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The time kill curve shows that Arenicin-3 exhibits a very potent and fast bactericidal activity with a bactericidal action within 30 minutes compared with gentamicin which exhibited bactericidal action after 60 minutes. Arenicin-3 did not display any heamolytic activity on human reed blood cells. In fact, Arenicin-3 could not be distinguished from the negative control. Conclusion • Arenicin-3 is very potent in vitro against multi-resistant Gram-negative bacteria. • Fast bactericidal action. • Low in vitro toxicity • Promising drug candidate Arenicin-3: A novel antimicrobial peptide showing potent in vitro activity against Gram-negative multi-resistant clinical isolates E. coli (n=148) 0.0625 0.125 0.25 0.5 1 2 4 8 16 32 64 128 256 512 10 24 0 10 20 30 40 50 60 70 80 90 100 Arenicin-3 C iprofloxacin Gentamicin M IC ( g/m l) A ccum ulated frequencies Abstract Background Arenicin-3 is an antimicrobial peptide isolated using Transposon-Assisted-Signal -Trapping from the lugworm Arenicola marina living on sediments in the tidal water. Structural analysis showed that Arenicin-3 belonged to the beta-hairpin peptides. This class of AMPs are known to exhibit cidal activities towards a diverse number of microorganisms. Interestingly, susceptibility data on clinical isolates of Klebsiella pneumoniae, Salmonella enterica, Pseudomonas aeruginosa and Escherichia coli showed very potent activities of Arenicin. Methods. Minimal inhibitory concentrations were performed according to the general guidelines for susceptibility measurements using micro-broth dilution provided by CLSI/ NCCLS (M7-A5) All isolates were tested by a standard time-kill methodology as described by CLSI document M26-A: Methods for Determining Bactericidal Activity of Antimicrobial Agents; approved guideline , with the exception of taking earlier time points than normal due to the rapid bactericidal nature of Arenicin-3. Results Gram-negative bacteria including multi-resistant clinical relevant isolates of Escherichia coli, Klebsiella pneumoniae, Salmonella Typhimurium, Pseudomonas aeruginosa and Stenotrophomonas maltophilia were susceptibility tested to Arenicin-3. The results for isolates of both Enterobactericeae (n=148) and non fermentors (n=53) populations, MIC90 was < 1 mg/ml. The antimicrobial activity is markedly bactericidal (MBC~1-4xMIC), causing 3-log (99.9%) reduction in the viable bacteria population within 1-2 hours of Arenicin-3 exposure. In conclusion, Arenicin-3 has shown potential antimicrobial activity, even against multi resistant clinical isolates (ESBL positive, fluoroquinolone resistant, aminoglycoside resistant) Introduction The Arenicin family consists of three members Arenicin-1 and -2 which were characterized by a Russian research group (Ovchinnikova et al., 2004) and Arenicin-3 which is a novel member of the family. Arenicin-3 was isolated from the marine lugworm Arenicola marina and shown to contain two disulfide bonds between Cys3, Cys20 and Cys7, Cys16. Together with 4 positively charged arginines, and 9 hydrophobic residues the peptide was shown to form a 21-residue amphipathic beta hairpin structure. Activity assays revealed that Arenicin-3 is highly active in vitro against a variety of Gram negative bacteria. MIC analysis showed that more than 90 % of the tested E. coli and Pseudomonas strains exhibited a MIC below 1ug/ml. Methods MIC MIC testing of clinical isolates of multi resistant ESBL positive Escherichia coli (n:148) and Pseudomonas spp. (n:33) were performed by broth micro-dilution, according to CLSI-defined methodology, M7-A5 (2003), against Arenicin-3, ampicillin, gentamicin and ciprofloxacin. MBC testing was performed according to CLSI M26-A (1999), against Arenicin-3. Quality Control (QC): Results for all MIC testing were within the acceptable standards based on the CLSI recommended QC ranges for each comparator agent and the appropriate ATCC control strains. The second strain collection (Table) contained 115 different Gram negative bacteria to include a wide variety of species. These strains were mostly isolated from clinical material and the results for Arenicin-3 susceptibility were revealed in table. Time Kill Time-kill assays were performed as suggested by CLSI guidelines (NCCLS, 1999) using a final