Understanding Biofilms

AboutBacteriality AbouttheMarshallProtocol NEWSFLASH(archives)

Biofilmsformwhenbacteriaadheretosurfacesinaqueousenvironmentsandbegintoexcreteaslimy,gluelikesubstancethatcananchorthemtoallkindsofmaterial

Ashumans,ourenvironmentconsistentlyexposesustoavarietyofdangers.Tornadoes,lightning,floodingandhurricanescanallhamperoursurvival.Nottomentionthefactthatmostofuscanencounterswervingcarsorillintentionedpeopleatanygivenmoment.

Thousandsofyearsago,humansrealizedthattheycouldbettersurviveadangerousworldiftheyformedintocommunities,particularlycommunitiesconsistingofpeoplewithdifferenttalents.Theyrealizedthatacommunityisfarmorelikelytosurvivethroughdivisionoflaboronepersonmakesfood,anothergathersresources,stillanotherprotectsthecommunityagainstinvaders.Workingtogetherinthismannerrequirescommunicationandcooperation.

Inhabitantsofacommunityliveincloseproximityandcreatevariousformsofshelterinordertoprotectthemselvesfromexternalthreats.Webuildhousesthatprotectourfamiliesandlargerbuildingsthatprotecttheentirecommunity.Groupingtogetherinsideplacesofshelterisalogicalwaytoenhancesurvival.

Withtheaboveinmind,itshouldcomeasnosurprisethatthepathogensweharborareseldomfoundassingleentities.Althoughthepathogensthatcauseacuteinfectionaregenerallyfreefloatingbacteriaalsoreferredtoasplanktonicbacteriathosechronicbacterialformsthatstickaroundfordecadeslongagoevolvedwaystojointogetherintocommunities.Why?Becausebydoingso,theyarebetterabletocombatthecellsofourimmunesystembentupondestroyingthem.

Itturnsoutthatavastnumberofthepathogensweharboraregroupedintocommunitiescalledbiofilms.InanarticletitledBacterialBiofilms:ACommonCauseofPersistentInfections,JWCostertonattheCenterforBiofilmEngineeringinMontanadefinesabacterialbiofilmasastructuredcommunityofbacterialcellsenclosedinaselfproducedpolymericmatrixandadherenttoaninertorlivingsurface. Inlaymansterms,thatmeansthatbacteriacanjointogetheronessentiallyanysurfaceandstarttoformaprotectivematrixaroundtheirgroup.Thematrixismadeofpolymerssubstancescomposedofmoleculeswithrepeatingstructuralunitsthatareconnectedbychemicalbonds.

AccordingtotheCenterforBiofilmEngineeringatMontanaStateUniversity,biofilmsformwhenbacteriaadheretosurfacesinaqueousenvironmentsandbegintoexcreteaslimy,gluelikesubstancethatcananchorthemtoallkindsofmaterialsuchasmetals,plastics,soilparticles,medicalimplantmaterialsand,mostsignificantly,humanoranimaltissue.Thefirstbacterialcoloniststoadheretoasurfaceinitiallydosobyinducingweak,reversiblebondscalledvanderWaalsforces.Ifthecolonistsarenotimmediatelyseparatedfromthesurface,theycananchorthemselvesmorepermanentlyusingcelladhesionmolecules,proteinsontheirsurfacesthatbindothercellsinaprocesscalledcelladhesion.

Thesebacterialpioneersfacilitatethearrivalofotherpathogensbyprovidingmorediverseadhesionsites.Theyalsobegintobuildthematrixthatholdsthebiofilmtogether.Iftherearespeciesthatareunabletoattachtoasurfaceontheirown,theyareoftenabletoanchorthemselvestothematrixordirectlytoearliercolonists.

Duringcolonization,thingsstarttogetinteresting.Multiplestudieshaveshown

Understanding BiofilmsAuthor:AmyProal

26MAY2008

[1]

NEWS FLASH

April4,2009:,MilkconsumptiontiedtoParkinsonsdiseaseMarch21,2009:,Heythere,howsyourKineosphaeramholdingup?

PeerReviewed Papers

Autoimmunediseaseintheeraofthemetagenome(PDF)VitaminD:thealternativehypothesis(PDF)DysregulationoftheVitaminDNuclearReceptormaycontributetothehigherprevalenceofsomeautoimmunediseasesinwomen(PDF)VitaminDmetabolitesasclinicalmarkersinautoimmuneandchronicdisease(PDF)

Amy's Conference Presentations

MetagenomicsymbiosisbetweenbacterialandviralpathogensinautoimmunediseaseNotesfromthe2009InternationalCongressofAntibodiesNotesfromthe2008InternationalCongressonAutoimmunity

Featured Articles

UpdateontoneandotherissuesWhypatientswithchronicdiseasearedisaffectedandhowonlinesocialnetworksmeettheirneedsSunblockingcultureamongtheChineseSecondguessingtheconsensusonvitaminDTravels,papers,andmoreanupdateThreedaysattheJ.CraigVenterInstituteThebacteriaboomimplicationsoftheHumanMicrobiomeProjectUnderstandingBiofilmsInterviewwithDr.RandallWolcott,bacterialbiofilmwoundspecialistInsightsintohorizontalgenetransfer:conversationswithDr.PeterGogartenandDr.JamesLakeVoicesofreasoninthevitaminDdebateInterviewwithevolutionarybiologistPaulEwaldInterviewwithDr.GregBlaney:MPphysicianBacteriaandcancer:aninterviewwithDr.AlanCantwellInterviewwithNadyaMarkova:LformExpertGeraldDomingue:PioneerofAtypicalBacteriaAHistoryofCellWallDeficientBacteria:ASelectionofResearchersWhoHaveWorkedwiththeLform

Patient Interviews

InterviewwithGeneJohnsonsarcoidosis,bladdercancerInterviewwithBonnieBlupus,SjogrensSyndromeInterviewwithChrisEastlunddiabetes,sarcoidosis,irritablebowelsyndrome

Abiofilminthegut.

Sessilecellsinabiofilmtalktoeachotherviaquorumsensingtobuildmicrocoloniesandtokeepwaterchannelsopen.

thatduringthetimeabiofilmisbeingcreated,thepathogensinsideitcan

communicatewitheachotherthankstoaphenomenoncalledquorumsensing.Althoughthemechanismsbehindquorumsensingarenotfullyunderstood,thephenomenonallowsasinglecelledbacteriumtoperceivehowmanyotherbacteriaareincloseproximity.Ifabacteriumcansensethatitissurroundedbyadensepopulationofotherpathogens,itismoreinclinedtojointhemandcontributetotheformationofabiofilm.

Bacteriathatengageinquorumsensingcommunicatetheirpresencebyemittingchemicalmessagesthattheirfellowinfectiousagentsareabletorecognize.Whenthemessagesgrowstrongenough,thebacteriarespondenmasse,behavingasagroup.Quorumsensingcanoccurwithinasinglebacterialspeciesaswellasbetweendiversespecies,andcanregulateahostofdifferentprocesses,essentiallyservingasasimplecommunicationnetwork.Avarietyofdifferentmoleculescanbeusedassignals.

Diseasecausingbacteriatalktoeachotherwithachemicalvocabulary,saysDougHibbinsofPrincetonUniversity.AgraduatestudentinthelabofPrincetonUniversitymicrobiologistDr.BonnieBassler,Hibbinswaspartofaresearcheffortwhichshedlightonhowthebacteriathatcausecholeraformbiofilmsandcommunicateviaquorumsensing.

Formingabiofilmisoneofthecrucialstepsincholerasprogression,statesBassler.They[bacteria]coverthemselvesinasortofgoopthatsashieldagainstantibiotics,allowingthemtogrowrapidly.Whentheysensethereareenoughofthem,theytrytoleavethebody.

Althoughcholerabacteriausetheintestinesasabreedingground,afterenoughbiofilmshaveformed,planktonicbacteriainsidethebiofilmseektoleavethebodyinordertoinfectanewhost.ItdidnttakelongforBasslerandteamtorealizethatthebacteriainsidecholerabiofilmsmustsignaleachotherinordertocommunicatethatitstimeforthecolonytostopreproducingandfocusinsteadonleavingthebody.

Wegenericallyunderstoodthatbacteriatalktoeachotherwithquorumsensing,butwedidntknowthespecificchemicalwordsthatcholerauses,Basslersaid.

ThenHigginsisolatedtheCAI1achemicalwhichoccursnaturallyincholera.Anothergraduatestudentfiguredouthowtomakethemoleculeinthelaboratory.BymoderatingthelevelofCAI1incontactwithcholerabacteria,Higginswassuccessfullyabletochemicallycontrolcholerasbehaviorinlabtests.HisteameventuallyconfirmedthatwhenCAI1isabsent,cholerabacteriaattachinbiofilmstotheircurrenthost.Butwhenthebacteriadetectenoughofthechemical,theystopmakingbiofilmsandreleasingtoxins,perceivingthatitistimetoleavethebodyinstead.Thus,CAI1mayverywellbethesinglemoleculethatallowthebacteriainsideacholerabiofilmtocommunicate.AlthoughitislikelythatthebacteriainacholerabiofilmmaycommunicatewithothersignalsbesidesCAI1,thestudyisagoodexampleofthefactthatsignalingmoleculesserveakeyroleindeterminingthestateofabiofilm.

Similarly,researchersattheUniversityofIowa(severalofwhomarenowattheUniversityofWashington)havespentthelastdecadeidentifyingthemoleculesthatallowthebacterialspeciesP.aeruginosatoformbiofilmsinthelungsofpatientswithcysticfibrosis. AlthoughtheP.auruginosaisolatedfromthelungsofpatientswithcysticfibrosislookslikeabiofilmandactslikeabiofilm,upuntilrecently,therewerenoobjectivetestsavailabletoconfirmthatthebacterialspeciesdidindeedformbiofilmsinthelungsofpatientswiththedisease,norwasthereawaytotellwhatproportionofP.aeruginosainthelungswereactuallyinbiofilmmode.

WeneededawaytoshowthattheP.auruginosaincysticfibrosislungswascommunicatinglikeabiofilm.ThatcouldtellusabouttheP.auruginosalifestyle,saidPradeepSingh,M.D.,aleadauthoronthestudywhoisnowattheUniversityofWashington.

