AboutBacteriality

26
MAY

2008

AbouttheMarshallProtocol

NEWSFLASH(archives)

Understanding Biofilms

NEWS FLASH

Author:AmyProal

April4,2009:,Milkconsumptiontiedto

Ashumans,ourenvironmentconsistentlyexposesustoavarietyofdangers.
Tornadoes,lightning,floodingandhurricanescanallhamperoursurvival.Not
tomentionthefactthatmostofuscanencounterswervingcarsorill
intentionedpeopleatanygivenmoment.
Thousandsofyearsago,humans
realizedthattheycouldbettersurvivea
dangerousworldiftheyformedinto
communities,particularlycommunities
consistingofpeoplewithdifferent
talents.Theyrealizedthatacommunity
isfarmorelikelytosurvivethrough
divisionoflaboronepersonmakes
food,anothergathersresources,still
anotherprotectsthecommunityagainst
invaders.Workingtogetherinthis
mannerrequirescommunicationand
cooperation.
Inhabitantsofacommunityliveinclose
proximityandcreatevariousformsof
shelterinordertoprotectthemselves
fromexternalthreats.Webuildhouses
thatprotectourfamiliesandlarger
buildingsthatprotecttheentire
community.Groupingtogetherinside
placesofshelterisalogicalwayto
enhancesurvival.

Parkinsonsdisease
March21,2009:,Heythere,howsyour

Kineosphaeramholdingup?

PeerReviewed Papers
Autoimmunediseaseintheeraofthe
metagenome(PDF)
VitaminD:thealternativehypothesis(PDF)
DysregulationoftheVitaminDNuclear
Receptormaycontributetothehigher
prevalenceofsomeautoimmunediseasesin
women(PDF)
VitaminDmetabolitesasclinicalmarkersin
autoimmuneandchronicdisease(PDF)

Amy's Conference Presentations

Metagenomicsymbiosisbetweenbacterial
andviralpathogensinautoimmunedisease
Notesfromthe2009InternationalCongress
ofAntibodies

Biofilmsformwhenbacteriaadhereto
surfacesinaqueousenvironmentsand
begintoexcreteaslimy,gluelike
substancethatcananchorthemtoall
kindsofmaterial

Withtheaboveinmind,itshouldcome
asnosurprisethatthepathogensweharborareseldomfoundassingle
entities.Althoughthepathogensthatcauseacuteinfectionaregenerallyfree
floatingbacteriaalsoreferredtoasplanktonicbacteriathosechronic
bacterialformsthatstickaroundfordecadeslongagoevolvedwaystojoin
togetherintocommunities.Why?Becausebydoingso,theyarebetterableto
combatthecellsofourimmunesystembentupondestroyingthem.
Itturnsoutthatavastnumberofthepathogensweharboraregroupedinto
communitiescalledbiofilms.InanarticletitledBacterialBiofilms:ACommon
CauseofPersistentInfections,JWCostertonattheCenterforBiofilm
EngineeringinMontanadefinesabacterialbiofilmasastructuredcommunity
ofbacterialcellsenclosedinaselfproducedpolymericmatrixandadherentto
aninertorlivingsurface. [1]Inlaymansterms,thatmeansthatbacteriacan
jointogetheronessentiallyanysurfaceandstarttoformaprotectivematrix
aroundtheirgroup.Thematrixismadeofpolymerssubstancescomposedof
moleculeswithrepeatingstructuralunitsthatareconnectedbychemical
bonds.
AccordingtotheCenterforBiofilmEngineeringatMontanaStateUniversity,
biofilmsformwhenbacteriaadheretosurfacesinaqueousenvironmentsand
begintoexcreteaslimy,gluelikesubstancethatcananchorthemtoallkinds
ofmaterialsuchasmetals,plastics,soilparticles,medicalimplantmaterials
and,mostsignificantly,humanoranimaltissue.Thefirstbacterialcoloniststo
adheretoasurfaceinitiallydosobyinducingweak,reversiblebondscalled
vanderWaalsforces.Ifthecolonistsarenotimmediatelyseparatedfromthe
surface,theycananchorthemselvesmorepermanentlyusingcelladhesion
molecules,proteinsontheirsurfacesthatbindothercellsinaprocesscalled
celladhesion.
Thesebacterialpioneersfacilitatethe
arrivalofotherpathogensbyproviding
morediverseadhesionsites.Theyalso
begintobuildthematrixthatholdsthe
biofilmtogether.Iftherearespecies
thatareunabletoattachtoasurfaceon
theirown,theyareoftenabletoanchor
themselvestothematrixordirectlyto
earliercolonists.
Duringcolonization,thingsstarttoget
interesting.Multiplestudieshaveshown

Notesfromthe2008InternationalCongress
onAutoimmunity

Featured Articles
Updateontoneandotherissues
Whypatientswithchronicdiseaseare
disaffectedandhowonlinesocialnetworks
meettheirneeds
SunblockingcultureamongtheChinese
Secondguessingtheconsensusonvitamin
D
Travels,papers,andmoreanupdate
ThreedaysattheJ.CraigVenterInstitute
Thebacteriaboomimplicationsofthe
HumanMicrobiomeProject
UnderstandingBiofilms
InterviewwithDr.RandallWolcott,bacterial
biofilmwoundspecialist
Insightsintohorizontalgenetransfer:
conversationswithDr.PeterGogartenand
Dr.JamesLake
VoicesofreasoninthevitaminDdebate
InterviewwithevolutionarybiologistPaul
Ewald
InterviewwithDr.GregBlaney:MPphysician
Bacteriaandcancer:aninterviewwithDr.
AlanCantwell
InterviewwithNadyaMarkova:Lform
Expert
GeraldDomingue:PioneerofAtypical
Bacteria
AHistoryofCellWallDeficientBacteria:A
SelectionofResearchersWhoHaveWorked
withtheLform

Patient Interviews
InterviewwithGeneJohnsonsarcoidosis,
bladdercancer
InterviewwithBonnieBlupus,Sjogrens
Syndrome
InterviewwithChrisEastlunddiabetes,
sarcoidosis,irritablebowelsyndrome

thatduringthetimeabiofilmisbeing
created,thepathogensinsideitcan
communicatewitheachotherthankstoaphenomenoncalledquorumsensing.
Althoughthemechanismsbehindquorumsensingarenotfullyunderstood,the
phenomenonallowsasinglecelledbacteriumtoperceivehowmanyother
bacteriaareincloseproximity.Ifabacteriumcansensethatitissurrounded
byadensepopulationofotherpathogens,itismoreinclinedtojointhemand
contributetotheformationofabiofilm.
Abiofilminthegut.

Bacteriathatengageinquorumsensingcommunicatetheirpresenceby
emittingchemicalmessagesthattheirfellowinfectiousagentsareableto
recognize.Whenthemessagesgrowstrongenough,thebacteriaresponden
masse,behavingasagroup.Quorumsensingcanoccurwithinasingle
bacterialspeciesaswellasbetweendiversespecies,andcanregulateahost
ofdifferentprocesses,essentiallyservingasasimplecommunicationnetwork.
Avarietyofdifferentmoleculescanbeusedassignals.
Diseasecausingbacteriatalktoeachotherwithachemicalvocabulary,says
DougHibbinsofPrincetonUniversity.Agraduatestudentinthelabof
PrincetonUniversitymicrobiologistDr.BonnieBassler,Hibbinswaspartofa
researcheffortwhichshedlightonhowthebacteriathatcausecholeraform
biofilmsandcommunicateviaquorumsensing. [2]
Formingabiofilmisoneofthecrucialstepsincholerasprogression,states
Bassler.They[bacteria]coverthemselvesinasortofgoopthatsashield
againstantibiotics,allowingthemtogrowrapidly.Whentheysensethereare
enoughofthem,theytrytoleavethebody.
Althoughcholerabacteriausetheintestinesasabreedingground,after
enoughbiofilmshaveformed,planktonicbacteriainsidethebiofilmseekto
leavethebodyinordertoinfectanewhost.ItdidnttakelongforBasslerand
teamtorealizethatthebacteriainsidecholerabiofilmsmustsignaleachother
inordertocommunicatethatitstimeforthecolonytostopreproducingand
focusinsteadonleavingthebody.
Wegenericallyunderstoodthatbacteriatalktoeachotherwithquorum
sensing,butwedidntknowthespecificchemicalwordsthatcholerauses,
Basslersaid.

InterviewwithRoyP.sarcoidosis,
rheumatoidarthritis
InterviewwithPeterdeJager:chronic
fatiguesyndrome,multiplechemical
sensitivity
InterviewwithKenL.PostTreatment
LymeDiseaseSyndrome(PTLDS)
InterviewwithDoreenV.autism,ADHD
depression,severeanxiety,CFS
InterviewwithJST:Neurosarcoidosis
InterviewwithMelindaStilesLyme,
Irritablebowelsyndrome/colitis,Radiculitis
(inflammationofthenerveroots)
InterviewwithFreddieAshSarcoidosisof
theheart,coronaryarterydisease,atrial
fibrillation
InterviewwithP.BearR.N.Chronic
Borreliosis(Lyme),MCS(multiplechemical
sensitivities),ChronicSpinalInflammation,
PeripheralNeuropathy
InterviewwithSherryCookSarcoidosis,
CatScratchFever,RestlessLegSyndrome
InterviewwithLeesaShanahan
Sarcoidosis(HeerfordtsSyndrome),Uveitis
InterviewwithMirekWozgasarcoidosis
InterviewwithPaulAlbertCFS,depression,
foodsensitivities
InterviewwithCaroleMorganSarcoidosis,
fibromyalgia,CFS
InterviewwithShirleyJ.(Saj)Sarcoidosis
InterviewwithSueAndornLyme,Babesia
InterviewwithIvalMeyerArthritis,
dyslexia

About Amy Proal

ThenHigginsisolatedtheCAI1achemicalwhichoccursnaturallyincholera.
Anothergraduatestudentfiguredouthowtomakethemoleculeinthe
laboratory.BymoderatingthelevelofCAI1incontactwithcholerabacteria,
Higginswassuccessfullyabletochemicallycontrolcholerasbehaviorinlab
tests.HisteameventuallyconfirmedthatwhenCAI1isabsent,cholera
bacteriaattachinbiofilmstotheircurrenthost.Butwhenthebacteriadetect
enoughofthechemical,theystopmakingbiofilmsandreleasingtoxins,
perceivingthatitistimetoleavethebodyinstead.Thus,CAI1mayverywell
bethesinglemoleculethatallowthebacteriainsideacholerabiofilmto
communicate.Althoughitislikelythatthebacteriainacholerabiofilmmay
communicatewithothersignalsbesidesCAI1,thestudyisagoodexampleof
thefactthatsignalingmoleculesserveakeyroleindeterminingthestateofa
biofilm.