concentration of antimicrobial agent at 10 times the MIC for the E. Coli reference strain. Bactericidal activity was defined as a reduction of 99.9% (≥3 log10) of the total count of CFU/ml (NCCLS, 1999). Haemolytic activity Freshly prepared human erythrocytes was used in the haemolytic activity test. The substance under investigation was added to the 8% erythrocytes to determine the influence of the substance on the cytoplasmic membrane of the red blood cells. The haemolytic activity was measured photometric by the absorbance of oxyhaemoglobin. Results The alignment shows the peptides related to Arenicin. Interestingly, the closest related peptide is Tachyplesin-1 identified in horseshoe crab with an identity of 57%. Tachyplesin-1 is also a beta-hairpin peptide stabilized with 2 cysteines bridges and is also highly charged with 6 positively charged residues. The MIC testing of the E. coli and Pseudomonas strains clearly show the potency of Arenicin-3 with a MIC50 value below 0,5 μg/ml and a MIC90 value below 1 μg/ml for the E. coli strains. This should be compared to a MIC 90 value for both comparators which is above 8 μg/ml. For the pseudomonas strains arenicin-3 showed a MIC 50 value below 0,125 μg/ml. The same trend were seen for other gram negative bacteria (table) F1-3986 Contact information: [email protected] Phone: +45 44460663 H C 50 Heam olysis ofArenicin-3 2 4 8 16 32 64 128 256 512 1024 0 10 20 30 40 60 70 80 90 100 H C 50 Arenicin-3 W T SDS PBS mg/L % Heam olysis Name Origin aa-sequence ARENICIN-3 Sandworm, Areni col a mari na) GFWYVVYRNGVRVYRRN ARENICIN-1 Sandworm, Areni col a mari na) RWVYAYVRVRGVLVRYRRARENICIN-2 Sandworm, Areni col a mari na) RWVYAYVRIRGVLVRYRRTACHYPLESIN-1 Horseshoe crab, Tachypl eus t r i dent at us ) KWFRVYRG....YRRTACHYPLECIN-3 Horseshoe crab, Tachypl eus t r i dent at us ) KWFRVYRG....YRKPOLYPHEMUSIN-1 Horseshoe crab, Li mul us pol yphemus ) RRWFRVYRG....YRKPOLYPHEMUSIN-2 Horseshoe crab, Li mul us pol yphemus ) RRWFRVYKG....YRKANDROCTONIN Scorpion, Andr oc t onus aus t r al i s ) RSVRQIKIRRR.GGYYKTNRPY GOMESIN Spider, Acant hos cur r i a gomes i ana) XRRLYKQRVTYRGR PROTEGRIN Pig, Sus scrofa) RGGGLYRRRFVGR 50% consensus W.RVYR R....YRRName Origin aa-sequence ARENICIN-3 Sandworm, Areni col a mari na) GFWYVVYRNGVRVYRRN ARENICIN-1 Sandworm, Areni col a mari na) RWVYAYVRVRGVLVRYRRARENICIN-2 Sandworm, Areni col a mari na) RWVYAYVRIRGVLVRYRRTACHYPLESIN-1 Horseshoe crab, Tachypl eus t r i dent at us ) KWFRVYRG....YRRName Origin aa-sequence ARENICIN-3 Sandworm, Areni col a mari na) GFCWYVCVYRNGVRVCYRRCN ARENICIN-1 Sandworm, Areni col a mari na) RWCVYAYVRVRGVLVRYRRCW ARENICIN-2 Sandworm, Areni col a mari na) RWCVYAYVRIRGVLVRYRRCW TACHYPLESIN-1 Horseshoe crab, Tachypl eus t r i dent at us ) KWCFRVCYRG....ICYRRCR TACHYPLECIN-3 Horseshoe crab, Tachypl eus t r i dent at us ) KWFRVYRG....YRKPOLYPHEMUSIN-1 Horseshoe crab, Li mul us pol yphemus ) RRWFRVYRG....YRKPOLYPHEMUSIN-2 Horseshoe crab, Li mul us pol yphemus ) RRWFRVYKG....YRKANDROCTONIN Scorpion, Andr oc t onus aus t r al i s ) RSVRQIKIRRR.GGYYKTNRPY GOMESIN Spider, Acant hos cur r i a gomes i ana) XRRLYKQRTACHYPLECIN-3 Horseshoe crab, Tachypl eus t r i dent at us ) KWCFRVCYRG....ICYRKCR POLYPHEMUSIN-1 Horseshoe crab, Li mul us pol yphemus ) RRWCFRVCYRG....FCYRKCR POLYPHEMUSIN-2 Horseshoe crab, Li mul us pol yphemus ) RRWCFRVCYKG....FCYRKCR ANDROCTONIN Scorpion, Andr oc t onus aus t r al i s ) RSVCRQIKICRRR.GGCYYKCTNRPY GOMESIN Spider, Acant hos cur r i a gomes i ana) XCRRLCYKQRCVTYCRGR PROTEGRIN Pig, Sus scrofa) RGGGLCYCRRRFCVCVGR 50% consensus WC.RVCYR R....CYRRC Reference Ovchinnikova el al., FEBS Letters, Vol. 577 (1-2), 209-214, 2004 D. SANDVANG, S. NEVE & H-H. KRISTENSEN Novozymes A/S, Bagsvaerd, Denmark Susceptibility data forArenicin-3 -M IC susceptibility determ ination fora variety ofG ram N egative (n:115) M inim alinhibitory concentration (µg/m l)for A renicin-3 Bacterial S trains N o tested 0.063 0.125 0.25 0.5 1 2 4 8 16 32 E nterobactericeae C itrobacter 7 2 5 E nterobacter 5 1 3 1 E scherichia coli 24 1 11 9 3 1 Hafnia alvei 1 1 Klebsiella . 9 4 4 1 P roteus mirabilis 3 1 1 1 S almonella enterica 8 2 6 S erratia marcescens 2 2 S higella . 2 2 Achromobacter . 