SinghandhiscolleaguesfinallydiscoveredthatP.aeruginosausesoneoftwoparticularquorumsensingmoleculestoinitiatetheformationofbiofilms.InNovember1999,hisresearchteamscreenedtheentirebacterialgenome,identifying39genesthatarestronglycontrolledbythequorumsensingsystem.

Ina2000studypublishedinNature,SinghandcolleaguesdevelopedasensitivetestwhichshowsP.auruginosafromcysticfibrosislungsproducesthetelltale,quorumsensingmoleculesthatarethesignalsforbiofilmformation.

[2]

[3]

[3]

InterviewwithRoyP.sarcoidosis,rheumatoidarthritisInterviewwithPeterdeJager:chronicfatiguesyndrome,multiplechemicalsensitivityInterviewwithKenL.PostTreatmentLymeDiseaseSyndrome(PTLDS)InterviewwithDoreenV.autism,ADHDdepression,severeanxiety,CFSInterviewwithJST:NeurosarcoidosisInterviewwithMelindaStilesLyme,Irritablebowelsyndrome/colitis,Radiculitis(inflammationofthenerveroots)InterviewwithFreddieAshSarcoidosisoftheheart,coronaryarterydisease,atrialfibrillationInterviewwithP.BearR.N.ChronicBorreliosis(Lyme),MCS(multiplechemicalsensitivities),ChronicSpinalInflammation,PeripheralNeuropathyInterviewwithSherryCookSarcoidosis,CatScratchFever,RestlessLegSyndromeInterviewwithLeesaShanahanSarcoidosis(HeerfordtsSyndrome),UveitisInterviewwithMirekWozgasarcoidosisInterviewwithPaulAlbertCFS,depression,foodsensitivitiesInterviewwithCaroleMorganSarcoidosis,fibromyalgia,CFSInterviewwithShirleyJ.(Saj)SarcoidosisInterviewwithSueAndornLyme,BabesiaInterviewwithIvalMeyerArthritis,dyslexia

About Amy Proal

AmyProalgraduatedfromGeorgetownUniversityin2005withadegreeinbiology.WhileatGeorgetown,shewroteherseniorthesisonChronicFatigueSyndromeandtheMarshallProtocol.

Amyhasspokenatseveralinternationalconferencesandauthoredseveralpeerreviewedpapersontheintersectionofbacteriaandchronicdisease.

IfyouhavequestionsabouttheMP,pleasevisitCureMyTh1.orgwherevolunteerpatientadvocateswillansweryourquestions.AnothergoodresourceistheMPKnowledgeBase,whichisscheduledtobecompletedwithinthenextyear.

Categories

biofilmscancercardiovasculardiseasecognitivedysfunctionconferencesandtrainingsdietfamilialaggregationfeaturedarticleshistoryhorizontalgenetransferinterview(doctor/researcher)interview(patient)lformbacteriamarshallprotocolmedicalresearchmentalillnessmicrobiomeNewsFlashobesitypersonalpresentations

Biofilmbacteriacanmoveinnumerousways:Collectively,byripplingorrollingacrossthesurface,orbydetachinginclumps.Individually,throughaswarmingandseedingdispersal.

ItturnsoutthatP.aerugnosasecretestwosignalingmolecules,onethatislong,andanotherthatisshort.Usingthenewtest,theteamwasabletoshowthatplanktonicformsofP.aeruginosaproducemorelongsignalingmolecules.Alternately,whentheytestedtheP.aeruginosastrainsisolatedfromthelungsofpatientswithcysticfibrosis(whichwereinbiofilmform),allofthestrainsproducedthesignalingmolecules,butintheoppositeratiomoreshortthanlong.

Interestingly,whenthebiofilmstrainsofP.aeruginosawereseparatedinbrothintoindividualbacterialforms,theyrevertedtoproducingmorelongsignalmoleculesthanshortones.Doesthismeanthatachangeinsignalingmolecularlengthcanindicatewhetherbacteriaremainasplanktonicformsordevelopintobiofilms?

Tofindout,theteamtookthebacteriafromthebrothandmadethemgrowasabiofilmagain.Sureenough,thosestrainsofbacteriainbiofilmformproducedmoreshortsignalmoleculesthanlong.

ThefactthattheP.aeruginosain[thelungsofcysticfibrosispatients]ismakingthesignalsintheratiosthatweseetellsusthatthereisabiofilmandthatmostoftheP.aeruginosainthelungisinthebiofilmstate,statesGreenberg,anothermemberoftheresearchteam.Hebelievesthatthefindingsallowforaclearbiochemicaldefinitionofwhetherbacteriaareinabiofilm.Techniquessimilartothoseusedbyhisgroupwilllikelybeusedtodeterminethepropertiesofotherbiofilmsignalingmolecules.

Development

Oncecolonizationhasbegun,thebiofilmgrowsthroughacombinationofcelldivisionandrecruitment.Thefinalstageofbiofilmformationisknownasdevelopmentandisthestageinwhichthebiofilmisestablishedandmayonlychangeinshapeandsize.Thisdevelopmentofabiofilmallowsforthecellsinsidetobecomemoreresistanttoantibioticsadministeredinastandardfashion.Infact,dependingontheorganismandtypeofantimicrobialandexperimentalsystem,biofilmbacteriacanbeuptoathousandtimesmoreresistanttoantimicrobialstressthanfreeswimmingbacteriaofthesamespecies.

Biofilmsgrowslowly,indiverselocations,andbiofilminfectionsareoftenslowtoproduceovertsymptoms.However,biofilmbacteriacanmoveinnumerouswaysthatallowthemtoeasilyinfectnewtissues.Biofilmsmaymovecollectively,byripplingorrollingacrossthesurface,orbydetachinginclumps.Sometimes,inadispersalstrategyreferredtoasswarming/seeding,abiofilmcolonydifferentiatestoformanouterwallofstationarybacteria,whiletheinnerregionofthebiofilmliquefies,allowingplanktoniccellstoswimoutofthebiofilmandleavebehindahollowmound.

Researchonthemolecularandgeneticbasisofbiofilmdevelopmenthasmadeitclearthatwhencellsswitchfromplanktonictocommunitymode,theyalsoundergoashiftinbehaviorthatinvolvesalterationsintheactivityofnumerousgenes.Thereisevidencethatspecificgenesmustbetranscribedduringtheattachmentphaseofbiofilmdevelopment.Inmanycases,theactivationofthesegenesisrequiredforsynthesisoftheextracellularmatrixthatprotectsthepathogensinside.

AccordingtoCosterton,thegenesthatallowabiofilmtodevelopareactivatedafterenoughcellsattachtoasolidsurface.Thus,itappearsthatattachmentitselfiswhatstimulatessynthesisoftheextracellularmatrixinwhichthesessilebacteriaareembedded,statesthemolecularbiologist.Thisnotionthatbacteriahaveasenseoftouchthatenablesdetectionofasurfaceandtheexpressionofspecificgenesisinitselfanexcitingareaofresearch

Certaincharacteristicsmayalsofacilitatetheabilityofsomebacteriatoformbiofilms.ScientistsattheDepartmentofMicrobiologyandMolecularGenetics,HarvardMedicalSchool,performedastudyinwhichtheycreatedamutantformofthebacterialspeciesP.aeguinosa(PA). Themutantslackedgenesthatcodeforhairlikeappendagescalledpili.Interestingly,themutantswereunabletoformbiofilms.SincethepiliofPAareinvolvedinatypeofsurfaceassociatedmotilitycalledtwitching,theteamhypothesizedthistwitchingmightberequiredfortheaggregationofcellsintothemicrocoloniesthatsubsequentlyformastablebiofilm.

Onceabiofilmhasofficiallyformed,itoftencontainschannelsinwhichnutrientscancirculate.Cellsindifferentregionsofabiofilmalsoexhibitdifferentpatternsofgeneexpression.Becausebiofilmsoftendeveloptheirownmetabolism,theyaresometimescomparedtothetissuesofhigher

[4]

[1]

[5]

statinsUncategorizedvideosvitaminD

Thebiofilmlifecycleinthreesteps:attachment,growthofcolonies(development),andperiodicdetachmentofplanktoniccells.

Levchenkoandteamusedthisdevicetoobservebacteriagrowingincrampedconditions.

organisms,inwhichcloselypackedcellsworktogetherandcreateanetworkinwhichmineralscanflow.

Thereisaperceptionthatsinglecelledorganismsareasocial,butthatismisguided,saidAndreLevchenko,assistantprofessorofbiomedicalengineeringinJohnsHopkinsUniversitysWhitingSchoolofEngineeringandanaffiliateoftheUniversitysInstituteforNanoBioTechnology.Whenbacteriaareunderstresswhichisthestoryoftheirlivestheyteamupandformthiscollectivecalledabiofilm.Ifyoulookatnaturallyoccurringbiofilms,theyhaveverycomplicatedarchitecture.Theyarelikecitieswithchannelsfornutrientstogoinandwastetogoout.

Understandinghowsuchcooperationamongpathogensevolvesandismaintainedrepresentsoneofevolutionarybiologysthorniestproblems.Thisstemsfromtherealitythat,innature,freeloadingcheatsinevitablyevolvetoexploitanycooperativegroupthatdoesntdefenditself,leadingtothebreakdownofcooperation.Sowhatcausesthebacteriainabiofilmtocontributetoandshareresourcesratherthansteal

them?Recently,Dr.MichaelBrockhurstoftheUniversityofLiverpoolandcolleaguesattheUniversitMontpellierandtheUniversityofOxfordconductedseveralstudiesinanefforttounderstandwhythebacteriainabiofilmcooperateandshareresourcesratherthanhordethem.

TheteamtookacloserlookatP.fluorescensbiofilms,whichareformedwhenindividualcellsoverproduceapolymerthatsticksthecellstogether,allowingthecolonizationofliquidsurfaces.Whileproductionofthepolymerismetabolicallycostlytoindividualcells,thebiofilmgroupbenefitsfromtheincreasedaccesstooxygenthatsurfacecolonizationprovides.Yet,evolutionarilyspeaking,suchasetupallowspossiblecheaterstoenterthebiofilm.Suchcheatscantakeadvantageoftheprotectivematrixwhilefailingtocontributeenergytoactuallybuildingthematrix.Iftoomanycheatersenterabiofilm,itwillweakenandeventuallybreakapart.

