understanding biofilms
DESCRIPTION
Overview of Biofilms.TRANSCRIPT
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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
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NEWS FLASH
April4,2009:,MilkconsumptiontiedtoParkinsonsdiseaseMarch21,2009:,Heythere,howsyourKineosphaeramholdingup?
PeerReviewed Papers
Autoimmunediseaseintheeraofthemetagenome(PDF)VitaminD:thealternativehypothesis(PDF)DysregulationoftheVitaminDNuclearReceptormaycontributetothehigherprevalenceofsomeautoimmunediseasesinwomen(PDF)VitaminDmetabolitesasclinicalmarkersinautoimmuneandchronicdisease(PDF)
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MetagenomicsymbiosisbetweenbacterialandviralpathogensinautoimmunediseaseNotesfromthe2009InternationalCongressofAntibodiesNotesfromthe2008InternationalCongressonAutoimmunity
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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.
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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.
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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
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statinsUncategorizedvideosvitaminD
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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
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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,
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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.
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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
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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
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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
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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
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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
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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
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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
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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]
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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.
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