumannii, one of the most characterized RND-efflux pump is the AdeABC system, composed of the AdeA, AdeB, and AdeC proteins. AdeB is a member of the RND superfamily, whereas AdeA and AdeC are homologous to membrane fusion proteins and to the outer membrane protein OprM from Pseudomonas aeruginosa respectively. This AdeABC pump confers resistance to various antibiotic classes including aminoglycosides and its overexpression plays a major role in acquired resistance in A. baumannii but it is not clear so far if it can be involved in the regulation of the AHL-mediated QS system in A. baumannii.
In this work, we characterize the biofilm formation by different strains of A. baumannii: A. baumannii ATCC 17978, an abaI AHL-synthase isogenic mutant, an adeB gene isogenic mutant and a clinical isolate, by using the crystal violet and resazurine methods. Moreover, we report the effect of the wide-spectrum, thermostable lactonase Aii20J, from Tenacibaculum sp. 20J on biofilm formation by these strains. The abaI mutant formed less than half of wild type biofilm. However, the adeB mutant showed a similar behaviour with the wild type. The addition of the Aii20J lactonase reduced the biofilm formation as well as the viable cells measured by resazurine in the wild type and in the adeB mutant, in contrast with the abaI mutant in which enzyme did not affect biofilm formation.
Acknowledgements: This work has been supported by Insituto de Salud Carlos III (PI13/02390).
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
59
Identification and cloning of enzymes responsible for quorum quenching activity in the marine bacterium Maribacter ulvicola
Diego Rey1, Celia Mayer1, Andrea Muras1, Manuel Romero2, Ana Otero1
1Departamento de Microbioloxía e Parasitoloxía, Facultade de Bioloxía-CIBUS, Universidade de Santiago de Compostela, Spain, 2Present address: Centre for
Biomolecular Sciences, School of Life Sciences, University of Nottingham NG7 2RD Nottingham, UK. E-mail: [email protected], [email protected]
Keywords: quorum sensing, quorum quenching, AHL, lactonase, acylase, genome analysis
Abstract Numerous bacterial functions, such as virulence and biofilm formation, are controlled by small diffusible molecules released to the medium, allowing bacteria to coordinate their behaviour once a minimal effective quorum has been reached, phenomenon known as quorum sensing (QS). Recently, with the increasing problem of antibiotic multiresistance the number of explored strategies to tackle this problem has increased quickly. One of these strategies is quorum quenching (QQ), consisting on the interference with QS systems by signal degradation or modification, e. g. the use of lactonase and acylase enzymes to degrade N-acyl homoserine lactones (AHLs) produced by Gram-negative bacteria.
In this work, we tried to identify the enzyme(s) responsible for the QQ activity observed in Maribacter ulvicola strain 139, a marine strain isolated from Atlantic Ocean sample at 10-m depth presenting a wide-spectrum AHL-QQ activity (1). The genome of M. ulvicola strain 139 was sequenced using Illumina technology and a whole genomic analysis was performed to detect sequences for known lactonases and acylases. As a result 4 candidate ORFs were identified: three potential acylases and one lactonase. Their sequences were amplified, cloned and overexpressed in E. coli in order to check their enzymatic activity against AHLs (C6 and C10-HSL) using a violacein production bioassay with Chromobacterium violaceum biosensors.
Only one of the 4 sequences, corresponding to a lactonase, demonstrated AHL degradation activity when over-expressed in E. coli. This enzyme AiiMu presented the domain HXHXDH belonging to the metallo-β-lactamase superfamily and a 30% homology with AiiA from Bacillus sp. 240B1. Additionally, the temperature resistance and substrate specificity of over-expressed AiiMu were studied and characterized.
Acknowledgement This work has been supported by Fundación Ramón Areces (CIVP16A1814).
References 1. Romero M, Martin-Cuadrado AB, Otero A. 2012. Determination of whether quorum
quenching is a common activity in marine bacteria by analysis of cultivable bacteria and metagenomic sequences. Appl Environ Microbiol 78:6345-6348.
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
58
Effect of the quorum quenching lactonase Aii20J on biofilm formation by different strains and mutants of Acinetobacter
baumannii Celia Mayer1, Manuel Romero1,2, Andrea Muras1, Soraya Rumbo3, Eva Gato3,
María Tomás3, Ana Otero1
1Departamento de Microbioloxía e Parasitoloxía, Facultade de Bioloxía-CIBUS, Universidade de Santiago de Compostela, Spain, 2Present address: Centre for
Biomolecular Sciences, School of Life Sciences, University of Nottingham NG7 2RD Nottingham, UK, 3Departamento de Microbioloxía, Complexo Hospitalario Universitario
A Coruña-INIBIC, Spain. E-mail: [email protected], [email protected]
Keywords: Acinetobacter baumannii, quorum sensing, biofilm, quorum quenching, acyl-homoserine-lactones, lactonase
Abstract Acinetobacter baumannii is an important Gram-negative nosocomial pathogen responsible for numerous healthcare-associated infections worldwide with an increasing interest due to the emergence of multidrug-resistant (MDR) strains. Among the virulence factors, one of the main causes for the persistence of A. baumannii is the biofilm formation, which has been reported to be regulated by quorum sensing (QS). A. baumannii presents a typical LuxI/LuxR-type quorum-sensing network that involves the abaI synthase and the abaR receptor and is mediated by long substituted N-acyl homoserine lactones (AHLs) such as OHC12-HSL. Therefore, the interference with QS systems, a process generally termed quorum quenching (QQ), represents an interesting anti-pathogenic strategy to control biofilm formation in this bacterium.
Additionally, several reports have demonstrated the contribution of resistance-nodulation-cell division (RND)-type efflux systems in conferring multidrug resistance in this pathogen. RND efflux pump have been related to QS regulation by the selection of AHLs in P. aeruginosa via the efflux system MexAb-OprM. In A. baumannii, one of the most characterized RND-efflux pump is the AdeABC system, composed of the AdeA, AdeB, and AdeC proteins. AdeB is a member of the RND superfamily, whereas AdeA and AdeC are homologous to membrane fusion proteins and to the outer membrane protein OprM from Pseudomonas aeruginosa respectively. This AdeABC pump confers resistance to various antibiotic classes including aminoglycosides and its overexpression plays a major role in acquired resistance in A. baumannii but it is not clear so far if it can be involved in the regulation of the AHL-mediated QS system in A. baumannii.
In this work, we characterize the biofilm formation by different strains of A. baumannii: A. baumannii ATCC 17978, an abaI AHL-synthase isogenic mutant, an adeB gene isogenic mutant and a clinical isolate, by using the crystal violet and resazurine methods. Moreover, we report the effect of the wide-spectrum, thermostable lactonase Aii20J, from Tenacibaculum sp. 20J on biofilm formation by these strains. The abaI mutant formed less than half of wild type biofilm. However, the adeB mutant showed a similar behaviour with the wild type. The addition of the Aii20J lactonase reduced the biofilm formation as well as the viable cells measured by resazurine in the wild type and in the adeB mutant, in contrast with the abaI mutant in which enzyme did not affect biofilm formation.
Acknowledgements: This work has been supported by Insituto de Salud Carlos III (PI13/02390).
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
59
Identification and cloning of enzymes responsible for quorum quenching activity in the marine bacterium Maribacter ulvicola
Diego Rey1, Celia Mayer1, Andrea Muras1, Manuel Romero2, Ana Otero1
1Departamento de Microbioloxía e Parasitoloxía, Facultade de Bioloxía-CIBUS, Universidade de Santiago de Compostela, Spain, 2Present address: Centre for
Biomolecular Sciences, School of Life Sciences, University of Nottingham NG7 2RD Nottingham, UK. E-mail: [email protected], [email protected]
Keywords: quorum sensing, quorum quenching, AHL, lactonase, acylase, genome analysis
Abstract Numerous bacterial functions, such as virulence and biofilm formation, are controlled by small diffusible molecules released to the medium, allowing bacteria to coordinate their behaviour once a minimal effective quorum has been reached, phenomenon known as quorum sensing (QS). Recently, with the increasing problem of antibiotic multiresistance the number of explored strategies to tackle this problem has increased quickly. One of these strategies is quorum quenching (QQ), consisting on the interference with QS systems by signal degradation or modification, e. g. the use of lactonase and acylase enzymes to degrade N-acyl homoserine lactones (AHLs) produced by Gram-negative bacteria.
In this work, we tried to identify the enzyme(s) responsible for the QQ activity observed in Maribacter ulvicola strain 139, a marine strain isolated from Atlantic Ocean sample at 10-m depth presenting a wide-spectrum AHL-QQ activity (1). The genome of M. ulvicola strain 139 was sequenced using Illumina technology and a whole genomic analysis was performed to detect sequences for known lactonases and acylases. As a result 4 candidate ORFs were identified: three potential acylases and one lactonase. Their sequences were amplified, cloned and overexpressed in E. coli in order to check their enzymatic activity against AHLs (C6 and C10-HSL) using a violacein production bioassay with Chromobacterium violaceum biosensors.
Only one of the 4 sequences, corresponding to a lactonase, demonstrated AHL degradation activity when over-expressed in E. coli. This enzyme AiiMu presented the domain HXHXDH belonging to the metallo-β-lactamase superfamily and a 30% homology with AiiA from Bacillus sp. 240B1. Additionally, the temperature resistance and substrate specificity of over-expressed AiiMu were studied and characterized.
Acknowledgement This work has been supported by Fundación Ramón Areces (CIVP16A1814).
References 1. Romero M, Martin-Cuadrado AB, Otero A. 2012. Determination of whether quorum
quenching is a common activity in marine bacteria by analysis of cultivable bacteria and metagenomic sequences. Appl Environ Microbiol 78:6345-6348.
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
60
Disabling Pseudomonas aeruginosa quorum sensing by bio-silver nanoparticles
Brahma N. Singh
Pharmacognosy & Ethnopharmacology Division, CSIR-National Botanical Research Institute, Lucknow-226001, Uttar Pradesh, India.
E-mail: [email protected]
Keywords: Mycofabricated silver nanoparticles; Quorum sensing; Virulence genes; Anti-biofilm; Pseudomonas aeruginosa PAO1
Abstract Quorum sensing is a chemical communication process that Pseudomonas aeruginosa uses to regulate collective behaviours including virulence factors, biofilm, and antibiotic resistance. Interrupting quorum sensing signals with its inhibitors has been proposed as a potential approach to prevent this bacterium virulence. In this study, we biosynthesize surface modified silver nanoparticles (denoted as “mfAgNPs”) using biomass aqueous extract of Rhizopus arrhizus BRS-07 and analyse them for inhibition of the P. aeruginosa PAO1 quorum sensing signalling. After internalization into the cells, mfAgNPs inhibited expression of virulence genes, those encoding proteases (lasA and lasB), pyocyanin (phzA1), and rhamnolipids (rhlA). The mfAgNPs also showed a potential to interfere with bacterial quorum sensing systems: las-rhl including the associated N-acyl homoserine lactones production. The results revealed that mfAgNPs showed anti-biofilm activity, inhibiting biofilm formation and also enhanced susceptibility of preformed biofilm to tobramycin. We have also established that this anti-biofilm activity is reliant on suppression of polyphosphate kinase activity, alginate production, swimming, and swarming motilities by mfAgNPs. Eventually, it was also observed that mfAgNPs enhanced the susceptibility of Staphylococcus aureus to erythromycin. Our study reveals the antimicrobial property of mfAgNPs able to interfere with P. aeruginosa quorum sensing systems and biofilms.
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
61
Non-invasive plasmonic detection and imaging of quorum sensing in biofilms of Pseudomonas aeruginosa
Gustavo Bodelón1, Verónica Montes-García1, Vanesa López-Puente1, Sergio Rodal-Cedeira1, Celina Costas1, Jorge Pérez-Juste1, Isabel Pastoriza-Santos1,
Luis M. Liz-Marzán1,2,3
1Departmento de Química Física, Universidade de Vigo. 36310 Vigo (Spain). 2Bionanoplasmonics Laboratory, CIC biomaGUNE. Paseo de Miramón 182, 20009
Donostia - San Sebastián (Spain).3Ikerbasque, Basque Foundation for Science. 48011 Bilbao (Spain). E-mail: [email protected], [email protected]
Keywords: Nanoparticles, plasmonics, quorum-sensing, biofilm, Pseudomonas aeruginosa, pyocyanin
Abstract In nature, most bacteria exist as organized communities termed biofilms, which are assumed to support Quorum Sensing (QS)-mediated processes. Importantly, QS and biofilms and believed to influence the capacity of bacteria to cause disease, and given their widespread presence, the development of novel methods for in situ and non-invasive study of these processes has become as a research priority. Herein, we describe nanostructured materials for plasmonic detection of QS in biofilms of the human opportunistic pathogen Pseudomonas aeruginosa. Sensors consisting in pNIPAM hydrogels or mesoporous titania thin films doped with gold nanoparticles were used as substrates for sustaining bacterial growth as biofilms and simultaneous surface-enhanced Raman spectroscopy (SERS) detection of pyocyanin as a proxy of QS. This study shows for the first time in situ plasmonic imaging and monitoring of QS in biofilm communities, providing a new analytical tool for direct detection of diffusible metabolites as signalling molecules. In addition we present preliminary experimental data showing the potential use of such plasmonic sensors as implanted materials for studying QS in animal models of infection.
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
60
Disabling Pseudomonas aeruginosa quorum sensing by bio-silver nanoparticles
Brahma N. Singh
Pharmacognosy & Ethnopharmacology Division, CSIR-National Botanical Research Institute, Lucknow-226001, Uttar Pradesh, India.
E-mail: [email protected]
Keywords: Mycofabricated silver nanoparticles; Quorum sensing; Virulence genes; Anti-biofilm; Pseudomonas aeruginosa PAO1
Abstract Quorum sensing is a chemical communication process that Pseudomonas aeruginosa uses to regulate collective behaviours including virulence factors, biofilm, and antibiotic resistance. Interrupting quorum sensing signals with its inhibitors has been proposed as a potential approach to prevent this bacterium virulence. In this study, we biosynthesize surface modified silver nanoparticles (denoted as “mfAgNPs”) using biomass aqueous extract of Rhizopus arrhizus BRS-07 and analyse them for inhibition of the P. aeruginosa PAO1 quorum sensing signalling. After internalization into the cells, mfAgNPs inhibited expression of virulence genes, those encoding proteases (lasA and lasB), pyocyanin (phzA1), and rhamnolipids (rhlA). The mfAgNPs also showed a potential to interfere with bacterial quorum sensing systems: las-rhl including the associated N-acyl homoserine lactones production. The results revealed that mfAgNPs showed anti-biofilm activity, inhibiting biofilm formation and also enhanced susceptibility of preformed biofilm to tobramycin. We have also established that this anti-biofilm activity is reliant on suppression of polyphosphate kinase activity, alginate production, swimming, and swarming motilities by mfAgNPs. Eventually, it was also observed that mfAgNPs enhanced the susceptibility of Staphylococcus aureus to erythromycin. Our study reveals the antimicrobial property of mfAgNPs able to interfere with P. aeruginosa quorum sensing systems and biofilms.
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
61
Non-invasive plasmonic detection and imaging of quorum sensing in biofilms of Pseudomonas aeruginosa
Gustavo Bodelón1, Verónica Montes-García1, Vanesa López-Puente1, Sergio Rodal-Cedeira1, Celina Costas1, Jorge Pérez-Juste1, Isabel Pastoriza-Santos1,
Luis M. Liz-Marzán1,2,3
1Departmento de Química Física, Universidade de Vigo. 36310 Vigo (Spain). 2Bionanoplasmonics Laboratory, CIC biomaGUNE. Paseo de Miramón 182, 20009
Donostia - San Sebastián (Spain).3Ikerbasque, Basque Foundation for Science. 48011 Bilbao (Spain). E-mail: [email protected], [email protected]
Keywords: Nanoparticles, plasmonics, quorum-sensing, biofilm, Pseudomonas aeruginosa, pyocyanin
Abstract In nature, most bacteria exist as organized communities termed biofilms, which are assumed to support Quorum Sensing (QS)-mediated processes. Importantly, QS and biofilms and believed to influence the capacity of bacteria to cause disease, and given their widespread presence, the development of novel methods for in situ and non-invasive study of these processes has become as a research priority. Herein, we describe nanostructured materials for plasmonic detection of QS in biofilms of the human opportunistic pathogen Pseudomonas aeruginosa. Sensors consisting in pNIPAM hydrogels or mesoporous titania thin films doped with gold nanoparticles were used as substrates for sustaining bacterial growth as biofilms and simultaneous surface-enhanced Raman spectroscopy (SERS) detection of pyocyanin as a proxy of QS. This study shows for the first time in situ plasmonic imaging and monitoring of QS in biofilm communities, providing a new analytical tool for direct detection of diffusible metabolites as signalling molecules. In addition we present preliminary experimental data showing the potential use of such plasmonic sensors as implanted materials for studying QS in animal models of infection.
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
62
Analysis of the interactions between Pseudomonas aeruginosa
LasR and quorum sensing modulators by SERS Celina Costas1, Vanesa López-Puente1, Gustavo Bodelón1, Concepción
González-Bello2, Jorge Pérez-Juste1, Isabel Pastoriza-Santos1, Luis M. Liz-Marzán3, 4
1Departamento de Química Física, Universidade de Vigo, 36301 Vigo, Spain, 2Centro Singular de Investigación en Química Biológica y Materiales Moleculares (CIQUS), Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain,
3Bionanoplasmonics Laboratory, CIC biomaGUNE, Paseo de Miramón 182, 20009 Donostia-San Sebastian, Spain 4Ikerbasque, Basque Foundation for Science, 48013
Bilbao, Spain. E-mail: [email protected]
Keywords: Quorum sensing, Pseudomonas, receptors, protein interactions, SERS spectroscopy, nanoplasmonics
Abstract Many members of the LuxR family of QS transcriptional activators, including LasR of Pseudomonas aeruginosa, are believed to require appropriate acyl homoserine lactone (acyl-HSL) ligands to fold into an active conformation. The understanding of such protein-ligand interactions provides a molecular framework for the development of QS modulators. Surface-enhanced Raman scattering (SERS) is a vibrational spectroscopy technique that can be performed in proteins at extremely low concentrations in their active state. Taking advantage of its high sensitivity, we demonstrate the application of SERS spectroscopy for detecting molecular interactions between the ligand-binding domain of LasR (LasRLBD) purified as a soluble apoprotein and modulators of Pseudomonas aeruginosa QS. We found that QS activators and inhibitors produce differential SERS fingerprints in LasRLBD, and in combination with molecular docking analysis, we provide insight into their possible mechanism of action. This study reveals signal-specific structural rearrangements in LasR upon ligand binding, thereby confirming SERS spectroscopy as a powerful tool for the analysis of ligand-induced conformational changes in proteins and for screening modulators of QS as potential therapeutic agents.
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
6
New insights into the QS regulatory network of Stenotrophomonas maltophilia
Pol Huedo1,2, Daniel Yero1,2, Sonia Martinez-Servat1,2, Xavier Daura1,3, Isidre Gibert1,2
1Institut de Biotecnologia i de Biomedicina (IBB), Universitat Autònoma de Barcelona (UAB), Cerdanyola del Vallès (Barcelona), Spain. 2Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona (UAB), Cerdanyola del Vallès
(Barcelona), Spain. 3Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain.
Abstract Stenotrophomonas maltophilia is a ubiquitous gram-negative bacterium that has emerged as an important human opportunistic pathogen affecting mainly immunocompromised patients. To date, the quorum sensing (QS) system described for this microorganism is based on the signalling molecule DSF (Diffusible Signal factor) or acid 11-cis-dodecenoic. The components that govern this QS system are encoded in the rpf cluster (Regulation of Pathogenicity Factors). We have recently demonstrated that two populations of S. maltophilia can be distinguished depending on the rpf cluster (rpf-1 or rpf-2) they harbour. The two clusters differ in the genes that encode the synthase RpfF and the sensor RpfC. Moreover, there is a strict association between the two components, such that only the pairs RpfF-1/RpfC-1 (rpf-1 variant) and RpfF-2/RpfC-2 (rpf-2 variant) are found in any population. Additionally, only those strains harbouring the rpf-1 variant produce detectable levels of DSF, which seems to, participate in the regulation of bacterial motility, biofilm development and virulence. Strains harbouring the rpf-2 variant need extra copies of rpfF-2 or the absence of rpfC-2 to achieve detectable levels of DSF. In this case, DSF-QS seems to control only some virulence-related phenotypes in very specific environments (e.g., zebrafish infection). We have also shown that in S. maltophilia DSF is produced in a positive feedback-manner and that DSF production and virulence properties of a mixed population are the result of synergies between the two rpf-variant groups.
Another fatty acid-mediated QS system that has raised interest in the last years is that based on the cis-decenoic signal (cis-DA), which has been described only in Pseudomonas aeruginosa. It has been shown that cis-DA induces biofilm dispersion of numerous bacteria. The putative enoyl-CoA hydratase (ECH) DspI (PA0745) is required for cis-DA synthesis and for the development of the full virulence capacity of P. aeruginosa. Preliminary results obtained by our group show that the genome of S. maltophilia contains the dspI orthologous gene smlt0266, which encodes for an ECH that similarly regulates biofilm dispersion in this bacterium. Furthermore, we have observed that the downstream gene encoding for the alternative ECH Smlt0267 in S. maltophilia and PA0744 in P. aeruginosa regulates more diverse virulence factors than the synthase DspI, including biofilm formation, bacterial motility and infection invivo. Altogether, it suggests that cis-DA QS is active in S. maltophilia and could be widely conserved among related bacteria.
Interestingly, we have also observed that besides fatty-acid-mediated QS systems, S.maltophilia responds also to exogenous acyl homoserine lactone signals (AHL) through the LuxR solo SmoR (Smlt1839), modulating swarming motility.
Overall, our findings evidence that S. maltophilia presents a complex QS regulatory network and provide valuable information for the design of new QS inhibitors as well as new antimicrobial treatments.
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
62
Analysis of the interactions between Pseudomonas aeruginosa
LasR and quorum sensing modulators by SERS Celina Costas1, Vanesa López-Puente1, Gustavo Bodelón1, Concepción
González-Bello2, Jorge Pérez-Juste1, Isabel Pastoriza-Santos1, Luis M. Liz-Marzán3, 4
1Departamento de Química Física, Universidade de Vigo, 36301 Vigo, Spain, 2Centro Singular de Investigación en Química Biológica y Materiales Moleculares (CIQUS), Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain,
3Bionanoplasmonics Laboratory, CIC biomaGUNE, Paseo de Miramón 182, 20009 Donostia-San Sebastian, Spain 4Ikerbasque, Basque Foundation for Science, 48013
Bilbao, Spain. E-mail: [email protected]
Keywords: Quorum sensing, Pseudomonas, receptors, protein interactions, SERS spectroscopy, nanoplasmonics
Abstract Many members of the LuxR family of QS transcriptional activators, including LasR of Pseudomonas aeruginosa, are believed to require appropriate acyl homoserine lactone (acyl-HSL) ligands to fold into an active conformation. The understanding of such protein-ligand interactions provides a molecular framework for the development of QS modulators. Surface-enhanced Raman scattering (SERS) is a vibrational spectroscopy technique that can be performed in proteins at extremely low concentrations in their active state. Taking advantage of its high sensitivity, we demonstrate the application of SERS spectroscopy for detecting molecular interactions between the ligand-binding domain of LasR (LasRLBD) purified as a soluble apoprotein and modulators of Pseudomonas aeruginosa QS. We found that QS activators and inhibitors produce differential SERS fingerprints in LasRLBD, and in combination with molecular docking analysis, we provide insight into their possible mechanism of action. This study reveals signal-specific structural rearrangements in LasR upon ligand binding, thereby confirming SERS spectroscopy as a powerful tool for the analysis of ligand-induced conformational changes in proteins and for screening modulators of QS as potential therapeutic agents.
ISQSI, Santiago de Compostela 2015 Poster communications – session 1
6
New insights into the QS regulatory network of Stenotrophomonas maltophilia
Pol Huedo1,2, Daniel Yero1,2, Sonia Martinez-Servat1,2, Xavier Daura1,3, Isidre Gibert1,2
1Institut de Biotecnologia i de Biomedicina (IBB), Universitat Autònoma de Barcelona (UAB), Cerdanyola del Vallès (Barcelona), Spain. 2Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona (UAB), Cerdanyola del Vallès
(Barcelona), Spain. 3Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain.