3 2 1 Acinetobacter 4 2 1 A eromonas 10 2 2 4 2 Alcaligenes 5 2 2 1 Pseudomonas 24 6 7 7 3 1 S tenotrophomonas 8 3 1 2 2 MIC determination were made in the range 0,06-128 (µg/ml) Arenicin 3 including gentamicin, ampicillin and ciprofloxacin as comparator antibiotics according to CLSI guidelines (M7-A5) and determined twice. MIC was performed on clinical isolates originating from Europe. Strain collection included sensitive and multi resistant isolates, most of which are know human pathogens Pseudom onas (n=33) 0.0625 0.125 0.25 0. 5 1 2 4 8 16 32 64 12 8 256 512 102 4 0 10 20 30 40 50 60 70 80 90 100 Arenicin-3 C iprofloxacin Gentamicin M IC ( g/m l) A ccum ulated frequencies Tim e kill forE . coli A TC C 25922 A renicin-3 in ca-M üllerH inton broth 0 20 40 60 80 100 120 140 10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 G rowth control 10 x M IC gentam icin 10 x M IC Arenicin-3 Tim e (m inutes) CFU/m l
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Transcript of Photo edit Edit: Change the photo by marking the background plane (picture) in the grouped objects....
The time kill curve shows that Arenicin-3 exhibits a very potent and fast bactericidal activity with a bactericidal action within 30 minutes compared with gentamicin which exhibited
bactericidal action after 60 minutes.
Arenicin-3 did not display any heamolytic activity on human reed blood cells. In fact, Arenicin-3 could not be distinguished from the negative control.
Conclusion
• Arenicin-3 is very potent in vitro against multi-resistant Gram-negative bacteria.
• Fast bactericidal action. • Low in vitro toxicity• Promising drug candidate
Arenicin-3: A novel antimicrobial peptide showing potent in vitro activity against Gram-negative multi-resistant clinical isolates
E. coli (n=148)
0.06
250.
125
0.25 0.
5 1 2 4 8 16 32 64 128
256
51210
240
10
20
30
40
50
60
70
80
90
100
Arenicin-3CiprofloxacinGentamicin
MIC (g/ml)
Acc
um
ula
ted
fre
qu
enci
es
Abstract
Background Arenicin-3 is an antimicrobial peptide isolated using Transposon-Assisted-Signal -Trapping from the lugworm Arenicola marina living on sediments in the tidal water.Structural analysis showed that Arenicin-3 belonged to the beta-hairpin peptides. This class of AMPs are known to exhibit cidal activities towards a diverse number of microorganisms. Interestingly, susceptibility data on clinical isolates of Klebsiella pneumoniae, Salmonella enterica, Pseudomonas aeruginosa and Escherichia coli showed very potent activities of Arenicin.Methods.Minimal inhibitory concentrations were performed according to the general guidelines for susceptibility measurements using micro-broth dilution provided by CLSI/ NCCLS (M7-A5)All isolates were tested by a standard time-kill methodology as described by CLSI document M26-A: Methods for Determining Bactericidal Activity of Antimicrobial Agents; approved guideline, with the exception of taking earlier time points than normal due to the rapid bactericidal nature of Arenicin-3.ResultsGram-negative bacteria including multi-resistant clinical relevant isolates of Escherichia coli, Klebsiella pneumoniae, Salmonella Typhimurium, Pseudomonas aeruginosa and Stenotrophomonas maltophilia were susceptibility tested to Arenicin-3. The results for isolates of both Enterobactericeae (n=148) and non fermentors (n=53) populations, MIC90 was < 1 mg/ml. The antimicrobial activity is markedly bactericidal (MBC~1-4xMIC), causing 3-log (99.9%) reduction in the viable bacteria population within 1-2 hours of Arenicin-3 exposure. In conclusion, Arenicin-3 has shown potential antimicrobial activity, even against multi resistant clinical isolates (ESBL positive, fluoroquinolone resistant, aminoglycoside resistant)
IntroductionThe Arenicin family consists of three members Arenicin-1 and -2 which were characterized by a Russian research group (Ovchinnikova et al., 2004) and Arenicin-3 which is a novel member of the family. Arenicin-3 was isolated from the marine lugworm Arenicola marina and shown to contain two disulfide bonds between Cys3, Cys20 and Cys7, Cys16. Together with 4 positively charged arginines, and 9 hydrophobic residues the peptide was shown to form a 21-residue amphipathic beta hairpin structure. Activity assays revealed that Arenicin-3 is highly active in vitro against a variety of Gram negative bacteria. MIC analysis showed that more than 90 % of the tested E. coli and Pseudomonas strains exhibited a MIC below 1ug/ml.