Afterseveralyearsofstudy,Brockhurstandteamrealizedthattheshorttermevolutionofdiversitywithinabiofilmisamajorfactorinhowsuccessfullyitsmemberscooperate.Theteamfoundthatonceinsideabiofilm,P.fluorescensdifferentiatesintovariousforms,eachofwhichusesdifferentnutrientresources.Thefactthatthesediversecooperatorsdontallcompeteforthesamechemicalsandnutrientssubstantiallyreducescompetitionforresourceswithinthebiofilm.

Whentheteammanipulateddiversitywithinexperimentalbiofilms,theyfoundthatdiversebiofilmscontainedfewercheatersandproducedlargergroupsthannondiversebiofilms.

Similarly,thisyear,researchersfromJohnsHopkinsVirginiaTechtheUniversityofCalifornia,SanDiegoandLundUniversityinSwedenrecentlyreleasedtheresultsofastudywhichfoundthatoncebacteriacooperateandformabiofilm,packingtightlytogetherfurtherenhancestheirsurvival.

TheteamcreatedanewdeviceinordertoobservethebehaviorofE.colibacteriaforcedtogrowinthecrampedconditions.Thedevice,whichallowsscientiststouseextremelysmallvolumesofcellsinsolution,containsaseriesoftinychambersofvariousshapesandsizesthatkeepthebacteriauniformlysuspendedinaculturemedium.

Notsurprisingly,thecrampedbacteriainthedevicebegantoformabiofilm.Theteamcapturedthedevelopmentofthebiofilmonvideo,andwereabletoobservethegradualselforganizationandeventualconstructionofbacterialbiofilmsovera24hourperiod.

First,AndreLevchenkoandHojungChoofJohnsHopkinsrecordedthebehaviorofsinglelayersofE.colicellsusingrealtimemicroscopy.Weweresurprisedtofindthatcellsgrowinginchambersofallsortsofshapesgraduallyorganizedthemselvesintohighlyregularstructures,Levchenkosaid.

Furtherobservationsusingmicroscopyrevealedthatthelongerthepackedcellpopulationresidedinthechambers,themoreorderedthebiofilmstructurebecame.Asthecellsinthebiofilmbecamemoreorderedand

[6]

[7]

[6]

Dr.LevchenkoofJohnsHopkinsandHojungCho,abiomedicalengineeringdoctoralstudent

Planktonicbacteriaareperiodicallyreleasedfromabiofilm

AntonvanLeeuwenhoek.

tightlypacked,thebiofilmbecameharderandhardertopenetrate.

LevchenkoalsonotedthatrodshapedE.colithatweretooshortortoolongtypicallydidnotorganizewellintothedense,circularmainhubofthebiofilm.Instead,thebacteriaofoddshapesorhighlydisorderedgroupsofcellswerefoundontheedgesofthebiofilm,wheretheyformedsharpcorners.

Nodes of relapsing infection?

Researchersoftennotethat,oncebiofilmsareestablished,planktonicbacteriamayperiodicallyleavethebiofilmontheirown.Whentheydo,theycanrapidlymultiplyanddisperse.

AccordingtoCosterton,thereisanaturalpatternofprogrammeddetachmentofplanktoniccellsfrombiofilms.ThismeansthatbiofilmscanactaswhatCostertonreferstoasnidusesofacuteinfection.Becausethebacteriainabiofilmareprotectedbyamatrix,thehostimmunesystemislesslikelytomountaresponsetotheirpresence.

Butifplanktonicbacteriaareperiodicallyreleasedfromthebiofilms,eachtimesinglebacterialformsenterthetissues,theimmunesystemsuddenlybecomesawareoftheirpresence.Itmayproceedtomountaninflammatoryresponsethatleadstoheightenedsymptoms.Thus,theperiodicreleaseofplanktonicbacteriafromsomebiofilmsmaybewhatcausesmanychronicrelapsinginfections.

AsMatthewR.ParsekofNorthwesternUniversitydescribesina2003paperintheAnnualReviewofMicrobiology,anypathogenthatsurvivesinachronicformbenefitsbykeepingthehostalive. Afterall,ifachronicbacterialformsimplykillsitshost,itwillnolongerhaveaplacetolive.SoaccordingtoParsek,chronicinfectionoften

resultsinadiseasestalematewherebacteriaofmoderatevirulencearesomewhatcontainedbythedefensesofthehost.Theinfectiousagentsneveractuallykillthehost,butthehostisneverabletofullykilltheinvadingpathogenseither.

Parsekbelievesthattheoptimalwayforbacteriatosurviveundersuchcircumstancesisinabiofilm,statingthatIncreasingevidencesuggeststhatthebiofilmmodeofgrowthmayplayakeyroleinbothoftheseadaptations.Biofilmgrowthincreasestheresistanceofbacteriatokillingandmaymakeorganismslessconspicuoustotheimmunesystemultimatelythismoderationofvirulencemayservethebacteriasinterestbyincreasingthelongevityofthehost.

The acceptance of biofilms as infectious entities

Perhapsbecausemanybiofilmsaresufficientlythicktobevisibletothenakedeye,themicrobialcommunitieswereamongthefirsttobestudiedbyearlymicrobiologists.AntonvanLeeuwenhoekscrapedtheplaquebiofilmfromhisteethandobservedwhathedescribedastheanimalculiinsidethemunderhisprimitivemicroscope.However,accordingtoCostertonandteamattheCenterforBiofilmResearchatMontanaStateUniversity,itwasnotuntilthe1970sthatscientistsbegantoappreciatethatbacteriainthebiofilmmodeofexistenceconstitutesuchamajorcomponentofthebacterialbiomassinmostenvironments.Then,itwasnotuntilthe1980sand1990sthatscientiststrulybegantounderstandhow

elaboratelyorganizedabacterialbiofilmcommunitycanbe.

AsRobertKolter,professorofmicrobiologyandmoleculargeneticsatHarvardMedicalSchool,andoneofthefirstscientiststostudyhowbiofilmsdevelopstates,Atfirst,however,studyingbiofilmswasaradicaldeparturefrompreviouswork.

Likemostmicrobialgeneticists,KolterhadbeentrainedinthetraditiondatingbacktoRobertKochandLouisPasteur,namelythatbacteriologyisbestconductedbystudyingpurestrainsofplanktonicbacteria.Whilethiswasatremendousadvanceformodernmicrobiology,italsodistractedmicrobiologistsfromamoreorganismicviewofbacteria,Kolteradds,

[1]

[8]

[1]

Biofilminaswampgasreactor.

BiofilminacidicpoolsatYellowstoneNationalPark.

Certainlywefeltthatpure,planktonicculturesweretheonlywaytowork.Yetinnaturebacteriadontlivelikethat,hesays.Infact,mostofthemoccurinmixed,surfacedwellingcommunities.

Althoughresearchonbiofilmshassurgedoverthepastfewdecades,themajorityofbiofilmresearchtodatehasfocusedonexternalbiofilms,orthosethatformonvarioussurfacesinournaturalenvironment.

Overthepastyears,asscientistsdevelopedbettertoolstoanalyzeexternalbiofilms,theyquicklydiscoveredthatbiofilmscancauseawiderangeofproblemsinindustrialenvironments.Forexample,biofilmscandevelopontheinteriorsofpipes,whichcanleadtocloggingandcorrosion.Biofilmsonfloorsandcounterscanmakesanitationdifficultinfoodpreparationareas.

Sincebiofilmshavetheabilitytoclogpipes,watersheds,storageareas,andcontaminatefoodproducts,largecompanieswithfacilitiesthatarenegativelyimpactedbytheirpresencehavenaturallytakenaninterestinsupportingbiofilmresearch,particularlyresearchthatspecifieshowbiofilmscanbeeliminated.

Thismeansthatmanyrecentadvancesinbiofilmdetectionhaveresultedfromcollaborationsbetweenmicrobialecologists,environmentalengineers,andmathematicians.Thisresearchhasgeneratednewanalyticaltoolsthathelpscientistsidentifybiofilms.

Forexample,theCanadiancompanyFASInternationalLtd.hasjustcreatedanendoluminalbrush,whichwillbelaunchedthisspring.Physicianscanusethebrushtoobtainsamplesfromtheinteriorofcatheters.Samplestakenfromcatheterscanbesenttoalab,whereresearchersdetermineifbiofilmsarepresentinthesample.Ifbiofilmsaredetected,thecatheterisimmediatelyreplaced,sincetheinsertionofcatheterswithbiofilmscancausethepatienttosufferfromnumerousinfections,someofwhicharepotentiallylifethreatening.

Scientistsnowrealizethatbiofilmsarenotjustcomposedofbacteria.Nearlyeveryspeciesofmicroorganismincludingviruses,fungi,andArchaeahavemechanismsbywhichtheycanadheretosurfacesandtoeachother.Furthermore,itisnowunderstoodthatbiofilmsareextremelydiverse.Forexample,upwardof300differentspeciesofbacteriacaninhabitthebiofilmsthatformdentalplaque.

Furthermore,biofilmshavebeenfoundliterallyeverywhereinnature,tothepointwhereanymainstreammicrobiologistwouldacknowledgethattheirpresenceisubiquitous.Theycanbefoundonrocksandpebblesatthebottomofmoststreamsorriversandoftenformonthesurfaceofstagnantpoolsofwater.Infact,biofilmsareimportantcomponentsoffoodchainsinriversandstreamsandaregrazeduponbytheaquaticinvertebratesuponwhichmanyfishfeed.Biofilmsevengrowinthehot,acidicpoolsatYellowstoneNationalParkandonglaciersinAntarctica.

Itisalsonowunderstoodthatthebiofilmmodeofexistencehasbeenaroundformillenia.Forexample,filamentousbiofilmshavebeenidentifiedinthe3.2billionyearolddeepseahydrothermalrocksofthePilbaraCraton,Australia.Accordingtoa2004articleinNatureReviewsMicrobiology,Biofilmformationappearsearlyinthefossilrecord(approximately3.25billionyearsago)andiscommonthroughoutadiverserangeoforganismsinboththeArchaeaandBacterialineages.Itisevidentthatbiofilmformationisanancientandintegralcomponentoftheprokaryoticlifecycle,andisakeyfactorforsurvivalindiverseenvironments.