Abstract Stenotrophomonas maltophilia is a ubiquitous gram-negative bacterium that has emerged as an important human opportunistic pathogen affecting mainly immunocompromised patients. To date, the quorum sensing (QS) system described for this microorganism is based on the signalling molecule DSF (Diffusible Signal factor) or acid 11-cis-dodecenoic. The components that govern this QS system are encoded in the rpf cluster (Regulation of Pathogenicity Factors). We have recently demonstrated that two populations of S. maltophilia can be distinguished depending on the rpf cluster (rpf-1 or rpf-2) they harbour. The two clusters differ in the genes that encode the synthase RpfF and the sensor RpfC. Moreover, there is a strict association between the two components, such that only the pairs RpfF-1/RpfC-1 (rpf-1 variant) and RpfF-2/RpfC-2 (rpf-2 variant) are found in any population. Additionally, only those strains harbouring the rpf-1 variant produce detectable levels of DSF, which seems to, participate in the regulation of bacterial motility, biofilm development and virulence. Strains harbouring the rpf-2 variant need extra copies of rpfF-2 or the absence of rpfC-2 to achieve detectable levels of DSF. In this case, DSF-QS seems to control only some virulence-related phenotypes in very specific environments (e.g., zebrafish infection). We have also shown that in S. maltophilia DSF is produced in a positive feedback-manner and that DSF production and virulence properties of a mixed population are the result of synergies between the two rpf-variant groups.
Another fatty acid-mediated QS system that has raised interest in the last years is that based on the cis-decenoic signal (cis-DA), which has been described only in Pseudomonas aeruginosa. It has been shown that cis-DA induces biofilm dispersion of numerous bacteria. The putative enoyl-CoA hydratase (ECH) DspI (PA0745) is required for cis-DA synthesis and for the development of the full virulence capacity of P. aeruginosa. Preliminary results obtained by our group show that the genome of S. maltophilia contains the dspI orthologous gene smlt0266, which encodes for an ECH that similarly regulates biofilm dispersion in this bacterium. Furthermore, we have observed that the downstream gene encoding for the alternative ECH Smlt0267 in S. maltophilia and PA0744 in P. aeruginosa regulates more diverse virulence factors than the synthase DspI, including biofilm formation, bacterial motility and infection invivo. Altogether, it suggests that cis-DA QS is active in S. maltophilia and could be widely conserved among related bacteria.
Interestingly, we have also observed that besides fatty-acid-mediated QS systems, S.maltophilia responds also to exogenous acyl homoserine lactone signals (AHL) through the LuxR solo SmoR (Smlt1839), modulating swarming motility.
Overall, our findings evidence that S. maltophilia presents a complex QS regulatory network and provide valuable information for the design of new QS inhibitors as well as new antimicrobial treatments.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
64
Inhibition of biofilm formation of Gram-negative bacteria using quorum-quenching compounds
Tomohiro Morohoshi1, Shunsuke Shiogai1, Seiji Ochiai1, Takaki Azuma1, Miwa Ishizuka2, Tsukasa Ikeda1
1Department of Material and Environmental Chemistry, Graduate School of Engineering, Utsunomiya University, 7-1-2 Yoto, Utsunomiya, 321-8585, Japan,
2Toshiba Co., Ltd. E-mail: [email protected]
Keywords: quorum sensing, N-acylhomoserine lactone, inhibitor, cyclodextrin, biofilm
Abstract Biofilms have often been a problem for chronic infection or biofouling of membrane bioreactor. Many Gram-negative bacteria regulate their biofilm formation by N-acylhomoserine lactone (AHL)-mediated quorum sensing. In previous study, some natural and artificial inhibitors have been reported to prevent the expression of genes controlled by quorum sensing. We have previously reported two different quorum-quenching compounds. Cyclodextrin (CD) is a natural product that possesses a hydrophilic external surface and a hydrophobic internal cavity. We have demonstrated that the cavity of CD could accept the acyl chain of AHL. The activation of quorum sensing-controlled genes in some Gram-negative bacteria could be blocked in the presence of CD (1). In addition, we have found the novel AHL analog compound, N-acyl cyclopentylamide (Cn-CPA), which can inhibit expression of virulence factors regulated by quorum sensing in Pseudomonas aeruginosa and Serratia marcescens (2). In this study, we evaluated the inhibitory effects of CD and Cn-CPA on biofilm formation by two bacterial strains, P. aeruginosa PAO1 and Aeromonas hydrophila R2. A. hydrophila R2 has been isolated fromthe activated sludge as an AHL producer in previous study. Cn-CPA showed certain degree ofinhibitory effect on biofilm formation of PAO1, but did not inhibit biofilm formation of R2. On theother hand, CD showed higher inhibitory effect on biofilm formation of both PAO1 and R2. Thethree-dimensional structure of biofilm of PAO1 was observed in flow cell system. The biofilm ofPAO1 was tighter with high cell density. When CD or Cn-CPA was added, the biofilm of PAO1was comparatively scattering with low cell density. These results demonstrated that CD and Cn-CPA are effective for inhibit biofilm formation of Gram-negative bacteria.
Acknowledgements: This work was supported by Core Research for Evolutional Science and Technology (CREST) from the Japan Science and Technology Agency (JST).
References 1. Morohoshi T, Tokita K, Ito S, Saito Y, Maeda S, Kato N, Ikeda T. 2013. Inhibition of
quorum sensing in Gram-negative bacteria by alkylamine-modified cyclodextrins. J BiosciBioeng. 116:175-179.
2. Morohoshi T, Shiono T, Takidouchi K, Kato M, Kato N, Kato J, Ikeda T. 2007. Inhibitionof quorum sensing in Serratia marcescens AS-1 by the synthetic analogs of N-acylhomoserine lactone. Appl Environ Microbiol. 73:6339-6344.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
65
Quinolone-based PqsR antagonists provide in vivo proof-of-concept for PQS-targeting anti-virulence strategy
C. Lu1, C. Maurer1, B. Kirsch1, A. Steinbach1, S. Weiß2, V. Pawar2, R.W.Hartmann1
1 Helmholtz-Institute for Pharmaceutical Research Saarland, Campus C2.3, 66123 Saarbrücken, Germany., 2 Helmholtz-Zentrum für Infektionsforschung GmbH,
Inhoffenstraße 7, 38124 Braunschweig, Germany. E-mail: [email protected]; [email protected]
Keywords: PqsR antagonists, anti-virulence agent, medicinal chemistry, resistance
Abstract Increasing antibiotic resistance urgently requires novel therapeutic strategies to combat microbial infections (1). One such promising approach is based on the selective blockade of bacterial virulence (2, 3). An attractive drug target in the human pathogen Pseudomonas aeruginosa is the virulence regulator PqsR (4) (Fig. 1). We discovered the affine PqsR antagonist 1, which, however, did not efficiently attenuate virulence of P. aeruginosa cells. A detailed investigation of this phenomenon, revealed a cell-mediated metabolism as the critical cause for the decreased activity. Blockade of the metabolic hot spot resulted in potent compound 3 that strongly reduced virulence and fully protected Galleria mellonella larvae from lethal P. aeruginosa infections at low nanomolar concentration (Fig. 2). Additionally, the developed anti-virulence agent showed promising results in a murine chronic infection model, therefore, providing a proof-of-concept for PqsR-targeted anti-virulence therapy.
Figure 1: Role of PqsR within the QS system Figure 2: Antagonist development strategy
References 1. Arias CA, Murray BE. 2010. Antibiotic-resistant bugs in the 21st century--a clinical super-
challenge. N Engl J Med 360:439-43.
2. Cegelski L, Marshall G, Eldridge G, and Hultgren SJ. 2008. The biology and futureprospects of antivirulence therapies. Nat Rev Microbiol 6:17-27.
3. Rasko DA, Sperandio V. 2010. Anti-virulence strategies to combat bacteria-mediateddisease. Nat Rev Drug Discov 9:117-28.
4. Cao H, Krishnan G, Goumnerov B, Tsongalis J, Tompkins R, Rahme LG. 2001. Aquorum sensing-associated virulence gene of Pseudomonas aeruginosa encodes a LysR-like transcription regulator with a unique self-regulatory mechanism. Proc Natl Acad SciUSA 98:14613-8.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
64
Inhibition of biofilm formation of Gram-negative bacteria using quorum-quenching compounds
Tomohiro Morohoshi1, Shunsuke Shiogai1, Seiji Ochiai1, Takaki Azuma1, Miwa Ishizuka2, Tsukasa Ikeda1
1Department of Material and Environmental Chemistry, Graduate School of Engineering, Utsunomiya University, 7-1-2 Yoto, Utsunomiya, 321-8585, Japan,
2Toshiba Co., Ltd. E-mail: [email protected]
Keywords: quorum sensing, N-acylhomoserine lactone, inhibitor, cyclodextrin, biofilm
Abstract Biofilms have often been a problem for chronic infection or biofouling of membrane bioreactor. Many Gram-negative bacteria regulate their biofilm formation by N-acylhomoserine lactone (AHL)-mediated quorum sensing. In previous study, some natural and artificial inhibitors have been reported to prevent the expression of genes controlled by quorum sensing. We have previously reported two different quorum-quenching compounds. Cyclodextrin (CD) is a natural product that possesses a hydrophilic external surface and a hydrophobic internal cavity. We have demonstrated that the cavity of CD could accept the acyl chain of AHL. The activation of quorum sensing-controlled genes in some Gram-negative bacteria could be blocked in the presence of CD (1). In addition, we have found the novel AHL analog compound, N-acyl cyclopentylamide (Cn-CPA), which can inhibit expression of virulence factors regulated by quorum sensing in Pseudomonas aeruginosa and Serratia marcescens (2). In this study, we evaluated the inhibitory effects of CD and Cn-CPA on biofilm formation by two bacterial strains, P. aeruginosa PAO1 and Aeromonas hydrophila R2. A. hydrophila R2 has been isolated fromthe activated sludge as an AHL producer in previous study. Cn-CPA showed certain degree ofinhibitory effect on biofilm formation of PAO1, but did not inhibit biofilm formation of R2. On theother hand, CD showed higher inhibitory effect on biofilm formation of both PAO1 and R2. Thethree-dimensional structure of biofilm of PAO1 was observed in flow cell system. The biofilm ofPAO1 was tighter with high cell density. When CD or Cn-CPA was added, the biofilm of PAO1was comparatively scattering with low cell density. These results demonstrated that CD and Cn-CPA are effective for inhibit biofilm formation of Gram-negative bacteria.
Acknowledgements: This work was supported by Core Research for Evolutional Science and Technology (CREST) from the Japan Science and Technology Agency (JST).
References 1. Morohoshi T, Tokita K, Ito S, Saito Y, Maeda S, Kato N, Ikeda T. 2013. Inhibition of
quorum sensing in Gram-negative bacteria by alkylamine-modified cyclodextrins. J BiosciBioeng. 116:175-179.
2. Morohoshi T, Shiono T, Takidouchi K, Kato M, Kato N, Kato J, Ikeda T. 2007. Inhibitionof quorum sensing in Serratia marcescens AS-1 by the synthetic analogs of N-acylhomoserine lactone. Appl Environ Microbiol. 73:6339-6344.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
65
Quinolone-based PqsR antagonists provide in vivo proof-of-concept for PQS-targeting anti-virulence strategy
C. Lu1, C. Maurer1, B. Kirsch1, A. Steinbach1, S. Weiß2, V. Pawar2, R.W.Hartmann1
1 Helmholtz-Institute for Pharmaceutical Research Saarland, Campus C2.3, 66123 Saarbrücken, Germany., 2 Helmholtz-Zentrum für Infektionsforschung GmbH,
Inhoffenstraße 7, 38124 Braunschweig, Germany. E-mail: [email protected]; [email protected]
Keywords: PqsR antagonists, anti-virulence agent, medicinal chemistry, resistance
Abstract Increasing antibiotic resistance urgently requires novel therapeutic strategies to combat microbial infections (1). One such promising approach is based on the selective blockade of bacterial virulence (2, 3). An attractive drug target in the human pathogen Pseudomonas aeruginosa is the virulence regulator PqsR (4) (Fig. 1). We discovered the affine PqsR antagonist 1, which, however, did not efficiently attenuate virulence of P. aeruginosa cells. A detailed investigation of this phenomenon, revealed a cell-mediated metabolism as the critical cause for the decreased activity. Blockade of the metabolic hot spot resulted in potent compound 3 that strongly reduced virulence and fully protected Galleria mellonella larvae from lethal P. aeruginosa infections at low nanomolar concentration (Fig. 2). Additionally, the developed anti-virulence agent showed promising results in a murine chronic infection model, therefore, providing a proof-of-concept for PqsR-targeted anti-virulence therapy.
Figure 1: Role of PqsR within the QS system Figure 2: Antagonist development strategy
References 1. Arias CA, Murray BE. 2010. Antibiotic-resistant bugs in the 21st century--a clinical super-
challenge. N Engl J Med 360:439-43.
2. Cegelski L, Marshall G, Eldridge G, and Hultgren SJ. 2008. The biology and futureprospects of antivirulence therapies. Nat Rev Microbiol 6:17-27.
3. Rasko DA, Sperandio V. 2010. Anti-virulence strategies to combat bacteria-mediateddisease. Nat Rev Drug Discov 9:117-28.
4. Cao H, Krishnan G, Goumnerov B, Tsongalis J, Tompkins R, Rahme LG. 2001. Aquorum sensing-associated virulence gene of Pseudomonas aeruginosa encodes a LysR-like transcription regulator with a unique self-regulatory mechanism. Proc Natl Acad SciUSA 98:14613-8.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
66
Biological evaluation of an in vivo-potent dual target PQS-Quorum Sensing inhibitor that hinders biofilm formation
Andreas Thomann1, Antonio G. Martins1, Christian Brengel1, Elisabeth Weidel1, Alberto Plaza1, Carsten Börger2, Martin Empting1, Rolf W. Hartmann1,3
1Helmholtz Institute for Pharmaceutical Research Saarland, Campus C 2.3, 66123 Saarbrücken, Germany; 2PharmBioTec GmbH, Sciencepark 1, Universtät des
Saarlandes, 66123 Saarbrücken, Germany; 3Department for Pharmaceutical and Medicinal Chemistry, Campus C 2.3, Saarland University, 66123 Saarbrücken,
Germany. E-mail: [email protected]
Keywords: Pseudomonas aeruginosa, PQS, Dual Target QS Inhibitors, PqsD, PqsR
Abstract Emergence of Pseudomonas aeruginosa (PA) as a leading cause of nosocomial infections and morbidity in immunocompromised patients has consolidated it in the race for novel antimicrobial compounds. PA infections are notoriously difficult to eradicate due to intrinsic resistance to a variety of available antibiotics. Its distinguished ability to form biofilms amplifies resistance and promotes immune response evasion.
The PA quorum-sensing (QS) system is a sophisticated network of genome-wide regulation. A major player is the pseudomonas quinolone signal system (PQS-QS) that regulates the production of several non-vital virulence and biofilm-related determinants. Therefore, QS circuitry is an attractive target for anti-virulence therapeutics with lowered resistance development potential (1-3). We have developed a dual-inhibitor compound (cmpd. A) of low molecular weight and high solubility that targets PQS transcriptional regulator (PqsR) and PqsD, a key enzyme in the biosynthesis of PQS-QS signal molecules (HHQ and PQS).
In this context, cmpd. A markedly reduced virulence factor production without affecting bacterial growth. Additionally, ciprofloxacin co-administration in vitro increased susceptibility of PA14 to antibiotic administration under biofilm conditions. Disruption of pathogenicity mechanisms was assessed in vivo, with significant increased survival of challenged larvae in an established Galleria mellonella infection model. Finally, we observed considerable reduction of biofilm volume and extracellular DNA.
Favourable physicochemical properties and effects on virulence/biofilm establish a promising starting point for further optimization. In particular, interference with biofilm holds great promise in lowering pathogenicity and increasing susceptibility to pharmacological treatment and immune responses in chronic and persistent infections.
References 1. Storz MP, Maurer CK, Zimmer C, Wagner N, Brengel C, de Jong JC, Lucas S, Müsken
M, Häussler S, Steinbach A, Hartmann RW. 2012. Validation of PqsD as an anti-biofilmtarget in Pseudomonas aeruginosa by development of small-molecule inhibitors. J AmChem Soc 134:16143-16143.
2. Lu C, Maurer, CK, Kirsch B, Steinbach A, Hartmann RW. 2014. Overcoming theUnexpected functional inversion of a PqsR antagonist in Pseudomonas aeruginosa: An invivo potent antivirulence agent targeting pqs Quorum Sensing. Angew Chem 126:1127-1130.
3. Weidel E, Negri M, Empting M, Hinsberger S, Hartmann RW, 2014. Composingcompound libraries for hit discovery – rationality-driven preselection or random choice bystructural diversity? Future Med Chem 6:2057-72.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
67
Discovery of novel small-molecules for the inhibition of Pseudomonas quinolone signalling
Ivan Lafayette1, Jean-Frédéric Dubern1, Quoc Tuan Do2, James Lazenby1, Nigel Halliday1, Stephan Heeb1, Paul Williams1, Miguel Cámara1
1Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom, 2 Greenpharma S. A., Orléans, France.
Abstract Pseudomonas aeruginosa is a major opportunistic pathogen in cystic fibrosis, wound and nosocomial infections due to its high levels of intrinsic and acquired resistance to antibiotics, presenting a serious public health problem. Hence there is an urgent need to develop novel antimicrobial therapies which can alleviate the health burden caused by this bacterium. We have developed a high-throughput screening test to identify compounds that can inhibit the PqsR quorum sensing (QS) regulator from P. aeruginosa which controls the expression of the pqsABCDE genes required for the biosynthesis of the 2-alkyl-4-quinolones (AQ)-dependent QS system and the switch on of virulence. This biosensor is based on the use of a chromosomal lux-based transcriptional fusion to the pqsA promoter region. Using this approach we have identified, within an extensive library of natural and synthetic compounds, a number hits which reduce psqA expression with IC50s down to the nM range. These compounds inhibit AQ biosynthesis, pyocyanin, protease and elastase production controlled by the AQ QS system. They also abolish biofilm formation in combination with ciprofloxacin. Preliminary experiments show that some of these compounds are non-cytotoxic at above inhibitory concentrations. Further work is being carried out to investigate the suitability of these compounds for pre-clinical experiments.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
66
Biological evaluation of an in vivo-potent dual target PQS-Quorum Sensing inhibitor that hinders biofilm formation
Andreas Thomann1, Antonio G. Martins1, Christian Brengel1, Elisabeth Weidel1, Alberto Plaza1, Carsten Börger2, Martin Empting1, Rolf W. Hartmann1,3
1Helmholtz Institute for Pharmaceutical Research Saarland, Campus C 2.3, 66123 Saarbrücken, Germany; 2PharmBioTec GmbH, Sciencepark 1, Universtät des
Saarlandes, 66123 Saarbrücken, Germany; 3Department for Pharmaceutical and Medicinal Chemistry, Campus C 2.3, Saarland University, 66123 Saarbrücken,
Germany. E-mail: [email protected]
Keywords: Pseudomonas aeruginosa, PQS, Dual Target QS Inhibitors, PqsD, PqsR
Abstract Emergence of Pseudomonas aeruginosa (PA) as a leading cause of nosocomial infections and morbidity in immunocompromised patients has consolidated it in the race for novel antimicrobial compounds. PA infections are notoriously difficult to eradicate due to intrinsic resistance to a variety of available antibiotics. Its distinguished ability to form biofilms amplifies resistance and promotes immune response evasion.
The PA quorum-sensing (QS) system is a sophisticated network of genome-wide regulation. A major player is the pseudomonas quinolone signal system (PQS-QS) that regulates the production of several non-vital virulence and biofilm-related determinants. Therefore, QS circuitry is an attractive target for anti-virulence therapeutics with lowered resistance development potential (1-3). We have developed a dual-inhibitor compound (cmpd. A) of low molecular weight and high solubility that targets PQS transcriptional regulator (PqsR) and PqsD, a key enzyme in the biosynthesis of PQS-QS signal molecules (HHQ and PQS).
In this context, cmpd. A markedly reduced virulence factor production without affecting bacterial growth. Additionally, ciprofloxacin co-administration in vitro increased susceptibility of PA14 to antibiotic administration under biofilm conditions. Disruption of pathogenicity mechanisms was assessed in vivo, with significant increased survival of challenged larvae in an established Galleria mellonella infection model. Finally, we observed considerable reduction of biofilm volume and extracellular DNA.
Favourable physicochemical properties and effects on virulence/biofilm establish a promising starting point for further optimization. In particular, interference with biofilm holds great promise in lowering pathogenicity and increasing susceptibility to pharmacological treatment and immune responses in chronic and persistent infections.
References 1. Storz MP, Maurer CK, Zimmer C, Wagner N, Brengel C, de Jong JC, Lucas S, Müsken
M, Häussler S, Steinbach A, Hartmann RW. 2012. Validation of PqsD as an anti-biofilmtarget in Pseudomonas aeruginosa by development of small-molecule inhibitors. J AmChem Soc 134:16143-16143.
2. Lu C, Maurer, CK, Kirsch B, Steinbach A, Hartmann RW. 2014. Overcoming theUnexpected functional inversion of a PqsR antagonist in Pseudomonas aeruginosa: An invivo potent antivirulence agent targeting pqs Quorum Sensing. Angew Chem 126:1127-1130.
3. Weidel E, Negri M, Empting M, Hinsberger S, Hartmann RW, 2014. Composingcompound libraries for hit discovery – rationality-driven preselection or random choice bystructural diversity? Future Med Chem 6:2057-72.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
67
Discovery of novel small-molecules for the inhibition of Pseudomonas quinolone signalling
Ivan Lafayette1, Jean-Frédéric Dubern1, Quoc Tuan Do2, James Lazenby1, Nigel Halliday1, Stephan Heeb1, Paul Williams1, Miguel Cámara1
1Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom, 2 Greenpharma S. A., Orléans, France.
Abstract Pseudomonas aeruginosa is a major opportunistic pathogen in cystic fibrosis, wound and nosocomial infections due to its high levels of intrinsic and acquired resistance to antibiotics, presenting a serious public health problem. Hence there is an urgent need to develop novel antimicrobial therapies which can alleviate the health burden caused by this bacterium. We have developed a high-throughput screening test to identify compounds that can inhibit the PqsR quorum sensing (QS) regulator from P. aeruginosa which controls the expression of the pqsABCDE genes required for the biosynthesis of the 2-alkyl-4-quinolones (AQ)-dependent QS system and the switch on of virulence. This biosensor is based on the use of a chromosomal lux-based transcriptional fusion to the pqsA promoter region. Using this approach we have identified, within an extensive library of natural and synthetic compounds, a number hits which reduce psqA expression with IC50s down to the nM range. These compounds inhibit AQ biosynthesis, pyocyanin, protease and elastase production controlled by the AQ QS system. They also abolish biofilm formation in combination with ciprofloxacin. Preliminary experiments show that some of these compounds are non-cytotoxic at above inhibitory concentrations. Further work is being carried out to investigate the suitability of these compounds for pre-clinical experiments.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
68
Development of novel antagonists of PqsR, an important regulator of Pseudomonas aeruginosa virulence
Michael Zender1, Benjamin Kirsch1, Christine K. Maurer1, Martin Empting1, Rolf W. Hartmann1,2
1Helmholtz-Institute for Pharmaceutical Research Saarland, Department of Drug Design and Optimization, Campus C2.3, 66123 Saarbrücken, Germany, 2Department
of Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, 66123 Saarbrücken, Germany. E-mail: [email protected]
Keywords: Pseudomonas aeruginosa, PqsR, Medicinal Chemistry, Fragment-based drug-discovery
Abstract Pseudomonas aeruginosa is a highly adaptable opportunistic pathogen causing chronic persistent lung infections in cystic fibrosis patients. It coordinates group behaviours via a cell-density-dependent communication system known as quorum sensing (QS). Among others the alkyl quinolones HHQ and PQS are utilized for cell-to-cell communication. Both signalling molecules bind to the transcriptional regulator PqsR, thereby, stimulating the expression of various virulence factors. Therefore, we consider the development of PqsR antagonists as a novel promising strategy to limit the pathogenicity of P. aeruginosa without affecting bacterial viability.