MethodsMICMIC testing of clinical isolates of multi resistant ESBL positive Escherichia coli (n:148) and Pseudomonas spp. (n:33) were performed by broth micro-dilution, according to CLSI-defined methodology, M7-A5 (2003), against Arenicin-3, ampicillin, gentamicin and ciprofloxacin. MBC testing was performed according to CLSI M26-A (1999), against Arenicin-3.Quality Control (QC): Results for all MIC testing were within the acceptable standards based on the CLSI recommended QC ranges for each comparator agent and the appropriate ATCC control strains.The second strain collection (Table) contained 115 different Gram negative bacteria to include a wide variety of species. These strains were mostly isolated from clinical material and the results for Arenicin-3 susceptibility were revealed in table.
Time KillTime-kill assays were performed as suggested by CLSI guidelines (NCCLS, 1999) using a final concentration of antimicrobial agent at 10 times the MIC for the E. Coli reference strain. Bactericidal activity was defined as a reduction of 99.9% (≥3 log10) of the total count of CFU/ml (NCCLS, 1999).
Haemolytic activityFreshly prepared human erythrocytes was used in the haemolytic activity test. The substance under investigation was added to the 8% erythrocytes to determine the influence of the substance on the cytoplasmic membrane of the red blood cells. The haemolytic activity was measured photometric by the absorbance of oxyhaemoglobin.
Results
The alignment shows the peptides related to Arenicin. Interestingly, the closest related peptide is Tachyplesin-1 identified in horseshoe crab with an identity of 57%. Tachyplesin-1 is also a beta-hairpin peptide stabilized with 2 cysteines bridges and is also highly charged with 6 positively charged residues.
The MIC testing of the E. coli and Pseudomonas strains clearly show the potency of Arenicin-3 with a MIC50 value below 0,5 μg/ml and a MIC90 value below 1 μg/ml for the E. coli strains. This should be compared to a MIC90 value for both comparators which is above 8 μg/ml. For the pseudomonas strains arenicin-3 showed aMIC 50 value below 0,125 μg/ml. The same trend were seen for other gram negative bacteria (table)
F1-3986 Contact information:[email protected]: +45 44460663
HC50
Heamolysis of Arenicin-3
2 4 8 16 32 64 128 256 512 10240
10
20
30
40
60
70
80
90
100
HC50
Arenicin-3 WTSDSPBS
mg/L
% H
eam
oly
sis
Name Origin aa-sequence
ARENICIN-3 Sandworm, Arenicola marina) GFCWYVCVYRNGVRVCYRRCNARENICIN-1 Sandworm, Arenicola marina) RWCVYAYVRVRGVLVRYRRCWARENICIN-2 Sandworm, Arenicola marina) RWCVYAYVRIRGVLVRYRRCWTACHYPLESIN-1 Horseshoe crab, Tachypleus tridentatus) KWCFRVCYRG....ICYRRCRTACHYPLECIN-3 Horseshoe crab, Tachypleus tridentatus) KWCFRVCYRG....ICYRKCRPOLYPHEMUSIN-1 Horseshoe crab, Limulus polyphemus) RRWCFRVCYRG....FCYRKCRPOLYPHEMUSIN-2 Horseshoe crab, Limulus polyphemus) RRWCFRVCYKG....FCYRKCRANDROCTONIN Scorpion, Androctonus australis) RSVCRQIKICRRR.GGCYYKCTNRPYGOMESIN Spider, Acanthoscurria gomesiana) XCRRLCYKQRCVTYCRGRPROTEGRIN Pig, Sus scrofa) RGGGLCYCRRRFCVCVGR