Biofilms and disease

Thefactthatexternalbiofilmsareubiquitousraisesthequestionifbiofilmscanformonessentiallyeverysurfaceinourexternalenvironments,cantheydothesameinsidethe

humanbody?Theanswerseemstobeyes,andoverthepastfewyears,researchoninternalbiofilmshasfinallystartedtopickuppace.Afterall,itseasyforbiofilmresearcherstoseethatthehumanbody,withitswiderangeofmoistsurfacesandmucosaltissue,isanexcellentplaceforbiofilmstothrive.Nottomentionthefactthatthosebacteriawhichjoinabiofilmhaveasignificantlygreaterchanceofevadingthebatteryofimmunesystemcellsthatmoreeasilyattackplanktonicforms.

[9]

[10]

Commonsitesofbiofilminfection.Onebiofilmreachthebloodstreamtheycanspreadtoanymoistsurfaceofthehumanbody.

Hundredsofmicrobialbiofilmcolonizethehumanmouth,causingtoothdecayandgumdisease.

Manywouldarguethatresearchoninternalbiofilmshasbeenlargelyneglected,despitethefactthatbacterialbiofilmsseemtohavegreatpotentialforcausinghumandisease.

PaulStoodleyoftheCenterforBiofilmEngineeringatMontanaStateUniversity,attributesmuchofthelaginstudyingbiofilmstothedifficultiesofworkingwithheterogeneousbiofilmscomparedwithhomogeneousplanktonicpopulations.Ina2004paperinNatureReviews,themolecularbiologistdescribesmanyreasonswhybiofilmsareextremelydifficulttoculture,suchasthefactthatthediffusionofliquidthroughabiofilmandthefluidforcesactingonabiofilmmustbecarefullycalculatedifitistobeculturedcorrectly.AccordingtoStoodley,theneedtomastersuchdifficultlaboratorytechniqueshasdeterredmanyscientistsfromattemptingtoworkwithbiofilms.

Also,sincemuchofthetechnologyneededtodetectinternalbiofilmswascreatedatthesametimeasthesequencingofthehumangenome,interestinbiofilmbacteria,andtheresearchgrantsthatwouldaccompanysuchinterest,havebeenlargelydivertedtoprojectswithadecidedlygeneticfocus.However,sincegeneticresearchhasfailedtouncoverthecauseofanyofthecommonchronicdiseases,biofilmsarefinallyjustoverthepastfewyearsbeingstudiedmoreintensely,andbeinggiventhecredittheydeserveasseriousinfectiousentities,capableofcausingawidearrayofchronicillnesses.

Injustashortperiodoftime,researchersstudyinginternalbiofilmshavealreadypeggedthemasthecauseofnumerouschronicinfectionsanddiseases,andthelistofillnessesattributedtothesebacterialcoloniescontinuestogrowrapidly.

AccordingtoarecentpublicstatementfromtheNationalInstitutesofHealth,morethan65%ofallmicrobialinfectionsarecausedbybiofilms.Thisnumbermightseemhigh,butaccordingtoKimLewisoftheDepartmentofChemicalandBiologicalEngineeringatTuftsUniversity,Ifonerecallsthatsuchcommoninfectionsasurinarytractinfections(causedbyE.coliandotherpathogens),catheterinfections(causedbyStaphylococcusaureusandothergrampositivepathogens),childmiddleearinfections(causedbyHaemophilusinfluenzae,forexample),commondentalplaqueformation,andgingivitis,allofwhicharecausedbybiofilms,arehardtotreatorfrequentlyrelapsing,thisfigureappearsrealistic.

AsLewismentions,perhapsthemostwellstudiedbiofilmsarethosethatmakeupwhatiscommonlyreferredtoasdentalplaque.Plaqueisabiofilmonthesurfacesoftheteeth,statesParsek.Thisaccumulationofmicroorganismssubjecttheteethandgingivaltissuestohighconcentrationsofbacterialmetaboliteswhichresultsindentaldisease.

Ithasalsorecentlybeenshownthatbiofilmsarepresentontheremovedtissueof80%ofpatientsundergoingsurgeryfor

chronicsinusitis.AccordingtoParsek,biofilmsmayalsocauseosteomyelitis,adiseaseinwhichthebonesandbonemarrowbecomeinfected.Thisissupportedbythefactthatmicroscopystudieshaveshownbiofilmformationoninfectedbonesurfacesfromhumansandexperimentalanimalmodels.Parsekalsoimplicatesbiofilmsinchronicprostatitissincemicroscopystudieshavealsodocumentedbiofilmsonthesurfaceoftheprostaticduct.Microbesthatcolonizevaginaltissueandtamponfiberscanalsoformintobiofilms,causinginflammationanddiseasesuchasToxicShockSyndrome.

Biofilmsalsocausetheformationofkidneystones.Thestonescausediseasebyobstructingurineflowandbyproducinginflammationandrecurrentinfectionthatcanleadtokidneyfailure.Approximately15%20%ofkidneystonesoccurinthesettingofurinarytractinfection.AccordingtoParsek,thesestonesareproducedbytheinterplaybetweeninfectingbacteriaandmineralsubstratesderivedfromtheurine.Thisinteractionresultsinacomplexbiofilmcomposedofbacteria,bacterialexoproducts,andmineralizedstonematerial.

Perhapsthefirsthintoftheroleof

[10]

[11]

[12]

Microbesthatcolonizevaginaltissueandtamponfiberscanbecomepathogenic,causinginflammationanddiseasesuchasToxicShockSyndrome.

CellsofStaphylococcusepidermidiscausingdevastatingdiseaseastheygrowonthecuffatamechanicalheartvalve.

bacteriainthesestonescamein1938whenHellstromexaminedstonespassedbyhispatientsandfoundbacteriaembeddeddeepinsidethem.Microscopicanalysisofstonesremovedfrominfectedpatientshasrevealedfeaturesthatcharacterizebiofilmgrowth.Foronething,bacteriaonthesurfaceandinsidethestonesareorganizedinmicrocoloniesandsurroundedbyamatrixcomposedofcrystallized(struvite)minerals.

Thentheresendocarditis,adiseasethatinvolvesinflammationoftheinnerlayersoftheheart.Theprimaryinfectiouslesioninendocarditisisacomplexbiofilmcomposedofbothbacterialandhostcomponentsthatislocatedonacardiacvalve.Thisbiofilm,knownasavegetation,causesdiseasebythreebasicmechanisms.First,thevegetationphysicallydisruptsvalvefunction,causingleakagewhenthevalveisclosedandinducingturbulenceanddiminishedflowwhenthevalveisopen.Second,thevegetationprovidesasourcefornearcontinuousinfectionofthebloodstreamthatpersistsevenduringantibiotictreatment.Thiscausesrecurrentfever,chronicsystemicinflammation,andotherinfections.Third,piecesoftheinfectedvegetationcanbreakoffandbecarriedtoaterminalpointinthecirculationwheretheyblocktheflowofblood(aprocessknownasembolization).Thebrain,kidney,andextremitiesareparticularlyvulnerabletotheeffectsofembolization.

Avarietyofpathogenicbiofimsarealsocommonlyfoundonmedicaldevicessuchasjointprosthesesandheartvalves.AccordingtoParsek,electronmicroscopyofthesurfacesofmedicaldevicesthathavebeenfociofdevicerelatedinfectionsshowsthepresenceoflargenumbersofslimeencasedbacteria.Tissuestakenfromnondevicerelatedchronicinfectionsalsoshowthepresenceofbiofilmbacteriasurroundedbyanexopolysaccharidematrix.Thesebiofilminfectionsmaybecausedbyasinglespeciesorbyamixtureofspeciesofbacteriaorfungi.

AccordingtoDr.PateloftheMayoClinic,individualswithprostheticjointsareoftenoblivioustothefactthattheirprostheticjointsharborbiofilminfections.

Whenpeoplethinkofinfection,theymaythinkoffeverorpuscomingoutofawound,explainsDr.Patel.However,thisisnotthecasewithprostheticjointinfection.Patientswilloftenexperiencepain,butnotothersymptomsusuallyassociatedwithinfection.Oftenwhathappensisthatthebacteriathatcauseinfectiononprostheticjointsarethesameasbacteriathatliveharmlesslyonourskin.However,onaprostheticjointtheycanstick,growandcauseproblemsoverthelongterm.

Manyofthesebacteriawouldnotinfectthejointwereitnotfortheprosthesis.

BiofilmsalsocauseLeptospirosis,aseriousbutneglectedemergingdiseasethatinfectshumansthroughcontaminatedwater.NewresearchpublishedintheMayissueofthejournalMicrobiologyshowsforthefirsttimehowbacteriathatcausethediseasesurviveintheenvironment.

LeptospirosisisamajorpublichealthprobleminsoutheastAsiaandSouthAmerica,withover500,000severecaseseveryyear.Between5%and20%ofthesecasesarefatal.RatsandothermammalscarrythediseasecausingpathogenLeptospirainterrogansintheirkidneys.Whentheyurinate,theycontaminatesurfacewaterwiththebacteria,whichcansurviveintheenvironmentforlongperiods.

Thisledustoseeifthebacteriabuildaprotectivecasingaroundthemselvesforprotection,saidProfessorMathieuPicardeaufromtheInstitutPasteurinParis,France.

Previously,scientistsbelievedthebacteriawereplanktonic.ButProfessorPicardeauandhisteamhaveshownthatL.interroganscanmakebiofilms,whichcouldbeoneofthemainfactorscontrollingsurvivalanddiseasetransmission.90%ofthespeciesofLeptospirawetestedcouldformbiofilms.IttakesL.interrogansanaverageof20daystomakeabiofilm,saysPicardeau.

Biofilmshavealsobeenimplicatedinawidearrayofveterinarydiseases.Forexample,researchersattheVirginiaMarylandRegionalCollegeofVeterinary

[13]

[14]

Whentheimmuneresponseiscompromised,Pseudomonasaeruginosabiofilmsareabletocolonizethealveoli,andtoformbiofilms.

