Biofilms - UCLouvain · 2012-10-07 · Biofilms have been found to be involved in a wide variety...
Transcript of Biofilms - UCLouvain · 2012-10-07 · Biofilms have been found to be involved in a wide variety...
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Biofilms:Biofilms:
understandingunderstandingphysiologyfordevelopingphysiologyfordevelopingnewtherapeuticstrategiesnewtherapeuticstrategies
MohammadShahrooeiMohammadShahrooei&&
ProfessorJohanVanEldereProfessorJohanVanEldereOctober5October5thth,2012,2012
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ContentsContents
• Introductiontobiofilms
• Biofilmsininfectionsanddevice‐relatedinfections(DRI)
• Staphylococcusspp.andstaphylococcalbiofilmsinDRI
• Preventionandtreatmentofstaphylococcalbiofilms
• Immunologicalapproaches:summaryofourresearch
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Whatisabiofilm?Whatisabiofilm?
Structuredcommunitiesofbacterialcellsenclosedinaself‐producedpolymericmatrixandadherenttoaninertoralivingsurface.
Abiofilmislikeatinycityinwhichmicrobialcellsformtowers.The"streets“betweenthetowersarefluid‐filledchannelsthatbringinnutrients,oxygenandothernecessitiesforlivebiofilmcommunities.
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KeycharacteristicsofbiofilmsKeycharacteristicsofbiofilms
• Biofilmsareheterogeneous,complex,dynamic structures,responsivetotheirenvironment
• Biofilmcellshavealteredgeneandproteinexpression profilesandpatternscomparedtotheirplanktonic
counterparts
• Biofilmcellscancoordinatebehaviorviaintercellular communicationusingbiochemicalsignalingmolecules (QuorumSensing)
• Biofilmsarelesssusceptibletoantimicrobialagents
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MechanismsofbiofilmresistanceMechanismsofbiofilmresistance
• Barrierpropertiesofthematrix(restrictedpenetration)
• Lowmetabolicactivity,slowgrowthandstress response
• Antimicrobialdestroyingenzymesandgenetransfer
• Quorumsensing(QS)
andheterogeneity
• Persisters,phenotypicsubpopulationofbacteriathat survivesantibiotictreatment
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ClinicalimportanceofbiofilmsClinicalimportanceofbiofilms
• Notoriouslyresistanttoimmunesystemattackand antimicrobialagents(upto1500timesmoreresistant)
• Biofilmshavebeenfoundtobeinvolvedinawide variety(upto80%)
ofmicrobialinfections
o Biofilmsleadto5millioninfectionsand150,000deathsin
USAandEU
annually
• Regularly,antimicrobialtherapyfailswithoutremoval oftheimplanteddevice
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BiofilmsininfectionsBiofilmsininfectionsInfectiousprocessesinwhichbiofilmshavebeenimplicatedinclude:
o urinarytractinfectionso catheterinfectionso middle‐earinfectionso sinusitiso formationofdentalplaque,gingivitiso coatingcontactlenseso endocarditiso infectionsincysticfibrosiso infectionsofpermanentindwellingdevicessuchasjoint
prosthesesandheartvalves
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DeviceDevice‐‐relatedrelated infections(DRI)infections(DRI)
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DeviceDevice‐‐relatedrelated infections(DRI)infections(DRI)
• Staphylococcusaureus
andcoagulase‐negativestaphylococci (CoNS),inparticular,S.epidermidis,haveemergedasmajor
nosocomialpathogensassociatedwithDRI,duetothefacts that:
o theyarethemostabundantskin‐colonizingbacteriao theyareabletoadheretothesurfaceandformabiofilm
• Biofilmformationisoneofthemajorvirulencefactorfor Staphylococcusspp.
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Staphylococcusspp.Staphylococcusspp.
• Gram‐positive,nonmotile,non‐sporeforming,spherical bacterium,coagulasenegativeorpositive
• Arrangegrape‐likeclusters
• Formwhitecolonies
1‐2mmØ after24h
• Mostareharmlessandnormalinhabitantofhumanskin andmucousmembranes
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BiofilmdevelopmentinBiofilmdevelopmentinStaphylococcusStaphylococcus
spp.spp.
, and extracellular DNA
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BiofilmdevelopmentinBiofilmdevelopmentinStaphylococcusStaphylococcus
spp.spp.