AmyProalgraduated
fromGeorgetown
Universityin2005with
adegreeinbiology.
WhileatGeorgetown,
shewrotehersenior
thesisonChronic
FatigueSyndromeand
theMarshallProtocol.

Similarly,researchersatthe
UniversityofIowa(severalof
whomarenowatthe
UniversityofWashington)have
spentthelastdecade
identifyingthemoleculesthat
allowthebacterialspeciesP.
aeruginosatoformbiofilmsin
thelungsofpatientswithcystic
fibrosis. [3]AlthoughtheP.
auruginosaisolatedfromthe
Sessilecellsinabiofilmtalktoeachothervia
lungsofpatientswithcystic
quorumsensingtobuildmicrocoloniesandto
fibrosislookslikeabiofilmand
keepwaterchannelsopen.
actslikeabiofilm,upuntil
recently,therewereno
objectivetestsavailabletoconfirmthatthebacterialspeciesdidindeedform
biofilmsinthelungsofpatientswiththedisease,norwasthereawaytotell
whatproportionofP.aeruginosainthelungswereactuallyinbiofilmmode.

IfyouhavequestionsabouttheMP,pleasevisit
CureMyTh1.orgwherevolunteerpatient
advocateswillansweryourquestions.Another
goodresourceistheMPKnowledgeBase,which
isscheduledtobecompletedwithinthenext
year.

Amyhasspokenat
severalinternational
conferencesandauthoredseveralpeerreviewed
papersontheintersectionofbacteriaand
chronicdisease.

Categories
biofilms
cancer
cardiovasculardisease
cognitivedysfunction
conferencesandtrainings
diet
familialaggregation
featuredarticles

WeneededawaytoshowthattheP.auruginosaincysticfibrosislungswas
communicatinglikeabiofilm.ThatcouldtellusabouttheP.
auruginosalifestyle,saidPradeepSingh,M.D.,aleadauthoronthestudywho
isnowattheUniversityofWashington.

history

SinghandhiscolleaguesfinallydiscoveredthatP.aeruginosausesoneoftwo
particularquorumsensingmoleculestoinitiatetheformationofbiofilms.In
November1999,hisresearchteamscreenedtheentirebacterialgenome,
identifying39genesthatarestronglycontrolledbythequorumsensing
system.

lformbacteria

Ina2000studypublishedinNature,Singhandcolleaguesdevelopeda
sensitivetestwhichshowsP.auruginosafromcysticfibrosislungsproduces
thetelltale,quorumsensingmoleculesthatarethesignalsforbiofilm
formation. [3]

horizontalgenetransfer
interview(doctor/researcher)
interview(patient)
marshallprotocol
medicalresearch
mentalillness
microbiome
NewsFlash
obesity
personal
presentations

ItturnsoutthatP.aerugnosasecretestwosignalingmolecules,onethatis
long,andanotherthatisshort.Usingthenewtest,theteamwasabletoshow
thatplanktonicformsofP.aeruginosaproducemorelongsignalingmolecules.
Alternately,whentheytestedtheP.aeruginosastrainsisolatedfromthelungs
ofpatientswithcysticfibrosis(whichwereinbiofilmform),allofthestrains
producedthesignalingmolecules,butintheoppositeratiomoreshortthan
long.
Interestingly,whenthebiofilmstrainsofP.aeruginosawereseparatedin
brothintoindividualbacterialforms,theyrevertedtoproducingmorelong
signalmoleculesthanshortones.Doesthismeanthatachangeinsignaling
molecularlengthcanindicatewhetherbacteriaremainasplanktonicformsor
developintobiofilms?
Tofindout,theteamtookthebacteriafromthebrothandmadethemgrowas
abiofilmagain.Sureenough,thosestrainsofbacteriainbiofilmform
producedmoreshortsignalmoleculesthanlong.
ThefactthattheP.aeruginosain[thelungsofcysticfibrosispatients]is
makingthesignalsintheratiosthatweseetellsusthatthereisabiofilmand
thatmostoftheP.aeruginosainthelungisinthebiofilmstate,states
Greenberg,anothermemberoftheresearchteam.Hebelievesthatthe
findingsallowforaclearbiochemicaldefinitionofwhetherbacteriaareina
biofilm.Techniquessimilartothoseusedbyhisgroupwilllikelybeusedto
determinethepropertiesofotherbiofilmsignalingmolecules.

Development
Oncecolonizationhasbegun,thebiofilmgrowsthroughacombinationofcell
divisionandrecruitment.Thefinalstageofbiofilmformationisknownas
developmentandisthestageinwhichthebiofilmisestablishedandmayonly
changeinshapeandsize.Thisdevelopmentofabiofilmallowsforthecells
insidetobecomemoreresistanttoantibioticsadministeredinastandard
fashion.Infact,dependingontheorganismandtypeofantimicrobialand
experimentalsystem,biofilmbacteriacanbeuptoathousandtimesmore
resistanttoantimicrobialstressthanfreeswimmingbacteriaofthesame
species.
Biofilmsgrowslowly,indiverselocations,andbiofilminfectionsareoftenslow
toproduceovertsymptoms.However,biofilmbacteriacanmoveinnumerous
waysthatallowthemtoeasilyinfectnewtissues.Biofilmsmaymove
collectively,byripplingorrollingacrossthesurface,orbydetachingin
clumps.Sometimes,inadispersalstrategyreferredtoasswarming/seeding,
abiofilmcolonydifferentiatestoformanouterwallofstationarybacteria,
whiletheinnerregionofthebiofilmliquefies,allowingplanktoniccellsto
swimoutofthebiofilmandleavebehindahollowmound. [4]
Researchonthemolecularand
geneticbasisofbiofilm
developmenthasmadeitclear
thatwhencellsswitchfrom
planktonictocommunitymode,
theyalsoundergoashiftin
behaviorthatinvolves
alterationsintheactivityof
numerousgenes.Thereis
evidencethatspecificgenes
mustbetranscribedduringthe
attachmentphaseofbiofilm
development.Inmanycases,
theactivationofthesegenesis
requiredforsynthesisofthe
extracellularmatrixthat
protectsthepathogensinside.

Biofilmbacteriacanmoveinnumerousways:
Collectively,byripplingorrollingacrossthe
surface,orbydetachinginclumps.Individually,
throughaswarmingandseedingdispersal.

AccordingtoCosterton,the
genesthatallowabiofilmtodevelopareactivatedafterenoughcellsattachto
asolidsurface.Thus,itappearsthatattachmentitselfiswhatstimulates
synthesisoftheextracellularmatrixinwhichthesessilebacteriaare
embedded,statesthemolecularbiologist.Thisnotionthatbacteriahavea
senseoftouchthatenablesdetectionofasurfaceandtheexpressionof
specificgenesisinitselfanexcitingareaofresearch [1]
Certaincharacteristicsmayalsofacilitatetheabilityofsomebacteriatoform
biofilms.ScientistsattheDepartmentofMicrobiologyandMolecularGenetics,
HarvardMedicalSchool,performedastudyinwhichtheycreatedamutant
formofthebacterialspeciesP.aeguinosa(PA). [5]Themutantslackedgenes
thatcodeforhairlikeappendagescalledpili.Interestingly,themutantswere
unabletoformbiofilms.SincethepiliofPAareinvolvedinatypeofsurface
associatedmotilitycalledtwitching,theteamhypothesizedthistwitchingmight
berequiredfortheaggregationofcellsintothemicrocoloniesthat
subsequentlyformastablebiofilm.
Onceabiofilmhasofficiallyformed,itoftencontainschannelsinwhich
nutrientscancirculate.Cellsindifferentregionsofabiofilmalsoexhibit
differentpatternsofgeneexpression.Becausebiofilmsoftendeveloptheir
ownmetabolism,theyaresometimescomparedtothetissuesofhigher