A ligand-based design strategy starting from the natural effector HHQ led to the identification of the first potent PqsR antagonists (1). In order to expand the chemical diversity and to address the poor physicochemical properties of these quinolone derivatives a surface plasmon resonance (SPR) screening against a library of low MW compounds was conducted (2,3). The thermodynamic profiles of the top five SPR hit compounds were evaluated using ITC methodology. The discovered hits showed enthalpy-driven binding and excellent LEs. Furthermore, the functional properties of the best fragments were analysed in a ß-galactosidase reporter gene assay as an additional guidepost for hit selection. A thiadiazole-2-amine was chosen as starting point for fragment-modification which afforded an oxadiazole derivative showing improved affinity and potency with consistently high LE. A fragment-growing approach led to compounds showing antagonistic activities as well as pyocyanin inhibition in the nanomolar range being even more potent than the quinolone derived antagonists while keeping superior physicochemical properties.
References 1. Lu C, Kirsch B, Zimmer C, de Jong JC, Henn C, Maurer CK, Müsken M, Häussler S,
Steinbach A, Hartmann RW. 2012. Discovery of Antagonists of PqsR, a Key Player in 2-Alkyl-4-quinolone-Dependent Quorum Sensing in Pseudomonas aeruginosa. Chem Biol19:381-390.
2. Klein T, Henn C, de Jong JC, Zimmer C, Kirsch B, Maurer CK, Pistorius D, Müller R,Steinbach A, Hartmann RW. 2012. Identification of small-molecule antagonists of thePseudomonas aeruginosa transcriptional regulator PqsR: biophysically guided hitdiscovery and optimization. ACS Chem Biol 7:1496-1501.
3. Zender M, Klein T, Henn C, Kirsch B, Maurer CK, Kail D, Ritter C, Dolezal O,Steinbach A, Hartmann RW. 2013. Discovery and Biophysical Characterization of 2-Amino-oxadiazoles as Novel Antagonists of PqsR, an Important Regulator ofPseudomonas aeruginosa Virulence J Med Chem 56:6761-6774.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
69
Inhibition of quorum sensing controlled virulence phenotypes in Pseudomonas aeruginosa PAO1 by using eucalyptus oil
Manmohit Kalia1, Vivek Kumar1, Pradeep Kumar Singh1, Suhaga Dohare1, S.S. Narvi2,Vishnu Agarwal1
1Department of Biotechnology, MotiLal Nehru National Institute of Technology, Allahabad, India 2Department of Chemistry, Motilal Nehru National Institute of
Technology, Allahabad, India. E-mail: [email protected]
Keywords: Quorum Sensing, Pseudomonas aeruginosa PAO1, 3-oxo-C12 HSL, lasI/lasR, rhlI/rhlR
Abstract Quorum sensing is a bacterial communication process in which bacteria communicates with each other using chemical messengers. Bacteria like Pseudomonas aeruginosa which is one of most common pathogen in nosocomial infections utilize quorum sensing to regulate various virulence phenotypes and biofilm formation. Pseudomonas aeruginosa utilizes three signalling pathways which are hierarchally related to each other two of which are luxI/luxR based (lasI/lasR, rhlI/rhlR) and one is Pseudomonas quinolone signal (PQS). 3-oxo-C12HSL is the major signalling molecule in the quorum sensing of Pseudomonas aeruginosa. Bacteria in biofilm mode are more resistant to the antibiotics so blocking the quorum sensing pathways is an alternative to traditional antibiotics. Inhibitors from natural products are an attractive alternative to combat antibiotic resistance. Eucalyptus oil is especially used for the treatment of respiratory ailments such as bronchitis and croup. Eucalyptus oil is used on large scale for preparation of liniments, cough syrups, tooth pastes and as pharmaceutical flavours in dentistry. Eucalyptus oil was tested for its quorum sensing activity using Chromobacterium violaceum CV026 and it was found that eucalyptus oil reduces the quorum sensing at sublethal concentration. Further eucalyptus oil can be used for inhibition of quorum sensing controlled virulence phenotypes of Pseudomonas aeruginosa PAO1.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
68
Development of novel antagonists of PqsR, an important regulator of Pseudomonas aeruginosa virulence
Michael Zender1, Benjamin Kirsch1, Christine K. Maurer1, Martin Empting1, Rolf W. Hartmann1,2
1Helmholtz-Institute for Pharmaceutical Research Saarland, Department of Drug Design and Optimization, Campus C2.3, 66123 Saarbrücken, Germany, 2Department
of Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, 66123 Saarbrücken, Germany. E-mail: [email protected]
Keywords: Pseudomonas aeruginosa, PqsR, Medicinal Chemistry, Fragment-based drug-discovery
Abstract Pseudomonas aeruginosa is a highly adaptable opportunistic pathogen causing chronic persistent lung infections in cystic fibrosis patients. It coordinates group behaviours via a cell-density-dependent communication system known as quorum sensing (QS). Among others the alkyl quinolones HHQ and PQS are utilized for cell-to-cell communication. Both signalling molecules bind to the transcriptional regulator PqsR, thereby, stimulating the expression of various virulence factors. Therefore, we consider the development of PqsR antagonists as a novel promising strategy to limit the pathogenicity of P. aeruginosa without affecting bacterial viability.
A ligand-based design strategy starting from the natural effector HHQ led to the identification of the first potent PqsR antagonists (1). In order to expand the chemical diversity and to address the poor physicochemical properties of these quinolone derivatives a surface plasmon resonance (SPR) screening against a library of low MW compounds was conducted (2,3). The thermodynamic profiles of the top five SPR hit compounds were evaluated using ITC methodology. The discovered hits showed enthalpy-driven binding and excellent LEs. Furthermore, the functional properties of the best fragments were analysed in a ß-galactosidase reporter gene assay as an additional guidepost for hit selection. A thiadiazole-2-amine was chosen as starting point for fragment-modification which afforded an oxadiazole derivative showing improved affinity and potency with consistently high LE. A fragment-growing approach led to compounds showing antagonistic activities as well as pyocyanin inhibition in the nanomolar range being even more potent than the quinolone derived antagonists while keeping superior physicochemical properties.
References 1. Lu C, Kirsch B, Zimmer C, de Jong JC, Henn C, Maurer CK, Müsken M, Häussler S,
Steinbach A, Hartmann RW. 2012. Discovery of Antagonists of PqsR, a Key Player in 2-Alkyl-4-quinolone-Dependent Quorum Sensing in Pseudomonas aeruginosa. Chem Biol19:381-390.
2. Klein T, Henn C, de Jong JC, Zimmer C, Kirsch B, Maurer CK, Pistorius D, Müller R,Steinbach A, Hartmann RW. 2012. Identification of small-molecule antagonists of thePseudomonas aeruginosa transcriptional regulator PqsR: biophysically guided hitdiscovery and optimization. ACS Chem Biol 7:1496-1501.
3. Zender M, Klein T, Henn C, Kirsch B, Maurer CK, Kail D, Ritter C, Dolezal O,Steinbach A, Hartmann RW. 2013. Discovery and Biophysical Characterization of 2-Amino-oxadiazoles as Novel Antagonists of PqsR, an Important Regulator ofPseudomonas aeruginosa Virulence J Med Chem 56:6761-6774.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
69
Inhibition of quorum sensing controlled virulence phenotypes in Pseudomonas aeruginosa PAO1 by using eucalyptus oil
Manmohit Kalia1, Vivek Kumar1, Pradeep Kumar Singh1, Suhaga Dohare1, S.S. Narvi2,Vishnu Agarwal1
1Department of Biotechnology, MotiLal Nehru National Institute of Technology, Allahabad, India 2Department of Chemistry, Motilal Nehru National Institute of
Technology, Allahabad, India. E-mail: [email protected]
Keywords: Quorum Sensing, Pseudomonas aeruginosa PAO1, 3-oxo-C12 HSL, lasI/lasR, rhlI/rhlR
Abstract Quorum sensing is a bacterial communication process in which bacteria communicates with each other using chemical messengers. Bacteria like Pseudomonas aeruginosa which is one of most common pathogen in nosocomial infections utilize quorum sensing to regulate various virulence phenotypes and biofilm formation. Pseudomonas aeruginosa utilizes three signalling pathways which are hierarchally related to each other two of which are luxI/luxR based (lasI/lasR, rhlI/rhlR) and one is Pseudomonas quinolone signal (PQS). 3-oxo-C12HSL is the major signalling molecule in the quorum sensing of Pseudomonas aeruginosa. Bacteria in biofilm mode are more resistant to the antibiotics so blocking the quorum sensing pathways is an alternative to traditional antibiotics. Inhibitors from natural products are an attractive alternative to combat antibiotic resistance. Eucalyptus oil is especially used for the treatment of respiratory ailments such as bronchitis and croup. Eucalyptus oil is used on large scale for preparation of liniments, cough syrups, tooth pastes and as pharmaceutical flavours in dentistry. Eucalyptus oil was tested for its quorum sensing activity using Chromobacterium violaceum CV026 and it was found that eucalyptus oil reduces the quorum sensing at sublethal concentration. Further eucalyptus oil can be used for inhibition of quorum sensing controlled virulence phenotypes of Pseudomonas aeruginosa PAO1.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
70
Regulatory effect of fatty acids from seeds (Amaranthus
hypochondriacus L., Salvia hispanica L. and Helianthus annuus L.) in the quorum sensing system of Chromobacterium
violaceum M. Pérez-López1, M. Soto-Hernández1, R. García-Contreras2, M. Martínez-
Vázquez3, R. San Miguel-Chávez1, I. Castillo-Juárez1
1 Laboratorio de Fitoquímica, Colegio de Postgraduados, Montecillo, Texcoco, Estado de México, México. 2Facultad de Medicina, Universidad Nacional Autónoma de México,
Ciudad Universitaria, Coyoacán, DF, México. 3Instituto de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, Coyoacán, México, DF, México.
E-mail: [email protected]
Keywords: fatty acids, free fatty acids, seeds, quorum sensing, quorum quenching, diffusible signal factor, Chromobacterium violaceum
Abstract Antibiotics resistance is a public health problem around the world, which has generated the search of new kind of antimicrobials with novel mechanisms of action to treat bacterial infections. Quorum sensing (QS) is a process that depends of population density, regulated by molecules called autoinducers (AI´s). Recently it has been described a family of Al’s, that belongs to kind of fatty acids known as DSF ("diffusible signal factor"), which has been shown to be involved in the regulation (activation / inhibition) of virulence factors expressed by QS. In this work, the effect of seed oils "amaranth" (Amaranthus hypochondriacus L.), "chia" (Salviahispanica L.) and "sunflower" (Helianthus annuus L.) and free fatty acids (FFA´s) was determined in the QS system of Chromobacterium violaceum. In addition, an infection model was developed in mice to determine the in vivo effect. The three oils at concentrations of 250 and 500 ug mL-1, reduced the production of violacein and alkaline protease production in a range of 20 to 60% without showing an effect on bacterial viability. Similarly three FFA´s inhibited violacein production at ranges from 15 to 91% at concentrations of 100 and 1000 uM. The intraperitoneal inoculation of bacteria into mice caused the death of animals, however supplementation in the diet of chia seeds and sunflower increased the percentage of survival by 16-33% in relation with those fed the standard diet. We demonstrated the participation of FFA in the inhibition of QS systems in vitro, and show that a diet rich in fatty acids increase the survival of infected mice.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
71
Phytochemical study and evaluation of quorum sensing system inhibition of barks Ceiba spp.
N. Muñoz-Cazares1, M. Soto-Hernández1, R. García-Contreras2, M. Martínez-Vázquez3, S. Aguilar-Rodríguez4, M. Montes de Oca-Mejía2, I. Castillo-Juárez1
1 Laboratorio de Fitoquímica, Colegio de Postgraduados, Montecillo, Texcoco, Estado de México, México. 2Facultad de Medicina, Universidad Nacional Autónoma de
México, Ciudad Universitaria, Coyoacán, DF, México. 3Instituto de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, Coyoacán, México,
DF, México. 4Laboratorio de Botánica, UMF, Facultad de Estudios Superiores-Iztacala, Universidad Nacional Autónoma de México, Tlanepantla, Estado de México, México.
E-mail: [email protected]
Keywords: bacterial communication, quorum-sensing inhibitors, Ceiba aesculifolia, Ceiba pentandra, antibiotic resistance, anti-quorum Mexican plant, Pseudomonas aeruginosa,Chromobacterium violaceum
Abstract Bacterial communication or quorum sensing is a process of gene expression at the population level, bacterial density dependent and mediated by molecules called autoinducers that regulate among other phenotypes, the expression of virulence factors. Quorum sensing systems (QSS) are widely used by bacterial pathogens to regulate mechanisms that mediate damage to the host. The inhibition of QSS (QSS-I) may be a useful alternative to antibiotics since quorum sensing controlled phenotypes are considered as a new target for antimicrobial chemotherapy. The aim of this study was identify plant sources that produce metabolites with inhibitory activity of bacterial quorum sensing system (QSS-I).
We determined the effect of hexane and dichloromethane extracts of Ceiba aesculifolia and Ceiba pentandra about phenotypes-QSS regulated of Chromobacterium violaceum and Pseudomonas aeruginosa. Extracts were fractionated and the main metabolites were identified. An anatomical study of the bark was made to define distinctive features to facilitate the identification of the samples.
QSS-I activity was found in the extracts of both species of Ceiba. Four fractions were identified with the ability to attenuate the production of pyocyanin and alkaline protease activity in a range of 30-40% and 65-80% respectively. These extracts and fractions are rich in terpenoids and steroidal compounds. Histological analysis revealed some differences that facilitate the identification of the two species.
It was confirmed that traditional use of these plants to fight infections through a mechanism complementary to antibiotic effect. Similarly, they are important sources for obtaining of QSS-I metabolites.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
70
Regulatory effect of fatty acids from seeds (Amaranthus
hypochondriacus L., Salvia hispanica L. and Helianthus annuus L.) in the quorum sensing system of Chromobacterium
violaceum M. Pérez-López1, M. Soto-Hernández1, R. García-Contreras2, M. Martínez-
Vázquez3, R. San Miguel-Chávez1, I. Castillo-Juárez1
1 Laboratorio de Fitoquímica, Colegio de Postgraduados, Montecillo, Texcoco, Estado de México, México. 2Facultad de Medicina, Universidad Nacional Autónoma de México,
Ciudad Universitaria, Coyoacán, DF, México. 3Instituto de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, Coyoacán, México, DF, México.
E-mail: [email protected]
Keywords: fatty acids, free fatty acids, seeds, quorum sensing, quorum quenching, diffusible signal factor, Chromobacterium violaceum
Abstract Antibiotics resistance is a public health problem around the world, which has generated the search of new kind of antimicrobials with novel mechanisms of action to treat bacterial infections. Quorum sensing (QS) is a process that depends of population density, regulated by molecules called autoinducers (AI´s). Recently it has been described a family of Al’s, that belongs to kind of fatty acids known as DSF ("diffusible signal factor"), which has been shown to be involved in the regulation (activation / inhibition) of virulence factors expressed by QS. In this work, the effect of seed oils "amaranth" (Amaranthus hypochondriacus L.), "chia" (Salviahispanica L.) and "sunflower" (Helianthus annuus L.) and free fatty acids (FFA´s) was determined in the QS system of Chromobacterium violaceum. In addition, an infection model was developed in mice to determine the in vivo effect. The three oils at concentrations of 250 and 500 ug mL-1, reduced the production of violacein and alkaline protease production in a range of 20 to 60% without showing an effect on bacterial viability. Similarly three FFA´s inhibited violacein production at ranges from 15 to 91% at concentrations of 100 and 1000 uM. The intraperitoneal inoculation of bacteria into mice caused the death of animals, however supplementation in the diet of chia seeds and sunflower increased the percentage of survival by 16-33% in relation with those fed the standard diet. We demonstrated the participation of FFA in the inhibition of QS systems in vitro, and show that a diet rich in fatty acids increase the survival of infected mice.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
71
Phytochemical study and evaluation of quorum sensing system inhibition of barks Ceiba spp.
N. Muñoz-Cazares1, M. Soto-Hernández1, R. García-Contreras2, M. Martínez-Vázquez3, S. Aguilar-Rodríguez4, M. Montes de Oca-Mejía2, I. Castillo-Juárez1
1 Laboratorio de Fitoquímica, Colegio de Postgraduados, Montecillo, Texcoco, Estado de México, México. 2Facultad de Medicina, Universidad Nacional Autónoma de
México, Ciudad Universitaria, Coyoacán, DF, México. 3Instituto de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, Coyoacán, México,
DF, México. 4Laboratorio de Botánica, UMF, Facultad de Estudios Superiores-Iztacala, Universidad Nacional Autónoma de México, Tlanepantla, Estado de México, México.
E-mail: [email protected]
Keywords: bacterial communication, quorum-sensing inhibitors, Ceiba aesculifolia, Ceiba pentandra, antibiotic resistance, anti-quorum Mexican plant, Pseudomonas aeruginosa,Chromobacterium violaceum
Abstract Bacterial communication or quorum sensing is a process of gene expression at the population level, bacterial density dependent and mediated by molecules called autoinducers that regulate among other phenotypes, the expression of virulence factors. Quorum sensing systems (QSS) are widely used by bacterial pathogens to regulate mechanisms that mediate damage to the host. The inhibition of QSS (QSS-I) may be a useful alternative to antibiotics since quorum sensing controlled phenotypes are considered as a new target for antimicrobial chemotherapy. The aim of this study was identify plant sources that produce metabolites with inhibitory activity of bacterial quorum sensing system (QSS-I).
We determined the effect of hexane and dichloromethane extracts of Ceiba aesculifolia and Ceiba pentandra about phenotypes-QSS regulated of Chromobacterium violaceum and Pseudomonas aeruginosa. Extracts were fractionated and the main metabolites were identified. An anatomical study of the bark was made to define distinctive features to facilitate the identification of the samples.
QSS-I activity was found in the extracts of both species of Ceiba. Four fractions were identified with the ability to attenuate the production of pyocyanin and alkaline protease activity in a range of 30-40% and 65-80% respectively. These extracts and fractions are rich in terpenoids and steroidal compounds. Histological analysis revealed some differences that facilitate the identification of the two species.
It was confirmed that traditional use of these plants to fight infections through a mechanism complementary to antibiotic effect. Similarly, they are important sources for obtaining of QSS-I metabolites.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
72
Quorum sensing inhibition and attenuation of virulence by atmospheric pressure non-thermal plasma
Alessandro Busetti1, Padrig Flynn1, W.G. Graham2, Brendan F. Gilmore1
1School of Pharmacy, Queen’s University Belfast, 97 Lisburn Rd, Belfast BT9 7BL, UK; 2Centre for Plasma Physics, Queen’s University Belfast, University Road Belfast, BT7
1NN, UK. E-mail: [email protected]
Keywords: Atmospheric pressure non-thermal plasma, quorum sensing inhibition, acyl homoserine lactones, virulence, wound treatment
Abstract The antimicrobial efficacy of Atmospheric pressure non-thermal plasma APNTP against microorganisms in both the planktonic and biofilm modes of growth strongly suggests its applicability for the treatment of viable tissues for conditions such as skin infections and biofilm-related chronic wounds. This study represents the first investigation of the effects of APNTP exposure on the acyl homoserine lactones (AHLs), a common class of autoinducers employed by several Gram negative human pathogens to regulate virulence factor production, biofilm formation and antibiotic susceptibility.
Solutions of the four acyl homoserine lactones N-butryl-dl-homoserine lactone (C4-AHL), N-hexanoyl-dl-homoserine lactone (C6-AHL), N-octanoyl-dl-homoserine lactone (C8 AHL) and N-(3-oxododecanoyl)-homoserine lactone (C12-AHL) (100 µM in PBS) were individually exposed to a Helium/Oxygen 0.5% plasma plume generated using a dielectric barrier atmospheric pressure plasma jet (2 standard litre per minute) for 30, 60, 120, and 240 seconds. Following exposure to APNTP, the AHL solutions were ran on C18 RP-TLC plates and overlaid with reporter strains CV026 and A. tumefaciens NTL4 (pZLR4). The exposure time to APNTP was found to be inversely proportional to the residual activity of the C6-AHL solution; violacein biosynthesis by reporter CV026 gradually decreased with increasing exposure time. Samples of AHLs treated for 120s and 240s failed to restore any visible violacein synthesis in CV026 or induce β–galactosidase expression in A. tumefaciens NTL4(pZLR4), suggesting the complete degradation of the signalling molecules. Luminescence-based assays using reporter strains E.coli JM109 pSB401 and E. coli JM109 pSB1142 confirmed the gradual inactivation of the C6, C8 and C12-AHL molecules in solutions exposed to APNTP over time with samples treated for 60s showing a significant decrease in luminescence, independently of growth. Samples treated for 120 and 240s were unable to elicit a significant luminesce response independently of growth confirming the complete inactivation of the AHLs in the samples.
This study demonstrates the ability of non-thermal plasma to disrupt the biological activity of AHLs in vitro, supporting the applicability of APNTP as an ‘anti-virulence’ approach with the potential of increasing susceptibility to host clearance-mechanisms and antibiotics for the treatment of chronic infections on viable tissues.
Acknowledgement All luminescence-based QS reporter strains used in this study were provided by Prof. Paul Williams and Dr. Paul Fletcher, Department of Molecular Microbiology, University of Nottingham.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
7
Bacterial communication and climate change: consequences for the coral ecosystem
Karen Tait1, Emma Ransome1,2,3, Colin Munn2
1Plymouth Marine Laboratory, Plymouth, PL1 3DH, UK. 2School of Marine Science and Engineering, Plymouth University, Plymouth, UK; 3Smithsonian National Museum of
Natural History, Washington, DC, USA. E-mail: [email protected]
Keywords: coral, Stylophora pistillata, temperature stress, QSI, community profiling
Abstract Research has highlighted the conserved and species-specific nature of coral-associated bacterial communities, but little is known about their interactions and ecological roles. With increases in coral disease correlating with climate change and the proposal that driving forces include compromised natural bacterial communities and increased pathogenicity of specific microbes, it is essential to understand how climate change is affecting the role of these communities in the coral ecosystem. AHL-mediated quorum sensing regulate processes including biofilm formation, motility and the production of virulence factors, such as antibiotics on a population level; processes that structure multicellular communities and which are expected to have significant roles in the maintenance of bacterial communities within the coral niche. Preliminary experiments established the presence of conserved, species specific AHL signal profiles in a number of corals, including in experimental and wild Stylophora pistillata colonies from the Red Sea. Further experiments established the degradation of the conserved signalling profile found in S. pistillata with exposure to 7 days of temperature stress (present at 22°C, variable at 26°C and absent at 30°C), indicating for the first time a link between bacterial communication and climate change. Although the increased temperature may have degraded the AHL signal molecules produced by the coral, it is also possible that the causative agent was a shift in community composition. When the impact of increased temperature on S. pistillata-associated bacterial communities were investigated by in depth sequencing of bacterial 16S rRNA genes, the relative abundance of several groups increased within S. pistillata colonies exposed to higher temperatures, including known AHL degrading groups such as Bacillus sp. Possible links between AHL signalling and the maintenance of coral health requires further investigation.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
72
Quorum sensing inhibition and attenuation of virulence by atmospheric pressure non-thermal plasma
Alessandro Busetti1, Padrig Flynn1, W.G. Graham2, Brendan F. Gilmore1
1School of Pharmacy, Queen’s University Belfast, 97 Lisburn Rd, Belfast BT9 7BL, UK; 2Centre for Plasma Physics, Queen’s University Belfast, University Road Belfast, BT7
1NN, UK. E-mail: [email protected]
Keywords: Atmospheric pressure non-thermal plasma, quorum sensing inhibition, acyl homoserine lactones, virulence, wound treatment
Abstract The antimicrobial efficacy of Atmospheric pressure non-thermal plasma APNTP against microorganisms in both the planktonic and biofilm modes of growth strongly suggests its applicability for the treatment of viable tissues for conditions such as skin infections and biofilm-related chronic wounds. This study represents the first investigation of the effects of APNTP exposure on the acyl homoserine lactones (AHLs), a common class of autoinducers employed by several Gram negative human pathogens to regulate virulence factor production, biofilm formation and antibiotic susceptibility.
