biofilms - uclouvain · 2012-10-07 · biofilms have been found to be involved in a wide variety...

Biofilms:Biofilms:

understandingunderstandingphysiologyfordevelopingphysiologyfordevelopingnewtherapeuticstrategiesnewtherapeuticstrategies

MohammadShahrooeiMohammadShahrooei&&

ProfessorJohanVanEldereProfessorJohanVanEldereOctober5October5thth,2012,2012

ContentsContents

• Introductiontobiofilms

• Biofilmsininfectionsanddevice‐relatedinfections(DRI)

• Staphylococcusspp.andstaphylococcalbiofilmsinDRI

• Preventionandtreatmentofstaphylococcalbiofilms

• Immunologicalapproaches:summaryofourresearch

Whatisabiofilm?Whatisabiofilm?

Structuredcommunitiesofbacterialcellsenclosedinaself‐producedpolymericmatrixandadherenttoaninertoralivingsurface.

Abiofilmislikeatinycityinwhichmicrobialcellsformtowers.The"streets“betweenthetowersarefluid‐filledchannelsthatbringinnutrients,oxygenandothernecessitiesforlivebiofilmcommunities.

KeycharacteristicsofbiofilmsKeycharacteristicsofbiofilms

• Biofilmsareheterogeneous,complex,dynamic structures,responsivetotheirenvironment

• Biofilmcellshavealteredgeneandproteinexpression profilesandpatternscomparedtotheirplanktonic

counterparts

• Biofilmcellscancoordinatebehaviorviaintercellular communicationusingbiochemicalsignalingmolecules (QuorumSensing)

• Biofilmsarelesssusceptibletoantimicrobialagents

MechanismsofbiofilmresistanceMechanismsofbiofilmresistance

• Barrierpropertiesofthematrix(restrictedpenetration)

• Lowmetabolicactivity,slowgrowthandstress response

• Antimicrobialdestroyingenzymesandgenetransfer

• Quorumsensing(QS)

andheterogeneity

• Persisters,phenotypicsubpopulationofbacteriathat survivesantibiotictreatment

ClinicalimportanceofbiofilmsClinicalimportanceofbiofilms

• Notoriouslyresistanttoimmunesystemattackand antimicrobialagents(upto1500timesmoreresistant)

• Biofilmshavebeenfoundtobeinvolvedinawide variety(upto80%)

ofmicrobialinfections

o Biofilmsleadto5millioninfectionsand150,000deathsin

USAandEU

annually

• Regularly,antimicrobialtherapyfailswithoutremoval oftheimplanteddevice

BiofilmsininfectionsBiofilmsininfectionsInfectiousprocessesinwhichbiofilmshavebeenimplicatedinclude:

o urinarytractinfectionso catheterinfectionso middle‐earinfectionso sinusitiso formationofdentalplaque,gingivitiso coatingcontactlenseso endocarditiso infectionsincysticfibrosiso infectionsofpermanentindwellingdevicessuchasjoint

prosthesesandheartvalves

DeviceDevice‐‐relatedrelated infections(DRI)infections(DRI)

DeviceDevice‐‐relatedrelated infections(DRI)infections(DRI)

• Staphylococcusaureus

andcoagulase‐negativestaphylococci (CoNS),inparticular,S.epidermidis,haveemergedasmajor

nosocomialpathogensassociatedwithDRI,duetothefacts that:

o theyarethemostabundantskin‐colonizingbacteriao theyareabletoadheretothesurfaceandformabiofilm

• Biofilmformationisoneofthemajorvirulencefactorfor Staphylococcusspp.

Staphylococcusspp.Staphylococcusspp.

• Gram‐positive,nonmotile,non‐sporeforming,spherical bacterium,coagulasenegativeorpositive

• Arrangegrape‐likeclusters

• Formwhitecolonies

1‐2mmØ after24h

• Mostareharmlessandnormalinhabitantofhumanskin andmucousmembranes

BiofilmdevelopmentinBiofilmdevelopmentinStaphylococcusStaphylococcus

spp.spp.

, and extracellular DNA

BiofilmdevelopmentinBiofilmdevelopmentinStaphylococcusStaphylococcus

spp.spp.

RoleofRoleoficaica

operoninstaphylococcalbiofilmsoperoninstaphylococcalbiofilms

‐1,6‐linkedN‐

acetylglucosamine

SchematicprocedureofPIAsynthesis(a)andthegenearrangementintheica

operon(b)

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

PreventivestrategiesPreventivestrategies

• Improvementofspecificclinicalpracticeguidelineso candecreasetheincidenceofDRI

• Antimicrobialbiomaterialo induction,generationandselectionofresistance

• Antimicrobialprophylaxiso highprevalenceofantimicrobialresistance

• Targetingessentialbiofilmfactorso inhibitionofenzymesinvolvedinbiofilm

biosynthesiso Immunoprophylaxis(needavaccine)

TreatmentofbiofilmsTreatmentofbiofilms

• Traditionalapproachisadministrationofantimicrobial agents

o Currently,theonlyeffectivetreatmentforbiofilminfectionsisto

removetheimplant,fighttheinfectionwithantibiotics,and

replacetheimplant,arisky,costlyandstressfulprocedure

• QSperturbationtorevertestablishedbiofilmso Inabiofilm,agr

expressionislimitedtosurface‐exposedarea

andagr

mutantsoccurnaturallyindeeperlayers

• Immunologicalapproaches

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

IdentificationofpotentialvaccinetargetsforvaccinationIdentificationofpotentialvaccinetargetsforvaccination againstagainstS.epidermidisS.epidermidis

biofilmformationbiofilmformation

• InsilicoselectionofS.epidermidissurface(Ses)proteins.SP,signal

peptide;