statins
Uncategorized
videos
vitaminD

organisms,inwhichcloselypackedcellsworktogetherandcreateanetwork
inwhichmineralscanflow.
Thereisaperceptionthatsinglecelledorganismsareasocial,butthatis
misguided,saidAndreLevchenko,assistantprofessorofbiomedical
engineeringinJohnsHopkinsUniversitysWhitingSchoolofEngineeringandan
affiliateoftheUniversitysInstituteforNanoBioTechnology.Whenbacteria
areunderstresswhichisthestoryoftheirlivestheyteamupandformthis
collectivecalledabiofilm.Ifyoulookatnaturallyoccurringbiofilms,they
haveverycomplicatedarchitecture.Theyarelikecitieswithchannelsfor
nutrientstogoinandwastetogoout. [6]
Understandinghowsuch
cooperationamongpathogens
evolvesandismaintained
representsoneofevolutionary
biologysthorniestproblems.
Thisstemsfromthereality
that,innature,freeloading
cheatsinevitablyevolveto
exploitanycooperativegroup
thatdoesntdefenditself,
leadingtothebreakdownof
Thebiofilmlifecycleinthreesteps:
cooperation.Sowhatcauses
attachment,growthofcolonies(development),
thebacteriainabiofilmto
andperiodicdetachmentofplanktoniccells.
contributetoandshare
resourcesratherthansteal
them?Recently,Dr.MichaelBrockhurstoftheUniversityofLiverpooland
colleaguesattheUniversitMontpellierandtheUniversityofOxfordconducted
severalstudiesinanefforttounderstandwhythebacteriainabiofilm
cooperateandshareresourcesratherthanhordethem. [7]
TheteamtookacloserlookatP.fluorescensbiofilms,whichareformedwhen
individualcellsoverproduceapolymerthatsticksthecellstogether,allowing
thecolonizationofliquidsurfaces.Whileproductionofthepolymeris
metabolicallycostlytoindividualcells,thebiofilmgroupbenefitsfromthe
increasedaccesstooxygenthatsurfacecolonizationprovides.Yet,
evolutionarilyspeaking,suchasetupallowspossiblecheaterstoenterthe
biofilm.Suchcheatscantakeadvantageoftheprotectivematrixwhilefailing
tocontributeenergytoactuallybuildingthematrix.Iftoomanycheaters
enterabiofilm,itwillweakenandeventuallybreakapart.
Afterseveralyearsofstudy,Brockhurstandteamrealizedthattheshortterm
evolutionofdiversitywithinabiofilmisamajorfactorinhowsuccessfullyits
memberscooperate.Theteamfoundthatonceinsideabiofilm,P.
fluorescensdifferentiatesintovariousforms,eachofwhichusesdifferent
nutrientresources.Thefactthatthesediversecooperatorsdontallcompete
forthesamechemicalsandnutrientssubstantiallyreducescompetitionfor
resourceswithinthebiofilm.
Whentheteammanipulateddiversitywithinexperimentalbiofilms,theyfound
thatdiversebiofilmscontainedfewercheatersandproducedlargergroups
thannondiversebiofilms.
Similarly,thisyear,researchersfromJohns
HopkinsVirginiaTechtheUniversityof
California,SanDiegoandLundUniversityin
Swedenrecentlyreleasedtheresultsofa
studywhichfoundthatoncebacteria
cooperateandformabiofilm,packingtightly
togetherfurtherenhancestheirsurvival. [6]
Theteamcreatedanewdeviceinorderto
observethebehaviorofE.colibacteriaforced
togrowinthecrampedconditions.The
device,whichallowsscientiststouse
extremelysmallvolumesofcellsinsolution,
containsaseriesoftinychambersofvarious
shapesandsizesthatkeepthebacteria
uniformlysuspendedinaculturemedium.
Notsurprisingly,thecrampedbacteriainthe
devicebegantoformabiofilm.Theteam
capturedthedevelopmentofthebiofilmon
video,andwereabletoobservethegradual
selforganizationandeventualconstructionof
bacterialbiofilmsovera24hourperiod.

Levchenkoandteamusedthis
devicetoobservebacteria
growingincrampedconditions.

First,AndreLevchenkoandHojungChoofJohnsHopkinsrecordedthebehavior
ofsinglelayersofE.colicellsusingrealtimemicroscopy.Weweresurprised
tofindthatcellsgrowinginchambersofallsortsofshapesgradually
organizedthemselvesintohighlyregularstructures,Levchenkosaid.
Furtherobservationsusingmicroscopy
revealedthatthelongerthepackedcell
populationresidedinthechambers,themore
orderedthebiofilmstructurebecame.Asthe
cellsinthebiofilmbecamemoreorderedand

tightlypacked,thebiofilmbecameharderand
hardertopenetrate.

Dr.LevchenkoofJohns
HopkinsandHojungCho,a
biomedicalengineering
doctoralstudent

LevchenkoalsonotedthatrodshapedE.
colithatweretooshortortoolongtypically
didnotorganizewellintothedense,circular
mainhubofthebiofilm.Instead,thebacteria
ofoddshapesorhighlydisorderedgroupsof
cellswerefoundontheedgesofthebiofilm,
wheretheyformedsharpcorners.

Nodes of relapsing infection?

Researchersoftennotethat,oncebiofilmsare
established,planktonicbacteriamayperiodicallyleavethebiofilmontheir
own.Whentheydo,theycanrapidlymultiplyanddisperse.
AccordingtoCosterton,thereisanaturalpatternofprogrammeddetachment
ofplanktoniccellsfrombiofilms.Thismeansthatbiofilmscanactaswhat
Costertonreferstoasnidusesofacuteinfection.Becausethebacteriaina
biofilmareprotectedbyamatrix,thehostimmunesystemislesslikelyto
mountaresponsetotheirpresence. [1]
Butifplanktonicbacteriaareperiodicallyreleasedfromthebiofilms,each
timesinglebacterialformsenterthetissues,theimmunesystemsuddenly
becomesawareoftheirpresence.Itmayproceedtomountaninflammatory
responsethatleadstoheightenedsymptoms.Thus,theperiodicreleaseof
planktonicbacteriafromsomebiofilmsmaybewhatcausesmanychronic
relapsinginfections.
AsMatthewR.Parsekof
NorthwesternUniversity
describesina2003paperin
theAnnualReviewof
Microbiology,anypathogenthat
survivesinachronicform
benefitsbykeepingthehost
alive. [8]Afterall,ifachronic
bacterialformsimplykillsits
Planktonicbacteriaareperiodicallyreleased
host,itwillnolongerhavea
fromabiofilm
placetolive.Soaccordingto
Parsek,chronicinfectionoften
resultsinadiseasestalematewherebacteriaofmoderatevirulenceare
somewhatcontainedbythedefensesofthehost.Theinfectiousagentsnever
actuallykillthehost,butthehostisneverabletofullykilltheinvading
pathogenseither.
Parsekbelievesthattheoptimalwayforbacteriatosurviveundersuch
circumstancesisinabiofilm,statingthatIncreasingevidencesuggeststhat
thebiofilmmodeofgrowthmayplayakeyroleinbothoftheseadaptations.
Biofilmgrowthincreasestheresistanceofbacteriatokillingandmaymake
organismslessconspicuoustotheimmunesystemultimatelythismoderation
ofvirulencemayservethebacteriasinterestbyincreasingthelongevityof
thehost.

The acceptance of biofilms as infectious entities

Perhapsbecausemanybiofilmsare
sufficientlythicktobevisibletothenaked
eye,themicrobialcommunitieswereamong
thefirsttobestudiedbyearly
microbiologists.AntonvanLeeuwenhoek
scrapedtheplaquebiofilmfromhisteethand
observedwhathedescribedasthe
animalculiinsidethemunderhisprimitive
microscope.However,accordingtoCosterton
andteamattheCenterforBiofilmResearch
atMontanaStateUniversity,itwasnotuntil
the1970sthatscientistsbegantoappreciate
thatbacteriainthebiofilmmodeofexistence
constitutesuchamajorcomponentofthe
bacterialbiomassinmostenvironments.
AntonvanLeeuwenhoek.
Then,itwasnotuntilthe1980sand1990s
thatscientiststrulybegantounderstandhow
elaboratelyorganizedabacterialbiofilmcommunitycanbe. [1]
AsRobertKolter,professorofmicrobiologyandmoleculargeneticsatHarvard
MedicalSchool,andoneofthefirstscientiststostudyhowbiofilms
developstates,Atfirst,however,studyingbiofilmswasaradicaldeparture
frompreviouswork.
Likemostmicrobialgeneticists,Kolterhadbeentrainedinthetraditiondating
backtoRobertKochandLouisPasteur,namelythatbacteriologyisbest
conductedbystudyingpurestrainsofplanktonicbacteria.Whilethiswasa
tremendousadvanceformodernmicrobiology,italsodistracted
microbiologistsfromamoreorganismicviewofbacteria,Kolteradds,

Certainlywefeltthatpure,planktonicculturesweretheonlywaytowork.Yet
innaturebacteriadontlivelikethat,hesays.Infact,mostofthemoccurin
mixed,surfacedwellingcommunities.
Althoughresearchonbiofilmshassurgedoverthepastfewdecades,the
majorityofbiofilmresearchtodatehasfocusedonexternalbiofilms,orthose
thatformonvarioussurfacesinournaturalenvironment.
Overthepastyears,asscientistsdevelopedbettertoolstoanalyzeexternal
biofilms,theyquicklydiscoveredthatbiofilmscancauseawiderangeof
problemsinindustrialenvironments.Forexample,biofilmscandeveloponthe
interiorsofpipes,whichcanleadtocloggingandcorrosion.Biofilmsonfloors
andcounterscanmakesanitationdifficultinfoodpreparationareas.
Sincebiofilmshavetheabilitytoclogpipes,watersheds,storageareas,and
contaminatefoodproducts,largecompanieswithfacilitiesthatarenegatively
impactedbytheirpresencehavenaturallytakenaninterestinsupporting
biofilmresearch,particularlyresearchthatspecifieshowbiofilmscanbe
eliminated.
Thismeansthatmanyrecentadvancesinbiofilmdetectionhaveresultedfrom
collaborationsbetweenmicrobialecologists,environmentalengineers,and
mathematicians.Thisresearchhasgeneratednewanalyticaltoolsthathelp
scientistsidentifybiofilms.
Forexample,theCanadiancompanyFAS
InternationalLtd.hasjustcreatedan
endoluminalbrush,whichwillbe
launchedthisspring.Physicianscanuse
thebrushtoobtainsamplesfromthe
interiorofcatheters.Samplestaken
fromcatheterscanbesenttoalab,
whereresearchersdetermineifbiofilms
arepresentinthesample.Ifbiofilmsare
detected,thecatheterisimmediately
replaced,sincetheinsertionofcatheters
withbiofilmscancausethepatientto
sufferfromnumerousinfections,some
ofwhicharepotentiallylifethreatening.
Scientistsnowrealizethatbiofilmsare
notjustcomposedofbacteria.Nearly
everyspeciesofmicroorganism
Biofilminaswampgasreactor.
includingviruses,fungi,andArchaea
havemechanismsbywhichtheycan
adheretosurfacesandtoeachother.Furthermore,itisnowunderstoodthat
biofilmsareextremelydiverse.Forexample,upwardof300differentspecies
ofbacteriacaninhabitthebiofilmsthatformdentalplaque. [9]
Furthermore,biofilmshavebeenfoundliterallyeverywhereinnature,tothe
pointwhereanymainstreammicrobiologistwouldacknowledgethattheir
presenceisubiquitous.Theycanbefoundonrocksandpebblesatthebottom
ofmoststreamsorriversandoftenformonthesurfaceofstagnantpoolsof
water.Infact,biofilmsareimportantcomponentsoffoodchainsinriversand
streamsandaregrazeduponbytheaquaticinvertebratesuponwhichmany
fishfeed.Biofilmsevengrowinthehot,acidicpoolsatYellowstoneNational
ParkandonglaciersinAntarctica.
Itisalsonowunderstoodthatthebiofilm
modeofexistencehasbeenaroundfor
millenia.Forexample,filamentousbiofilms
havebeenidentifiedinthe3.2billionyearold
deepseahydrothermalrocksofthePilbara
Craton,Australia.Accordingtoa2004article
inNatureReviewsMicrobiology,Biofilm
formationappearsearlyinthefossilrecord
(approximately3.25billionyearsago)andis
commonthroughoutadiverserangeof
organismsinboththeArchaeaandBacteria
lineages.Itisevidentthatbiofilmformationis
anancientandintegralcomponentofthe
prokaryoticlifecycle,andisakeyfactorfor
survivalindiverseenvironments. [10]