Solutions of the four acyl homoserine lactones N-butryl-dl-homoserine lactone (C4-AHL), N-hexanoyl-dl-homoserine lactone (C6-AHL), N-octanoyl-dl-homoserine lactone (C8 AHL) and N-(3-oxododecanoyl)-homoserine lactone (C12-AHL) (100 µM in PBS) were individually exposed to a Helium/Oxygen 0.5% plasma plume generated using a dielectric barrier atmospheric pressure plasma jet (2 standard litre per minute) for 30, 60, 120, and 240 seconds. Following exposure to APNTP, the AHL solutions were ran on C18 RP-TLC plates and overlaid with reporter strains CV026 and A. tumefaciens NTL4 (pZLR4). The exposure time to APNTP was found to be inversely proportional to the residual activity of the C6-AHL solution; violacein biosynthesis by reporter CV026 gradually decreased with increasing exposure time. Samples of AHLs treated for 120s and 240s failed to restore any visible violacein synthesis in CV026 or induce β–galactosidase expression in A. tumefaciens NTL4(pZLR4), suggesting the complete degradation of the signalling molecules. Luminescence-based assays using reporter strains E.coli JM109 pSB401 and E. coli JM109 pSB1142 confirmed the gradual inactivation of the C6, C8 and C12-AHL molecules in solutions exposed to APNTP over time with samples treated for 60s showing a significant decrease in luminescence, independently of growth. Samples treated for 120 and 240s were unable to elicit a significant luminesce response independently of growth confirming the complete inactivation of the AHLs in the samples.
This study demonstrates the ability of non-thermal plasma to disrupt the biological activity of AHLs in vitro, supporting the applicability of APNTP as an ‘anti-virulence’ approach with the potential of increasing susceptibility to host clearance-mechanisms and antibiotics for the treatment of chronic infections on viable tissues.
Acknowledgement All luminescence-based QS reporter strains used in this study were provided by Prof. Paul Williams and Dr. Paul Fletcher, Department of Molecular Microbiology, University of Nottingham.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
7
Bacterial communication and climate change: consequences for the coral ecosystem
Karen Tait1, Emma Ransome1,2,3, Colin Munn2
1Plymouth Marine Laboratory, Plymouth, PL1 3DH, UK. 2School of Marine Science and Engineering, Plymouth University, Plymouth, UK; 3Smithsonian National Museum of
Natural History, Washington, DC, USA. E-mail: [email protected]
Keywords: coral, Stylophora pistillata, temperature stress, QSI, community profiling
Abstract Research has highlighted the conserved and species-specific nature of coral-associated bacterial communities, but little is known about their interactions and ecological roles. With increases in coral disease correlating with climate change and the proposal that driving forces include compromised natural bacterial communities and increased pathogenicity of specific microbes, it is essential to understand how climate change is affecting the role of these communities in the coral ecosystem. AHL-mediated quorum sensing regulate processes including biofilm formation, motility and the production of virulence factors, such as antibiotics on a population level; processes that structure multicellular communities and which are expected to have significant roles in the maintenance of bacterial communities within the coral niche. Preliminary experiments established the presence of conserved, species specific AHL signal profiles in a number of corals, including in experimental and wild Stylophora pistillata colonies from the Red Sea. Further experiments established the degradation of the conserved signalling profile found in S. pistillata with exposure to 7 days of temperature stress (present at 22°C, variable at 26°C and absent at 30°C), indicating for the first time a link between bacterial communication and climate change. Although the increased temperature may have degraded the AHL signal molecules produced by the coral, it is also possible that the causative agent was a shift in community composition. When the impact of increased temperature on S. pistillata-associated bacterial communities were investigated by in depth sequencing of bacterial 16S rRNA genes, the relative abundance of several groups increased within S. pistillata colonies exposed to higher temperatures, including known AHL degrading groups such as Bacillus sp. Possible links between AHL signalling and the maintenance of coral health requires further investigation.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
74
Analysis of N-acylhomoserine lactone-degrading mechanism in the coagulase-negative staphylococci
Ryusuke Sato1, Takahito Yamaguchi1, Nobutaka Someya2, Tsukasa Ikeda1,Tomohiro Morohoshi1
1Department of Material and Environmental Chemistry, Graduate School of Engineering, Utsunomiya University, 7-1-2 Yoto, Utsunomiya, 321-8585, Japan,
2National Agriculture and Food Research Organization (NARO), 3-1-1 Kannondai, Tsukuba, Ibaraki 305-8517, Japan. E-mail: [email protected]
Keywords: quorum sensing, N-acylhomoserine lactone, lactonase, coagulase-negative staphylococci
Abstract Many gram-negative bacteria have a quorum-sensing system and produce N-acylhomoserine lactones (AHLs) that they use as a quorum-sensing signal molecule. AHL-degrading genes have been cloned and characterized from various bacteria. Coagulase-negative staphylococci (CNS) are present on the skin of animals and considered less virulent. In this study, the novel AHL-degrading gene was screened from the genome sequences of CNS species. In previous study, we have identified a novel AHL-lactonase gene (ahlS) from potato leaf-associated Solibacillus silvestris. The ahlS homologues were present in the genome of CNS strains, which were Staphylococcus carnosus, S. haemolyticus, and S. saprophyticus. To check the AHL-degrading activity, these three CNS strains were cultivated in LB medium containing 10 µM C6-HSL or C10-HSL. After incubation for 9 h, AHLs were completely degraded by these strains. The ahlS genes on the genome of CNS strains were amplified by PCR and cloned into the pBBR1MCS5 vector. After checking AHL-degrading activity, Escherichia coli harbouring the ahlS-expressing plasmids showed obvious AHL-degrading activity. Especially, E. coli harbouring ahlS from S. haemolyticus showed the strongest AHL-degrading activity and completely degraded 10 µM C6-HSL within 6 hours, and C10-HSL within 9 hours. These results demonstrated that AhlS from CNS showed a high level of degrading activity against AHLs with short acyl chains. AHL restoration assay revealed that AhlS from CNS works as AHL lactonase as well as AhlS from S. silvestris. The ahlS-expressing plasmid was transformed into Pseudomonas aeruginosa PAO1. AHL production in PAO1 harbouring ahlS-expressing plasmid was checked by cross-streak assay using Chromobacterium violaceum CV026 and VIR07 as AHL-reporter strains. As the results, short-chain AHLs were degraded in PAO1 harbouring ahlS-expressing plasmid, but long-chain AHLs were not. These results assumed that AhlS selectively inhibit short-chain AHL-dependent quorum sensing in PAO1.
Acknowledgements This work was supported by the Grant-in-Aid for Young Scientists (B) (No. 24760643) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
References 1. Morohoshi T, Tominaga Y, Someya N, Ikeda T. 2012. Complete genome sequence and
characterization of the N-acyl homoserine lactone-degrading gene of the potato leaf-associated Solibacillus silvestris. J Biosci Bioeng 113:20-25.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
75
SHP-RovS cell-cell communication mechanism, a target for quorum quenching?
David Pérez-Pascual, Coralie Metton, Colette Besset, Véronique Monnet, Rozenn Gardan
INRA, Unité MICALIS, Bâtiment 526, F-78352 Jouy-en-Josas, France.
Keywords: Streptococcus agalactiae, Rgg, peptides
Abstract Rgg regulators and their cognate pheromones, called small hydrophobic peptides (SHPs), are present in nearly all streptococcal species and are involved in cell-cell communication. The general pathway of this cell-to-cell communication system is well understood. Streptococcus agalactiae is an opportunistic pathogenic bacterium responsible for fatal infections in neonates and of increasing diseases in immunocompromised adults. S. agalactiae is also an important pathogen in animals, responsible for mastitis in cows, and considered as an emerging pathogen in fish farms. Up today no vaccine is available to prevent its infection. S. agalactiae has one copy of the shp/rgg locus and the SHP-associated Rgg is called RovS in this species. We have demonstrated the implication of the SHP/RovS mechanism in the virulence, as well as its host organ specificity, in mouse model (1). Hence, the SHP/RovS cell-to-cell communication system appears to be an essential mechanism that regulates pathogenicity in S. agalactiae and represents an attractive target for the development of new therapeutic treatments. We will present the different strategies that could be used to target this mechanism in order to control the virulence of this bacterium in different organisms.
References 1. Pérez-Pascual D, Gaudu P, Fleuchot B, Besset C, Rosinski-Chupin I, Guillot A,
Monnet V, Gardan R. 2015. RovS and its associated signaling peptide form a cell-to-cell communication system required for Streptococcus agalactiae pathogenesis. MBio 6.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
74
Analysis of N-acylhomoserine lactone-degrading mechanism in the coagulase-negative staphylococci
Ryusuke Sato1, Takahito Yamaguchi1, Nobutaka Someya2, Tsukasa Ikeda1,Tomohiro Morohoshi1
1Department of Material and Environmental Chemistry, Graduate School of Engineering, Utsunomiya University, 7-1-2 Yoto, Utsunomiya, 321-8585, Japan,
2National Agriculture and Food Research Organization (NARO), 3-1-1 Kannondai, Tsukuba, Ibaraki 305-8517, Japan. E-mail: [email protected]
Keywords: quorum sensing, N-acylhomoserine lactone, lactonase, coagulase-negative staphylococci
Abstract Many gram-negative bacteria have a quorum-sensing system and produce N-acylhomoserine lactones (AHLs) that they use as a quorum-sensing signal molecule. AHL-degrading genes have been cloned and characterized from various bacteria. Coagulase-negative staphylococci (CNS) are present on the skin of animals and considered less virulent. In this study, the novel AHL-degrading gene was screened from the genome sequences of CNS species. In previous study, we have identified a novel AHL-lactonase gene (ahlS) from potato leaf-associated Solibacillus silvestris. The ahlS homologues were present in the genome of CNS strains, which were Staphylococcus carnosus, S. haemolyticus, and S. saprophyticus. To check the AHL-degrading activity, these three CNS strains were cultivated in LB medium containing 10 µM C6-HSL or C10-HSL. After incubation for 9 h, AHLs were completely degraded by these strains. The ahlS genes on the genome of CNS strains were amplified by PCR and cloned into the pBBR1MCS5 vector. After checking AHL-degrading activity, Escherichia coli harbouring the ahlS-expressing plasmids showed obvious AHL-degrading activity. Especially, E. coli harbouring ahlS from S. haemolyticus showed the strongest AHL-degrading activity and completely degraded 10 µM C6-HSL within 6 hours, and C10-HSL within 9 hours. These results demonstrated that AhlS from CNS showed a high level of degrading activity against AHLs with short acyl chains. AHL restoration assay revealed that AhlS from CNS works as AHL lactonase as well as AhlS from S. silvestris. The ahlS-expressing plasmid was transformed into Pseudomonas aeruginosa PAO1. AHL production in PAO1 harbouring ahlS-expressing plasmid was checked by cross-streak assay using Chromobacterium violaceum CV026 and VIR07 as AHL-reporter strains. As the results, short-chain AHLs were degraded in PAO1 harbouring ahlS-expressing plasmid, but long-chain AHLs were not. These results assumed that AhlS selectively inhibit short-chain AHL-dependent quorum sensing in PAO1.
Acknowledgements This work was supported by the Grant-in-Aid for Young Scientists (B) (No. 24760643) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
References 1. Morohoshi T, Tominaga Y, Someya N, Ikeda T. 2012. Complete genome sequence and
characterization of the N-acyl homoserine lactone-degrading gene of the potato leaf-associated Solibacillus silvestris. J Biosci Bioeng 113:20-25.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
75
SHP-RovS cell-cell communication mechanism, a target for quorum quenching?
David Pérez-Pascual, Coralie Metton, Colette Besset, Véronique Monnet, Rozenn Gardan
INRA, Unité MICALIS, Bâtiment 526, F-78352 Jouy-en-Josas, France.
Keywords: Streptococcus agalactiae, Rgg, peptides
Abstract Rgg regulators and their cognate pheromones, called small hydrophobic peptides (SHPs), are present in nearly all streptococcal species and are involved in cell-cell communication. The general pathway of this cell-to-cell communication system is well understood. Streptococcus agalactiae is an opportunistic pathogenic bacterium responsible for fatal infections in neonates and of increasing diseases in immunocompromised adults. S. agalactiae is also an important pathogen in animals, responsible for mastitis in cows, and considered as an emerging pathogen in fish farms. Up today no vaccine is available to prevent its infection. S. agalactiae has one copy of the shp/rgg locus and the SHP-associated Rgg is called RovS in this species. We have demonstrated the implication of the SHP/RovS mechanism in the virulence, as well as its host organ specificity, in mouse model (1). Hence, the SHP/RovS cell-to-cell communication system appears to be an essential mechanism that regulates pathogenicity in S. agalactiae and represents an attractive target for the development of new therapeutic treatments. We will present the different strategies that could be used to target this mechanism in order to control the virulence of this bacterium in different organisms.
References 1. Pérez-Pascual D, Gaudu P, Fleuchot B, Besset C, Rosinski-Chupin I, Guillot A,
Monnet V, Gardan R. 2015. RovS and its associated signaling peptide form a cell-to-cell communication system required for Streptococcus agalactiae pathogenesis. MBio 6.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
76
Anti-biofilm effect of Rosmarinus officinalis against MRSAStaphylococcus aureus
E.H. Endo, T.U. Nakamura, C.V. Nakamura, B.P. Dias Filho
Universidade Estadual de Maringá. Avenida Colombo, 5790, Maringá, PR, Brazil. E-mail: [email protected]
Keywords: MRSA, anti-biofilm, Rosmarinus officinalis
Abstract Methicillin-resistant Staphylococcus aureus (MRSA) is a Gram-positive bacterium which causes community- and hospital-acquired infections. Biofilm formation is one defense mechanism, since bacteria embedded in biofilms are more difficult to eradicate than planktonic cells. Single-drug treatment for MRSA related infections is becoming less effective and natural products may be an alternative for future antibacterial medicine development (1-3). Factors that lead to growth inhibition of biofilm or its breakup or eradication is required. In order to prevent biofilm formation, several studies have been performed to find antimicrobial agents that affect the viability of bacteria in biofilms. Natural products from plants have been shown to influence microbial biofilm formation. In this study, activity of Rosemary (Rosmarinus officinalis) extract was determined against planktonic and biofilm cells of Staphylococcus aureus ATCC 29213 and 13 clinical isolates, being 10 MRSA and 3 MSSA. Congo Red agar (CRA) method was used to screening for biofilm formation by Staphylococcus isolates. The effect of these extracts on pre-formed biofilms of MRSA was determined by MTT reduction assay. All these strains showed black colonies on CRA plates. Good antibacterial effect was obtained, MICs values ranged from 15.6 to 125 µg/ml and MBC values were the same or two fold above the MIC. Pre-formed biofilms were also inhibited by rosemary extract at concentrations of 62.5 to 125 µg/ml. Further studies are necessary to investigate possible mechanism of action involved, however, these data indicated a potential application of R. officinalis in the development of strategies for staphylococcal infections.
Acknowledgement CNPq, CAPES, Fundação Araucária, Programa de Pós-graduação em Ciências Farmacêuticas.
References: 1. Davis SL, Perri MB, Donabedian SM, Manierski C, Singh A, Vager D, Haque NZ, Speirs
K, Muder RR, Robinson-Dunn B, Hayden MK, Zervos. 2007. Epidemiology and outcomes of community-associated methicillin-resistant Staphylococcus aureus infection. J Clin Microbiol 45:1705-1711.
2. Zuo GY, Wang GC, Zhao YB, Xu GL, Hao XY, Han J, Zhao Q. 2008. Screening of Chinese medicinal plants for inhibition against clinical isolates of methicillin- resistant Staphylococcus aureus (MRSA). J Ethnopharmacol 120: 287-290.
3. Patel R. 2005. Biofilms and antimicrobial resistance. Clin Orthop Relat Res; 437: 41-47.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
77
Application of quorum sensing inhibitors to prevent biofilm formation of periodontopathogens
Eun-Ju Ryu, Jaehyun Sim, Yun-Ki Ko, Kwang-Soo Jung, Bong-Kyu Choi
Department of Oral Microbiology and Immunology, School of Dentistry, Seoul National University, Seoul, Republic of Korea.
E-mail: [email protected]
Keywords: Quorum Sensing, Autoinducer(AI), Periodontopathogen, Periodontitis, Biofilm
Abstract Periodontitis is caused by subgingival biofilms, which are composed of multi-species oral bacteria, mostly of Gram-negative anaerobes along with over-aggressive immune response leading to tooth loss. Quorum sensing (QS) is a cell-to-cell signalling system comprising small signalling molecules secreted by various microbial species, affecting various phenotypes including bacterial virulence and biofilm formation. Autoinducer 2 (AI-2) is an universal QS molecule that plays an important role in dental biofilm formation. The objective of this study was to evaluate QS inhibitors (QSIs) including furanone compound and D-ribose to inhibit biofilm formation of periodontopathogens.
The inhibitory effect of the QSIs on AI-2 activity and biofilm formation of periodontopathogens was evaluated. AI-2 activity was assessed using AI-2 reporter strain Vibrio harveyi BB170 and semi-purified Fusobacterium nucleatum AI-2. Biofilm formation was evaluated by crystal violet staining and confocal scanning laser microscopy.
Furanone compound and D-ribose significantly inhibited AI-2 activity of F. nucleatum, the major coaggregation bridge organism that connects early colonizing commensals and late pathogenic colonizers in dental biofilms. They also inhibited biofilm formation of periodontopathogens including F. nucleatum, Porphyromonas gingivalis, Tannerella forsythia and Aggregatibacter actinomycetemcomitans.
AI-2 of periodontopathogens may be a target for the inhibition of pathogenic dental biofilm formation and QSIs can be applied as preventive agents against biofilm formation of periodontopathogens.
Acknowledgement This study was supported by a grant (HI12C0675) of the Korea Healthcare Technology R&D, Ministry of Health and Welfare, Republic of Korea.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
76
Anti-biofilm effect of Rosmarinus officinalis against MRSAStaphylococcus aureus
E.H. Endo, T.U. Nakamura, C.V. Nakamura, B.P. Dias Filho
Universidade Estadual de Maringá. Avenida Colombo, 5790, Maringá, PR, Brazil. E-mail: [email protected]
Keywords: MRSA, anti-biofilm, Rosmarinus officinalis
Abstract Methicillin-resistant Staphylococcus aureus (MRSA) is a Gram-positive bacterium which causes community- and hospital-acquired infections. Biofilm formation is one defense mechanism, since bacteria embedded in biofilms are more difficult to eradicate than planktonic cells. Single-drug treatment for MRSA related infections is becoming less effective and natural products may be an alternative for future antibacterial medicine development (1-3). Factors that lead to growth inhibition of biofilm or its breakup or eradication is required. In order to prevent biofilm formation, several studies have been performed to find antimicrobial agents that affect the viability of bacteria in biofilms. Natural products from plants have been shown to influence microbial biofilm formation. In this study, activity of Rosemary (Rosmarinus officinalis) extract was determined against planktonic and biofilm cells of Staphylococcus aureus ATCC 29213 and 13 clinical isolates, being 10 MRSA and 3 MSSA. Congo Red agar (CRA) method was used to screening for biofilm formation by Staphylococcus isolates. The effect of these extracts on pre-formed biofilms of MRSA was determined by MTT reduction assay. All these strains showed black colonies on CRA plates. Good antibacterial effect was obtained, MICs values ranged from 15.6 to 125 µg/ml and MBC values were the same or two fold above the MIC. Pre-formed biofilms were also inhibited by rosemary extract at concentrations of 62.5 to 125 µg/ml. Further studies are necessary to investigate possible mechanism of action involved, however, these data indicated a potential application of R. officinalis in the development of strategies for staphylococcal infections.
Acknowledgement CNPq, CAPES, Fundação Araucária, Programa de Pós-graduação em Ciências Farmacêuticas.
References: 1. Davis SL, Perri MB, Donabedian SM, Manierski C, Singh A, Vager D, Haque NZ, Speirs
K, Muder RR, Robinson-Dunn B, Hayden MK, Zervos. 2007. Epidemiology and outcomes of community-associated methicillin-resistant Staphylococcus aureus infection. J Clin Microbiol 45:1705-1711.
2. Zuo GY, Wang GC, Zhao YB, Xu GL, Hao XY, Han J, Zhao Q. 2008. Screening of Chinese medicinal plants for inhibition against clinical isolates of methicillin- resistant Staphylococcus aureus (MRSA). J Ethnopharmacol 120: 287-290.
3. Patel R. 2005. Biofilms and antimicrobial resistance. Clin Orthop Relat Res; 437: 41-47.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
77
Application of quorum sensing inhibitors to prevent biofilm formation of periodontopathogens
Eun-Ju Ryu, Jaehyun Sim, Yun-Ki Ko, Kwang-Soo Jung, Bong-Kyu Choi
Department of Oral Microbiology and Immunology, School of Dentistry, Seoul National University, Seoul, Republic of Korea.
E-mail: [email protected]
Keywords: Quorum Sensing, Autoinducer(AI), Periodontopathogen, Periodontitis, Biofilm
Abstract Periodontitis is caused by subgingival biofilms, which are composed of multi-species oral bacteria, mostly of Gram-negative anaerobes along with over-aggressive immune response leading to tooth loss. Quorum sensing (QS) is a cell-to-cell signalling system comprising small signalling molecules secreted by various microbial species, affecting various phenotypes including bacterial virulence and biofilm formation. Autoinducer 2 (AI-2) is an universal QS molecule that plays an important role in dental biofilm formation. The objective of this study was to evaluate QS inhibitors (QSIs) including furanone compound and D-ribose to inhibit biofilm formation of periodontopathogens.
The inhibitory effect of the QSIs on AI-2 activity and biofilm formation of periodontopathogens was evaluated. AI-2 activity was assessed using AI-2 reporter strain Vibrio harveyi BB170 and semi-purified Fusobacterium nucleatum AI-2. Biofilm formation was evaluated by crystal violet staining and confocal scanning laser microscopy.
Furanone compound and D-ribose significantly inhibited AI-2 activity of F. nucleatum, the major coaggregation bridge organism that connects early colonizing commensals and late pathogenic colonizers in dental biofilms. They also inhibited biofilm formation of periodontopathogens including F. nucleatum, Porphyromonas gingivalis, Tannerella forsythia and Aggregatibacter actinomycetemcomitans.
AI-2 of periodontopathogens may be a target for the inhibition of pathogenic dental biofilm formation and QSIs can be applied as preventive agents against biofilm formation of periodontopathogens.
Acknowledgement This study was supported by a grant (HI12C0675) of the Korea Healthcare Technology R&D, Ministry of Health and Welfare, Republic of Korea.
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
78
Interference with quorum sensing signals to prevent biofilm formation by Streptococcus mutans
Andrea Muras1, Celia Mayer1, Manuel Romero1,2, Mariam Ferrer3, Alex Mira3 Ana Otero1
1Department of Microbiology and Parasitology, Faculty of Biology-CIBUS, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain.