50% consensus WC.RVCYR R....CYRRC
Name Origin aa-sequence
ARENICIN-3 Sandworm, Arenicola marina) GFCWYVCVYRNGVRVCYRRCNARENICIN-1 Sandworm, Arenicola marina) RWCVYAYVRVRGVLVRYRRCWARENICIN-2 Sandworm, Arenicola marina) RWCVYAYVRIRGVLVRYRRCWTACHYPLESIN-1 Horseshoe crab, Tachypleus tridentatus) KWCFRVCYRG....ICYRRCR
Name Origin aa-sequence
ARENICIN-3 Sandworm, Arenicola marina) GFCWYVCVYRNGVRVCYRRCNARENICIN-1 Sandworm, Arenicola marina) RWCVYAYVRVRGVLVRYRRCWARENICIN-2 Sandworm, Arenicola marina) RWCVYAYVRIRGVLVRYRRCWTACHYPLESIN-1 Horseshoe crab, Tachypleus tridentatus) KWCFRVCYRG....ICYRRCRTACHYPLECIN-3 Horseshoe crab, Tachypleus tridentatus) KWCFRVCYRG....ICYRKCRPOLYPHEMUSIN-1 Horseshoe crab, Limulus polyphemus) RRWCFRVCYRG....FCYRKCRPOLYPHEMUSIN-2 Horseshoe crab, Limulus polyphemus) RRWCFRVCYKG....FCYRKCRANDROCTONIN Scorpion, Androctonus australis) RSVCRQIKICRRR.GGCYYKCTNRPYGOMESIN Spider, Acanthoscurria gomesiana) XCRRLCYKQRC
TACHYPLECIN-3 Horseshoe crab, Tachypleus tridentatus) KWCFRVCYRG....ICYRKCRPOLYPHEMUSIN-1 Horseshoe crab, Limulus polyphemus) RRWCFRVCYRG....FCYRKCRPOLYPHEMUSIN-2 Horseshoe crab, Limulus polyphemus) RRWCFRVCYKG....FCYRKCRANDROCTONIN Scorpion, Androctonus australis) RSVCRQIKICRRR.GGCYYKCTNRPYGOMESIN Spider, Acanthoscurria gomesiana) XCRRLCYKQRCVTYCRGRPROTEGRIN Pig, Sus scrofa) RGGGLCYCRRRFCVCVGR
50% consensus WC.RVCYR R....CYRRC
Reference
Ovchinnikova el al., FEBS Letters, Vol. 577 (1-2), 209-214, 2004
D. SANDVANG, S. NEVE & H-H. KRISTENSENNovozymes A/S, Bagsvaerd, Denmark
Susceptibility data for Arenicin-3 - MIC susceptibility determination for a variety of Gram Negative (n:115)
Minimal inhibitory concentration (µg/ml) for Arenicin-3
Bacterial Strains
No tested
0.063
0.125
0.25
0.5
1 2
4
8
16
32
Enterobactericeae
Citrobacter 7 2 5
Enterobacter 5 1 3 1
Escherichia coli 24 1 11 9 3 1
Hafnia alvei
1 1
Klebsiella . 9 4 4 1
Proteus mirabilis
3 1 1 1
Salmonella enterica 8 2 6
Serratia marcescens
2 2
Shigella . 2 2
Achromobacter . 3 2 1
Acinetobacter 4 2 1
Aeromonas 10 2 2 4 2
Alcaligenes 5 2 2 1
Pseudomonas 24 6 7 7 3 1
Stenotrophomonas 8 3 1 2 2
MIC determination were made in the range 0,06-128 (µg/ml) Arenicin 3 including gentamicin, ampicillin and ciprofloxacin as comparator antibiotics according to CLSI guidelines (M7-A5) and determined twice.MIC was performed on clinical isolates originating from Europe. Strain collection included sensitive and multi resistant isolates, most of which are know human pathogens
Pseudomonas (n=33)
0.06
250.
125
0.25 0.
5 1 2 4 8 16 32 64 128
256
51210
240
10
20
30
40
50
60
70
80
90
100
Arenicin-3CiprofloxacinGentamicin
MIC (g/ml)
Acc
um
ula
ted
fre
qu
enci
es
Time kill for E. coli ATCC25922Arenicin-3 in ca-Müller Hinton broth
0 20 40 60 80 100 120 140100
101
102
103
104
105
106
107
108
109
1010
Growth control
10 x MIC gentamicin
10 x MIC Arenicin-3
Time (minutes)
CF
U/m
l