Biofilms and disease

Thefactthatexternalbiofilmsareubiquitous
raisesthequestionifbiofilmscanformon
essentiallyeverysurfaceinourexternal
environments,cantheydothesameinsidethe
humanbody?Theanswerseemstobeyes,andoverthepastfewyears,
researchoninternalbiofilmshasfinallystartedtopickuppace.Afterall,its
easyforbiofilmresearcherstoseethatthehumanbody,withitswiderange
ofmoistsurfacesandmucosaltissue,isanexcellentplaceforbiofilmsto
thrive.Nottomentionthefactthatthosebacteriawhichjoinabiofilmhavea
significantlygreaterchanceofevadingthebatteryofimmunesystemcellsthat
moreeasilyattackplanktonicforms.
Biofilminacidicpoolsat
YellowstoneNationalPark.

Manywouldarguethatresearchoninternalbiofilmshasbeenlargely
neglected,despitethefactthatbacterialbiofilmsseemtohavegreatpotential
forcausinghumandisease.
PaulStoodleyoftheCenterfor
BiofilmEngineeringatMontana
StateUniversity,attributes
muchofthelaginstudying
biofilmstothedifficultiesof
workingwithheterogeneous
biofilmscomparedwith
homogeneousplanktonic
populations.Ina2004paperin
NatureReviews,themolecular
biologistdescribesmany
reasonswhybiofilmsare
extremelydifficulttoculture,
suchasthefactthatthe
diffusionofliquidthrougha
Commonsitesofbiofilminfection.Onebiofilm
biofilmandthefluidforces
reachthebloodstreamtheycanspreadtoany
actingonabiofilmmustbe
moistsurfaceofthehumanbody.
carefullycalculatedifitistobe
culturedcorrectly.Accordingto
Stoodley,theneedtomastersuchdifficultlaboratorytechniqueshasdeterred
manyscientistsfromattemptingtoworkwithbiofilms. [10]
Also,sincemuchofthetechnologyneededtodetectinternalbiofilmswas
createdatthesametimeasthesequencingofthehumangenome,interestin
biofilmbacteria,andtheresearchgrantsthatwouldaccompanysuchinterest,
havebeenlargelydivertedtoprojectswithadecidedlygeneticfocus.
However,sincegeneticresearchhasfailedtouncoverthecauseofanyofthe
commonchronicdiseases,biofilmsarefinallyjustoverthepastfewyears
beingstudiedmoreintensely,andbeinggiventhecredittheydeserveas
seriousinfectiousentities,capableofcausingawidearrayofchronicillnesses.
Injustashortperiodoftime,researchersstudyinginternalbiofilmshave
alreadypeggedthemasthecauseofnumerouschronicinfectionsand
diseases,andthelistofillnessesattributedtothesebacterialcolonies
continuestogrowrapidly.
AccordingtoarecentpublicstatementfromtheNationalInstitutesofHealth,
morethan65%ofallmicrobialinfectionsarecausedbybiofilms.Thisnumber
mightseemhigh,butaccordingtoKimLewisoftheDepartmentofChemical
andBiologicalEngineeringatTuftsUniversity,Ifonerecallsthatsuch
commoninfectionsasurinarytractinfections(causedbyE.coliandother
pathogens),catheterinfections(causedbyStaphylococcusaureusandother
grampositivepathogens),childmiddleearinfections(causedbyHaemophilus
influenzae,forexample),commondentalplaqueformation,andgingivitis,all
ofwhicharecausedbybiofilms,arehardtotreatorfrequentlyrelapsing,this
figureappearsrealistic. [11]
AsLewismentions,perhapsthe
mostwellstudiedbiofilmsare
thosethatmakeupwhatis
commonlyreferredtoasdental
plaque.Plaqueisabiofilmon
thesurfacesoftheteeth,
statesParsek.This
accumulationof
microorganismssubjectthe
teethandgingivaltissuesto
highconcentrationsofbacterial
metaboliteswhichresultsin
dentaldisease. [12]
Hundredsofmicrobialbiofilmcolonizethe
humanmouth,causingtoothdecayandgum
disease.

Ithasalsorecentlybeenshown
thatbiofilmsarepresentonthe
removedtissueof80%of
patientsundergoingsurgeryfor
chronicsinusitis.AccordingtoParsek,biofilmsmayalsocauseosteomyelitis,a
diseaseinwhichthebonesandbonemarrowbecomeinfected.Thisis
supportedbythefactthatmicroscopystudieshaveshownbiofilmformationon
infectedbonesurfacesfromhumansandexperimentalanimalmodels.Parsek
alsoimplicatesbiofilmsinchronicprostatitissincemicroscopystudieshave
alsodocumentedbiofilmsonthesurfaceoftheprostaticduct.Microbesthat
colonizevaginaltissueandtamponfiberscanalsoformintobiofilms,causing
inflammationanddiseasesuchasToxicShockSyndrome.
Biofilmsalsocausetheformationofkidneystones.Thestonescausedisease
byobstructingurineflowandbyproducinginflammationandrecurrent
infectionthatcanleadtokidneyfailure.Approximately15%20%ofkidney
stonesoccurinthesettingofurinarytractinfection.AccordingtoParsek,these
stonesareproducedbytheinterplaybetweeninfectingbacteriaandmineral
substratesderivedfromtheurine.Thisinteractionresultsinacomplexbiofilm
composedofbacteria,bacterialexoproducts,andmineralizedstonematerial.
Perhapsthefirsthintoftheroleof

bacteriainthesestonescamein1938
whenHellstromexaminedstonespassed
byhispatientsandfoundbacteria
embeddeddeepinsidethem.
Microscopicanalysisofstonesremoved
frominfectedpatientshasrevealed
featuresthatcharacterizebiofilm
growth.Foronething,bacteriaonthe
surfaceandinsidethestonesare
organizedinmicrocoloniesand
surroundedbyamatrixcomposedof
Microbesthatcolonizevaginaltissue
crystallized(struvite)minerals.
andtamponfiberscanbecome
Thentheresendocarditis,adiseasethat pathogenic,causinginflammationand
involvesinflammationoftheinner
diseasesuchasToxicShockSyndrome.
layersoftheheart.Theprimary
infectiouslesioninendocarditisisa
complexbiofilmcomposedofbothbacterialandhostcomponentsthatis
locatedonacardiacvalve.Thisbiofilm,knownasavegetation,causesdisease
bythreebasicmechanisms.First,thevegetationphysicallydisruptsvalve
function,causingleakagewhenthevalveisclosedandinducingturbulenceand
diminishedflowwhenthevalveisopen.Second,thevegetationprovidesa
sourcefornearcontinuousinfectionofthebloodstreamthatpersistseven
duringantibiotictreatment.Thiscausesrecurrentfever,chronicsystemic
inflammation,andotherinfections.Third,piecesoftheinfectedvegetationcan
breakoffandbecarriedtoaterminalpointinthecirculationwheretheyblock
theflowofblood(aprocessknownasembolization).Thebrain,kidney,and
extremitiesareparticularlyvulnerabletotheeffectsofembolization.
Avarietyofpathogenicbiofimsarealsocommonlyfoundonmedicaldevices
suchasjointprosthesesandheartvalves.AccordingtoParsek,electron
microscopyofthesurfacesofmedicaldevicesthathavebeenfociofdevice
relatedinfectionsshowsthepresenceoflargenumbersofslimeencased
bacteria.Tissuestakenfromnondevicerelatedchronicinfectionsalsoshow
thepresenceofbiofilmbacteriasurroundedbyanexopolysaccharidematrix.
Thesebiofilminfectionsmaybecausedbyasinglespeciesorbyamixtureof
speciesofbacteriaorfungi.
AccordingtoDr.PateloftheMayoClinic,individualswithprostheticjointsare
oftenoblivioustothefactthattheirprostheticjointsharborbiofilminfections.
[13]