2 School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, NG7 2RD Nottingham. UK. 3FISABIO Foundation, Center for
Advanced Research in Public Health, 46020 Valencia, Spain. E-mail: [email protected], [email protected]
Keywords: biofilm, quorum sensing, quorum quenching, dental cavities, AI-2
Abstract Streptococcus mutans is the primary causative agent for dental cavities in humans and is usually present as part of a complex biofilm on the surface of the tooth (dental plaque). Therefore, the removal or inhibition of biofilm formation by S. mutans could lead to a decrease in the incidence of dental cavities. Previous studies have demonstrated the role of interspecies signalling in biofilm formation and suggested that streptococci regulate their own biofilm formation through quorum sensing (QS) processes that include a peptide pheromone signal called competence stimulating peptide (CSP) and the universal signalling molecule AI-2. Mutation of QS genes of both systems affect the formation and architecture in S. mutans biofilms indicating that the inhibition of QS processes, known as quorum quenching (QQ), may represent an interesting anti-pathogenic strategy to prevent dental cavities and to control bacterial infections by S. mutans. The objective of this work was to evaluate the effect of purified cell extracts (PCE) of Tenacibaculum sp. strain 20J, a marine bacterium with a wide-spectrum QQ activity, on biofilm formation by S. mutans ATCC25175 measured by the xCELLigence system RTCA SP (ACEA Biosciencies Inc.). Extracts from the QQ strain Bacillus thuringiensis serovar Berliner ATCC10792 and the non-QQ strain T. maritimum NCBI2154Twere also tested. Furanone C30, a molecule previously described to interfere with QS systems was used as a control in biofilm assays. Results showed that PCE from Tenacibaculum sp. 20J reduced by 50% the biofilm formation by S. mutans without affecting growth, indicating the presence of compound(s) with anti-biofilm activity against S. mutans ATCC25175. No effect on biofilm formation was observed for PCE from Bacillus and T. maritimum. Additional experiments carried out with the indicator strains Vibrio harveyi BB170 and V. harveyi JMH597 suggested the presence of a non-protein molecule(s) that is capable to interfere with the AI-2 QS system being the responsible for the inhibition of the biofilm formation. Moreover, the addition of the purified Aii20J, an AHL-lactonase from 20J showed no effect on S. mutans biofilm formation.
Acknowledgement
This work was supported by Fundación Ramón Areces (CIVP16A1814).
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015
79
Satellite Meeting on Novel Antifouling Strategies
Invited lectures
ISQSI, Santiago de Compostela 2015 Poster communications – session 2
78
Interference with quorum sensing signals to prevent biofilm formation by Streptococcus mutans
Andrea Muras1, Celia Mayer1, Manuel Romero1,2, Mariam Ferrer3, Alex Mira3 Ana Otero1
1Department of Microbiology and Parasitology, Faculty of Biology-CIBUS, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain.
2 School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, NG7 2RD Nottingham. UK. 3FISABIO Foundation, Center for
Advanced Research in Public Health, 46020 Valencia, Spain. E-mail: [email protected], [email protected]
Keywords: biofilm, quorum sensing, quorum quenching, dental cavities, AI-2
Abstract Streptococcus mutans is the primary causative agent for dental cavities in humans and is usually present as part of a complex biofilm on the surface of the tooth (dental plaque). Therefore, the removal or inhibition of biofilm formation by S. mutans could lead to a decrease in the incidence of dental cavities. Previous studies have demonstrated the role of interspecies signalling in biofilm formation and suggested that streptococci regulate their own biofilm formation through quorum sensing (QS) processes that include a peptide pheromone signal called competence stimulating peptide (CSP) and the universal signalling molecule AI-2. Mutation of QS genes of both systems affect the formation and architecture in S. mutans biofilms indicating that the inhibition of QS processes, known as quorum quenching (QQ), may represent an interesting anti-pathogenic strategy to prevent dental cavities and to control bacterial infections by S. mutans. The objective of this work was to evaluate the effect of purified cell extracts (PCE) of Tenacibaculum sp. strain 20J, a marine bacterium with a wide-spectrum QQ activity, on biofilm formation by S. mutans ATCC25175 measured by the xCELLigence system RTCA SP (ACEA Biosciencies Inc.). Extracts from the QQ strain Bacillus thuringiensis serovar Berliner ATCC10792 and the non-QQ strain T. maritimum NCBI2154Twere also tested. Furanone C30, a molecule previously described to interfere with QS systems was used as a control in biofilm assays. Results showed that PCE from Tenacibaculum sp. 20J reduced by 50% the biofilm formation by S. mutans without affecting growth, indicating the presence of compound(s) with anti-biofilm activity against S. mutans ATCC25175. No effect on biofilm formation was observed for PCE from Bacillus and T. maritimum. Additional experiments carried out with the indicator strains Vibrio harveyi BB170 and V. harveyi JMH597 suggested the presence of a non-protein molecule(s) that is capable to interfere with the AI-2 QS system being the responsible for the inhibition of the biofilm formation. Moreover, the addition of the purified Aii20J, an AHL-lactonase from 20J showed no effect on S. mutans biofilm formation.
Acknowledgement
This work was supported by Fundación Ramón Areces (CIVP16A1814).
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015
79
Satellite Meeting on Novel Antifouling Strategies
Invited lectures
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015
Invited lectures
81
Towards commercialisation of a novel marine nuclease for biofilm removal and prevention
J. Grant Burgess
School of Marine Science and Technology, Newcastle University, UK. E-mail: [email protected]
Abstract The majority of antibiotics in use today are derived from natural products produced by the microbes themselves following billions of years of evolution. Thus bacteria are best at killing other bacteria. We applied a similar approach and asked the question: how do bacteria combat the biofilms of their competitors? This lead to the discovery of a Bacillus secreted protein NucB, in supernatants, which has the ability to degrade and break up bacterial biofilms. We have applied this enzyme to dispersal of biofilms from a number of industrial and medical situations including joint replacement infections, oral biofilms, as well as marine biofilms. Breaking up the biofilms may allow more effective treatment with antibiotics or biocides. NucB can break down biofilms by degrading extracellular DNA, an important structural component of the extracellular matrix. NucB is also effective against biofilms in chronic rhinosinusitis and tracheoesophageal speech valves. The properties of this enzyme and the implications for fouling control and treatment will be presented.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015
Invited lectures
81
Towards commercialisation of a novel marine nuclease for biofilm removal and prevention
J. Grant Burgess
School of Marine Science and Technology, Newcastle University, UK. E-mail: [email protected]
Abstract The majority of antibiotics in use today are derived from natural products produced by the microbes themselves following billions of years of evolution. Thus bacteria are best at killing other bacteria. We applied a similar approach and asked the question: how do bacteria combat the biofilms of their competitors? This lead to the discovery of a Bacillus secreted protein NucB, in supernatants, which has the ability to degrade and break up bacterial biofilms. We have applied this enzyme to dispersal of biofilms from a number of industrial and medical situations including joint replacement infections, oral biofilms, as well as marine biofilms. Breaking up the biofilms may allow more effective treatment with antibiotics or biocides. NucB can break down biofilms by degrading extracellular DNA, an important structural component of the extracellular matrix. NucB is also effective against biofilms in chronic rhinosinusitis and tracheoesophageal speech valves. The properties of this enzyme and the implications for fouling control and treatment will be presented.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Invited lectures
82
Biomimetics approaches for the development of new antifouling strategies
Claire Hellio
UBO/IUEM/LEMAR Bioprospection platform), Université de Bretagne Occidentale. France. E-mail: [email protected]
Abstract
The growing demand to develop a novel, environmentally friendly antifouling (AF) material is ever increasing. Bioinspiration is an attractive alternative in developing such a material, learning from nature’s own designs and solutions and transferring them to solve particular problems. In order to achieve this goal, the actual mechanisms and strategies that evolution has produced needs to be elucidated from the subject species. The work presented has investigated the role of surface topography and chemistry combined in a single material – a property that exists naturally in some common marine organisms such as algae and sponges. Isolation and characterisation of new anti-QS compounds were performed from sponges and their epibionts collected in Mexico and French Polynesia. The most active compounds were then incorporated in various paint matrixes. Lab-assays and field results will be presented. Origins of the most active compounds (sponge vs epibionts) and implication for sustainable production will be discussed.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015
8
Satellite Meeting on Novel Antifouling Strategies
Oral communications
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Invited lectures
82
Biomimetics approaches for the development of new antifouling strategies
Claire Hellio
UBO/IUEM/LEMAR Bioprospection platform), Université de Bretagne Occidentale. France. E-mail: [email protected]
Abstract
The growing demand to develop a novel, environmentally friendly antifouling (AF) material is ever increasing. Bioinspiration is an attractive alternative in developing such a material, learning from nature’s own designs and solutions and transferring them to solve particular problems. In order to achieve this goal, the actual mechanisms and strategies that evolution has produced needs to be elucidated from the subject species. The work presented has investigated the role of surface topography and chemistry combined in a single material – a property that exists naturally in some common marine organisms such as algae and sponges. Isolation and characterisation of new anti-QS compounds were performed from sponges and their epibionts collected in Mexico and French Polynesia. The most active compounds were then incorporated in various paint matrixes. Lab-assays and field results will be presented. Origins of the most active compounds (sponge vs epibionts) and implication for sustainable production will be discussed.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015
8
Satellite Meeting on Novel Antifouling Strategies
Oral communications
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Oral communications
85
The FP7-Ocean EU project BYEFOULING: an interdisciplinary approach to novel anti-fouling strategies
João Tedim1,2, Christian Simon3
1Smallmatek - Small Materials and Technologies, Lda., Rua Canhas, 3810-075 Aveiro, Portugal.2Department of Materials and Ceramic Engineering, CICECO-Aveiro Institute of Materials, University of Aveiro, 3810-193 Aveiro, Portugal3SINTEF, Department of
Nano and Hybrid Materials, P.O. Box 124, Blindern, 0314 Oslo, Norway. E-mail: [email protected]
Keywords: Anti-fouling materials, nanotechnology, environmentally-friendly, drag reduction, maritime, aquaculture
Abstract The BYEFOULING project addresses high volume production of low toxic and environmentally-friendly antifouling coatings for mobile and stationary maritime applications. The technology will fulfil the coating requirements as a result of the incorporation of novel antifouling agents and a new set of binders into coating formulations for maritime transportation and fishing vessels, floating devices and aquaculture.
The main vision of BYEFOULING is to provide the means for industrial, cost-effective and robust manufacturing of antifouling coatings in Europe, where SMEs are coating components developers, production technology providers and end users. A set of procedures, guidelines and fabrication tools are under development, enabling short time to market for new coating concepts.
The main goal of BYEFOULING is to design, develop and upscale antifouling coatings with enhanced performance compared to current available products. The approach in BYEFOULING is to tackle the different stages of the biofouling process using innovative antifouling agents, covering surface-structured materials, protein adsorption inhibitors, quorum sensing inhibitors, natural biocides and microorganisms with antifouling properties. Encapsulation of the innovative compounds in smart nanostructured materials will be implemented to optimize coating performance and cost all along their life cycle. A proof-of-concept for the most promising candidates will be developed and demonstrators will be produced and tested on fields.
BYEFOULING combines a multidisciplinary leading research team from 11 European countries, which are already acting worldwide in the scientific community, with highly relevant and skilled technological partners, to build a consortium able to develop a full production line for antifouling coatings in Europe.
Acknowledgement This research is supported by the EU FP7 Project “Low-toxic cost-efficient environment-friendly antifouling materials” (OCEAN for Tomorrow) under Grant Agreement no. 612717.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Oral communications
85
The FP7-Ocean EU project BYEFOULING: an interdisciplinary approach to novel anti-fouling strategies
João Tedim1,2, Christian Simon3
1Smallmatek - Small Materials and Technologies, Lda., Rua Canhas, 3810-075 Aveiro, Portugal.2Department of Materials and Ceramic Engineering, CICECO-Aveiro Institute of Materials, University of Aveiro, 3810-193 Aveiro, Portugal3SINTEF, Department of
Nano and Hybrid Materials, P.O. Box 124, Blindern, 0314 Oslo, Norway. E-mail: [email protected]
Keywords: Anti-fouling materials, nanotechnology, environmentally-friendly, drag reduction, maritime, aquaculture
Abstract The BYEFOULING project addresses high volume production of low toxic and environmentally-friendly antifouling coatings for mobile and stationary maritime applications. The technology will fulfil the coating requirements as a result of the incorporation of novel antifouling agents and a new set of binders into coating formulations for maritime transportation and fishing vessels, floating devices and aquaculture.
The main vision of BYEFOULING is to provide the means for industrial, cost-effective and robust manufacturing of antifouling coatings in Europe, where SMEs are coating components developers, production technology providers and end users. A set of procedures, guidelines and fabrication tools are under development, enabling short time to market for new coating concepts.
The main goal of BYEFOULING is to design, develop and upscale antifouling coatings with enhanced performance compared to current available products. The approach in BYEFOULING is to tackle the different stages of the biofouling process using innovative antifouling agents, covering surface-structured materials, protein adsorption inhibitors, quorum sensing inhibitors, natural biocides and microorganisms with antifouling properties. Encapsulation of the innovative compounds in smart nanostructured materials will be implemented to optimize coating performance and cost all along their life cycle. A proof-of-concept for the most promising candidates will be developed and demonstrators will be produced and tested on fields.
BYEFOULING combines a multidisciplinary leading research team from 11 European countries, which are already acting worldwide in the scientific community, with highly relevant and skilled technological partners, to build a consortium able to develop a full production line for antifouling coatings in Europe.
Acknowledgement This research is supported by the EU FP7 Project “Low-toxic cost-efficient environment-friendly antifouling materials” (OCEAN for Tomorrow) under Grant Agreement no. 612717.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Oral communications
86
Synergistic fouling control technologies – project overview and first results
Dorothea Stübing1, John van Haare2
1Fraunhofer IFAM, Wiener Str. 12, 28359 Bremen, Germany, 2Dutch Polymer Institute, P.O. Box 902, 5600 AX Eindhoven, Netherlands. E-mail: [email protected]
Keywords: Biofouling control, foul release, non-toxic, drag reduction
Abstract Marine biofouling, the unwanted colonization of marine organisms on surfaces immersed in seawater has a huge economic and environmental impact in terms of maintenance requirements for marine structures, increased vessel fuel consumption, operating costs, greenhouse gas emissions and spread of non-indigenous species. SEAFRONT is a European FP7 project and aims to significantly advance the control of biofouling and reduce hydrodynamic drag by integrating multiple technology concepts such as surface structure, surface chemistry and bio-active/bio-based fouling control methodologies into one environmentally benign and drag-reducing solution for mobile and stationary maritime applications. In parallel, a combination of laboratory-based performance benchmarking and end-user field trials will be undertaken in order to develop an enhanced fundamental/mechanistic understanding of the coating-biofouling interaction, the impact of this on hydrodynamic drag and to inform technology development and down-selection of promising fouling control solutions. This project aims to facilitate a leap forward in reducing greenhouse gas emissions from marine transport and the conservation of the marine ecosystem by adopting a multidisciplinary and synergistic approach to fouling control.
The present work outlines the general approach of SEAFRONT and highlights first results of selected developments with regard to surface structure and chemistry.
Acknowledgement The research leading to these results has received funding from the European Union's Seventh Framework Programme for research, technological development, and demonstration under grant agreement no 614034.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Oral communications
87
New biocoating for corrosion inhibition in metal surfaces Emilio Blas, Rocío Barros, Edith Guedella
ACCIONA Infraestructuras, Valportillo Segunda 8, 28108, Madrid, Spain. E-mail:[email protected]
Keywords: Corrosion, Fouling, Metal Surfaces, Infrastructures, Coating
Abstract Among the different types of corrosion, Microbial Influence Corrosion (MIC) is estimated to be involved in at least 10% of the corrosion problems of structures. MIC is a very aggressive form of corrosion with many proposed mechanisms for its prevention but these solutions include biocides and solutions not environmentally friendly.
ACCIONA Infrastructures coordinates the European project BIOCORIN “New biocoating for corrosion inhibition in metal surfaces”, within FP7-ENVIRONMENT program (FP7 ENV.2011 www.biocorin.eu). The project aims at developing an innovative biomimetic and eco-efficient environmental technology for inhibiting microbial induced corrosion (MIC) produced by biofouling through the integration of microorganisms in a sol-gel coating for metal surfaces of civil engineering structures in marine and terrestrial environments such as highway bridges, gas and liquid transmission pipelines, waterways and ports, airports and railroads.
Firstly, the diversity of microbial communities on metal surfaces exposed to different environment was described using metagenomic analysis. In addition, microorganisms most suitable to counteract the fouling and MIC threats in civil infrastructures affected by these phenomena under different types of conditions have been identified. Thereafter, a coating based on sol–gel technology has been designed for immobilization of living microorganisms that will prevent MIC corrosion. The sol-gel system developed has been highly biocompatible with the microorganism identified, allowing to incorporate living cells and very sensitive biomolecules. In addition, the design and formulation of sol-gel coatings has been compatible with the defined metal surfaces and must show very good adhesion to the metal surfaces.
The environmental performance of the solution has also been evaluated from the life cycle analysis (LCA) perspective. Preliminary results have shown that the innovative BIOCORIN coating presents significant reduction (46% of CO2 emission and 71% of methane emissions) resulted in a decrease of the impact category of Global Warming Potential (GWP) compared with an epoxy coating.
The technology is under validation by means of three case studies with different environmental conditions. These demonstrator sites are the port of Harlingen (The Netherlands), the port of Gijón (Spain) and the port of Naples (Italy).
It is expected that BIOCORIN solution will develop a coating for biofouling inhibition in infrastructures sector generating a longer lasting and more economic coating, increasing durability ratios and reduction of costs compared to commercial anticorrosion and anti-biofouling coatings, with better environmental properties compared without using organic or inorganic chemical compounds.
Acknowledgement The authors would like to thank all BIOCORIN partners for the development of the project. This work is sponsored by European Commission under the Seventh Framework Program.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Oral communications
86
Synergistic fouling control technologies – project overview and first results
Dorothea Stübing1, John van Haare2
1Fraunhofer IFAM, Wiener Str. 12, 28359 Bremen, Germany, 2Dutch Polymer Institute, P.O. Box 902, 5600 AX Eindhoven, Netherlands. E-mail: [email protected]
Keywords: Biofouling control, foul release, non-toxic, drag reduction
Abstract Marine biofouling, the unwanted colonization of marine organisms on surfaces immersed in seawater has a huge economic and environmental impact in terms of maintenance requirements for marine structures, increased vessel fuel consumption, operating costs, greenhouse gas emissions and spread of non-indigenous species. SEAFRONT is a European FP7 project and aims to significantly advance the control of biofouling and reduce hydrodynamic drag by integrating multiple technology concepts such as surface structure, surface chemistry and bio-active/bio-based fouling control methodologies into one environmentally benign and drag-reducing solution for mobile and stationary maritime applications. In parallel, a combination of laboratory-based performance benchmarking and end-user field trials will be undertaken in order to develop an enhanced fundamental/mechanistic understanding of the coating-biofouling interaction, the impact of this on hydrodynamic drag and to inform technology development and down-selection of promising fouling control solutions. This project aims to facilitate a leap forward in reducing greenhouse gas emissions from marine transport and the conservation of the marine ecosystem by adopting a multidisciplinary and synergistic approach to fouling control.
The present work outlines the general approach of SEAFRONT and highlights first results of selected developments with regard to surface structure and chemistry.
Acknowledgement The research leading to these results has received funding from the European Union's Seventh Framework Programme for research, technological development, and demonstration under grant agreement no 614034.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Oral communications
87
New biocoating for corrosion inhibition in metal surfaces Emilio Blas, Rocío Barros, Edith Guedella
ACCIONA Infraestructuras, Valportillo Segunda 8, 28108, Madrid, Spain. E-mail:[email protected]
Keywords: Corrosion, Fouling, Metal Surfaces, Infrastructures, Coating
Abstract Among the different types of corrosion, Microbial Influence Corrosion (MIC) is estimated to be involved in at least 10% of the corrosion problems of structures. MIC is a very aggressive form of corrosion with many proposed mechanisms for its prevention but these solutions include biocides and solutions not environmentally friendly.
ACCIONA Infrastructures coordinates the European project BIOCORIN “New biocoating for corrosion inhibition in metal surfaces”, within FP7-ENVIRONMENT program (FP7 ENV.2011 www.biocorin.eu). The project aims at developing an innovative biomimetic and eco-efficient environmental technology for inhibiting microbial induced corrosion (MIC) produced by biofouling through the integration of microorganisms in a sol-gel coating for metal surfaces of civil engineering structures in marine and terrestrial environments such as highway bridges, gas and liquid transmission pipelines, waterways and ports, airports and railroads.
Firstly, the diversity of microbial communities on metal surfaces exposed to different environment was described using metagenomic analysis. In addition, microorganisms most suitable to counteract the fouling and MIC threats in civil infrastructures affected by these phenomena under different types of conditions have been identified. Thereafter, a coating based on sol–gel technology has been designed for immobilization of living microorganisms that will prevent MIC corrosion. The sol-gel system developed has been highly biocompatible with the microorganism identified, allowing to incorporate living cells and very sensitive biomolecules. In addition, the design and formulation of sol-gel coatings has been compatible with the defined metal surfaces and must show very good adhesion to the metal surfaces.
The environmental performance of the solution has also been evaluated from the life cycle analysis (LCA) perspective. Preliminary results have shown that the innovative BIOCORIN coating presents significant reduction (46% of CO2 emission and 71% of methane emissions) resulted in a decrease of the impact category of Global Warming Potential (GWP) compared with an epoxy coating.
The technology is under validation by means of three case studies with different environmental conditions. These demonstrator sites are the port of Harlingen (The Netherlands), the port of Gijón (Spain) and the port of Naples (Italy).
It is expected that BIOCORIN solution will develop a coating for biofouling inhibition in infrastructures sector generating a longer lasting and more economic coating, increasing durability ratios and reduction of costs compared to commercial anticorrosion and anti-biofouling coatings, with better environmental properties compared without using organic or inorganic chemical compounds.
Acknowledgement The authors would like to thank all BIOCORIN partners for the development of the project. This work is sponsored by European Commission under the Seventh Framework Program.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Oral communications
88
Effects of novel anti-fouling paints on aquatic species of different trophic levels
Chen Piller1, Eldad Hoch-Gunter1,2, Michal Weis1, Severine Larroze3, Tânia Teixeira3, Leonid Goldenberg4, Alexie Antipov4, Yehuda Benayahu1
1Department of Zoology, Tel Aviv University, Ramat Aviv, Tel Aviv, 69978, Israel, 2Interuniversity Institute for Marine Sciences in Eilat, Eilat 88103, Israel,3AquaBioTech
Group, Central Complex, Naggar Street, Targa Gap, Mosta, MST 1761, Malta, 4PlasmaChem GmbH, Rudower Chaussee 29, D-12489, Berlin, Germany
E-mail: [email protected]
Keywords: Antimacrofouling, engineered paints, Bugula, mussels, settlement, efficacy
Abstract Biofouling is defined as a natural phenomenon comprising the undesirable accumulation of microorganisms, plants and invertebrates on submerged surfaces in the aquatic environment. Marine fouling may severely affect man-made structures, mainly ship hulls, resulting in damage to their stability, loss of speed and increased CO2 emissions in vessels, as well as overall increase in maintenance costs. The FP7 project “BYEFOULING” combines the development of diverse types of antifouling strategies and thus targets a variety of fouling stages. Novel engineered paints with antifouling properties are under development, based on hydrophobic siloxanes, fluorinated and quaternary ammonium siloxanes that have been enhanced by co-biocides.
The present study examined the efficacy of the above novel antifouling compounds, using the common macrofoulers Bugula neritina (bryozoan) and Brachidontes pharaonis (mussels). For intervalidation purposes the tests were carried out under the prevailing conditions of the Israeli Mediterranean, the Maltese Mediterranean and of the northern Gulf of Aqaba (Red Sea). Freshly released larvae of B. neritina and adults of B. pharaonis were tested in the laboratory for 72 hours, using tissue culture plates painted with the new paint formulations. Survivorship and settlement of both fouling species were monitored, in addition to metamorphosis for the Bugula larvae. On the novel paint plates, almost all larvae settled within 24 hours, but failed to metamorphose normally into zooids with an active lophophore. The commercial paints caused absolute and immediate mortality of the larvae, which was denoted by lysis. The results enabled us to present a visual scoring system of the paints' effects, based on the malformed developmental stages of the larvae. The B. pharaonis mussels developed a lower number of byssus threads on the experimental plates compared to the control, while on the commercial paints they did not settle and mortality ensued. The newly developed paints reflected a substantial efficacy regarding their ability to inhibit settlement of macrofoulers and seem to be less harmful than the available commercial paints.