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RoleofRoleoficaica
operoninstaphylococcalbiofilmsoperoninstaphylococcalbiofilms
‐1,6‐linkedN‐
acetylglucosamine
SchematicprocedureofPIAsynthesis(a)andthegenearrangementintheica
operon(b)
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RoleofRoleoficaica
operoninstaphylococcalbiofilmsoperoninstaphylococcalbiofilms
• PIAissynthesizedbyenzymesencodedbyica
operon
• PIAplayaroleinattachmentandaccumulationphases
• MostofclinicalisolatesofCoNSandS.epidermidis
are ica+,PIA‐dependentbiofilm‐formingstrains
• Sofar,allMRSA(methicillin‐resistantStaphylococcusaureus)
have beenshowntobeica+,proteinaceous(PIA‐independent)
biofilm‐formingstrains,whereasMSSA(methicillin‐resistant Staphylococcusaureus)
canbeica‐/+,PIAindependent/dependentbiofilmforming
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PreventivestrategiesPreventivestrategies
• Improvementofspecificclinicalpracticeguidelineso candecreasetheincidenceofDRI
• Antimicrobialbiomaterialo induction,generationandselectionofresistance
• Antimicrobialprophylaxiso highprevalenceofantimicrobialresistance
• Targetingessentialbiofilmfactorso inhibitionofenzymesinvolvedinbiofilm
biosynthesiso Immunoprophylaxis(needavaccine)
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TreatmentofbiofilmsTreatmentofbiofilms
• Traditionalapproachisadministrationofantimicrobial agents
o Currently,theonlyeffectivetreatmentforbiofilminfectionsisto
removetheimplant,fighttheinfectionwithantibiotics,and
replacetheimplant,arisky,costlyandstressfulprocedure
• QSperturbationtorevertestablishedbiofilmso Inabiofilm,agr
expressionislimitedtosurface‐exposedarea
andagr
mutantsoccurnaturallyindeeperlayers
• Immunologicalapproaches
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S.aureusS.aureus
andandS.epidermidisS.epidermidis
vaccinesvaccines• Activeimmunization
‐
Currentandfinishedclinicalvaccinetrialsusingactiveimmunization
‐
MerckV710vaccine
‐
StaphVaxdevelopedbyNabi
‐
EpiVAX™
‐
Vaccinesinpre‐clinicaldevelopmentusingactiveimmunization
‐
Alpha‐toxin
‐
Panton‐Valentineleukocidin(PVL)
‐
PentaStaph(Nabi)
‐
Multi‐componentadhesinvaccine
‐
Poly‐N‐acetylglucosamine(PNAG)
‐
Als3p(Novadigm):
‐
Iron‐regulatedproteins(Syntiron/SanofiPasteur)
• Passiveimmunization/therapeuticantibodies
‐
Passiveimmunizationstrategiesinclinicaltrials
‐
AltastaphfromindividualtreatedwithNabi’sStaphVax
‐
Clumpingfactor(ClfA)targetedantibodies
‐
Aurograb
‐
Pagibaximab
‐
Passiveimmunizationstrategiesinpre‐clinicaldevelopment
‐
Alphatoxin
‐
PVL
‐
Superantigens
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IdentificationofpotentialvaccinetargetsforvaccinationIdentificationofpotentialvaccinetargetsforvaccination againstagainstS.epidermidisS.epidermidis
biofilmformationbiofilmformation
• InsilicoselectionofS.epidermidissurface(Ses)proteins.SP,signal
peptide;
TM,transmembranehelix;
PBD,peptidoglycanbindingdomain;
CBD,choline‐bindingdomains
Idealanti‐biofilmvaccinetargetsaresurfacecomponentsthatwereconservedacrossthespecies,inparticularthosewhicharehighlyexpressedinthebloodstreamandinbiofilms,withapossibleroleinbiofilmformationoranessentialfunction
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SelectionofbestpotentialvaccinetargetsSelectionofbestpotentialvaccinetargets• FiveSesproteinswereselectedbasedontheproteinsize,the
numberofantigenicdeterminantsandtheimportanceoftheprotein
family,towhichthecandidateproteinbelongs,inS.epidermidis
biofilmformationandpathogenesis
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RecombinantSesandantiRecombinantSesandanti‐‐SesantibodyproductionSesantibodyproduction• Surface‐exposedpartofSesproteinswererecombinantlyexpressed
inE.coli
andpolyclonalanti‐Sesantibodieswereraisedagainst
themandspecificanti‐Sesantibodieswerepurifiedusingantigen‐
affinitypurification
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ValidationofexpressionofSesproteinsonthesurfaceValidationofexpressionofSesproteinsonthesurface
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SelectionofbestpotentialvaccinetargetSelectionofbestpotentialvaccinetarget• Biofilminhibitionwasassessedinvitro,usingthemicrotiterplate
assay
(Primary attachment) (Overnight biofilm formation)
Pre‐immune (□) Post‐immune (■)