Whenpeoplethinkofinfection,
theymaythinkoffeverorpus
comingoutofawound,
explainsDr.Patel.However,
thisisnotthecasewith
prostheticjointinfection.
Patientswilloftenexperience
pain,butnotothersymptoms
usuallyassociatedwith
infection.Oftenwhathappens
isthatthebacteriathatcause
infectiononprostheticjoints
arethesameasbacteriathat
liveharmlesslyonourskin.
CellsofStaphylococcusepidermidiscausing
However,onaprostheticjoint
devastatingdiseaseastheygrowonthecuffat
theycanstick,growandcause
amechanicalheartvalve.
problemsoverthelongterm.
Manyofthesebacteriawouldnotinfectthejointwereitnotforthe
prosthesis.
BiofilmsalsocauseLeptospirosis,aseriousbutneglectedemergingdisease
thatinfectshumansthroughcontaminatedwater.Newresearchpublishedin
theMayissueofthejournalMicrobiologyshowsforthefirsttimehowbacteria
thatcausethediseasesurviveintheenvironment.
LeptospirosisisamajorpublichealthprobleminsoutheastAsiaandSouth
America,withover500,000severecaseseveryyear.Between5%and20%of
thesecasesarefatal.Ratsandothermammalscarrythediseasecausing
pathogenLeptospirainterrogansintheirkidneys.Whentheyurinate,they
contaminatesurfacewaterwiththebacteria,whichcansurviveinthe
environmentforlongperiods.
Thisledustoseeifthebacteriabuildaprotectivecasingaroundthemselves
forprotection,saidProfessorMathieuPicardeaufromtheInstitutPasteurin
Paris,France. [14]
Previously,scientistsbelievedthebacteriawereplanktonic.ButProfessor
PicardeauandhisteamhaveshownthatL.interroganscanmakebiofilms,
whichcouldbeoneofthemainfactorscontrollingsurvivalanddisease
transmission.90%ofthespeciesofLeptospirawetestedcouldformbiofilms.
IttakesL.interrogansanaverageof20daystomakeabiofilm,says
Picardeau.
Biofilmshavealsobeenimplicatedinawidearrayofveterinarydiseases.For
example,researchersattheVirginiaMarylandRegionalCollegeofVeterinary

MedicineatVirginiaTechwerejustawardedagrantfromtheUnitedStates
DepartmentofAgriculturetostudytherolebiofilmsplayinthedevelopmentof
BovineRespiratoryDiseaseComplex(BRDC).Ifbiofilmsplayaroleinbovine
respiratorydisease,itslikelyonlyamatteroftimebeforetheywillbe
establishedasacauseofhumanrespiratorydiseasesaswell.
Asmentionedpreviously,infectionby
thebacteriumPseudomonasaeruginosa
(P.aeruginosa)isthemaincauseof
deathamongpatientswithcystic
fibrosis.Pseudomonasisabletosetup
permanentresidenceinthelungsof
patientswithcysticfibrosiswhere,if
youaskmostmainstreamresearchers,
itisimpossibletokill.Eventually,
chronicinflammationproducedbythe
immunesysteminresponseto
Whentheimmuneresponseis
Pseudomonasdestroysthelungand
compromised,Pseudomonas
causesrespiratoryfailure.Inthe
aeruginosabiofilmsareabletocolonize
permanentinfectionphase,P.
thealveoli,andtoformbiofilms.
aeruginosabiofilmsarethoughttobe
presentintheairway,althoughmuch
abouttheinfectionpathogenesisremainsunclear. [15]
Cysticfibrosisiscausedbymutationsintheproteinsofchannelsthatregulates
chloride.Howabnormalchloridechannelproteinleadstobiofilminfection
remainshotlydebated.Itisclear,however,thatcysticfibrosispatients
manifestsomekindofhostdefensedefectlocalizedtotheairwaysurface.
Somehowthisleadstoadebilitatingbiofilminfection.

Biofilms have the potential to cause a tremendous array of infections and diseases
Becauseinternalpathogenicbiofilmresearchcomprisessuchanewfieldof
study,theinfectionsdescribedabovealmostcertainlyrepresentjustthetipof
theicebergwhenitcomestothenumberofchronicdiseasesandinfections
currentlycausedbybiofilms.
Forexample,itwasntuntilJulyof2006thatresearchersrealizedthatthe
majorityofearinfectionsarecausedbybiofilmbacteria.Theseinfections,
whichcanbeeitheracuteorchronic,arereferredtocollectivelyasotitis
media(OM).Theyarethemostcommonillnessforwhichchildrenvisita
physician,receiveantibiotics,orundergosurgeryintheUnitedStates.
TherearetwosubtypesofchronicOM.RecurrentOM(ROM)isdiagnosedwhen
childrensufferrepeatedinfectionsoveraspanoftimeandduringwhich
clinicalevidenceofthediseaseresolvesbetweenepisodes.ChronicOMwith
effusionisdiagnosedwhenchildrenhavepersistentfluidintheearsthatlasts
formonthsintheabsenceofanyothersymptomsexceptconductivehearing
loss.
Ittookovertenyearsforresearcherstorealizethatotitismediaiscausedby
biofilms.Finally,in2002,Drs.EhrlichandJ.ChristopherPost,anAllegheny
GeneralHospitalpediatricearspecialistandmedicaldirectoroftheCenterfor
GenomicSciences,publishedthefirstanimalevidenceofbiofilmsinthemiddle
earintheJournaloftheAmericanMedicalAssociation,settingthestagefor
furtherclinicalinvestigation.
Inasubsequentstudy,EhrlichandPostobtainedmiddleearmucosaor
membranetissuebiopsiesfromchildrenundergoingaprocedureforotitis.
Theteamgathereduninfectedmucosalbiopsiesfromchildrenandadults
undergoingcochlearimplantationasacontrol. [16]
Usingadvancedconfocallaserscanningmicroscopy,LuanneHallStoodley,
Ph.D.andherASRIcolleaguesobtainedthreedimensionalimagesofthe
biopsiesandevaluatedthemforbiofilmmorphologyusinggenericstainsand
speciesspecificprobesforHaemophilusinfluenzae,Streptococcuspneumoniae
andMoraxellacatarrhalis.Effusions,whenpresent,werealsoevaluatedfor
evidenceofpathogenspecificnucleicacidsequences(indicatingpresenceof
livebacteria).
Thestudyfoundmucosalbiofilmsinthemiddleearsof46/50children(92%)
withbothformsofotitis.Biofilmswerenotobservedineightcontrolmiddle
earmucosaspecimensobtainedfromcochlearimplantpatients.
Infact,allofthechildreninthestudy
whosufferedfromchronicotitismedia
testedpositiveforbiofilmsinthemiddle
ear,eventhosewhowere
asymptomatic,causingErlichto
concludethat,Itappearsthatinmany
casesrecurrentdiseasestemsnotfrom
reinfectionaswaspreviouslythought
andwhichformsthebasisfor
conventionaltreatment,butfroma
persistentbiofilm.
Hewentontostatethatthediscovery

ofbiofilmsinthesettingofchronicotitis
mediarepresentedalandmark
evolutioninthemedicalcommunitys
understandingaboutadiseasethat
afflictsmillionsofchildrenworldwide
eachyearandfurtherendorsesthe
emergingbiofilmparadigmofchronic
infectiousdisease.
Theemergingbiofilmparadigmof
chronicdiseasereferstoanew
movementinwhichresearcherssuchas
Ehrlicharecallingforatremendousshift
inthewaythemedicalcommunityviews
bacterialbiofilms.Thosescientistswho
supportanemergingbiofilmparadigm
Otitismedia,orinflammationofthe
ofchronicdiseasefeelthatbiofilm
innerear,iscausedbybiofilm.
researchisofutmostimportance
becauseofthefactthattheinfectiousentitieshavethepotentialtocauseso
manyformsofchronicdisease.TheMarshallPathogenesisisanimportantpart
ofthisparadigmshift.
Itwasalsojustlastyearthatresearchersrealizedthatbiofilmscausemost
infectionsassociatedwithcontactlensuse.In2006,Bausch&Lombwithdrew
itsReNuwithMoistureLoccontactlenssolutionbecauseahighproportionof
cornealinfectionswereassociatedwithit.Itwasntlongbeforeresearchersat
theUniversityHospitalsCaseMedicalCenterfoundthattheinfectionswere
causedbybiofilms. [17]
Oncetheyliveinthattypeofstate[abiofilm],thecellsbecomeresistantto
lenssolutionsandimmunetothebodysowndefensesystem,saidMahmoud
A.Ghannoum,Ph.D,seniorinvestigatorofthestudy.Thisstudyshouldalert
contactlenswearerstotheimportanceofpropercareforcontactlensesto
protectagainstpotentiallyvirulenteyeinfections,hesaid.
ItturnsoutthatthebiofilmsdetectedbyGhannoumandteamwerecomposed
offungi,particularlyaspeciescalledFusarium.Histeamalsodiscoveredthat
thestrainoffungus(withthecatchyname,ATCC36031)usedfortestingthe
effectivenessoflenscaresolutionsisastrainthatdoesnotproducebiofilms
astheclinicalfungalstrainsdo.ReNucontactsolution,therefore,waseffective
inthelaboratory,butfailedwhenfacedwithstrainsinrealworldsituations.
Unfortunately,Ghannoumandteam
werenotabletocreateamethodto
targetanddestroythefungalbiofilms
thatplagueusersofReNuandsome
othercontactlenssolutions.
ThentheresDr.RandallWolcottwho
justrecentlydiscoveredandconfirmed
thatthesludgecoveringdiabeticwounds
islargelymadeupofbiofilms.Whereas
beforeWolcottsworksuchlimbs
generallyhadtobeamputated,nowthat
theyhavebeencorrectlylinkedto
biofilms,measuressuchasthose
describedinthisinterviewcanbetaken
tostopthespreadofinfectionandsave
thelimb.Wolcotthasfinallybeengiven Fungalbiofilmcanformincontactlens
solutionleadingtopotentiallyvirulent
agrantbytheNationalInstitutesof
eyeinfections
Healthtofurtherstudychronicbiofilms
andwounddevelopment.
Dr.GarthJamesandtheMedicalBiofilmLaboratoryteamatMontanaState
Universityarealsoresearchingwoundsandbiofilms.Theirlatestarticleandan
imageshowingwoundbiofilmwasfeaturedonthecoveroftheJanuary
February2008issueofWoundRepairandRegeneration. [18]