Acknowledgement This research is supported by the EU FP7 Project “Low-toxic cost-efficient environment-friendly antifouling materials” (OCEAN for Tomorrow) under Grant Agreement no. 612717.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Oral communications
89
Monitoring biofouling on hard substrata through DNA based approaches
Naiara Rodríguez-Ezpeleta, Iratxe Menchaca, Izaskun Zorita, Laura Alonso, Javier Franco
AZTI, Marine Research Division, Txatxarramendi ugartea z/g, Sukarrieta 48395, Bizkaia, Spain. E-mail:[email protected]
Keywords: biofouling, metabarcoding, genomics
Abstract Biofouling formation starts with a first phase in which microscopic organisms (bacteria and microbial eukaryotes) adhere to a submerged surface making it suitable for macroscopic organisms to attach. This initial biological settlement, which occurs during the first hours or days (depending on location and environmental variables) is crucial for the incrustation of other macroorganisms such as invertebrates and algae. Hence, understanding the first stages in biofouling formation is central to investigations on new biofouling prevention strategies, contributing to attenuate consequences of biofilm formation such as economic losses and introduction of non-indigenous species. Yet, identification of microscopic organisms and of macroscopic ones at early developmental stages (e.g. larvae) is a time and resource consuming task that can only be performed by expert taxonomists. We have studied the first stages of biofouling formation via metabarcoding, a potentially more cost-effective and accurate alternative to visual taxonomy for species identification. Metabarcoding consists on sequencing a fragment of the genomic DNA extracted from an environmental sample and comparing it to a reference database for taxonomic assignment. We have applied this technique to 5 x 5 cm polyethylene plates submerged for different time periods at 4 and 20 m depth. By amplification of the bacteria and eukaryotic specific small subunit ribosomal RNA genes (16S and 18S rRNA respectively), we have characterized the sequence of microorganisms responsible of biofouling formation under different conditions. Our results prove metabarcoding as a promising approach to monitor biofouling on hard substrata.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Oral communications
88
Effects of novel anti-fouling paints on aquatic species of different trophic levels
Chen Piller1, Eldad Hoch-Gunter1,2, Michal Weis1, Severine Larroze3, Tânia Teixeira3, Leonid Goldenberg4, Alexie Antipov4, Yehuda Benayahu1
1Department of Zoology, Tel Aviv University, Ramat Aviv, Tel Aviv, 69978, Israel, 2Interuniversity Institute for Marine Sciences in Eilat, Eilat 88103, Israel,3AquaBioTech
Group, Central Complex, Naggar Street, Targa Gap, Mosta, MST 1761, Malta, 4PlasmaChem GmbH, Rudower Chaussee 29, D-12489, Berlin, Germany
E-mail: [email protected]
Keywords: Antimacrofouling, engineered paints, Bugula, mussels, settlement, efficacy
Abstract Biofouling is defined as a natural phenomenon comprising the undesirable accumulation of microorganisms, plants and invertebrates on submerged surfaces in the aquatic environment. Marine fouling may severely affect man-made structures, mainly ship hulls, resulting in damage to their stability, loss of speed and increased CO2 emissions in vessels, as well as overall increase in maintenance costs. The FP7 project “BYEFOULING” combines the development of diverse types of antifouling strategies and thus targets a variety of fouling stages. Novel engineered paints with antifouling properties are under development, based on hydrophobic siloxanes, fluorinated and quaternary ammonium siloxanes that have been enhanced by co-biocides.
The present study examined the efficacy of the above novel antifouling compounds, using the common macrofoulers Bugula neritina (bryozoan) and Brachidontes pharaonis (mussels). For intervalidation purposes the tests were carried out under the prevailing conditions of the Israeli Mediterranean, the Maltese Mediterranean and of the northern Gulf of Aqaba (Red Sea). Freshly released larvae of B. neritina and adults of B. pharaonis were tested in the laboratory for 72 hours, using tissue culture plates painted with the new paint formulations. Survivorship and settlement of both fouling species were monitored, in addition to metamorphosis for the Bugula larvae. On the novel paint plates, almost all larvae settled within 24 hours, but failed to metamorphose normally into zooids with an active lophophore. The commercial paints caused absolute and immediate mortality of the larvae, which was denoted by lysis. The results enabled us to present a visual scoring system of the paints' effects, based on the malformed developmental stages of the larvae. The B. pharaonis mussels developed a lower number of byssus threads on the experimental plates compared to the control, while on the commercial paints they did not settle and mortality ensued. The newly developed paints reflected a substantial efficacy regarding their ability to inhibit settlement of macrofoulers and seem to be less harmful than the available commercial paints.
Acknowledgement This research is supported by the EU FP7 Project “Low-toxic cost-efficient environment-friendly antifouling materials” (OCEAN for Tomorrow) under Grant Agreement no. 612717.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Oral communications
89
Monitoring biofouling on hard substrata through DNA based approaches
Naiara Rodríguez-Ezpeleta, Iratxe Menchaca, Izaskun Zorita, Laura Alonso, Javier Franco
AZTI, Marine Research Division, Txatxarramendi ugartea z/g, Sukarrieta 48395, Bizkaia, Spain. E-mail:[email protected]
Keywords: biofouling, metabarcoding, genomics
Abstract Biofouling formation starts with a first phase in which microscopic organisms (bacteria and microbial eukaryotes) adhere to a submerged surface making it suitable for macroscopic organisms to attach. This initial biological settlement, which occurs during the first hours or days (depending on location and environmental variables) is crucial for the incrustation of other macroorganisms such as invertebrates and algae. Hence, understanding the first stages in biofouling formation is central to investigations on new biofouling prevention strategies, contributing to attenuate consequences of biofilm formation such as economic losses and introduction of non-indigenous species. Yet, identification of microscopic organisms and of macroscopic ones at early developmental stages (e.g. larvae) is a time and resource consuming task that can only be performed by expert taxonomists. We have studied the first stages of biofouling formation via metabarcoding, a potentially more cost-effective and accurate alternative to visual taxonomy for species identification. Metabarcoding consists on sequencing a fragment of the genomic DNA extracted from an environmental sample and comparing it to a reference database for taxonomic assignment. We have applied this technique to 5 x 5 cm polyethylene plates submerged for different time periods at 4 and 20 m depth. By amplification of the bacteria and eukaryotic specific small subunit ribosomal RNA genes (16S and 18S rRNA respectively), we have characterized the sequence of microorganisms responsible of biofouling formation under different conditions. Our results prove metabarcoding as a promising approach to monitor biofouling on hard substrata.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Oral communications
90
Bio-based and bio-active biofouling control strategies
Linda Wittig1, Ingo Grunwald1, Anke Wunder2, Dan Isaksson3
1Fraunhofer IFAM, Wiener Str. 12, 28359 Bremen, Germany,2BioLog Biotechnologie und Logistik GmbH, Max-Planck-Ring 45, 06188 Landsberg, Germany,3I-tech AB,
Pepparedsleden 1,SE-431 83, Mölndal, Sweden. E-mail: [email protected]
Keywords: Biofouling control, foul release, chitosan, selektope, polyglycerols
Abstract Marine fouling is well studied and often described to follow a specific pattern the beginning of which involves formation of an organic film and bacterial attachment and growth, which is considered to condition the surface for further settlement of fouling organisms. To prevent the bacterial fouling, different strategies have been applied in order to immobilise non-toxic, environmentally benign bioactive compounds: chitosan, Selektope and polyglycerols. Chitosans are polyaminosaccharides obtained by deacetylation of naturally occurring chitin, which shows a broad range of antimicrobial activity. Selektope has the ability to manipulate the settlement behaviour of certain fouling invertebrates (e.g. barnacles, tube worms). And polyglycerols are able to dramatically reduce the non-specific interaction of proteins with surfaces. For the immobilisation, special attention was given to identify suitable surface chemistries for each of the three used actives. Verification of the presence of the molecules on the coating surfaces was done by ATR-FTIR and XPS measurements. Furthermore, the antimicrobial activity and the ability of these functionalized surfaces to reduce bacterial adhesion were investigated.
Acknowledgement: The research leading to these results has received funding from the European Union's Seventh Framework Programme for research, technological development, and demonstration under grant agreement no 614034.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Oral communications
91
Novel quorum sensing disruptors and their antifouling implications
Alberto J. Martín-Rodríguez1,2, Sergio J. Álvarez-Méndez1, Víctor S. Martín2, José J. Fernández
1Institute for Bioorganic Chemistry “Antonio González”, Center for Biomedical Research of the Canary Islands (CIBICAN), Department of Organic Chemistry, University of La
Laguna. Av. Astrofísico Francisco Sánchez 2, 38206 La Laguna, Tenerife, Spain.2Oceanic Platform of the Canary Islands (PLOCAN). Carretera de Taliarte s/n,
35214 Telde, Gran Canaria, Spain. E-mail: [email protected]
Keywords: Phosphonium salts, vinyl ethers, Chromobacterium violaceum, Vibrio harveyi, Quorum Sensing, antifouling
Abstract Over the last few years our research has been focused on the identification of novel antifouling compounds. Since product availability is a must for practical antifouling solutions, emphasis has been placed on the screening of chemical libraries from organic synthesis, in particular as Quorum Sensing (QS) disruptors. Given that biofilm formation is an initial step in the biofouling process, the use of QS inhibitors has emerged as a non-toxic mechanism for the control of the early stages of this detrimental phenomenon. This screening program has led to the identification of two families of compounds with an attractive bioactive profile: a) phosphonium salts, and b) vinyl ethers.
Whereas ‘onium’ compounds have been widely employed as broad-spectrum biocides, their QS inhibitory properties and their ability to interfere with other relevant processes for the biological colonization of marine substrata have not been previously reported (1). Similarly, vinyl ethers are novel chemical entities (2) with an unprecedented nature as inhibitors of QS-regulated phenotypes. Here we present the keys for the biological activity of these chemical entities based on their structure-activity relationships, as well as the ongoing research regarding their modes of action.
Acknowledgements Financial support was provided by grants SAF2011-28883-C03-01 and CTQ2011-28417-C02-01/BQU (MINECO, Spain). A.J.M-R. thanks PLOCAN for a 2+2 fellowship. S.J.A-M. holds a FPU fellowship from MINECO. The authors are grateful to Dr. Tom Defoirdt (University of Ghent). We acknowledge funding granted to the ULL by the Canarian Agency for Research, Innovation and Information Society, financed 85% by the European Social Fund.
References
1. Martín-Rodríguez AJ, Babarro JMF, Lahoz F, Sansón M, Martín VS, Norte M, Fernández JJ. 2015. From Broad-Spectrum Biocides to Quorum Sensing Disruptors and Mussel Repellents: Antifouling Profile of Alkyl Triphenylphosphonium Salts. PLoS One (in press).
2. Tejedor D, Álvarez-Méndez SJ, López-Soria JM, Martín VSM, García-Tellado F. 2014. Eur J Org Chem 1:198–205.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Oral communications
90
Bio-based and bio-active biofouling control strategies
Linda Wittig1, Ingo Grunwald1, Anke Wunder2, Dan Isaksson3
1Fraunhofer IFAM, Wiener Str. 12, 28359 Bremen, Germany,2BioLog Biotechnologie und Logistik GmbH, Max-Planck-Ring 45, 06188 Landsberg, Germany,3I-tech AB,
Pepparedsleden 1,SE-431 83, Mölndal, Sweden. E-mail: [email protected]
Keywords: Biofouling control, foul release, chitosan, selektope, polyglycerols
Abstract Marine fouling is well studied and often described to follow a specific pattern the beginning of which involves formation of an organic film and bacterial attachment and growth, which is considered to condition the surface for further settlement of fouling organisms. To prevent the bacterial fouling, different strategies have been applied in order to immobilise non-toxic, environmentally benign bioactive compounds: chitosan, Selektope and polyglycerols. Chitosans are polyaminosaccharides obtained by deacetylation of naturally occurring chitin, which shows a broad range of antimicrobial activity. Selektope has the ability to manipulate the settlement behaviour of certain fouling invertebrates (e.g. barnacles, tube worms). And polyglycerols are able to dramatically reduce the non-specific interaction of proteins with surfaces. For the immobilisation, special attention was given to identify suitable surface chemistries for each of the three used actives. Verification of the presence of the molecules on the coating surfaces was done by ATR-FTIR and XPS measurements. Furthermore, the antimicrobial activity and the ability of these functionalized surfaces to reduce bacterial adhesion were investigated.
Acknowledgement: The research leading to these results has received funding from the European Union's Seventh Framework Programme for research, technological development, and demonstration under grant agreement no 614034.
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Novel quorum sensing disruptors and their antifouling implications
Alberto J. Martín-Rodríguez1,2, Sergio J. Álvarez-Méndez1, Víctor S. Martín2, José J. Fernández
1Institute for Bioorganic Chemistry “Antonio González”, Center for Biomedical Research of the Canary Islands (CIBICAN), Department of Organic Chemistry, University of La
Laguna. Av. Astrofísico Francisco Sánchez 2, 38206 La Laguna, Tenerife, Spain.2Oceanic Platform of the Canary Islands (PLOCAN). Carretera de Taliarte s/n,
35214 Telde, Gran Canaria, Spain. E-mail: [email protected]
Keywords: Phosphonium salts, vinyl ethers, Chromobacterium violaceum, Vibrio harveyi, Quorum Sensing, antifouling
Abstract Over the last few years our research has been focused on the identification of novel antifouling compounds. Since product availability is a must for practical antifouling solutions, emphasis has been placed on the screening of chemical libraries from organic synthesis, in particular as Quorum Sensing (QS) disruptors. Given that biofilm formation is an initial step in the biofouling process, the use of QS inhibitors has emerged as a non-toxic mechanism for the control of the early stages of this detrimental phenomenon. This screening program has led to the identification of two families of compounds with an attractive bioactive profile: a) phosphonium salts, and b) vinyl ethers.
Whereas ‘onium’ compounds have been widely employed as broad-spectrum biocides, their QS inhibitory properties and their ability to interfere with other relevant processes for the biological colonization of marine substrata have not been previously reported (1). Similarly, vinyl ethers are novel chemical entities (2) with an unprecedented nature as inhibitors of QS-regulated phenotypes. Here we present the keys for the biological activity of these chemical entities based on their structure-activity relationships, as well as the ongoing research regarding their modes of action.
Acknowledgements Financial support was provided by grants SAF2011-28883-C03-01 and CTQ2011-28417-C02-01/BQU (MINECO, Spain). A.J.M-R. thanks PLOCAN for a 2+2 fellowship. S.J.A-M. holds a FPU fellowship from MINECO. The authors are grateful to Dr. Tom Defoirdt (University of Ghent). We acknowledge funding granted to the ULL by the Canarian Agency for Research, Innovation and Information Society, financed 85% by the European Social Fund.
References
1. Martín-Rodríguez AJ, Babarro JMF, Lahoz F, Sansón M, Martín VS, Norte M, Fernández JJ. 2015. From Broad-Spectrum Biocides to Quorum Sensing Disruptors and Mussel Repellents: Antifouling Profile of Alkyl Triphenylphosphonium Salts. PLoS One (in press).
2. Tejedor D, Álvarez-Méndez SJ, López-Soria JM, Martín VSM, García-Tellado F. 2014. Eur J Org Chem 1:198–205.
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Polymer mediated bacterial clustering: cell-adhesive properties of cationic homo- and copolymers
Iria Louzao1, Cheng Sui1, Klaus Winzer2, Francisco Fernandez-Trillo3, Cameron Alexander1
1 School of Pharmacy, The University of Nottingham, University Park, Nottingham NG7 2RD, UK; 2 School of Molecular Medical Sciences, Centre for Biomolecular Sciences, The University of Nottingham, University Park, Nottingham NG7 2RD, UK; 3 School of
Chemistry, University of Birmingham, Birmingham B15 2TT, UK.
Keywords: polymers, gram-positive and gram-negative bacteria, anti-infective materials, adhesion, polycations, catechols
Abstract New anti-infective materials are needed urgently as alternatives to conventional biocides (1). It has been recently reported that polymer materials designed to bind to the surface of bacteria can induce the formation of cell clusters which enhance the expression of quorum sensing controlled phenotypes. These materials are relevant for infection. They have the potential to inhibit adhesion and to modulate Quorum Sensing (QS) controlled virulence at the same time (2). Here we carefully evaluate the role that charge and catechol moieties present in these polymers play on the binding. Assessment of the ability of the cationic poly(N-[3-(dimethylamino)propyl] methacrylamide) (pDMAPMAm, P1), poly(N-dopamine methacrylamide-co-N-[3-(dimethylamino)propyl] methacrylamide) (pDMAm-co- pDMAPMAm, P2) and p(3,4-dihydroxy-L-phenylalanine methacrylamide), p(L-DMAm, P3) to cluster a range of bacteria (both Gram-negative and Gram-positive) under conditions of different pH and polymer concentration is investigated. We identify that clustering ability is strongly dependent on the balance between charge and hydrophobicity. Moreover, our results suggest that catechol moieties have an enhanced effect on adhesive properties, but only in the presence of cationic residues such as for P2. Overall, our results reveal the need to consider synergistic structure-property relationship when designing antimicrobial polymers.
Acknowledgements We thank Sheyda Azimi and Dr. Vincenzo Taresco for assistance with confocal microscopy and helpful discussions respectively and Christy Grainger-Boultby for technical support. We gratefully acknowledge financial support from the Engineering and Physical Sciences Research Council (EP/G042462/1, and EP/H005625/1 Leadership Fellowship to CA).
References 1. Siedenbiedel F, Tiller JC. 2012. Antimicrobial Polymers in Solution and on Surfaces:
Overview and Functional Principles. Polymers 4:46-71.
2. Lui LT, Xue X, Sui C, Brown A, Pritchard DI, Halliday N, Winzer K, Howdle SM, Fernandez-Trillo F, Krasnogor N, Alexander C. 2013. Bacteria clustering by polymers induces the expression of quorum-sensing-controlled phenotypes. Nature Chem 5:1058-1065.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Oral communications
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Test on the environmental impacts of two commercial antifouling coating systems
G. Matteucci1, M. Esposito2, F. Fiesoletti1, V. James2, L. Napolano2, C. Pascale2, P. Rossini1, A. Testoni1
1Istituto di Ricerca Gruppo CSA, via Al Torrente 22, 47923 Rimini, Italy,1Stress S.c.ar.l, Vico II San Nicola alla Dogana 9, 80133, Napoli,Italy.
E-mail: [email protected]
Keywords: marine systems, oxic/anoxic conditions, biotoxicity, inorganic contaminants, organic contaminants, Life Cycle Assessment, Environmental Product Declaration
Abstract In the framework of BIOCORIN project (“New biocoating for corrosion inhibition in metal surfaces”, within FP7-ENVIRONMENT program (FP7 ENV.2011 www.biocorin.eu), two commercial coating systems, each one consisting of both a primer and an antifouling (“System1” based on Cu2O and “System2” based on ZnO), were analysed in order to investigate their environmental impacts on humans and environment through lab tests and life cycle analysis (LCA).
With regard to lab tests, several incubations were performed in marine water (Gulf of Naples, Mediterranean Sea, Italy) as critical environment favouring metal corrosion and biofouling generation. Dissolved oxygen was chosen as the most representative parameter to test the response of the coating at the environmental conditions. Oxygen content affects bacterial species presence and activities, organic matter degradation, nutrient and oxides/hydroxides concentrations. In natural environments oxygen ranges from oxic to hypoxic values and, in critical conditions, it could reach anoxic values (<1 mg/l) causing strong environmental effects/impacts. For these reasons a specific test has been set up alternating the environment conditions (aerobic/anaerobic) for 2 months in the RISE incubator system. The RISE incubator allows simulating in situ environmental conditions. The main parameters characterizing marine water environment have been monitored in the microcosms at the beginning, during and at the end of the experiments to verify significant environmental variations.
The Life Cycle Assessment (LCA) has been also performed. A cradle to grave system boundary has been considered for each coating; SimaPro software and Environmental Product Declaration (EPD) standard have been used for the LCA analysis, enabling quantification of the environmental impacts of the two coating options by means of 6 environmental indicators. Firstly, the aim of carried out LCA, was to quantify (separately) the environmental footprint of coating solutions; indeed, for each of them, the interpretation analysis has been conducted in order to define which element, material, or process mainly influenced the LCA results. Moreover, the second objective of this analysis was to compare all the above options to determine the most environmentally sustainable choice. The final purpose is to test the toxicity, chemical and environmental impacts of commercial anti-corrosion coatings considering the new European perspectives and the new green products demand.
Acknowledgement The authors would like to acknowledge the BIOCORIN Consortium and the financial support of the European Commission under the Seventh Framework Program.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Oral communications
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Polymer mediated bacterial clustering: cell-adhesive properties of cationic homo- and copolymers
Iria Louzao1, Cheng Sui1, Klaus Winzer2, Francisco Fernandez-Trillo3, Cameron Alexander1
1 School of Pharmacy, The University of Nottingham, University Park, Nottingham NG7 2RD, UK; 2 School of Molecular Medical Sciences, Centre for Biomolecular Sciences, The University of Nottingham, University Park, Nottingham NG7 2RD, UK; 3 School of
Chemistry, University of Birmingham, Birmingham B15 2TT, UK.
Keywords: polymers, gram-positive and gram-negative bacteria, anti-infective materials, adhesion, polycations, catechols
Abstract New anti-infective materials are needed urgently as alternatives to conventional biocides (1). It has been recently reported that polymer materials designed to bind to the surface of bacteria can induce the formation of cell clusters which enhance the expression of quorum sensing controlled phenotypes. These materials are relevant for infection. They have the potential to inhibit adhesion and to modulate Quorum Sensing (QS) controlled virulence at the same time (2). Here we carefully evaluate the role that charge and catechol moieties present in these polymers play on the binding. Assessment of the ability of the cationic poly(N-[3-(dimethylamino)propyl] methacrylamide) (pDMAPMAm, P1), poly(N-dopamine methacrylamide-co-N-[3-(dimethylamino)propyl] methacrylamide) (pDMAm-co- pDMAPMAm, P2) and p(3,4-dihydroxy-L-phenylalanine methacrylamide), p(L-DMAm, P3) to cluster a range of bacteria (both Gram-negative and Gram-positive) under conditions of different pH and polymer concentration is investigated. We identify that clustering ability is strongly dependent on the balance between charge and hydrophobicity. Moreover, our results suggest that catechol moieties have an enhanced effect on adhesive properties, but only in the presence of cationic residues such as for P2. Overall, our results reveal the need to consider synergistic structure-property relationship when designing antimicrobial polymers.
Acknowledgements We thank Sheyda Azimi and Dr. Vincenzo Taresco for assistance with confocal microscopy and helpful discussions respectively and Christy Grainger-Boultby for technical support. We gratefully acknowledge financial support from the Engineering and Physical Sciences Research Council (EP/G042462/1, and EP/H005625/1 Leadership Fellowship to CA).
References 1. Siedenbiedel F, Tiller JC. 2012. Antimicrobial Polymers in Solution and on Surfaces:
Overview and Functional Principles. Polymers 4:46-71.
2. Lui LT, Xue X, Sui C, Brown A, Pritchard DI, Halliday N, Winzer K, Howdle SM, Fernandez-Trillo F, Krasnogor N, Alexander C. 2013. Bacteria clustering by polymers induces the expression of quorum-sensing-controlled phenotypes. Nature Chem 5:1058-1065.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Oral communications
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Test on the environmental impacts of two commercial antifouling coating systems
G. Matteucci1, M. Esposito2, F. Fiesoletti1, V. James2, L. Napolano2, C. Pascale2, P. Rossini1, A. Testoni1
1Istituto di Ricerca Gruppo CSA, via Al Torrente 22, 47923 Rimini, Italy,1Stress S.c.ar.l, Vico II San Nicola alla Dogana 9, 80133, Napoli,Italy.
E-mail: [email protected]
Keywords: marine systems, oxic/anoxic conditions, biotoxicity, inorganic contaminants, organic contaminants, Life Cycle Assessment, Environmental Product Declaration
Abstract In the framework of BIOCORIN project (“New biocoating for corrosion inhibition in metal surfaces”, within FP7-ENVIRONMENT program (FP7 ENV.2011 www.biocorin.eu), two commercial coating systems, each one consisting of both a primer and an antifouling (“System1” based on Cu2O and “System2” based on ZnO), were analysed in order to investigate their environmental impacts on humans and environment through lab tests and life cycle analysis (LCA).