MedicineatVirginiaTechwerejustawardedagrantfromtheUnitedStatesDepartmentofAgriculturetostudytherolebiofilmsplayinthedevelopmentofBovineRespiratoryDiseaseComplex(BRDC).Ifbiofilmsplayaroleinbovinerespiratorydisease,itslikelyonlyamatteroftimebeforetheywillbeestablishedasacauseofhumanrespiratorydiseasesaswell.

Asmentionedpreviously,infectionbythebacteriumPseudomonasaeruginosa(P.aeruginosa)isthemaincauseofdeathamongpatientswithcysticfibrosis.Pseudomonasisabletosetuppermanentresidenceinthelungsofpatientswithcysticfibrosiswhere,ifyouaskmostmainstreamresearchers,itisimpossibletokill.Eventually,chronicinflammationproducedbytheimmunesysteminresponsetoPseudomonasdestroysthelungandcausesrespiratoryfailure.Inthepermanentinfectionphase,P.aeruginosabiofilmsarethoughttobepresentintheairway,althoughmuchabouttheinfectionpathogenesisremainsunclear.

Cysticfibrosisiscausedbymutationsintheproteinsofchannelsthatregulateschloride.Howabnormalchloridechannelproteinleadstobiofilminfectionremainshotlydebated.Itisclear,however,thatcysticfibrosispatientsmanifestsomekindofhostdefensedefectlocalizedtotheairwaysurface.Somehowthisleadstoadebilitatingbiofilminfection.

Biofilms have the potential to cause a tremendous array of infections and diseases

Becauseinternalpathogenicbiofilmresearchcomprisessuchanewfieldofstudy,theinfectionsdescribedabovealmostcertainlyrepresentjustthetipoftheicebergwhenitcomestothenumberofchronicdiseasesandinfectionscurrentlycausedbybiofilms.

Forexample,itwasntuntilJulyof2006thatresearchersrealizedthatthemajorityofearinfectionsarecausedbybiofilmbacteria.Theseinfections,whichcanbeeitheracuteorchronic,arereferredtocollectivelyasotitismedia(OM).Theyarethemostcommonillnessforwhichchildrenvisitaphysician,receiveantibiotics,orundergosurgeryintheUnitedStates.

TherearetwosubtypesofchronicOM.RecurrentOM(ROM)isdiagnosedwhenchildrensufferrepeatedinfectionsoveraspanoftimeandduringwhichclinicalevidenceofthediseaseresolvesbetweenepisodes.ChronicOMwitheffusionisdiagnosedwhenchildrenhavepersistentfluidintheearsthatlastsformonthsintheabsenceofanyothersymptomsexceptconductivehearingloss.

Ittookovertenyearsforresearcherstorealizethatotitismediaiscausedbybiofilms.Finally,in2002,Drs.EhrlichandJ.ChristopherPost,anAlleghenyGeneralHospitalpediatricearspecialistandmedicaldirectoroftheCenterforGenomicSciences,publishedthefirstanimalevidenceofbiofilmsinthemiddleearintheJournaloftheAmericanMedicalAssociation,settingthestageforfurtherclinicalinvestigation.

Inasubsequentstudy,EhrlichandPostobtainedmiddleearmucosaormembranetissuebiopsiesfromchildrenundergoingaprocedureforotitis.Theteamgathereduninfectedmucosalbiopsiesfromchildrenandadultsundergoingcochlearimplantationasacontrol.

Usingadvancedconfocallaserscanningmicroscopy,LuanneHallStoodley,Ph.D.andherASRIcolleaguesobtainedthreedimensionalimagesofthebiopsiesandevaluatedthemforbiofilmmorphologyusinggenericstainsandspeciesspecificprobesforHaemophilusinfluenzae,StreptococcuspneumoniaeandMoraxellacatarrhalis.Effusions,whenpresent,werealsoevaluatedforevidenceofpathogenspecificnucleicacidsequences(indicatingpresenceoflivebacteria).

Thestudyfoundmucosalbiofilmsinthemiddleearsof46/50children(92%)withbothformsofotitis.Biofilmswerenotobservedineightcontrolmiddleearmucosaspecimensobtainedfromcochlearimplantpatients.

Infact,allofthechildreninthestudywhosufferedfromchronicotitismediatestedpositiveforbiofilmsinthemiddleear,eventhosewhowereasymptomatic,causingErlichtoconcludethat,Itappearsthatinmanycasesrecurrentdiseasestemsnotfromreinfectionaswaspreviouslythoughtandwhichformsthebasisforconventionaltreatment,butfromapersistentbiofilm.

Hewentontostatethatthediscovery

[15]

[16]

Otitismedia,orinflammationoftheinnerear,iscausedbybiofilm.

Fungalbiofilmcanformincontactlenssolutionleadingtopotentiallyvirulenteyeinfections

ofbiofilmsinthesettingofchronicotitismediarepresentedalandmarkevolutioninthemedicalcommunitysunderstandingaboutadiseasethatafflictsmillionsofchildrenworldwideeachyearandfurtherendorsestheemergingbiofilmparadigmofchronicinfectiousdisease.

TheemergingbiofilmparadigmofchronicdiseasereferstoanewmovementinwhichresearcherssuchasEhrlicharecallingforatremendousshiftinthewaythemedicalcommunityviewsbacterialbiofilms.Thosescientistswhosupportanemergingbiofilmparadigmofchronicdiseasefeelthatbiofilmresearchisofutmostimportance

becauseofthefactthattheinfectiousentitieshavethepotentialtocausesomanyformsofchronicdisease.TheMarshallPathogenesisisanimportantpartofthisparadigmshift.

Itwasalsojustlastyearthatresearchersrealizedthatbiofilmscausemostinfectionsassociatedwithcontactlensuse.In2006,Bausch&LombwithdrewitsReNuwithMoistureLoccontactlenssolutionbecauseahighproportionofcornealinfectionswereassociatedwithit.ItwasntlongbeforeresearchersattheUniversityHospitalsCaseMedicalCenterfoundthattheinfectionswerecausedbybiofilms.

Oncetheyliveinthattypeofstate[abiofilm],thecellsbecomeresistanttolenssolutionsandimmunetothebodysowndefensesystem,saidMahmoudA.Ghannoum,Ph.D,seniorinvestigatorofthestudy.Thisstudyshouldalertcontactlenswearerstotheimportanceofpropercareforcontactlensestoprotectagainstpotentiallyvirulenteyeinfections,hesaid.

ItturnsoutthatthebiofilmsdetectedbyGhannoumandteamwerecomposedoffungi,particularlyaspeciescalledFusarium.Histeamalsodiscoveredthatthestrainoffungus(withthecatchyname,ATCC36031)usedfortestingtheeffectivenessoflenscaresolutionsisastrainthatdoesnotproducebiofilmsastheclinicalfungalstrainsdo.ReNucontactsolution,therefore,waseffectiveinthelaboratory,butfailedwhenfacedwithstrainsinrealworldsituations.

Unfortunately,GhannoumandteamwerenotabletocreateamethodtotargetanddestroythefungalbiofilmsthatplagueusersofReNuandsomeothercontactlenssolutions.

ThentheresDr.RandallWolcottwhojustrecentlydiscoveredandconfirmedthatthesludgecoveringdiabeticwoundsislargelymadeupofbiofilms.WhereasbeforeWolcottsworksuchlimbsgenerallyhadtobeamputated,nowthattheyhavebeencorrectlylinkedtobiofilms,measuressuchasthosedescribedinthisinterviewcanbetakentostopthespreadofinfectionandsavethelimb.WolcotthasfinallybeengivenagrantbytheNationalInstitutesofHealthtofurtherstudychronicbiofilmsandwounddevelopment.

Dr.GarthJamesandtheMedicalBiofilmLaboratoryteamatMontanaStateUniversityarealsoresearchingwoundsandbiofilms.TheirlatestarticleandanimageshowingwoundbiofilmwasfeaturedonthecoveroftheJanuaryFebruary2008issueofWoundRepairandRegeneration.

Biofilm bacteria and chronic inflammatory disease

Injustafewshortyears,thepotentialofbiofilmstocausedebilitatingchronicinfectionshasbecomesoclearthatthereislittledoubtthatbiofilmsarepartofthepathogenicmixorpeasoupthatcausemostorallchronicautoimmuneandinflammatorydiseases.

Infact,thanks,inlargepart,totheresearchofbiomedicalresearcherDr.TrevorMarshall,itisnowincreasinglyunderstoodthatchronicinflammatorydiseasesresultfrominfectionwithalargemicrobiotaofchronicbiofilmandLformbacteria(collectivelycalledtheTh1pathogens). Themicrobiotaisthoughttobecomprisedofnumerousbacterialspecies,someofwhichhaveyettobediscovered.However,mostofthepathogensthatcauseinflammatorydiseasehaveonethingincommontheyhavealldevelopedwaystoevadetheimmunesystemandpersistaschronicformsthatthebodyisunabletoeliminatenaturally.

SomeLformbacteriaareabletoevadetheimmunesystembecause,longago,theyevolvedtheabilitytoresideinsidemacrophages,theverywhite

[17]

[18]

[19] [20]

DiagramoftheVitaminDReceptorandcapnine.

bloodscellsoftheimmunesystemthataresupposedtokillinvadingpathogens.Uponformation,Lformbacteriaalsolosetheircellwalls,whichmakesthemimpervioustocomponentsoftheimmuneresponsethatdetectinvadingpathogensbyidentifyingtheproteinsontheircellwalls.ThefactthatLformbacterialackcellwallsalsomeansthatthebetalactamantibiotics,whichworkbytargetingthebacterialcellwall,arecompletelyineffectiveatkillingthem.

Clearly,transformingintotheLformoffersanypathogenasurvivaladvantage.ButamongthosepathogensnotinanLformstate,joiningabiofilmisjustaslikelytoenhancetheirabilitytoevadetheimmunesystem.Onceenoughchronicpathogenshavegroupedtogetherandformedastablecommunitywithastrongprotectivematrix,theyarelikelyabletoresideinanyareaofthebody,causingthehosttosufferfromchronicsymptomsthatarebothmentalandphysicalinnature.