Biofilm bacteria and chronic inflammatory disease

Injustafewshortyears,thepotentialofbiofilmstocausedebilitatingchronic
infectionshasbecomesoclearthatthereislittledoubtthatbiofilmsarepart
ofthepathogenicmixorpeasoupthatcausemostorallchronic
autoimmuneandinflammatorydiseases.
Infact,thanks,inlargepart,totheresearchofbiomedicalresearcherDr.
TrevorMarshall,itisnowincreasinglyunderstoodthatchronicinflammatory
diseasesresultfrominfectionwithalargemicrobiotaofchronicbiofilmandL
formbacteria(collectivelycalledtheTh1pathogens). [19] [20]Themicrobiotais
thoughttobecomprisedofnumerousbacterialspecies,someofwhichhave
yettobediscovered.However,mostofthepathogensthatcauseinflammatory
diseasehaveonethingincommontheyhavealldevelopedwaystoevade
theimmunesystemandpersistaschronicformsthatthebodyisunableto
eliminatenaturally.
SomeLformbacteriaareabletoevadetheimmunesystembecause,long
ago,theyevolvedtheabilitytoresideinsidemacrophages,theverywhite

bloodscellsoftheimmunesystemthataresupposedtokillinvading
pathogens.Uponformation,Lformbacteriaalsolosetheircellwalls,which
makesthemimpervioustocomponentsoftheimmuneresponsethatdetect
invadingpathogensbyidentifyingtheproteinsontheircellwalls.Thefactthat
Lformbacterialackcellwallsalsomeansthatthebetalactamantibiotics,
whichworkbytargetingthebacterialcellwall,arecompletelyineffectiveat
killingthem. [21]
Clearly,transformingintotheLformoffersanypathogenasurvival
advantage.ButamongthosepathogensnotinanLformstate,joiningabiofilm
isjustaslikelytoenhancetheirabilitytoevadetheimmunesystem.Once
enoughchronicpathogenshavegroupedtogetherandformedastable
communitywithastrongprotectivematrix,theyarelikelyabletoresidein
anyareaofthebody,causingthehosttosufferfromchronicsymptomsthat
arebothmentalandphysicalinnature.
Biofilmresearcherswillalsotellyouthat,notsurprisingly,biofilmsformwith
greatereaseinanimmunocompromisedhost.Marshallsresearchhasmadeit
clearthatmanyoftheTh1pathogensarecapableofcreatingsubstancesthat
bindandinactivatetheVitaminDReceptorafundamentalreceptorofthe
bodythatcontrolstheactivityoftheinnateimmunesystem,orthebodysfirst
lineofdefenseagainstintracellularinfection. [22]
Thus,aspatientsaccumulateagreater
numberoftheTh1pathogens,more
andmoreofthechronicbacterial
formscreatesubstancescapableof
disablingtheVDR.Thiscausesa
snowballeffect,inwhichthepatient
becomesincreasingly
immunocompromisedastheyacquirea
largerbacterialload.
Foronething,itspossiblethatmany
ofthebacteriathatsurviveinside
biofilmsarecapableofcreatingVDR
blockingsubstances.Thus,the
formationofbiofilmsmaycontributeto
immunedysfunction.Conversely,as
DiagramoftheVitaminDReceptorand
patientsacquireLformbacteriaand
capnine.
otherpersistentbacterialforms
capableofcreatingVDRblocking
substances,itbecomesexceptionally
easyforbiofilmstoformonanytissuesurfaceofthehumanbody.
Thus,patientswhobegintoacquireLformbacteriaalmostalwaysfallvictim
tobiofilminfectionsaswell,sinceitisalltooeasyforpathogenstogroup
togetherintoabiofilmwhentheimmunesystemisntworkinguptopar.
Todate,thereisalsonostrictcriteriathatseparateLformbacteriafrom
biofilmbacteriaoranyotherchronicpathogenicforms.ThismeansthatLform
bacteriamayalsoformintobiofilms,andbydoingsoenteramodeofsurvival
thatmakesthemtrulyimpervioustotheimmunesystem.SomeLform
bacteriamaynotformcompletebiofilms,yetmaystillpossesstheabilityto
surroundthemselvesinaprotectivematrix.Underthesecircumstancesone
mightsaytheyareinabiofilmlikestate.
Marshalloftenreferstothepathogensthatcauseinflammatorydiseaseasan
intraphgocytic,metagenomicmicrobiotaofbacteria,termswhichsuggestthat
mostchronicbacterialformspossesspropertiesofbothLformandbiofilm
bacteria.Intraphagocyticreferstothefactthatthepathogenscanbefound
insidethecellsoftheimmunesystem.Thetermmetagenomicindicatesthat
thereareatremendousnumberofdifferentspeciesofthesechronicbacterial
forms.Finally,microbiotareferstothefactthatbiofilmcommunitiessustain
theirpathogenicactivity.
Forexample,whenobservedunderadarkfieldmicroscope,Lformbacteria
areoftenencasedinprotectivebiofilmsheaths.Ifthebloodcontainingthe
pathogensareagedovernight,thebacterialcoloniesreachapointwherethey
expandandburstoutofthecell,causingthecelltoburstaswell.Thenthey
extendashuge,longbiofilmtubules,whicharepresumablyhelpingthe
pathogensspreadtoothercells.ThetubulesalsohelpspreadbacterialDNAto
neighboringcells.
Clearly,thereisagreatneedformoreresearchonhowdifferentchronic
bacterialformsinteract.Todate,Lformresearchershaveessentiallyfocused
soleyontheLform,whilefailingtoinvestigatehowfrequentlythewallless
pathogensformintobiofilmsorbecomepartsofbiofilmcommunitiestogether
withbacteriawithcellwalls.Conversely,mostbiofilmresearchersareintently
studyingthebiofilmmodeofgrowthwithoutconsideringthepresenceofL
formbacteria.So,itwilllikelytakeseveralyearsbeforewewillbebetterable
tounderstandprobableoverlapsbetweenthelifestylesofLformandbiofilm
bacteria.
Anyonewhoisskepticalaboutthefactthatbiofilmslikelyformalarge
percentageofthemicrobiotathatcauseinflammatorydiseaseshouldconsider
manyoftherecentstudiesthathavelinkedestablishedbiofilminfectionstoa
higherriskformultipleformsofchronicinflammatorydisease.Take,for

example,studiesthathavefoundalinkbetweenperiodontaldiseaseand
severalmajorinflammatoryconditions.A1989articlepublishedinBritish
MedicalJournalshowedacorrelationbetweendentaldiseaseandsystemic
disease(stroke,heartdisease,diabetes).Aftercorrectingforage,exercise,
diet,smoking,weight,bloodcholesterollevel,alcoholuseandhealthcare,
peoplewhohadperiodontaldiseasehadasignificantlyhigherincidenceof
heartdisease,strokeandprematuredeath.Morerecently,theseresultswere
confirmedinstudiesintheUnitedStates,Canada,GreatBritain,Sweden,and
Germany.Theeffectsarestriking.Forexample,researchersfromthe
CanadianHealthBureaufoundthatpeoplewithperiodontaldiseasehadatwo
timeshigherriskofdyingfromcardiovasculardisease. [23]
Sinceweknowthatperiodontaldisease
iscausedbybiofilmbacteria,themost
logicalexplanationforthefactthat
peoplewithdentalproblemsaremuch
morelikelytosufferfromheartdisease
andstrokeisthatthebiofilmsintheir
mouthshavegraduallyspreadtothe
moistsurfacesoftheircirculatory
systems.Orperhapsifthebacteriain
periodontalbiofilmscreateVDRbinding
substances,theirabilitytoslowinnate
immunefunctionallowsnewbiofilms
Dentalplaqueasseenundera
(andLformbacteriaaswell)tomore
scanningelectronmicrocroscope.
easilyformandinfecttheheartand
bloodvessels.Conversely,systemic
infectionwithVDRblockingbiofilmbacteriaisalsolikelytoweakenimmune
defensesinthegumsandfacilitateperiodontaldisease.
Infact,itappearsthatbiofilmbacteriainthemouthalsofacilitatethe
formationofbiofilmandLformbacteriainthebrain.Justlastyear,
researchersatVasantHiraniatUniversityCollegeLondonreleasedtheresults
ofastudywhichfoundthatelderlypeoplewhohavelosttheirteethareat
morethanthreefoldgreaterriskofmemoryproblemsanddementia. [24]
Atthemoment,AutoimmunityResearchFoundationdoesnothavethe
resourcestoculturebiofilmsfrompatientsonthetreatmentand,evenifthey
did,currentmethodsforculturinginternalbiofilmsremainunreliable.
AccordingtoStoodley,Thelackofstandardmethodsforgrowing,quantifying
andtestingbiofilmsincontinuouscultureresultsinincalculablevariability
betweenlaboratorysystems.Biofilmmicrobiologyiscomplexandnotwell
representedbyflaskcultures.Althoughhomogeneityallowsstatistical
enumeration,theextenttowhichitreflectsthereal,lessorderlyworldis
questionable. [10]

How else do we acquire biofilm bacteria?