With regard to lab tests, several incubations were performed in marine water (Gulf of Naples, Mediterranean Sea, Italy) as critical environment favouring metal corrosion and biofouling generation. Dissolved oxygen was chosen as the most representative parameter to test the response of the coating at the environmental conditions. Oxygen content affects bacterial species presence and activities, organic matter degradation, nutrient and oxides/hydroxides concentrations. In natural environments oxygen ranges from oxic to hypoxic values and, in critical conditions, it could reach anoxic values (<1 mg/l) causing strong environmental effects/impacts. For these reasons a specific test has been set up alternating the environment conditions (aerobic/anaerobic) for 2 months in the RISE incubator system. The RISE incubator allows simulating in situ environmental conditions. The main parameters characterizing marine water environment have been monitored in the microcosms at the beginning, during and at the end of the experiments to verify significant environmental variations.
The Life Cycle Assessment (LCA) has been also performed. A cradle to grave system boundary has been considered for each coating; SimaPro software and Environmental Product Declaration (EPD) standard have been used for the LCA analysis, enabling quantification of the environmental impacts of the two coating options by means of 6 environmental indicators. Firstly, the aim of carried out LCA, was to quantify (separately) the environmental footprint of coating solutions; indeed, for each of them, the interpretation analysis has been conducted in order to define which element, material, or process mainly influenced the LCA results. Moreover, the second objective of this analysis was to compare all the above options to determine the most environmentally sustainable choice. The final purpose is to test the toxicity, chemical and environmental impacts of commercial anti-corrosion coatings considering the new European perspectives and the new green products demand.
Acknowledgement The authors would like to acknowledge the BIOCORIN Consortium and the financial support of the European Commission under the Seventh Framework Program.
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Satallite Meeting on Novel Antifouling Strategies Poster communications
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015
95
Satallite Meeting on Novel Antifouling Strategies Poster communications
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A tool for controlling biofouling on membrane bioreactor (MBR) wastewater systems based on the selection of quorum
quenching bacteria L. Arregui1, A. Soler1, R. Liébana1, A. Santos1, D. Marquina1, F. Muñagorri2, S.
Serrano1
1Department of Microbiology III, Complutense University, Madrid, Spain. 2 Department I+D+i URBASER, Madrid, Spain. E-mail: [email protected]
Keywords: Quorum quenching, Quorum sensing, AHL, Biofilm formation, Membrane system, Wastewater treatment
Abstract Biofouling of membranes in membrane bioreactors (MBR) wastewater treatment systems could be considered as a consequence of a Quorum sensing (QS) effect since QS is one of the mechanisms controlling bacterial biofilm development. Bacterial community from the activated sludge of a full scale MBR plant treating urban wastewater was mainly constituted by gram negative bacteria. In this sense, to prevent biofouling through QS disruption strategies we first searched for N-acyl homoserine lactone producer strains using Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1 biosensors. Then, we studied their adhesion capability, the structure of the formed biofilms and the interactions among these strains. The obtained results showed that the isolated AHL producing bacteria were distributed in four different classes, being Gammaproteobacteria dominant. Among the Gammaproteobacteria class, Aeromonadales and Pseudomonadales conformed the main orders. Strains owning to the genera Aeromonas, Shewanella, Acinetobacter and Pseudomonas were classified as strongly adherent bacteria and the experiments performed with multi strain mixtures indicated that they probably act as primary colonizers subsequently favouring the attachment by co-adhesion of other strains. These gram negative genera were also isolated from MBR plants treating leachate from solid residues. In order to detect bacteria able to interfere with QS and inhibit biofilm development or reduce established biofilms, the AHL-degrading activity of 100 isolated strains is being investigated. Gram positive strains (68 isolates) were the dominant bacteria within this environment and, to date, 12 strains have been detected with potential Quorum quenching (QQ) activity. Among the strains showing QQ activity are included several species of Bacillus, genus which comprises, as described in the literature, species that are capable of degrading the AHLs by the action of a specific gene (aiiA, AutoInducer Inactivator), which encodes a lactonase that inactivates the AHL by hydrolysing the HSL ring.
Acknowledgements The authors want to acknowledge URBASER for the financial support (research fund project Biolix), the Cytometry and Fluorescence Microscopy Centre (Complutense University of Madrid) for CLSM expertise and Dr. A. Murciano for assistance with statistical analyses.
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A tool for controlling biofouling on membrane bioreactor (MBR) wastewater systems based on the selection of quorum
quenching bacteria L. Arregui1, A. Soler1, R. Liébana1, A. Santos1, D. Marquina1, F. Muñagorri2, S.
Serrano1
1Department of Microbiology III, Complutense University, Madrid, Spain. 2 Department I+D+i URBASER, Madrid, Spain. E-mail: [email protected]
Keywords: Quorum quenching, Quorum sensing, AHL, Biofilm formation, Membrane system, Wastewater treatment
Abstract Biofouling of membranes in membrane bioreactors (MBR) wastewater treatment systems could be considered as a consequence of a Quorum sensing (QS) effect since QS is one of the mechanisms controlling bacterial biofilm development. Bacterial community from the activated sludge of a full scale MBR plant treating urban wastewater was mainly constituted by gram negative bacteria. In this sense, to prevent biofouling through QS disruption strategies we first searched for N-acyl homoserine lactone producer strains using Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1 biosensors. Then, we studied their adhesion capability, the structure of the formed biofilms and the interactions among these strains. The obtained results showed that the isolated AHL producing bacteria were distributed in four different classes, being Gammaproteobacteria dominant. Among the Gammaproteobacteria class, Aeromonadales and Pseudomonadales conformed the main orders. Strains owning to the genera Aeromonas, Shewanella, Acinetobacter and Pseudomonas were classified as strongly adherent bacteria and the experiments performed with multi strain mixtures indicated that they probably act as primary colonizers subsequently favouring the attachment by co-adhesion of other strains. These gram negative genera were also isolated from MBR plants treating leachate from solid residues. In order to detect bacteria able to interfere with QS and inhibit biofilm development or reduce established biofilms, the AHL-degrading activity of 100 isolated strains is being investigated. Gram positive strains (68 isolates) were the dominant bacteria within this environment and, to date, 12 strains have been detected with potential Quorum quenching (QQ) activity. Among the strains showing QQ activity are included several species of Bacillus, genus which comprises, as described in the literature, species that are capable of degrading the AHLs by the action of a specific gene (aiiA, AutoInducer Inactivator), which encodes a lactonase that inactivates the AHL by hydrolysing the HSL ring.
Acknowledgements The authors want to acknowledge URBASER for the financial support (research fund project Biolix), the Cytometry and Fluorescence Microscopy Centre (Complutense University of Madrid) for CLSM expertise and Dr. A. Murciano for assistance with statistical analyses.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Poster communications
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Hidroboya®: The 'Sample&Hold' platform to overcome the fouling problem
Xulio Fernández Hermida
Telecommunication Engineering School, Rua Maxwell sn, University of Vigo. E-mail: [email protected]
Keywords: buoy, fouling, sensors, Hidroboya, remote sensing
Abstract When you place and use a sensor directly into the water for a period of a couple months, you will get a lot of life attached to the sensor but not any interesting data from it. If you put your sensor in the Hidroboya platform, the life will be surrounding the Hidroboya, but the sensor (like a pearl in an oyster) will keep providing good data for years.
Hidroboya ® is a platform to measure water quality parameters. It effectively overcomes the crucial problem of the fouling in the sensors. The main idea behind Hidroboya is to keep the sensors in a chamber (the sampling chamber) where the sensors are kept in a wet and dark environment between the time when the measuring operations are done.
In the time to do a measurement, the water is left to flow into the sampling chamber and to stay there for a time (a few seconds) to let the sensors to get stable in the new environment. Then the measuring operations are done. After that, using an air compressor, the water is flowed back to the point it has been taken from (a number of meters under the water surface), and the sensors, in the sampling chamber, are kept in the wet and dark conditions that exist naturally in the sampling chamber. These conditions (wet and dark) are ideal for them to perfectly be preserved until the next measuring cycle.
The sampling chamber has the same function as the capacitor in the electronic sample and hold circuit. Valves and pumps are the 'active elements' to move water to and from the sampling chamber.
The capacitor keeps the value of the signal during the time the Analog to Digital Converter needs to perform the conversion. The Sampling Chamber does the same, by holding the water during the time the sensors need to gather data from the sample.
Acknowledgement Hidroboya project has received the financial aid of regional government (Xunta de Galicia) under the INCITE program of 2010 (grant code: 10MDS322008PR, project period: 2010-2013).
References 1. Fernández-Hermida X, Durán-Neira C, Lago-Reguera MD, Posse-García I, Martín-
Rodríguez F. 2013. Is the Hidroboya™ a Suitable System to Read Water Parameters? Coastal Processes III. WIT Press. Wessex Institute of Technology, UK.
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Novel anti-fouling nanomaterials: Effects on macrofoulers from the Eastern Mediterranean and the northern Gulf of Aqaba
(Red Sea) Eldad Hoch-Gutner1,2‡, Chen Piller1, Francisco Avelelas3, Roberto Martins4,
Eliana Malheiro3, Frederico Maia3, João Tedim5, Yehuda Benayahu1
1Department of Zoology, Tel Aviv University, Ramat Aviv, Tel Aviv, 69978,Israel,2Interuniversity Institute for Marine Science in Eilat, Eilat 88103,
Israel,3Smallmatek - Small Materials and Technologies, Lda., Rua Canhas, 3810-075 Aveiro, Portugal, 4Department of Biology and CESAM, University of Aveiro, 3810-
193Aveiro, Portugal, 5Department of Materials and Ceramic Engineering, CICECO, University of Aveiro, 3810-193 Aveiro, Portugal. E-mail:[email protected]
Keywords: Engineered nanomaterials, antimacrofouling, Bugula, mussels, efficacy
Abstract Marine biofouling refers to the accumulation of microorganisms, algae and invertebrates on submerged unprotected substrates. This process may impact man-made maritime structures, such as ship hulls or offshore fish farms, with severe economic and environmental consequences, by requiring frequent and costly maintenance activities, by increasing carbon dioxide emission from ships and by contributing to the dispersion of alien species. To minimize this problem, “BYEFOULING” FP7 project was recently launched aimed to design, develop and upscale antifouling coatings with enhanced performance compared to the currently available products. The present study involved two partners from this consortium. The Portuguese SME Smallmatek develops novel engineered nanomaterials (ENMs) with antifouling properties and, within this scope some ENMs were loaded with commercially available biocides, zinc pyrithione and copper pyrithione, in order to achieve control over the leaching rate of the active compound. Tel Aviv University conducted efficacy laboratory tests on the above-mentioned ENMs, using larvae of Bugula netrinia (bryozoan) and adults of Brachidontes pharaonis (mussel). All assays were conducted under the environmental conditions prevailing in the Israeli Eastern Mediterranean Sea and in Eilat (Gulf of Aqaba, southern coast of Israel). The tested concentrations of the ENMs ranged between 100 g.L-1 and 100 mg.L-1. The antimacrofouling effects of all ENMs were concentration-dependent. The highest concentration induced the most deleterious effects for B. neritina larvae (larval mortality, or failure to settle) and B. pharaonis (failure to attach by byssus threads, number of produced threads and mortality). The control exhibited regular settlement and development for B. neritina and attachment by B. pharoanis. In general, the results supported further development of ENMs for implementation in new antifouling coatings. Undoubtedly, new developed antifouling coatings will demonstrate enhanced performance together with an eco-friendly design, compared to the latest currently available products.
Acknowledgement This research is supported by the EU FP7 Project “Low-toxic cost-efficient environment-friendly antifouling materials” (OCEAN for Tomorrow) under Grant Agreement no. 612717.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Poster communications
98
Hidroboya®: The 'Sample&Hold' platform to overcome the fouling problem
Xulio Fernández Hermida
Telecommunication Engineering School, Rua Maxwell sn, University of Vigo. E-mail: [email protected]
Keywords: buoy, fouling, sensors, Hidroboya, remote sensing
Abstract When you place and use a sensor directly into the water for a period of a couple months, you will get a lot of life attached to the sensor but not any interesting data from it. If you put your sensor in the Hidroboya platform, the life will be surrounding the Hidroboya, but the sensor (like a pearl in an oyster) will keep providing good data for years.
Hidroboya ® is a platform to measure water quality parameters. It effectively overcomes the crucial problem of the fouling in the sensors. The main idea behind Hidroboya is to keep the sensors in a chamber (the sampling chamber) where the sensors are kept in a wet and dark environment between the time when the measuring operations are done.
In the time to do a measurement, the water is left to flow into the sampling chamber and to stay there for a time (a few seconds) to let the sensors to get stable in the new environment. Then the measuring operations are done. After that, using an air compressor, the water is flowed back to the point it has been taken from (a number of meters under the water surface), and the sensors, in the sampling chamber, are kept in the wet and dark conditions that exist naturally in the sampling chamber. These conditions (wet and dark) are ideal for them to perfectly be preserved until the next measuring cycle.
The sampling chamber has the same function as the capacitor in the electronic sample and hold circuit. Valves and pumps are the 'active elements' to move water to and from the sampling chamber.
The capacitor keeps the value of the signal during the time the Analog to Digital Converter needs to perform the conversion. The Sampling Chamber does the same, by holding the water during the time the sensors need to gather data from the sample.
Acknowledgement Hidroboya project has received the financial aid of regional government (Xunta de Galicia) under the INCITE program of 2010 (grant code: 10MDS322008PR, project period: 2010-2013).
References 1. Fernández-Hermida X, Durán-Neira C, Lago-Reguera MD, Posse-García I, Martín-
Rodríguez F. 2013. Is the Hidroboya™ a Suitable System to Read Water Parameters? Coastal Processes III. WIT Press. Wessex Institute of Technology, UK.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Poster communications
99
Novel anti-fouling nanomaterials: Effects on macrofoulers from the Eastern Mediterranean and the northern Gulf of Aqaba
(Red Sea) Eldad Hoch-Gutner1,2‡, Chen Piller1, Francisco Avelelas3, Roberto Martins4,
Eliana Malheiro3, Frederico Maia3, João Tedim5, Yehuda Benayahu1
1Department of Zoology, Tel Aviv University, Ramat Aviv, Tel Aviv, 69978,Israel,2Interuniversity Institute for Marine Science in Eilat, Eilat 88103,
Israel,3Smallmatek - Small Materials and Technologies, Lda., Rua Canhas, 3810-075 Aveiro, Portugal, 4Department of Biology and CESAM, University of Aveiro, 3810-
193Aveiro, Portugal, 5Department of Materials and Ceramic Engineering, CICECO, University of Aveiro, 3810-193 Aveiro, Portugal. E-mail:[email protected]
Keywords: Engineered nanomaterials, antimacrofouling, Bugula, mussels, efficacy
Abstract Marine biofouling refers to the accumulation of microorganisms, algae and invertebrates on submerged unprotected substrates. This process may impact man-made maritime structures, such as ship hulls or offshore fish farms, with severe economic and environmental consequences, by requiring frequent and costly maintenance activities, by increasing carbon dioxide emission from ships and by contributing to the dispersion of alien species. To minimize this problem, “BYEFOULING” FP7 project was recently launched aimed to design, develop and upscale antifouling coatings with enhanced performance compared to the currently available products. The present study involved two partners from this consortium. The Portuguese SME Smallmatek develops novel engineered nanomaterials (ENMs) with antifouling properties and, within this scope some ENMs were loaded with commercially available biocides, zinc pyrithione and copper pyrithione, in order to achieve control over the leaching rate of the active compound. Tel Aviv University conducted efficacy laboratory tests on the above-mentioned ENMs, using larvae of Bugula netrinia (bryozoan) and adults of Brachidontes pharaonis (mussel). All assays were conducted under the environmental conditions prevailing in the Israeli Eastern Mediterranean Sea and in Eilat (Gulf of Aqaba, southern coast of Israel). The tested concentrations of the ENMs ranged between 100 g.L-1 and 100 mg.L-1. The antimacrofouling effects of all ENMs were concentration-dependent. The highest concentration induced the most deleterious effects for B. neritina larvae (larval mortality, or failure to settle) and B. pharaonis (failure to attach by byssus threads, number of produced threads and mortality). The control exhibited regular settlement and development for B. neritina and attachment by B. pharoanis. In general, the results supported further development of ENMs for implementation in new antifouling coatings. Undoubtedly, new developed antifouling coatings will demonstrate enhanced performance together with an eco-friendly design, compared to the latest currently available products.
Acknowledgement This research is supported by the EU FP7 Project “Low-toxic cost-efficient environment-friendly antifouling materials” (OCEAN for Tomorrow) under Grant Agreement no. 612717.
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Inhibitory activity in extracts of the marine microalga Isochrysis
aff. galbana clone T-ISO against different marine microalgae Isabel Freire, Andrea Muras, Isabel Reyero, Ana Otero
Department of Microbiology and Parasitology, University of Santiago de Compostela, Spain. E-mail: [email protected], [email protected]
Keywords: Biofouling, antifouling, inhibitory activity, eco-friendly, microalga, Isochrysis
Abstract Biofouling, the undesirable accumulation of organisms on submerged surfaces, is a major problem for all marine industries. Different antifouling paints are used to prevent biofouling on industrial applications; however, most of them use toxic biocides presenting many adverse ecotoxicological effects. Thus, there is a need for the development of “environmentally friendly” low-toxicity antifouling compounds. Sponges and macroalgae have been identified as sources of these compounds; however, the production of antifouling substances from these sources may not be sustainable. On the contrary, marine microorganisms may constitute a sustainable source of environmentally friendly compounds with antifouling activity. Among these, microalgae are especially interesting because their production is economically feasible and already stablished. The presence of compounds with inhibitory activity against bacteria, microalgae and larvae from invertebrates has been already described in several species of microalgae. Inhibitory activity against several microalgae has been described in the culture medium of one strain belonging to the genus Isochrysis (1), indicating a potential use as anti-fouling agent.
The aim of this work is the study of the presence of growth or biofilm formation inhibitory activity in the culture media of strain Isochrysis aff. galbana clone T-ISO. Ethyl acetate extracts of culture media obtained from late stationary phase of T-ISO cultures were tested against four different species of microalgae: Tetraselmis suecica, Nannochloropsis gaditana, Porphyridium cruentum and Nitzschia sp. Assays were carried out 2-mL cell culture plates under conditions promoting algal attachment. The addition of the T-ISO extract in a concentration 175 Pg/mL produced a reduction of biofilm formation of 40% for T. suecica, 26% for P. cruentum and 33% for Nitzschia sp. However, little effect was shown on the growth of the Eustigmatophyte N.gaditana, with a reduction of 9% of biofilm inhibition. The culture system used for T-ISO strongly affected the inhibitory activity retrieved.
Data confirm the presence of growth inhibitory activity in strain Isochrysis aff. galbana clon T-ISO against different microalgal species. Further research is required in order to characterize the molecules responsible for the activity and to establish culture conditions driving to maximal production at both, laboratory and large scales.
Acknowledgement This research is supported by the EU FP7 Project “Low-toxic cost-efficient environment-friendly antifouling materials” (OCEAN for Tomorrow) under Grant Agreement no. 612717.
References 1. Yingying S, Changhai W, Jing C. 2008. Growth inhibition of the eight species of microalgae
by growth inhibitor from the culture of Isochrysis galbana and its isolation and identification. J Appl Phycol 20:315-321.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Poster communications
101
Effect of Bacillus licheniformis CECT20T on biofilm formation by marine bacteria
Andrea Muras, Isabel Freire, Celia Mayer, Ana Otero
1Department of Microbiology and Parasitology, Faculty of Biology-CIBUS, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain.
E-mail: [email protected], [email protected]
Keywords: biofilm, biofouling, bacillus, quorum sensing, quorum quenching
Abstract Biofilms are microbial sessile communities characterized by cells that are attached to a substratum or to each other, are embedded in a self-produced matrix of extracellular polymeric substances and exhibit an altered phenotype compared to planktonic cells. The bacterial biofilm formation is the first step in the development of the biofouling process on submerged surfaces and is often dependent on a chemical cell-to-cell communication process known as Quorum Sensing (QS). Therefore, the interference with QS, a process known as Quorum Quenching (QQ) has been proposed as a novel anti-fouling strategy. Bacillus licheniformis is a Gram-positive endospore-forming organism considered a non-pathogenic bacterium to humans in general. B. licheniformis is found safe for use in food and agriculture industry and products for industrial use have been approved by authorities in the United States, Europe and Japan. Furthermore, some compounds derived from different strains are reported to be effective against bacterial biofilm. Therefore, we decide to test the anti-biofilm activity of biomass extracts (aqueous and methanolic) of B. licheniformis CECT20T. Several marine bacteria strains were selected to be tested for biofilm formation using the xCELLigence system and were characterized regarding the presence of Quorum Sensing (QS) or Quorum Quenching (QQ) systems. Four of them were selected (Vibrio aestuarianus, V. tubiashii, Pseudoalteromonas flavipulchra and P. maricaloris) on the basis of strong biofilm formation and sensitivity to QS inhibitors. The aqueous and methanolic cell extracts of B. licheniformis CECT20T caused a reduction of round 50% in biofilm formation by P. maricaloris whose biofilm was also sensitive to QQ enzymes that degrade N-acyl homoserine lactones (AHLs). Since no QQ activity could be detected in B. licheniformis CECT20T against AHLs, a different mechanism has to be the responsible of the anti-biofilm activity in this strain. As a conclusion, this work demonstrates the potential of B. licheniformis CECT20T to produce antifouling eco-friendly compounds useful not only in industry but also in biomedicine.
Acknowledgement This research is supported by the EU FP7 Project “Low-toxic cost-efficient environment-friendly antifouling materials” (OCEAN for Tomorrow) under Grant Agreement no. 612717.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Poster communications
100
Inhibitory activity in extracts of the marine microalga Isochrysis
aff. galbana clone T-ISO against different marine microalgae Isabel Freire, Andrea Muras, Isabel Reyero, Ana Otero
Department of Microbiology and Parasitology, University of Santiago de Compostela, Spain. E-mail: [email protected], [email protected]
Keywords: Biofouling, antifouling, inhibitory activity, eco-friendly, microalga, Isochrysis
Abstract Biofouling, the undesirable accumulation of organisms on submerged surfaces, is a major problem for all marine industries. Different antifouling paints are used to prevent biofouling on industrial applications; however, most of them use toxic biocides presenting many adverse ecotoxicological effects. Thus, there is a need for the development of “environmentally friendly” low-toxicity antifouling compounds. Sponges and macroalgae have been identified as sources of these compounds; however, the production of antifouling substances from these sources may not be sustainable. On the contrary, marine microorganisms may constitute a sustainable source of environmentally friendly compounds with antifouling activity. Among these, microalgae are especially interesting because their production is economically feasible and already stablished. The presence of compounds with inhibitory activity against bacteria, microalgae and larvae from invertebrates has been already described in several species of microalgae. Inhibitory activity against several microalgae has been described in the culture medium of one strain belonging to the genus Isochrysis (1), indicating a potential use as anti-fouling agent.
The aim of this work is the study of the presence of growth or biofilm formation inhibitory activity in the culture media of strain Isochrysis aff. galbana clone T-ISO. Ethyl acetate extracts of culture media obtained from late stationary phase of T-ISO cultures were tested against four different species of microalgae: Tetraselmis suecica, Nannochloropsis gaditana, Porphyridium cruentum and Nitzschia sp. Assays were carried out 2-mL cell culture plates under conditions promoting algal attachment. The addition of the T-ISO extract in a concentration 175 Pg/mL produced a reduction of biofilm formation of 40% for T. suecica, 26% for P. cruentum and 33% for Nitzschia sp. However, little effect was shown on the growth of the Eustigmatophyte N.gaditana, with a reduction of 9% of biofilm inhibition. The culture system used for T-ISO strongly affected the inhibitory activity retrieved.
Data confirm the presence of growth inhibitory activity in strain Isochrysis aff. galbana clon T-ISO against different microalgal species. Further research is required in order to characterize the molecules responsible for the activity and to establish culture conditions driving to maximal production at both, laboratory and large scales.
Acknowledgement This research is supported by the EU FP7 Project “Low-toxic cost-efficient environment-friendly antifouling materials” (OCEAN for Tomorrow) under Grant Agreement no. 612717.