Biofilmresearcherswillalsotellyouthat,notsurprisingly,biofilmsformwithgreatereaseinanimmunocompromisedhost.MarshallsresearchhasmadeitclearthatmanyoftheTh1pathogensarecapableofcreatingsubstancesthatbindandinactivatetheVitaminDReceptorafundamentalreceptorofthebodythatcontrolstheactivityoftheinnateimmunesystem,orthebodysfirstlineofdefenseagainstintracellularinfection.

Thus,aspatientsaccumulateagreaternumberoftheTh1pathogens,moreandmoreofthechronicbacterialformscreatesubstancescapableofdisablingtheVDR.Thiscausesasnowballeffect,inwhichthepatientbecomesincreasinglyimmunocompromisedastheyacquirealargerbacterialload.

Foronething,itspossiblethatmanyofthebacteriathatsurviveinsidebiofilmsarecapableofcreatingVDRblockingsubstances.Thus,theformationofbiofilmsmaycontributetoimmunedysfunction.Conversely,aspatientsacquireLformbacteriaandotherpersistentbacterialformscapableofcreatingVDRblockingsubstances,itbecomesexceptionallyeasyforbiofilmstoformonanytissuesurfaceofthehumanbody.

Thus,patientswhobegintoacquireLformbacteriaalmostalwaysfallvictimtobiofilminfectionsaswell,sinceitisalltooeasyforpathogenstogrouptogetherintoabiofilmwhentheimmunesystemisntworkinguptopar.

Todate,thereisalsonostrictcriteriathatseparateLformbacteriafrombiofilmbacteriaoranyotherchronicpathogenicforms.ThismeansthatLformbacteriamayalsoformintobiofilms,andbydoingsoenteramodeofsurvivalthatmakesthemtrulyimpervioustotheimmunesystem.SomeLformbacteriamaynotformcompletebiofilms,yetmaystillpossesstheabilitytosurroundthemselvesinaprotectivematrix.Underthesecircumstancesonemightsaytheyareinabiofilmlikestate.

Marshalloftenreferstothepathogensthatcauseinflammatorydiseaseasanintraphgocytic,metagenomicmicrobiotaofbacteria,termswhichsuggestthatmostchronicbacterialformspossesspropertiesofbothLformandbiofilmbacteria.Intraphagocyticreferstothefactthatthepathogenscanbefoundinsidethecellsoftheimmunesystem.Thetermmetagenomicindicatesthatthereareatremendousnumberofdifferentspeciesofthesechronicbacterialforms.Finally,microbiotareferstothefactthatbiofilmcommunitiessustaintheirpathogenicactivity.

Forexample,whenobservedunderadarkfieldmicroscope,Lformbacteriaareoftenencasedinprotectivebiofilmsheaths.Ifthebloodcontainingthepathogensareagedovernight,thebacterialcoloniesreachapointwheretheyexpandandburstoutofthecell,causingthecelltoburstaswell.Thentheyextendashuge,longbiofilmtubules,whicharepresumablyhelpingthepathogensspreadtoothercells.ThetubulesalsohelpspreadbacterialDNAtoneighboringcells.

Clearly,thereisagreatneedformoreresearchonhowdifferentchronicbacterialformsinteract.Todate,LformresearchershaveessentiallyfocusedsoleyontheLform,whilefailingtoinvestigatehowfrequentlythewalllesspathogensformintobiofilmsorbecomepartsofbiofilmcommunitiestogetherwithbacteriawithcellwalls.Conversely,mostbiofilmresearchersareintentlystudyingthebiofilmmodeofgrowthwithoutconsideringthepresenceofLformbacteria.So,itwilllikelytakeseveralyearsbeforewewillbebetterabletounderstandprobableoverlapsbetweenthelifestylesofLformandbiofilmbacteria.

Anyonewhoisskepticalaboutthefactthatbiofilmslikelyformalargepercentageofthemicrobiotathatcauseinflammatorydiseaseshouldconsidermanyoftherecentstudiesthathavelinkedestablishedbiofilminfectionstoahigherriskformultipleformsofchronicinflammatorydisease.Take,for

[21]

[22]

Dentalplaqueasseenunderascanningelectronmicrocroscope.

Abiofilmonapieceoflettuce

example,studiesthathavefoundalinkbetweenperiodontaldiseaseandseveralmajorinflammatoryconditions.A1989articlepublishedinBritishMedicalJournalshowedacorrelationbetweendentaldiseaseandsystemicdisease(stroke,heartdisease,diabetes).Aftercorrectingforage,exercise,diet,smoking,weight,bloodcholesterollevel,alcoholuseandhealthcare,peoplewhohadperiodontaldiseasehadasignificantlyhigherincidenceofheartdisease,strokeandprematuredeath.Morerecently,theseresultswereconfirmedinstudiesintheUnitedStates,Canada,GreatBritain,Sweden,andGermany.Theeffectsarestriking.Forexample,researchersfromtheCanadianHealthBureaufoundthatpeoplewithperiodontaldiseasehadatwotimeshigherriskofdyingfromcardiovasculardisease.

Sinceweknowthatperiodontaldiseaseiscausedbybiofilmbacteria,themostlogicalexplanationforthefactthatpeoplewithdentalproblemsaremuchmorelikelytosufferfromheartdiseaseandstrokeisthatthebiofilmsintheirmouthshavegraduallyspreadtothemoistsurfacesoftheircirculatorysystems.OrperhapsifthebacteriainperiodontalbiofilmscreateVDRbindingsubstances,theirabilitytoslowinnateimmunefunctionallowsnewbiofilms(andLformbacteriaaswell)tomoreeasilyformandinfecttheheartandbloodvessels.Conversely,systemic

infectionwithVDRblockingbiofilmbacteriaisalsolikelytoweakenimmunedefensesinthegumsandfacilitateperiodontaldisease.

Infact,itappearsthatbiofilmbacteriainthemouthalsofacilitatetheformationofbiofilmandLformbacteriainthebrain.Justlastyear,researchersatVasantHiraniatUniversityCollegeLondonreleasedtheresultsofastudywhichfoundthatelderlypeoplewhohavelosttheirteethareatmorethanthreefoldgreaterriskofmemoryproblemsanddementia.

Atthemoment,AutoimmunityResearchFoundationdoesnothavetheresourcestoculturebiofilmsfrompatientsonthetreatmentand,eveniftheydid,currentmethodsforculturinginternalbiofilmsremainunreliable.AccordingtoStoodley,Thelackofstandardmethodsforgrowing,quantifyingandtestingbiofilmsincontinuouscultureresultsinincalculablevariabilitybetweenlaboratorysystems.Biofilmmicrobiologyiscomplexandnotwellrepresentedbyflaskcultures.Althoughhomogeneityallowsstatisticalenumeration,theextenttowhichitreflectsthereal,lessorderlyworldisquestionable.

How else do we acquire biofilm bacteria?

Asdiscussedthusfar,biofilmsformspontaneouslyasbacteriainsidethehumanbodygrouptogether.Yetpeoplecanalsoingestbiofilmsbyeatingcontaminatedfood.

AccordingtoresearchersattheUniversityofGuelphinOntarioCanada,itisincreasinglysuspectedthatbiofilmsplayanimportantroleincontaminationofmeatduringprocessingandpackaging.ThegroupwarnsthatgreateractionmustbetakentoreducethepresenceoffoodbornepathogenslikeEscherichiacoliandListeriamonocytogenesandspoilagemicroorganismssuchasthePseudomonasspecies(allofwhichformbiofilms)throughoutthefoodprocessingchaintoensurethesafetyandshelflifeoftheproduct.Mostofthesemicroorganismsareubiquitousintheenvironmentorbroughtintoprocessingfacilitiesthroughhealthyanimalcarriers.

HansBlaschekoftheUniversityofIllinoishasdiscoveredthatbiofilmsformonmuchoftheotherfoodproductsweconsumeaswell.

Ifyoucouldseeapieceofcelerythatsbeenmagnified10,000times,youdknowwhatthescientistsfightingfoodbornepathogensareupagainst,saysBlaschek.

Itslikelookingatamoonscape,fullofcratersandcrevices.Andmanyofthepathogensthatcausefoodborneillness,suchasShigella,E.coli,andListeria,makesticky,sugarybiofilmsthatgetdowninthesecrevices,sticklikeglue,andhangonlikecrazy.

AccordingtoBlaschek,theproblemfacedbyproducesupplierscanbeatriplewhammy.Ifyoureunluckyenoughtobedealingwithapathogenandthepathogenhastheadditionalattributeofbeingabletoformbiofilmandyouredealingwithafoodproductthatsminimallyprocessed,well,youretriplyunlucky,thescientistsaid.Youmaybeabletoscrubtheorganismoffthe

[23]

[24]

[10]

surface,butthecellsinthesebiofilmsareverygoodataligningthemselvesinthesubsurfaceareasofproduce.

ScottMartin,aUniversityofIllinoisfoodscienceandhumannutritionprofessoragrees,stating,Oncethepathogenicorganismgetsontheproduct,noamountofwashingwillremoveit.Themicrobesattachtothesurfaceofproduceinastickybiofilm,andwashingjustisntveryeffective.

Biofilmscanevenbefoundinprocessedwater.Justthismonth,astudywasreleasedinwhichresearchersattheDepartmentofBiologicalSciences,atVirginiaPolytechnicInstituteisolatedM.aviumbiofilmfromtheshowerheadofawomanwithM.aviumpulmonarydisease. AmoleculartechniquecalledDNAfingerprintingdemonstratedthatM.aviumisolatesfromthewaterwerethesameformsthatwerecausingthewomansrespiratoryillness.

Effectively targeting biofilm infections

Althoughthemainstreammedicalcommunityisrapidlyacknowledgingthelargenumberofdiseasesandinfectionscausedbybiofilms,mostresearchersareconvincedthatbiofilmsaredifficultorimpossibletodestroy,particularlythosecellsthatformthedeeperlayersofathickbiofilm.Mostpapersonbiofilmsstatethattheyareresistanttoantibioticsadministeredinastandardmanner.Forexample,despitethefactthatEhrlichandteamdiscoveredthatbiofilmbacteriacauseotitismedia,theyareunabletoofferaneffectivesolutionthatwouldactuallyallowforthedestructionofbiofilmsintheearcanal.Otherteamshavealsocomeupshortincreatingmethodstobreakupthebiofilmstheyimplicateasthecauseofnumerousinfections.