Asdiscussedthusfar,biofilmsformspontaneouslyasbacteriainsidethe
humanbodygrouptogether.Yetpeoplecanalsoingestbiofilmsbyeating
contaminatedfood.
AccordingtoresearchersattheUniversityofGuelphinOntarioCanada,itis
increasinglysuspectedthatbiofilmsplayanimportantroleincontaminationof
meatduringprocessingandpackaging.Thegroupwarnsthatgreateraction
mustbetakentoreducethepresenceoffoodbornepathogenslikeEscherichia
coliandListeriamonocytogenesandspoilagemicroorganismssuchas
thePseudomonasspecies(allofwhichformbiofilms)throughoutthefood
processingchaintoensurethesafetyandshelflifeoftheproduct.Mostof
thesemicroorganismsareubiquitousintheenvironmentorbroughtinto
processingfacilitiesthroughhealthyanimalcarriers.
HansBlaschekoftheUniversityofIllinoishasdiscoveredthatbiofilmsformon
muchoftheotherfoodproductsweconsumeaswell.
Ifyoucouldseeapieceof
celerythatsbeenmagnified
10,000times,youdknowwhat
thescientistsfightingfoodborne
pathogensareupagainst,says
Blaschek.

Abiofilmonapieceoflettuce

Itslikelookingata
moonscape,fullofcratersand
crevices.Andmanyofthe
pathogensthatcausefoodborne
illness,suchasShigella,E.
coli,andListeria,makesticky,
sugarybiofilmsthatgetdown
inthesecrevices,sticklike
glue,andhangonlikecrazy.

AccordingtoBlaschek,theproblemfacedbyproducesupplierscanbeatriple
whammy.Ifyoureunluckyenoughtobedealingwithapathogenandthe
pathogenhastheadditionalattributeofbeingabletoformbiofilmandyoure
dealingwithafoodproductthatsminimallyprocessed,well,youretriply
unlucky,thescientistsaid.Youmaybeabletoscrubtheorganismoffthe

surface,butthecellsinthesebiofilmsareverygoodataligningthemselvesin
thesubsurfaceareasofproduce.
ScottMartin,aUniversityofIllinoisfoodscienceandhumannutritionprofessor
agrees,stating,Oncethepathogenicorganismgetsontheproduct,noamount
ofwashingwillremoveit.Themicrobesattachtothesurfaceofproduceina
stickybiofilm,andwashingjustisntveryeffective.
Biofilmscanevenbefoundinprocessedwater.Justthismonth,astudywas
releasedinwhichresearchersattheDepartmentofBiologicalSciences,at
VirginiaPolytechnicInstituteisolatedM.aviumbiofilmfromtheshowerhead
ofawomanwithM.aviumpulmonarydisease. [25]Amoleculartechnique
calledDNAfingerprintingdemonstratedthatM.aviumisolatesfromthewater
werethesameformsthatwerecausingthewomansrespiratoryillness.

Effectively targeting biofilm infections

Althoughthemainstreammedicalcommunityisrapidlyacknowledgingthe
largenumberofdiseasesandinfectionscausedbybiofilms,mostresearchers
areconvincedthatbiofilmsaredifficultorimpossibletodestroy,particularly
thosecellsthatformthedeeperlayersofathickbiofilm.Mostpaperson
biofilmsstatethattheyareresistanttoantibioticsadministeredinastandard
manner.Forexample,despitethefactthatEhrlichandteamdiscoveredthat
biofilmbacteriacauseotitismedia,theyareunabletoofferaneffective
solutionthatwouldactuallyallowforthedestructionofbiofilmsintheear
canal.Otherteamshavealsocomeupshortincreatingmethodstobreakup
thebiofilmstheyimplicateasthecauseofnumerousinfections.
Thismeanspatientswithbiofilminfectionsaregenerallytoldbymainstream
doctorsthattheyhaveanuntreatableinfection.Insomecases,adisease
causingbiofilmcanbecutoutofapatientstissues,oreffortsaremadeto
draincomponentsofthebiofilmoutofthebody.Forexample,doctorstreating
otitismediaoftentreatspatientswithmyringotomy,asurgicalprocedurein
whichsmalltubesareplacedintheeardrumtocontinuouslydraininfectious
fluid.
Whenitcomestoadministeringantibioticsinanefforttotargetbiofilms,one
thingiscertain.Mainstreamresearchershaverepeatedlytriedtokillbiofilms
bygivingpatientshigh,constantdosesofantibiotics.Unfortunately,when
administeredinhighdoses,theantibioticmaytemporarilyweakenthebiofilm
butisincapableofdestroyingit,ascertaincellsinevitablypersistandallow
thebiofilmtoregenerate.
Youcanputapatienton[ahighdose]antibiotics,anditmayseemthatthe
infectionhasdisappeared,saysLevchenko.Butinafewmonths,it
reappears,anditisusuallyinanantibioticresistantform.
Whatthevastmajorityofresearchersworkingwithbiofilmsfailtorealizeis
thatantibioticsarecapableofdestroyingbiofilms.Thecatchisthatantibiotics
areonlyeffectiveagainstbiofilmsifadministeredinaveryspecificmanner.
Furthermore,onlycertainantibioticsappeartoeffectivelytargetbiofilms.After
decadesofresearch,muchofwhichwasderivedfrommolecularmodeling
data,Marshallwasthefirsttocreateanantibioticregimenthatappearsto
effectivelytargetanddestroybiofilms.Centraltothetreatment,whichis
calledtheMarshallProtocol,isthefactthatbiofilmsandotherTh1pathogens
succumbtospecificbacteriostaticantibioticstakeninverylow,pulseddoses.
Itisonlywhenantibioticsareadministeredinthismannerthattheyappear
capableoffullyeradicatingbiofilms. [19] [20]
InapaperentitledTheRiddleofBiofilmResistance,Dr.KimLewisofTulane
Universitydiscussesthemechanismsbywhichpulsed,lowdoseantibioticsare
abletobreakupbiofilms,whileantibioticsadministeredinastandardmanner
(high,constantdoses)cannot.AccordingtoLewis,theuseofpulsed,lowdose
antibioticstotargetbiofilmbacteriaissupportedbyobservationssheandher
colleagueshavemadeinthelaboratory. [11]
Someresearchersclaimthatantibioticscannotpenetratethematrixthat
surroundsabiofilm.ButresearchbyLewisandotherscientistshasconfirmed
thattheinabilityofantibioticstopenetratethebiofilmmatrixismuchmoreof
anexceptionthanarule.AccordingtoLewis,Inmostcasesinvolvingsmall
antimicrobialmolecules,thebarrierofthepolysaccharidematrixshouldonly
postponethedeathofcellsratherthanaffordusefulprotection.
Forexample,arecentstudythatusedlowconcentrationsofanantibioticto
killP.aeruginosabiofilmbacteriafoundthatthemajorityofbiofilmcellswere
effectivelyeliminatedbyantibioticsinamannerthatdidnotdiffermuchfrom
whatisobservedwhenthesameantibioticconcentrationsareadministeredto
singleplanktoniccells. [26]
Thus,sinceantibioticscangenerallypenetrate
biofilms,someotherfactorisresponsiblefor
thefactthattheycannotbekilledbystandard
highdoseantibiotictherapy.Itturnsoutthat
afterantibioticsareappliedtoabiofilm,a
numberofcellscalledpersistersareleft
behind.Persistersaresimplycellsthatare
abletosurvivethefirstonslaughtof

antibiotics,andifleftunchecked,gradually
allowthebiofilmtoformagain.Accordingto
Lewis,persistercellsformwithparticular
easeinimmunocompromisedpatientsbecause
theimmunesystemisunabletohelpthe
antibioticmopupallthebiofilmcellsithas
targeted.

Afterantibioticsareapplied
toabiofilm,anumberofcells
calledpersistersareleft
behind.

Thissimpleobservationsuggestsanew
paradigmforexplaining,atleastinprinciple,
thephenomenonofbiofilmresistanceto
killingbyawiderangeofantimicrobials,
statesLewis.Themajorityofcellsina
biofilmarenotnecessarilymoreresistantto
killingthanplanktoniccellsanddierapidly
whentreatedwith[anantibiotic]thatcankill
slowlygrowingcells.

Thus,adoseofantibioticsparticularlyinimmunocompromisedpatients
eradicatesmostofthebiofilmpopulationbutleavesasmallfractionof
survivingpersistersbehind.Unfortunately,inthesamesensethatthebeta
lactamantibioticspromotetheformationofLformbacteria,persistercellsare
actuallypreservedbythepresenceofanantibioticthatinhibitstheirgrowth.
Thus,paradoxically,dosinganantibioticinaconstant,highdosemanner(in
whichtheantibioticisalwayspresent)helpspersisterspersevere.
Butinthecaseoflow,pulseddosing,whereanantibioticisadministered,
withdrawn,thenadministeredagain,thefirstapplicationofantibioticwill
eradicatethebulkofbiofilmcells,leavingpersistercellsbehind.Withdrawlof
theantibioticallowsthepersisterpopulationtostartgrowing.Since
administrationoftheantibioticistemporarilystopped,thesurvivalof
persistersisnotenhanced.Thiscausesthepersistercellstolosetheir
phenotype(theirshapeandbiochemicalproperties),meaningthattheyare
unabletoswitchbackintobiofilmmode.Asecondapplicationoftheantibiotic
shouldthencompletelyeliminatethepersistercells,whicharestillin
planktonicmode.
Lewishasfoundthatthefeasibilityofapulsed,orcyclicalbiofilmeradication
approachdependsontherateatwhichpersistersloseresistancetokillingand
regeneratenewpersisters.Italsodependsontheabilitytomanipulatethe
antibioticconcentrationsomethingthatisdonequiteeffectivelybypatients
ontheMarshallProtocolwhocarefullydosetheirantibioticsatdifferentlevels,
allowingconstantvariationinantibioticconcentration.AlthoughLewis
speculatesthatallowingtheconcentrationofanantibiotictodropcould
potentiallyleadtoresistancetowardstheantibiotic,sheisquicktoaddthatif
twoormoreantibioticsareusedtotargetabiofilmatonetime,such
resistancewouldnotoccur.Again,sincetheMarshallProtocolusesatotalof
fivebacteriostaticantibiotics,usuallytakentwoorthreeatatime,concernsof
resistanceareessentiallynegligible.
Itisentirelypossiblethat
successfulcasesof
antimicrobialtherapyofbiofilm
infectionsresultfroma
fortuitousoptimalcycling
[pulseddosing]ofanantibiotic
concentrationthateliminated
firstthebulkofthebiofilmand
thentheprogenyofthe
persistersthatbeganto
divide,statesLewis.