References 1. Yingying S, Changhai W, Jing C. 2008. Growth inhibition of the eight species of microalgae
by growth inhibitor from the culture of Isochrysis galbana and its isolation and identification. J Appl Phycol 20:315-321.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Poster communications
101
Effect of Bacillus licheniformis CECT20T on biofilm formation by marine bacteria
Andrea Muras, Isabel Freire, Celia Mayer, Ana Otero
1Department of Microbiology and Parasitology, Faculty of Biology-CIBUS, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain.
E-mail: [email protected], [email protected]
Keywords: biofilm, biofouling, bacillus, quorum sensing, quorum quenching
Abstract Biofilms are microbial sessile communities characterized by cells that are attached to a substratum or to each other, are embedded in a self-produced matrix of extracellular polymeric substances and exhibit an altered phenotype compared to planktonic cells. The bacterial biofilm formation is the first step in the development of the biofouling process on submerged surfaces and is often dependent on a chemical cell-to-cell communication process known as Quorum Sensing (QS). Therefore, the interference with QS, a process known as Quorum Quenching (QQ) has been proposed as a novel anti-fouling strategy. Bacillus licheniformis is a Gram-positive endospore-forming organism considered a non-pathogenic bacterium to humans in general. B. licheniformis is found safe for use in food and agriculture industry and products for industrial use have been approved by authorities in the United States, Europe and Japan. Furthermore, some compounds derived from different strains are reported to be effective against bacterial biofilm. Therefore, we decide to test the anti-biofilm activity of biomass extracts (aqueous and methanolic) of B. licheniformis CECT20T. Several marine bacteria strains were selected to be tested for biofilm formation using the xCELLigence system and were characterized regarding the presence of Quorum Sensing (QS) or Quorum Quenching (QQ) systems. Four of them were selected (Vibrio aestuarianus, V. tubiashii, Pseudoalteromonas flavipulchra and P. maricaloris) on the basis of strong biofilm formation and sensitivity to QS inhibitors. The aqueous and methanolic cell extracts of B. licheniformis CECT20T caused a reduction of round 50% in biofilm formation by P. maricaloris whose biofilm was also sensitive to QQ enzymes that degrade N-acyl homoserine lactones (AHLs). Since no QQ activity could be detected in B. licheniformis CECT20T against AHLs, a different mechanism has to be the responsible of the anti-biofilm activity in this strain. As a conclusion, this work demonstrates the potential of B. licheniformis CECT20T to produce antifouling eco-friendly compounds useful not only in industry but also in biomedicine.
Acknowledgement This research is supported by the EU FP7 Project “Low-toxic cost-efficient environment-friendly antifouling materials” (OCEAN for Tomorrow) under Grant Agreement no. 612717.
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Poster communications
102
Monitoring bacterial biofilm development with xCELLigence® technology
Andrea Muras, Celia Mayer, Isabel Freire, Ana Otero
Department of Microbiology and Parasitology, Faculty of Biology-CIBUS, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain.
E-mail: [email protected], [email protected]
Keywords: biofilm, xCELLigence, impedance, Staphylococcus aureus, Streptococcus mutans
Abstract Biofilms are complex microbial communities which usually have a vital role in virulence of many pathogenic bacteria and they can be a life-threatening in diseases especially when they are attached over medical devices. Indeed, bacterial biofilms were estimated to cause over 80% of microbial infections in the body including dental caries, periodontal disease, cardiovascular disease, obesity, pregnancy complications, diabetes and respiratory diseases. The biofilm-growing bacterial cells express different phenotypes and often exhibit different characteristics than planktonic cells, presenting increased resistance to antimicrobial agents 100- to 1000- fold. Among biofilm-forming pathogenic bacteria, the Gram-positive bacteria Staphylococcus aureus and Streptococcus mutans are important biofilm-forming pathogens in humans, a quantification method able to obtain the measurement in a fast, efficient and reproductive way is needed.
The crystal violet staining assay is one of the methods most commonly used to quantify the biofilm formation by bacteria despite of its low reproducibility. Moreover, this method stains indistinctly all the negatively charged molecules in the wells, producing a value not directly related to the number of cells. To avoid these handicaps the xCELLigence System RTCA SP (ACEA, Biosciences Inc.) is proposed to quantify biofilms due to its continuous monitoring of the cell adhesion and real-time access to data on biofilm formation. The equipment is based in a microelectronic biosensor allowing the capture of data with high sensitivity and accuracy. The microelectronic cell sensor arrays are integrated into the bottom of the special plates (E-plates) and therefore this method is suitable only for species forming biofilm on the bottom of the well. The growth of cells on the bottom of wells causes changes in the electrode impedance. These changes that represent cell adherence status are measured by a unitless parameter called Cell Index (CI) which is the impedance change divided by a background value. The electronic impedance measured between electrodes is influenced by the degree of cell adhesion, allowing the monitoring of biofilm formation in real-time. The electrode measurements are affected by salinity of the culture medium higher than 10 g of NaCl per and therefore, the use for anti-fouling compounds screening with marine bacteria is limited since the marine broth is not suitable for this equipment.
In order to test the ability of the xCELLigence system to measure biofilm formation, S. aureus and S. mutans were inoculated in the E-plates in Brain Heart Infusion (BHI) supplemented with glucose (0.25%) and sucrose (0.1%), respectively at a final optical density of 0.05. Furthermore, furanone (0.1 M), a molecule already described as anti-biofilm agent, was added to observe the effect on biofilm formation. Results demonstrate that the xCELLigence system is an effective method to quantify biofilm in low-salinity culture media and to use for the screening of anti-biofilm agents against humans pathogens or in environmental applications such as inhibitors of membrane fouling.
Acknowledgement This work was supported by Fundación Ramón Areces (CIVP16A1814).
Author index
10
Author index
Satellite meeting on Novel Antifouling Strategies, Santiago de Compostela 2015 Poster communications
102
Monitoring bacterial biofilm development with xCELLigence® technology
Andrea Muras, Celia Mayer, Isabel Freire, Ana Otero
Department of Microbiology and Parasitology, Faculty of Biology-CIBUS, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain.
E-mail: [email protected], [email protected]
Keywords: biofilm, xCELLigence, impedance, Staphylococcus aureus, Streptococcus mutans
Abstract Biofilms are complex microbial communities which usually have a vital role in virulence of many pathogenic bacteria and they can be a life-threatening in diseases especially when they are attached over medical devices. Indeed, bacterial biofilms were estimated to cause over 80% of microbial infections in the body including dental caries, periodontal disease, cardiovascular disease, obesity, pregnancy complications, diabetes and respiratory diseases. The biofilm-growing bacterial cells express different phenotypes and often exhibit different characteristics than planktonic cells, presenting increased resistance to antimicrobial agents 100- to 1000- fold. Among biofilm-forming pathogenic bacteria, the Gram-positive bacteria Staphylococcus aureus and Streptococcus mutans are important biofilm-forming pathogens in humans, a quantification method able to obtain the measurement in a fast, efficient and reproductive way is needed.
The crystal violet staining assay is one of the methods most commonly used to quantify the biofilm formation by bacteria despite of its low reproducibility. Moreover, this method stains indistinctly all the negatively charged molecules in the wells, producing a value not directly related to the number of cells. To avoid these handicaps the xCELLigence System RTCA SP (ACEA, Biosciences Inc.) is proposed to quantify biofilms due to its continuous monitoring of the cell adhesion and real-time access to data on biofilm formation. The equipment is based in a microelectronic biosensor allowing the capture of data with high sensitivity and accuracy. The microelectronic cell sensor arrays are integrated into the bottom of the special plates (E-plates) and therefore this method is suitable only for species forming biofilm on the bottom of the well. The growth of cells on the bottom of wells causes changes in the electrode impedance. These changes that represent cell adherence status are measured by a unitless parameter called Cell Index (CI) which is the impedance change divided by a background value. The electronic impedance measured between electrodes is influenced by the degree of cell adhesion, allowing the monitoring of biofilm formation in real-time. The electrode measurements are affected by salinity of the culture medium higher than 10 g of NaCl per and therefore, the use for anti-fouling compounds screening with marine bacteria is limited since the marine broth is not suitable for this equipment.
In order to test the ability of the xCELLigence system to measure biofilm formation, S. aureus and S. mutans were inoculated in the E-plates in Brain Heart Infusion (BHI) supplemented with glucose (0.25%) and sucrose (0.1%), respectively at a final optical density of 0.05. Furthermore, furanone (0.1 M), a molecule already described as anti-biofilm agent, was added to observe the effect on biofilm formation. Results demonstrate that the xCELLigence system is an effective method to quantify biofilm in low-salinity culture media and to use for the screening of anti-biofilm agents against humans pathogens or in environmental applications such as inhibitors of membrane fouling.
Acknowledgement This work was supported by Fundación Ramón Areces (CIVP16A1814).
Author index
10
Author index
Author index
105
A Capalash, N. 36
Aamdal-Scheie, A. 40 Castañeda, P. 48
Agarwal, V. 69 Castellanos de Figueroa, L.I. 27
Aguilar-Rodríquez, S. 71 Castillo-Juárez, I. 70,71
Alexander, C. 92 Cavaleiro, E. 38
Alonso, L. 89 Chan, W. 47
Al-Sohaibani, S. 46 Charlton, K. 33
Álvarez-Méndez, S.J. 91 Chianella, I. 38
Andrenacci, D. 55 Choi, B-K. 77
Antipov, A. 88 Cocotl-Yañez, M. 54
Arregui, L. 97 Coenye, T. 32,37,45,51
Ascenso, O.S. 41 Correa, E. 30
Avelelas, F. 99 Correia, A. 38
Azuma, T. 64 Costas, C. 61,62
B D
Barros, R. 87 Dafhnis-Calas, F. 54
Benayahu, Y. 88,89 Daura, X. 63
Benneche, T. 40 De la Fuente-Núñez, C. 32
Bertini, E.V. 27 Defoirdt, T. 18,39,40
Besset, C. 75 Dessaux, Y. 19
Bhargava, N. 36 Dohare, S. 69
Birmes, F.S. 31,52,53 Dias Filho, B.P. 76
Blas, E. 87 Do, Q.T. 67
Bodelón, G. 61 Downham, C. 33
Börger, C. 66 Duarte, A.S. 38
Bossier, P. 39 Dubern, J-F. 67
Brackman, G. 32,37,45,51
Brengel, C. 66 E
Broadbent, I. 33 Empting, M. 66,68
Busetti, A. 72 Endo, E.H. 76
Burgess, J.G. 81 Esposito,M. 93
Esteves, A.C. 38
C
Cámara, M. 17,20,54,67
Author index
105
A Capalash, N. 36
Aamdal-Scheie, A. 40 Castañeda, P. 48
Agarwal, V. 69 Castellanos de Figueroa, L.I. 27
Aguilar-Rodríquez, S. 71 Castillo-Juárez, I. 70,71
Alexander, C. 92 Cavaleiro, E. 38
Alonso, L. 89 Chan, W. 47
Al-Sohaibani, S. 46 Charlton, K. 33
Álvarez-Méndez, S.J. 91 Chianella, I. 38
Andrenacci, D. 55 Choi, B-K. 77
Antipov, A. 88 Cocotl-Yañez, M. 54
Arregui, L. 97 Coenye, T. 32,37,45,51
Ascenso, O.S. 41 Correa, E. 30
Avelelas, F. 99 Correia, A. 38
Azuma, T. 64 Costas, C. 61,62
B D
Barros, R. 87 Dafhnis-Calas, F. 54
Benayahu, Y. 88,89 Daura, X. 63
Benneche, T. 40 De la Fuente-Núñez, C. 32
Bertini, E.V. 27 Defoirdt, T. 18,39,40
Besset, C. 75 Dessaux, Y. 19
Bhargava, N. 36 Dohare, S. 69
Birmes, F.S. 31,52,53 Dias Filho, B.P. 76
Blas, E. 87 Do, Q.T. 67
Bodelón, G. 61 Downham, C. 33
Börger, C. 66 Duarte, A.S. 38
Bossier, P. 39 Dubern, J-F. 67
Brackman, G. 32,37,45,51
Brengel, C. 66 E
Broadbent, I. 33 Empting, M. 66,68
Busetti, A. 72 Endo, E.H. 76
Burgess, J.G. 81 Esposito,M. 93
Esteves, A.C. 38
C
Cámara, M. 17,20,54,67
Author index
106
F Hellio, C. 82
Faure, D. 19 Hoch-Gunter, E. 88
Federle, M.J. 21 Holzapfel, W. 42,43
Fernandes, M.M. 34 Huedo, P. 63
Fernández, J.J. 91
Fernández Hermida, X. 98 I
Fernández-Trillo, F. 92 Ikeda, T. 64,74
Ferrer, M. 78 Isaksson, D. 90
Fetzner, S. 31,52,53 Ishizuka, M. 64
Fiesoletti, F. 93 Ivanova, K. 34
Flórez, A. 30
Flórez, A.M. 30 J
Flynn, P. 72 Jaiyen, Y. 47
Franco, J. 89 James, V. 93
Freire, I. 57,100,101,102 Jung, K-S. 77
G K
Gato, E. 58 Kalia, M. 69
García-Contreras, R. 48,70,71 Kalinowski, J. 31
Gardan, R. 75 Kirsch, B. 65,68
Garge, S. 29 Ko, Y-K. 77
Gibert, I. 63 Krasnogor, N. 54
Gilmore, B.F. 72 Kumar Singh, P. 69
Goldenberg, L. 88 Kumar, U. 39
González-Bello, C. 62 Kumar, V. 69
Graham, W.G. 72
Grandclément, C. 19 L
Grunwald, I. 90 Lafayette, I. 67
Guedella, E. 87 Larroze, S. 88
Lazenby, J. 67
H Lee, K. 42,43
Halliday, N. 67 Leguina, C.V. 27
Hancock, R.E.W. 32 Lereclus, D. 44
Hartmann, R.W. 65,66,68 Liébana, R. 97
Havenhand, J. 20 Liz-Marzán, L.M. 61,62
Heeb, S. 54,67 Llamas, I. 26,56
Author index
107
López-Puente, V. 61 Muñoz-Cazares, N. 71
Louzao, I. 92 Muras, A. 28,57,58,59,
Lu, C. 65 78,100,101,102
Murugan, K. 46
M Murray, E. 47
Maeda, T. 48
Maia, F. 99 N
Malheiro, E. 99 Nakamura, C.V. 76
Manco, G. 55 Nakamura, T.U. 76
Marques, J.C. 41 Napolano, L. 93
Marquina, D. 97 Narvi, S.S. 69
Martín, V.S. 91 Nascimento, P. 57
Martínez-Servat, S. 63 Natrah, F.M.I. 39
Martínez-Vázquez, M. 70,71 Nerurkar, A. 29
Martín-Rodríguez, A.J. 91 Nessler, S. 44
Martins, A.G. 66 Nieto-Peñalver, C.G. 27
Martins, R. 99
Matteucci, G. 93 O
Maurer, C. 65, 68 Ochiai, S. 64
Mayer, C. 28,57,58,59, Ordúz, S. 30
78,101,102 Otero, A. 28,57,58,59,
Menchaca, I. 89 78,100,101,102
Metton, C. 75 P
Milhazes-Cunha, H. 57 Palliyil, S. 33
Miguel, A.S. 41 Pande, G.S.J. 39
Mira, A. 78 Park, H. 42,43
Monnet, V. 75 Pascale, C. 93
Montes de Oca-Mejía, M. 71 Pastoriza-Santos, I. 61,62
Montes-García, V. 61 Pawar, V. 65
Morohoshi, T. 64,74 Perchat, S. 44
Müller, C. 31,53 Pérez-Juste, J. 61,62
Munn, C. 73 Pérez-López, M. 70
Muñagorri, F. 97 Pérez-Pascual, D. 75
Author index
106
F Hellio, C. 82
Faure, D. 19 Hoch-Gunter, E. 88
Federle, M.J. 21 Holzapfel, W. 42,43
Fernandes, M.M. 34 Huedo, P. 63
Fernández, J.J. 91
Fernández Hermida, X. 98 I
Fernández-Trillo, F. 92 Ikeda, T. 64,74
Ferrer, M. 78 Isaksson, D. 90
Fetzner, S. 31,52,53 Ishizuka, M. 64
Fiesoletti, F. 93 Ivanova, K. 34
Flórez, A. 30
Flórez, A.M. 30 J
Flynn, P. 72 Jaiyen, Y. 47
Franco, J. 89 James, V. 93
Freire, I. 57,100,101,102 Jung, K-S. 77
G K
Gato, E. 58 Kalia, M. 69
García-Contreras, R. 48,70,71 Kalinowski, J. 31
Gardan, R. 75 Kirsch, B. 65,68
Garge, S. 29 Ko, Y-K. 77
Gibert, I. 63 Krasnogor, N. 54
Gilmore, B.F. 72 Kumar Singh, P. 69
Goldenberg, L. 88 Kumar, U. 39
González-Bello, C. 62 Kumar, V. 69
Graham, W.G. 72
Grandclément, C. 19 L
Grunwald, I. 90 Lafayette, I. 67
Guedella, E. 87 Larroze, S. 88
Lazenby, J. 67
H Lee, K. 42,43
Halliday, N. 67 Leguina, C.V. 27
Hancock, R.E.W. 32 Lereclus, D. 44
Hartmann, R.W. 65,66,68 Liébana, R. 97
Havenhand, J. 20 Liz-Marzán, L.M. 61,62
Heeb, S. 54,67 Llamas, I. 26,56
Author index
107
López-Puente, V. 61 Muñoz-Cazares, N. 71
Louzao, I. 92 Muras, A. 28,57,58,59,
Lu, C. 65 78,100,101,102
Murugan, K. 46
M Murray, E. 47
Maeda, T. 48
Maia, F. 99 N
Malheiro, E. 99 Nakamura, C.V. 76
Manco, G. 55 Nakamura, T.U. 76
Marques, J.C. 41 Napolano, L. 93
Marquina, D. 97 Narvi, S.S. 69
Martín, V.S. 91 Nascimento, P. 57
Martínez-Servat, S. 63 Natrah, F.M.I. 39
Martínez-Vázquez, M. 70,71 Nerurkar, A. 29
Martín-Rodríguez, A.J. 91 Nessler, S. 44
Martins, A.G. 66 Nieto-Peñalver, C.G. 27
Martins, R. 99
Matteucci, G. 93 O
Maurer, C. 65, 68 Ochiai, S. 64
Mayer, C. 28,57,58,59, Ordúz, S. 30
78,101,102 Otero, A. 28,57,58,59,
Menchaca, I. 89 78,100,101,102
Metton, C. 75 P
Milhazes-Cunha, H. 57 Palliyil, S. 33
Miguel, A.S. 41 Pande, G.S.J. 39
Mira, A. 78 Park, H. 42,43
Monnet, V. 75 Pascale, C. 93
Montes de Oca-Mejía, M. 71 Pastoriza-Santos, I. 61,62
Montes-García, V. 61 Pawar, V. 65
Morohoshi, T. 64,74 Perchat, S. 44
Müller, C. 31,53 Pérez-Juste, J. 61,62
Munn, C. 73 Pérez-López, M. 70
Muñagorri, F. 97 Pérez-Pascual, D. 75
Author index
108
Piletska, E.V. 38 Serrano, S. 97
Piletsky, S.A. 38 Sharma, P. 36
Piller, C. 88,98 Shin, H-K. 42,43
Plaza, A. 66 Shiogai, S. 64
Porter, A. J. 33 Sim, J 77
Porzio, E. 55 Simon, C. 85
Singh, B. N. 69
Q Slachmuylders, L. 37
Quax, W.J. 35 Someya, N. 74
Quesada, E. 26,56 Soler, A. 97
Soto-Hernández, M. 70,71
R Steele, V. 47
Ransome, E. 73 Steinbach, A. 65
Reffuveille, F. 32 Streit, W.R 25
Rey, D. 59 Stübing, D. 86
Reyero, I. 57,1 Suarez, M.O. 30
Rodal-Cedeira, S. 61 Sui, C. 92
Rodríguez, P. 14
Rodríguez-Ezpeleta, N. 89 T
Romero, M. 28,57,58,59, 78 Tait, K. 20,73
Romero, N. 30 Talagas, A. 44
Rossini, P. 93 Tedim, J. 85,99
Rückert, C. 31 Teixeira, T. 88
Rueda, N.J. 30 Testoni, A. 93
Rui, F. 41,47 Thomann, A. 66
Rumbo, S. 58 Tomas, M. 58
Ryu, E-J. 77 Torres, M. 26,56
Tzanov, T. 34
S
San Miguel-Chávez, R. 70 U
Santos, A. 97 Uroz, S. 26
Sato, R. 74 Utari, P.D. 35
Sekar, K. 46
Author index
109
V
Van den Driessche, F. 45
Van Haare, J. 86
Van Merkerk, R. 35
Ventura, M.R. 41
W Weidel, E. 66 Weis, M. 88 Weiβ, S. 65 Whitcombe, M.J. 38 Williams, P. 13,20,47,67
Winzer, K. 92 Wittig, L. 90 Wood, T.K. 48 Wunder, A. 90 X Xavier, K.B. 41 Y Yamaguchi, T. 74 Yang, Q. 40 Yero, D. 63 Z Zender,M. 68 Zhang, Y. 51 Zorita, I. 89
Author index
108
Piletska, E.V. 38 Serrano, S. 97
Piletsky, S.A. 38 Sharma, P. 36
Piller, C. 88,98 Shin, H-K. 42,43
Plaza, A. 66 Shiogai, S. 64
Porter, A. J. 33 Sim, J 77
Porzio, E. 55 Simon, C. 85
Singh, B. N. 69
Q Slachmuylders, L. 37
Quax, W.J. 35 Someya, N. 74
Quesada, E. 26,56 Soler, A. 97
Soto-Hernández, M. 70,71
R Steele, V. 47
Ransome, E. 73 Steinbach, A. 65
Reffuveille, F. 32 Streit, W.R 25
Rey, D. 59 Stübing, D. 86
Reyero, I. 57,1 Suarez, M.O. 30
Rodal-Cedeira, S. 61 Sui, C. 92
Rodríguez, P. 14
Rodríguez-Ezpeleta, N. 89 T
Romero, M. 28,57,58,59, 78 Tait, K. 20,73
Romero, N. 30 Talagas, A. 44
Rossini, P. 93 Tedim, J. 85,99
Rückert, C. 31 Teixeira, T. 88
Rueda, N.J. 30 Testoni, A. 93
Rui, F. 41,47 Thomann, A. 66
Rumbo, S. 58 Tomas, M. 58
Ryu, E-J. 77 Torres, M. 26,56
Tzanov, T. 34
S
San Miguel-Chávez, R. 70 U
Santos, A. 97 Uroz, S. 26
Sato, R. 74 Utari, P.D. 35
Sekar, K. 46
Author index
109
V
Van den Driessche, F. 45
Van Haare, J. 86
Van Merkerk, R. 35
Ventura, M.R. 41
W Weidel, E. 66 Weis, M. 88 Weiβ, S. 65 Whitcombe, M.J. 38 Williams, P. 13,20,47,67
Winzer, K. 92 Wittig, L. 90 Wood, T.K. 48 Wunder, A. 90 X Xavier, K.B. 41 Y Yamaguchi, T. 74 Yang, Q. 40 Yero, D. 63 Z Zender,M. 68 Zhang, Y. 51 Zorita, I. 89
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Research Advances in Quorum Sensing Inhibition
Supported by:
Book of abstracts
2015
SANTIAGO DE COMPOSTELA
I International Symposium on Quorum Sensing Inhibition
and Satellite Meeting on Novel Anti-Fouling Strategies
![Research Article Broad Spectrum Anti-Quorum Sensing ...downloads.hindawi.com/journals/scientifica/2016/5823013.pdf · isms is called quorum sensing (QS) []. Quorum sensing is a process](https://static.fdocuments.us/doc/165x107/5edbc5d7ad6a402d66662749/research-article-broad-spectrum-anti-quorum-sensing-isms-is-called-quorum-sensing.jpg)
![Natural Anti-Quorum Sensing agents against Pseudomonas ... · 2. Quorum Sensing: a Novel Target Vfr Quorum sensing (QS) is a population-dependent event [13]. The capability to sense](https://static.fdocuments.us/doc/165x107/5edbcc02ad6a402d66663060/natural-anti-quorum-sensing-agents-against-pseudomonas-2-quorum-sensing-a.jpg)