TM,transmembranehelix;

PBD,peptidoglycanbindingdomain;

CBD,choline‐bindingdomains

Idealanti‐biofilmvaccinetargetsaresurfacecomponentsthatwereconservedacrossthespecies,inparticularthosewhicharehighlyexpressedinthebloodstreamandinbiofilms,withapossibleroleinbiofilmformationoranessentialfunction

SelectionofbestpotentialvaccinetargetsSelectionofbestpotentialvaccinetargets• FiveSesproteinswereselectedbasedontheproteinsize,the

numberofantigenicdeterminantsandtheimportanceoftheprotein

family,towhichthecandidateproteinbelongs,inS.epidermidis

biofilmformationandpathogenesis

RecombinantSesandantiRecombinantSesandanti‐‐SesantibodyproductionSesantibodyproduction• Surface‐exposedpartofSesproteinswererecombinantlyexpressed

inE.coli

andpolyclonalanti‐Sesantibodieswereraisedagainst

themandspecificanti‐Sesantibodieswerepurifiedusingantigen‐

affinitypurification

ValidationofexpressionofSesproteinsonthesurfaceValidationofexpressionofSesproteinsonthesurface

SelectionofbestpotentialvaccinetargetSelectionofbestpotentialvaccinetarget• Biofilminhibitionwasassessedinvitro,usingthemicrotiterplate

assay

(Primary attachment) (Overnight biofilm formation)

Pre‐immune (□) Post‐immune (■) 

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

InvivoInvivo modelsmodels

Subcutaneouscatheter(SC)ratmodel

Jugularveincatheterized(JVC)mousemodel

ActiveandpassiveimmunizationActiveandpassiveimmunization• EffectofαSesC‐IgGson1‐dayoldbiofilmsinvivo(passiveimmunization)• EffectofimmunizationofratswithrSesConbiofilmformation(active

immunization)

Passive immunization Active immunization

60.42-fold

20-fold

EffectofantiEffectofanti‐‐SesConDRIinJVCmodelSesConDRIinJVCmodel24haftertheimplantation,JVCmicewereinoculatedwith1.0E+8CFU10bpre‐incubatedwithpre‐immuneorαSesC‐IgG’s.5daysafterinoculation,thenumberofbacteriacolonizingthecatheter,organsorinbloodstreamwasquantifiedbyCFUcounting.*P<0.05;**P<0.01;***P<0.001

MechanismoffunctionofantiMechanismoffunctionofanti‐‐SesCIgGSesCIgG’’ss

Invitro

neutralization

Invivo

opsonization

Semi‐quantitativemicrotiterplate

Invitroopsonophagocytosisassay

SesCisassociatedwithDRISesCisassociatedwithDRIinvivoinvivo24haftertheimplantation,JVCmicewereinoculatedwith1.0E+7CFUS.aureusviathecatheterlumen,5daysafterinoculation,thenumberofbacteriacolonizingthecatheter,organsorinbloodstreamwasquantifiedbyCFUcounting.Theerrorbarsindicatethestandarderrorsofthemean.*P<0.05;**P<0.01;***P<0.001

SesCisassociatedwithDRISesCisassociatedwithDRIinvivoinvivoEffectofpre‐incubationwithpre‐immuneorαSesC‐IgG’sonS.aureus8325‐4strainanditssesC‐positivetransformant

BiofilmdevelopmentinBiofilmdevelopmentinStaphylococcusStaphylococcus

spp.spp.

SesCswitchesmechanismofbiofilmformationSesCswitchesmechanismofbiofilmformationinvitroinvitroTransformationwithsesC

changesthephenotypeofbiofilmformationofPIA‐dependentbiofilm‐formingstrains

*

*

SesCswitchesmechanismofbiofilmformationSesCswitchesmechanismofbiofilmformationinvitroinvitroTransformationwithsesC

changesthephenotypeofbiofilmformationofPIA‐dependentbiofilm‐formingstrains

*

*

SesCswitchesmechanismofbiofilmformationSesCswitchesmechanismofbiofilmformationinvitroinvitroTransforamtionwithsesC

changesthephenotypeofbiofilmformationofPIA‐dependentbiofilm‐formingstrains

*

*

SesCswitchesmechanismofbiofilmformationSesCswitchesmechanismofbiofilmformationinvitroinvitroTransformationwithsesC

changesthephenotypeofbiofilmformationofPIA‐dependentbiofilm‐formingstrains

*

*

*

*

SesCswitchesmechanismofbiofilmformationSesCswitchesmechanismofbiofilmformationinvitroinvitroTransformationwithsesC

changesthephenotypeofbiofilmformationofPIA‐dependentbiofilm‐formingstrains

*

*

SesCswitchesmechanismofbiofilmformationSesCswitchesmechanismofbiofilmformationinvitroinvitroTransformationwithsesC

changesthephenotypeofbiofilmformationofPIA‐dependentbiofilm‐formingstrains

*

**

*

ConclusionsConclusions

• SesCplaysaroleinS.epidermidisbiofilmformation

• SesCmightencodeanessentialfunctioninS. epidermidis

• SesCmightbeapromisingtargetforvaccine developmentagainstS.epidermidisbiofilmformation

AcknowledgmentAcknowledgment• Prof.JohanVanEldere• Prof.JozefAnne• Prof.LieveVanmellaert• Prof.PeterHermans• Dr.BenoitStijlemans• Prof.JamesPO’Gara• Dr.ElainWaters• LalehandLadanKhodaparast• DorienHofmans• RitaMerckx• andothercolleaguesinourlab

QuestionsQuestions