Modelofbiofilmresistancebasedonpersister
Lewisworkhasbeensupported survival.Aninitialtreatmentofhighdose
byotherresearchteams.
constantantibiotickillsplanktoniccellsandthe
Recently,researchersatthe
majorityofbiofilmcells.Butpersistersremain
UniversityofIowafoundthat
aliveandresurrectthebiofilm,causingthe
subinhibitory(extremelylow
infectiontorelapse
dose)concentrationsofthe
bacteriostaticantibiotic
azithromycinsignificantlydecreasedbiomassandmaximalthicknessinboth
formingandestablishedbiofilms. [27]Theseextremelylowconcentrationsof
azithromycininhibitedbiofilmsinallbutthemosthighlyresistantisolates.In
contrast,subinhibitoryconcentrationsofgentamicin,whichisnota
bacteriostaticantibiotic,hadnoeffectonbiofilmformation.Infact,biofilms
actuallybecameresistanttogentamicinatconcentrationsfarabovethe
minimuminhibitoryconcentration.
ResearchersatTulaneUniversityrecentlyconfirmedyetagainthatlow,pulsed
dosingisasuperiorwayoftargetingtreatmentresistantbiofilmbacteria.
Accordingtotheteam,whomathematicallymodeledtheactionofantibiotics
onbacterialbiofilms,Exposingabiofilmtolowconcentrationdosesofan
antimicrobialagentforlongertimeismoreeffectivethanshorttimedosing
withhighantimicrobialagentconcentration. [28]
Similarly,abioengineerledteamattheUniversityofWashingtonrecently
createdanantibioticcontainingpolymerthatreleasesantibioticslowlyonto
thesurfaceofhospitaldevices,suchascathetersandprostheses,toreduce

theriskofbiofilmrelatedinfections.
Ratherthanmassivelydosingthepatientwithhighlevelsofreleased
antibiotic,thisstrategyallowsthereleaseofextremelylowlevelsofthisvery
potentantibioticoverlongperiodsoftime,explainedBuddyRatner,PhD,
ProfessorandDirectoroftheEngineeredBiomaterialsProgramatthe
UniversityofWashington,Seattle.Wecalculatedtheamountreleasedatthe
surfacethatwouldkill100%ofthebacteriaenteringthesurfacezone.
WhenchallengedbyDr.LeonardA.MermelfromBrownUniversitySchoolof
Medicineontheissuethatlongtermuseofpulsed,lowdoseantibioticsmight
allowforincreasedresistanceonthepartofthebacteriabeingtreated,Ratner
responded,Dr.Mermelsconcernsare,infact,whywedevelopedthissystem
for[antibiotic]release.Bacteriathatlivethroughantibioticdosingcangoonto
produceresistantstrains.If100%ofthebacteriaapproachingthesurfaceare
killed,theycantproduceresistantoffspring.Theclassicalphysicianapproach,
dosingthepatientsystemicallyandheavilytoridthepatientofpersistent
bacteria,canleadtothoseresistantstrains.Ourapproachreleasesminiscule
dosescomparedtowhataphysicianwoulduse,butreleasestheantibiotic
whereitwillbeoptimallyeffectiveandleastlikelytoleaveantibioticresistant
survivors.
Althoughtakenorally,theMPantibioticsaretakeninthesamemanneras
thoseadministeredbyRatnerandteam.Becausetheytooaredosedatoptimal
timesinextremelysmalldoses,thechancethatlongtermantibioticusemight
fosterresistantbacteriaisagain,essentiallynegligible,especiallywhen
multipleantibioticsaretypicallyused.
KeytotheabilityoftheMarshallProtocoltoeffectivelytargetbiofilmbacteria
isthefactthatthespecificpulsed,lowdosebacteriostaticantibioticsusedby
thetreatmentaretakeninconjunctionwithamedicationcalledBenicar.
BenicarbindsandactivatestheVitaminDReceptor,displacingbacterial
substancesand25Dfromthereceptor,sothatitcanonceagainactivatethe
innateimmunesystem. [29]Benicarissoeffectiveatstrengtheningtheinnate
immuneresponsethatthepatientsownimmunesystemultimatelyhelps
destroythebiofilmweakenedbypulsed,lowdoseantibiotics.
Thus,itisnotenoughforpatientsontheMarshallProtocoltosimplytake
specificpulsed,lowdoseantibiotics.Theactivityoftheirinnateimmune
systemmustalsoberestoredsothatthecellsoftheimmunesystemcan
activelycombatbiofilmbacteria,thematrixthatsurroundsthem,andpersister
cells.

Afterantibioticsareappliedtoa
biofilm,anumberofcellscalled
persistersareleftbehind

HowdoweknowthattheMarshall
Protocoleffectivelykillsbiofilm
bacteria?Namelybecausethosepatients
toreachthelaterstagesofthe
treatmentdonotreportsymptoms
associatedwithestablishedbiofilm
diseases.PatientsontheMPwhoonce
sufferedfromchronicearinfections
(OM),chronicsinusinfections,or
periodontaldiseasefindthatsuch
infectionsresolveoverthecourseof
treatment.Furthermore,sincewenow
understandthatbiofilmsalmost
certainlyformalargepartofthe
chronicmicrobiotaofpathogensthat
causechronicinflammatoryand
autoimmunediseases,thefactthat
patientscanusetheMarshallProtocolto
recoverfromsuchillnessesagain
suggeststhatthetreatmentmustbe
effectivelyallowingthemtotargetand

destroybiofilms.
BecauseallevidencepointstothefactthattheMPdoesindeedeffectively
targetbiofilmbacteria,itisofutmostimportancethatpeoplewhosufferfrom
anysortofbiofilminfectionstartthetreatment.KnowledgeoftheMarshall
Protocolhasyettoreachthecysticfibrosiscommunity,butthereisgreathope
thatifpeoplewiththediseaseweretostarttheMP,theycoulddestroytheP.
aeruginosabiofilmsthatcausetheiruntimelydeaths.Inthesamevein,people
withawiderangeofinfections,suchasthoseinfectedwithbiofilmduring
surgery,canlikelyrestoretheirhealthwiththeMP.
ItistobehopedthattheclinicaldataemergingfromtheMarshallProtocol
studysite,whichshowspatientsrecoveringfrombiofilmrelateddiseases,will
inspirefutureresearcherstoinvestagreatdealofenergyintofurther
researchaimedatidentifyingandstudyingthebiofilmbacteriabacteriathat
almostcertainlyformpartofthemicrobiotaofpathogensthatcause
inflammatorydisease.Inthecomingyears,asthetechnologytodetect
biofilmsbecomesevenmoresophisticated,itisalmostcertainthatagreat
numberofbiofilmswillbeofficiallydetectedanddocumentedinpatientswith
avastarrayofchronicdiseases.

REFERENCES

Costerton,J.W.,Stewart,P.S.,&Greenberg,E.P.(1999).Bacterialbiofilms:acommon
causeofpersistentinfections.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).ThemajorVibriocholeraeautoinduceranditsroleinvirulence
factorproduction.Nature,450(7171),8836.[

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

][

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

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

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

][

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

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

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

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

][

][

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

][

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

Trampuz,A.,Piper,K.E.,Jacobson,M.J.,Hanssen,A.D.,Unni,K.K.,Osmon,D.R.,et
al.(2007).SonicationofRemovedHipandKneeProsthesesforDiagnosisofInfection.N
EnglJMed,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).Pseudomonasaeruginosa
biofilmformationinthecysticfibrosisairway.Pulmonarypharmacology&therapeutics.[
]
HallStoodley,L.,Hu,F.Z.,Gieseke,A.,Nistico,L.,Nguyen,D.,Hayes,J.,etal.
(2006).DirectDetectionofBacterialBiofilmsontheMiddleEarMucosaofChildrenWith
ChronicOtitisMedia.JAMA,296(2),202211.[

Imamura,Y.,Chandra,J.,Mukherjee,P.K.,Lattif,A.A.,SzczotkaFlynn,L.B.,
Pearlman,E.,etal.(2008).FusariumandCandidaalbicansBiofilmsonSoftContact
Lenses:ModelDevelopment,InfluenceofLensType,andSusceptibilitytoLensCare
Solutions.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).ANewApproachtoTreatingIntraphagocyticCWDBacterial
PathogensinSarcoidosis,CFS,LymeandotherInflammatoryDiseases.[

][

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

][

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

Marshall,T.G.(2007).BacterialCapnineBlocksTranscriptionofHumanAntimicrobial
Peptides.NaturePrecedings.[

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

Stewart,R.,&Hirani,V.(2007).DentalHealthandCognitiveImpairmentinanEnglish
NationalSurveyPopulation.JournaloftheAmericanGeriatricsSociety,55(9),1410
1414.[

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

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

Starner,TimothyDetal.2008.SubinhibitoryConcentrationsofAzithromycinDecrease
NontypeableHaemophilusinfluenzaeBiofilmFormationandDiminishEstablished
Biofilms.Antimicrobialagentsandchemotherapy52(1):13745.[

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

Marshall,T.G.(2006).VDRNuclearReceptorCompetenceistheKeytoRecoveryfrom
ChronicInflammatoryandAutoimmuneDisease.[

Filedunder:biofilms,featuredarticles

RSSfeedforcommentsonthispost

Commentsareclosed.

Copyright2014Bacteriality