Thismeanspatientswithbiofilminfectionsaregenerallytoldbymainstreamdoctorsthattheyhaveanuntreatableinfection.Insomecases,adiseasecausingbiofilmcanbecutoutofapatientstissues,oreffortsaremadetodraincomponentsofthebiofilmoutofthebody.Forexample,doctorstreatingotitismediaoftentreatspatientswithmyringotomy,asurgicalprocedureinwhichsmalltubesareplacedintheeardrumtocontinuouslydraininfectiousfluid.

Whenitcomestoadministeringantibioticsinanefforttotargetbiofilms,onethingiscertain.Mainstreamresearchershaverepeatedlytriedtokillbiofilmsbygivingpatientshigh,constantdosesofantibiotics.Unfortunately,whenadministeredinhighdoses,theantibioticmaytemporarilyweakenthebiofilmbutisincapableofdestroyingit,ascertaincellsinevitablypersistandallowthebiofilmtoregenerate.

Youcanputapatienton[ahighdose]antibiotics,anditmayseemthattheinfectionhasdisappeared,saysLevchenko.Butinafewmonths,itreappears,anditisusuallyinanantibioticresistantform.

Whatthevastmajorityofresearchersworkingwithbiofilmsfailtorealizeisthatantibioticsarecapableofdestroyingbiofilms.Thecatchisthatantibioticsareonlyeffectiveagainstbiofilmsifadministeredinaveryspecificmanner.Furthermore,onlycertainantibioticsappeartoeffectivelytargetbiofilms.Afterdecadesofresearch,muchofwhichwasderivedfrommolecularmodelingdata,Marshallwasthefirsttocreateanantibioticregimenthatappearstoeffectivelytargetanddestroybiofilms.Centraltothetreatment,whichiscalledtheMarshallProtocol,isthefactthatbiofilmsandotherTh1pathogenssuccumbtospecificbacteriostaticantibioticstakeninverylow,pulseddoses.Itisonlywhenantibioticsareadministeredinthismannerthattheyappearcapableoffullyeradicatingbiofilms.

InapaperentitledTheRiddleofBiofilmResistance,Dr.KimLewisofTulaneUniversitydiscussesthemechanismsbywhichpulsed,lowdoseantibioticsareabletobreakupbiofilms,whileantibioticsadministeredinastandardmanner(high,constantdoses)cannot.AccordingtoLewis,theuseofpulsed,lowdoseantibioticstotargetbiofilmbacteriaissupportedbyobservationssheandhercolleagueshavemadeinthelaboratory.

Someresearchersclaimthatantibioticscannotpenetratethematrixthatsurroundsabiofilm.ButresearchbyLewisandotherscientistshasconfirmedthattheinabilityofantibioticstopenetratethebiofilmmatrixismuchmoreofanexceptionthanarule.AccordingtoLewis,Inmostcasesinvolvingsmallantimicrobialmolecules,thebarrierofthepolysaccharidematrixshouldonlypostponethedeathofcellsratherthanaffordusefulprotection.

Forexample,arecentstudythatusedlowconcentrationsofanantibiotictokillP.aeruginosabiofilmbacteriafoundthatthemajorityofbiofilmcellswereeffectivelyeliminatedbyantibioticsinamannerthatdidnotdiffermuchfromwhatisobservedwhenthesameantibioticconcentrationsareadministeredtosingleplanktoniccells.

Thus,sinceantibioticscangenerallypenetratebiofilms,someotherfactorisresponsibleforthefactthattheycannotbekilledbystandardhighdoseantibiotictherapy.Itturnsoutthatafterantibioticsareappliedtoabiofilm,anumberofcellscalledpersistersareleftbehind.Persistersaresimplycellsthatareabletosurvivethefirstonslaughtof

[25]

[19] [20]

[11]

[26]

Afterantibioticsareappliedtoabiofilm,anumberofcellscalledpersistersareleftbehind.

Modelofbiofilmresistancebasedonpersistersurvival.Aninitialtreatmentofhighdoseconstantantibiotickillsplanktoniccellsandthemajorityofbiofilmcells.Butpersistersremainaliveandresurrectthebiofilm,causingtheinfectiontorelapse

antibiotics,andifleftunchecked,graduallyallowthebiofilmtoformagain.AccordingtoLewis,persistercellsformwithparticulareaseinimmunocompromisedpatientsbecausetheimmunesystemisunabletohelptheantibioticmopupallthebiofilmcellsithastargeted.

Thissimpleobservationsuggestsanewparadigmforexplaining,atleastinprinciple,thephenomenonofbiofilmresistancetokillingbyawiderangeofantimicrobials,statesLewis.Themajorityofcellsinabiofilmarenotnecessarilymoreresistanttokillingthanplanktoniccellsanddierapidlywhentreatedwith[anantibiotic]thatcankillslowlygrowingcells.

Thus,adoseofantibioticsparticularlyinimmunocompromisedpatientseradicatesmostofthebiofilmpopulationbutleavesasmallfractionofsurvivingpersistersbehind.Unfortunately,inthesamesensethatthebetalactamantibioticspromotetheformationofLformbacteria,persistercellsareactuallypreservedbythepresenceofanantibioticthatinhibitstheirgrowth.Thus,paradoxically,dosinganantibioticinaconstant,highdosemanner(inwhichtheantibioticisalwayspresent)helpspersisterspersevere.

Butinthecaseoflow,pulseddosing,whereanantibioticisadministered,withdrawn,thenadministeredagain,thefirstapplicationofantibioticwilleradicatethebulkofbiofilmcells,leavingpersistercellsbehind.Withdrawloftheantibioticallowsthepersisterpopulationtostartgrowing.Sinceadministrationoftheantibioticistemporarilystopped,thesurvivalofpersistersisnotenhanced.Thiscausesthepersistercellstolosetheirphenotype(theirshapeandbiochemicalproperties),meaningthattheyareunabletoswitchbackintobiofilmmode.Asecondapplicationoftheantibioticshouldthencompletelyeliminatethepersistercells,whicharestillinplanktonicmode.

Lewishasfoundthatthefeasibilityofapulsed,orcyclicalbiofilmeradicationapproachdependsontherateatwhichpersistersloseresistancetokillingandregeneratenewpersisters.ItalsodependsontheabilitytomanipulatetheantibioticconcentrationsomethingthatisdonequiteeffectivelybypatientsontheMarshallProtocolwhocarefullydosetheirantibioticsatdifferentlevels,allowingconstantvariationinantibioticconcentration.AlthoughLewisspeculatesthatallowingtheconcentrationofanantibiotictodropcouldpotentiallyleadtoresistancetowardstheantibiotic,sheisquicktoaddthatiftwoormoreantibioticsareusedtotargetabiofilmatonetime,suchresistancewouldnotoccur.Again,sincetheMarshallProtocolusesatotaloffivebacteriostaticantibiotics,usuallytakentwoorthreeatatime,concernsofresistanceareessentiallynegligible.

Itisentirelypossiblethatsuccessfulcasesofantimicrobialtherapyofbiofilminfectionsresultfromafortuitousoptimalcycling[pulseddosing]ofanantibioticconcentrationthateliminatedfirstthebulkofthebiofilmandthentheprogenyofthepersistersthatbegantodivide,statesLewis.

Lewisworkhasbeensupportedbyotherresearchteams.Recently,researchersattheUniversityofIowafoundthatsubinhibitory(extremelylowdose)concentrationsofthebacteriostaticantibioticazithromycinsignificantlydecreasedbiomassandmaximalthicknessinbothformingandestablishedbiofilms. Theseextremelylowconcentrationsofazithromycininhibitedbiofilmsinallbutthemosthighlyresistantisolates.Incontrast,subinhibitoryconcentrationsofgentamicin,whichisnotabacteriostaticantibiotic,hadnoeffectonbiofilmformation.Infact,biofilmsactuallybecameresistanttogentamicinatconcentrationsfarabovetheminimuminhibitoryconcentration.

ResearchersatTulaneUniversityrecentlyconfirmedyetagainthatlow,pulseddosingisasuperiorwayoftargetingtreatmentresistantbiofilmbacteria.Accordingtotheteam,whomathematicallymodeledtheactionofantibioticsonbacterialbiofilms,Exposingabiofilmtolowconcentrationdosesofanantimicrobialagentforlongertimeismoreeffectivethanshorttimedosingwithhighantimicrobialagentconcentration.

Similarly,abioengineerledteamattheUniversityofWashingtonrecentlycreatedanantibioticcontainingpolymerthatreleasesantibioticslowlyontothesurfaceofhospitaldevices,suchascathetersandprostheses,toreduce

[27]

[28]

Afterantibioticsareappliedtoabiofilm,anumberofcellscalledpersistersareleftbehind

theriskofbiofilmrelatedinfections.

Ratherthanmassivelydosingthepatientwithhighlevelsofreleasedantibiotic,thisstrategyallowsthereleaseofextremelylowlevelsofthisverypotentantibioticoverlongperiodsoftime,explainedBuddyRatner,PhD,ProfessorandDirectoroftheEngineeredBiomaterialsProgramattheUniversityofWashington,Seattle.Wecalculatedtheamountreleasedatthesurfacethatwouldkill100%ofthebacteriaenteringthesurfacezone.

WhenchallengedbyDr.LeonardA.MermelfromBrownUniversitySchoolofMedicineontheissuethatlongtermuseofpulsed,lowdoseantibioticsmightallowforincreasedresistanceonthepartofthebacteriabeingtreated,Ratnerresponded,Dr.Mermelsconcernsare,infact,whywedevelopedthissystemfor[antibiotic]release.Bacteriathatlivethroughantibioticdosingcangoontoproduceresistantstrains.If100%ofthebacteriaapproachingthesurfacearekilled,theycantproduceresistantoffspring.Theclassicalphysicianapproach,dosingthepatientsystemicallyandheavilytoridthepatientofpersistentbacteria,canleadtothoseresistantstrains.Ourapproachreleasesminisculedosescomparedtowhataphysicianwoulduse,butreleasestheantibioticwhereitwillbeoptimallyeffectiveandleastlikelytoleaveantibioticresistantsurvivors.