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EffectofantiEffectofanti‐‐SesCIgGSesCIgG’’sonsonS.epidermidisS.epidermidis
biofilmsbiofilmsinvitroinvitro
Pre‐immune (□) Post‐immune (■)
S. epidermidis 10b S. epidermidis 1457 S. warneri
Primary attachment
Specific and dose-dependent effect
Effect on 1-day established biofilms
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InvivoInvivo modelsmodels
Subcutaneouscatheter(SC)ratmodel
Jugularveincatheterized(JVC)mousemodel
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ActiveandpassiveimmunizationActiveandpassiveimmunization• EffectofαSesC‐IgGson1‐dayoldbiofilmsinvivo(passiveimmunization)• EffectofimmunizationofratswithrSesConbiofilmformation(active
immunization)
Passive immunization Active immunization
60.42-fold
20-fold
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EffectofantiEffectofanti‐‐SesConDRIinJVCmodelSesConDRIinJVCmodel24haftertheimplantation,JVCmicewereinoculatedwith1.0E+8CFU10bpre‐incubatedwithpre‐immuneorαSesC‐IgG’s.5daysafterinoculation,thenumberofbacteriacolonizingthecatheter,organsorinbloodstreamwasquantifiedbyCFUcounting.*P<0.05;**P<0.01;***P<0.001
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MechanismoffunctionofantiMechanismoffunctionofanti‐‐SesCIgGSesCIgG’’ss
Invitro
neutralization
Invivo
opsonization
Semi‐quantitativemicrotiterplate
Invitroopsonophagocytosisassay
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SesCisassociatedwithDRISesCisassociatedwithDRIinvivoinvivo24haftertheimplantation,JVCmicewereinoculatedwith1.0E+7CFUS.aureusviathecatheterlumen,5daysafterinoculation,thenumberofbacteriacolonizingthecatheter,organsorinbloodstreamwasquantifiedbyCFUcounting.Theerrorbarsindicatethestandarderrorsofthemean.*P<0.05;**P<0.01;***P<0.001
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SesCisassociatedwithDRISesCisassociatedwithDRIinvivoinvivoEffectofpre‐incubationwithpre‐immuneorαSesC‐IgG’sonS.aureus8325‐4strainanditssesC‐positivetransformant
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BiofilmdevelopmentinBiofilmdevelopmentinStaphylococcusStaphylococcus
spp.spp.
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SesCswitchesmechanismofbiofilmformationSesCswitchesmechanismofbiofilmformationinvitroinvitroTransformationwithsesC
changesthephenotypeofbiofilmformationofPIA‐dependentbiofilm‐formingstrains
*
*
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SesCswitchesmechanismofbiofilmformationSesCswitchesmechanismofbiofilmformationinvitroinvitroTransformationwithsesC
changesthephenotypeofbiofilmformationofPIA‐dependentbiofilm‐formingstrains
*
*
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SesCswitchesmechanismofbiofilmformationSesCswitchesmechanismofbiofilmformationinvitroinvitroTransforamtionwithsesC
changesthephenotypeofbiofilmformationofPIA‐dependentbiofilm‐formingstrains
*
*
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SesCswitchesmechanismofbiofilmformationSesCswitchesmechanismofbiofilmformationinvitroinvitroTransformationwithsesC
changesthephenotypeofbiofilmformationofPIA‐dependentbiofilm‐formingstrains
*
*
*
*
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SesCswitchesmechanismofbiofilmformationSesCswitchesmechanismofbiofilmformationinvitroinvitroTransformationwithsesC
changesthephenotypeofbiofilmformationofPIA‐dependentbiofilm‐formingstrains
*
*
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SesCswitchesmechanismofbiofilmformationSesCswitchesmechanismofbiofilmformationinvitroinvitroTransformationwithsesC
changesthephenotypeofbiofilmformationofPIA‐dependentbiofilm‐formingstrains
*
**
*
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ConclusionsConclusions
• SesCplaysaroleinS.epidermidisbiofilmformation
• SesCmightencodeanessentialfunctioninS. epidermidis
• SesCmightbeapromisingtargetforvaccine developmentagainstS.epidermidisbiofilmformation
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AcknowledgmentAcknowledgment• Prof.JohanVanEldere• Prof.JozefAnne• Prof.LieveVanmellaert• Prof.PeterHermans• Dr.BenoitStijlemans• Prof.JamesPO’Gara• Dr.ElainWaters• LalehandLadanKhodaparast• DorienHofmans• RitaMerckx• andothercolleaguesinourlab
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QuestionsQuestions