Althoughtakenorally,theMPantibioticsaretakeninthesamemannerasthoseadministeredbyRatnerandteam.Becausetheytooaredosedatoptimaltimesinextremelysmalldoses,thechancethatlongtermantibioticusemightfosterresistantbacteriaisagain,essentiallynegligible,especiallywhenmultipleantibioticsaretypicallyused.

KeytotheabilityoftheMarshallProtocoltoeffectivelytargetbiofilmbacteriaisthefactthatthespecificpulsed,lowdosebacteriostaticantibioticsusedbythetreatmentaretakeninconjunctionwithamedicationcalledBenicar.BenicarbindsandactivatestheVitaminDReceptor,displacingbacterialsubstancesand25Dfromthereceptor,sothatitcanonceagainactivatetheinnateimmunesystem. Benicarissoeffectiveatstrengtheningtheinnateimmuneresponsethatthepatientsownimmunesystemultimatelyhelpsdestroythebiofilmweakenedbypulsed,lowdoseantibiotics.

Thus,itisnotenoughforpatientsontheMarshallProtocoltosimplytakespecificpulsed,lowdoseantibiotics.Theactivityoftheirinnateimmunesystemmustalsoberestoredsothatthecellsoftheimmunesystemcanactivelycombatbiofilmbacteria,thematrixthatsurroundsthem,andpersistercells.

HowdoweknowthattheMarshallProtocoleffectivelykillsbiofilmbacteria?Namelybecausethosepatientstoreachthelaterstagesofthetreatmentdonotreportsymptomsassociatedwithestablishedbiofilmdiseases.PatientsontheMPwhooncesufferedfromchronicearinfections(OM),chronicsinusinfections,orperiodontaldiseasefindthatsuchinfectionsresolveoverthecourseoftreatment.Furthermore,sincewenowunderstandthatbiofilmsalmostcertainlyformalargepartofthechronicmicrobiotaofpathogensthatcausechronicinflammatoryandautoimmunediseases,thefactthatpatientscanusetheMarshallProtocoltorecoverfromsuchillnessesagainsuggeststhatthetreatmentmustbeeffectivelyallowingthemtotargetand

destroybiofilms.

BecauseallevidencepointstothefactthattheMPdoesindeedeffectivelytargetbiofilmbacteria,itisofutmostimportancethatpeoplewhosufferfromanysortofbiofilminfectionstartthetreatment.KnowledgeoftheMarshallProtocolhasyettoreachthecysticfibrosiscommunity,butthereisgreathopethatifpeoplewiththediseaseweretostarttheMP,theycoulddestroytheP.aeruginosabiofilmsthatcausetheiruntimelydeaths.Inthesamevein,peoplewithawiderangeofinfections,suchasthoseinfectedwithbiofilmduringsurgery,canlikelyrestoretheirhealthwiththeMP.

ItistobehopedthattheclinicaldataemergingfromtheMarshallProtocolstudysite,whichshowspatientsrecoveringfrombiofilmrelateddiseases,willinspirefutureresearcherstoinvestagreatdealofenergyintofurtherresearchaimedatidentifyingandstudyingthebiofilmbacteriabacteriathatalmostcertainlyformpartofthemicrobiotaofpathogensthatcauseinflammatorydisease.Inthecomingyears,asthetechnologytodetectbiofilmsbecomesevenmoresophisticated,itisalmostcertainthatagreatnumberofbiofilmswillbeofficiallydetectedanddocumentedinpatientswithavastarrayofchronicdiseases.

REFERENCES

[29]

Costerton,J.W.,Stewart,P.S.,&Greenberg,E.P.(1999).Bacterialbiofilms:acommoncauseofpersistentinfections.Science(NewYork,N.Y.),284(5418),131822.[ ][ ][][ ]

Higgins,D.A.,Pomianek,M.E.,Kraml,C.M.,Taylor,R.K.,Semmelhack,M.F.,&Bassler,B.L.(2007).ThemajorVibriocholeraeautoinduceranditsroleinvirulencefactorproduction.Nature,450(7171),8836.[ ]

Singh,P.K.,Schaefer,A.L.,Parsek,M.R.,Moninger,T.O.,Welsh,M.J.,&Greenberg,E.P.(2000).Quorumsensingsignalsindicatethatcysticfibrosislungsareinfectedwithbacterialbiofilms.Nature,407(6805),7624.[ ][ ]

Stoodley,P.,PurevdorjGage,B.,&Costerton,J.W.(2005).Clinicalsignificanceofseedingdispersalinbiofilms:aresponse.Microbiology,151(11),3453.[ ]

Otoole,G.A.,&Kolter,R.(1998).FlagellarandTwitchingMotilityAreNecessaryforPseudomonasAeruginosaBiofilmDevelopment.MolecularMicrobiology,30(2),295304.[ ]

Cho,H.,Jnsson,H.,Campbell,K.,Melke,P.,Williams,J.W.,Jedynak,B.,etal.(2007).SelfOrganizationinHighDensityBacterialColonies:EfficientCrowdControl.PLoSBiology,5(11),e302EP.[ ][ ]

Brockhurst,M.A.,Hochberg,M.E.,Bell,T.,&Buckling,A.(2006).Characterdisplacementpromotescooperationinbacterialbiofilms.Currentbiology:CB,16(20),20304.[ ]

Parsek,M.R.,&Singh,P.K.(2003).Bacterialbiofilms:anemerginglinktodiseasepathogenesis.Annualreviewofmicrobiology,57,677701.[ ]

Kraigsley,A.,Ronney,P.,&Finkel,S.Hydrodynamiceffectsonbiofilmformation.RetrievedMay28,2008.[ ]

HallStoodley,L.,Costerton,J.W.,&Stoodley,P.(2004).Bacterialbiofilms:fromtheNaturalenvironmenttoinfectiousdiseases.NatRevMicro,2(2),95108.[ ][ ][ ]

Lewis,K.(2001).Riddleofbiofilmresistance.Antimicrobialagentsandchemotherapy,45(4),9991007.[ ][ ]

Parsek,M.R.,&Singh,P.K.(2003).Bacterialbiofilms:anemerginglinktodiseasepathogenesis.Annualreviewofmicrobiology,57,677701.[ ]

Trampuz,A.,Piper,K.E.,Jacobson,M.J.,Hanssen,A.D.,Unni,K.K.,Osmon,D.R.,etal.(2007).SonicationofRemovedHipandKneeProsthesesforDiagnosisofInfection.NEnglJMed,357(7),654663.[ ]

Ristow,P.,Bourhy,P.,Kerneis,S.,Schmitt,C.,Prevost,M.,Lilenbaum,W.,etal.(2008).Biofilmformationbysaprophyticandpathogenicleptospires.Microbiology,154(5),13091317.[ ]

MoreauMarquis,S.,Stanton,B.A.,&OToole,G.A.(2008).Pseudomonasaeruginosabiofilmformationinthecysticfibrosisairway.Pulmonarypharmacology&therapeutics.[]

HallStoodley,L.,Hu,F.Z.,Gieseke,A.,Nistico,L.,Nguyen,D.,Hayes,J.,etal.(2006).DirectDetectionofBacterialBiofilmsontheMiddleEarMucosaofChildrenWithChronicOtitisMedia.JAMA,296(2),202211.[ ]

Imamura,Y.,Chandra,J.,Mukherjee,P.K.,Lattif,A.A.,SzczotkaFlynn,L.B.,Pearlman,E.,etal.(2008).FusariumandCandidaalbicansBiofilmsonSoftContactLenses:ModelDevelopment,InfluenceofLensType,andSusceptibilitytoLensCareSolutions.Antimicrob.AgentsChemother.,52(1),171182.[ ]

James,G.A.,Swogger,E.,Wolcott,R.,Pulcini,E.D.,Secor,P.,Sestrich,J.,etal.(2008).BiofilmsinChronicWounds.WoundRepairandRegeneration,16(1),3744.[ ]

Marshall,T.G.(2006b).ANewApproachtoTreatingIntraphagocyticCWDBacterialPathogensinSarcoidosis,CFS,LymeandotherInflammatoryDiseases.[ ][ ]

Marshall,T.G.,&Marshall,F.E.(2004).Sarcoidosissuccumbstoantibioticsimplicationsforautoimmunedisease.Autoimmunityreviews,3(4),295300.[ ][ ]

Sr,G.J.D.,&Woody,H.B.(1997).Bacterialpersistenceandexpressionofdisease.ClinicalMicrobiologyReviews,10(2).[ ]

Marshall,T.G.(2007).BacterialCapnineBlocksTranscriptionofHumanAntimicrobialPeptides.NaturePrecedings.[ ]

Morrison,H.I.,Ellison,L.F.,&Taylor,G.W.(1999).Periodontaldiseaseandriskoffatalcoronaryheartandcerebrovasculardiseases.Journalofcardiovascularrisk,6(1),711.[ ]

Stewart,R.,&Hirani,V.(2007).DentalHealthandCognitiveImpairmentinanEnglishNationalSurveyPopulation.JournaloftheAmericanGeriatricsSociety,55(9),14101414.[ ]

FalkinhamIii,J.O.,Iseman,M.D.,Haas,P.D.,&Soolingen,D.V.(2008).Mycobacteriumaviuminashowerlinkedtopulmonarydisease.Journalofwaterandhealth,6(2),20913.[ ]

Lewis,K.(2001).Riddleofbiofilmresistance.Antimicrobialagentsandchemotherapy,45(4),9991007.[ ]

Starner,TimothyDetal.2008.SubinhibitoryConcentrationsofAzithromycinDecreaseNontypeableHaemophilusinfluenzaeBiofilmFormationandDiminishEstablishedBiofilms.Antimicrobialagentsandchemotherapy52(1):13745.[ ]

Cogan,N.G.,Cortez,R.,&Fauci,L.(2005).Modelingphysiologicalresistanceinbacterialbiofilms.Bulletinofmathematicalbiology,67(4),83153.[ ]

Marshall,T.G.(2006).VDRNuclearReceptorCompetenceistheKeytoRecoveryfromChronicInflammatoryandAutoimmuneDisease.[ ]

RSSfeedforcommentsonthispost

Commentsareclosed.

Filedunder:biofilms,featuredarticles

Copyright2014Bacteriality