Sundqvist G, Figdor D (2003) - Prime Endodontics

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SundqvistG,FigdorD(2003)|PrimeEndodontics
EndodonticTopics2003,6,328
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ENDODONTICTOPICS2003
Lifeasanendodonticpathogen
Ecologicaldifferencesbetweentheuntreatedandrootfilledrootcanals
GRANSUNDQVIST&DAVIDFIGDOR
This review describes the type of microbial flora in the untreated root canal and the rootfilled canal with persistent
infection. Recent contributions of molecular methods of microbial identification are outlined along with a discussion of
advantagesandlimitationsofthesemethods.Ecologicalandenvironmentalfactorsaretheprimereasonsfordifferences
in the microbial flora in these distinct habitats. Shared phenotypic traits and an ability to respond to the modified
environmentselectforthespeciesthatestablishapersistentrootcanalinfection.
Introduction
Lifeisnoteasyforanendodonticpathogen.Microbesseekingtoestablishintherootcanalmustleavethenutritionally
richanddiverseenvironmentoftheoralcavity,breachenamel,invadedentine,overwhelmtheimmuneresponseofthe
pulpandsettleintheremainingnecrotictissuewithintherootcanal.Duringthattimetheyhavetocompeteinalimited
space with other microbes for the available nutrition. It is no accident that microbes berth in a particular environment
thereareecologicaladvantagesforthemtoestablishandflourishifconditionsarefavorable.Throughgeneticexchange
and mutation, microbes have developed specialized systems that facilitate their ability to find, compete and survive in
theseveryspecificenvironments.
Bacteriaareeverywhere,buttheenvironmentselects
Intheoralcavity,thereareanestimated1010bacteria(1)consistingofmorethan500differentkindsofmicroorganisms
(2,3)andallseekanicheandnutrition.Oneoftheprimaryfunctionsoftoothenamelistoexcludethesemicroorganisms
from the underlying dentinepulp complex. As long as the enamel and cementum layers are intact, the pulp and root
canal are protected from invasion, but loss of these structures by caries, cracks or trauma opens an avenue for
penetration of bacteria through the dentinal tubules. All bacteria within the oral cavity share the same opportunities for
invading the root canal space however only a restricted group of species have been identified in infected root canals
(47).Thereasonforthedisproportionateratiobetweenpotentialandactualnumberofspeciesisthattherootcanalisa
uniqueenvironmentwherebiologicalselectiondrivesthetypeandcourseofinfection.Ananaerobicmilieu,interactions
between microbial factors and the availability of nutrition are principal elements that define the composition of the
microbialflora.
Intheinitialphaseofarootcanalinfection,thenumberofspeciesisusuallylow.Ifthewayofinvasionisviacaries,
the bacteria in the front of the carious process are the first to reach the pulp. In cases where there is no apparent
communicationwiththeoralcavityandthebacteriapenetratethroughdentinaltubules,asintraumacaseswithoutpulp
exposure,thereisnoclearpatternofprimarybacterialinvaders(4,5).Thenumberofbacterialspeciesinaninfectedroot
canal may vary from one to more than 12, and the number of bacterial cells varies from <102 to>108 per sample. A
correlationseemstoexistbetweenthesizeoftheperiapicallesionandthenumberofbacterialspeciesandcellsinthe
rootcanal.Teethwithlongstandinginfectionsandlargelesionsusuallyharbormorebacterialspeciesandhaveahigher
densityofbacteriaintheirrootcanalsthanteethwithsmalllesions.
Theoralandrootcanalflora
Most of the resident oral microbial flora is consistent with dental health. The predominant microbial diseases of the
oralcavity,cariesandperiodontaldisease,developatsiteswhereamicrobialbiofilm,plaque,isalreadyestablishedand
diseaseoccurswithachangeintheenvironmentalconditions,thetypeandmixofmicrobialflora.Thus,changesatthe
tooth surface with a buildup of acidogenic or aciduric bacteria result in demineralization at the tooth surface, leading to
caries. An increase in proteolytic bacteria at the gingival crevice is one of a sequence of factors leading to the
development of periodontal disease (8). Of the major dental diseases, infection of the root canal is unique for the oral
cavitysinceinfectionestablisheswherenomicroorganismshavepreviouslybeenpresent.
Therootcanalasauniquesiteofinfection
In 1894, WD Miller published his findings on the bacteriological investigation of pulps (9). He observed many different
microorganismsintheinfectedpulpspaceandrealizedthatsomewereuncultivablewhencomparedwiththefullrange
observedbymicroscopy,andthattheflorawasdifferentinthecoronal,middleandapicalpartsofthecanalsystem.Due
to limitations of his sampling and cultivation technique, Miller was unable to verify this observation and it was not until
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1982 that this could be shown by culturing (10). Differences in availability of nutrients and oxygen tension in the apical
regioncomparedwiththemainrootcanalareimportantreasonsforthedominanceofslowgrowing,obligatelyanaerobic
bacteriaintheapicalregion.
Studiesonthedynamicsofrootcanalinfectionshaveshownthattherelativeproportionsofanaerobicmicroorganisms
andbacterialcellsincreasewithtimeandthatthefacultativelyanaerobicbacteriaareoutnumberedwhenthecanalshave
beeninfectedfor3monthsormore(10).Whenacombinationofbacterialstrainsoriginallyisolatedfromaninfectedroot
canalwereinoculatedinequalquantitiesintofurthercanalsinexperimentalinfections,theoriginalproportionofbacterial
strains was reproduced and anaerobic bacteria dominated again (11). This illustrates that interactive mechanisms
operateamongstthesemicroorganisms,aconceptfurthersupportedbythefindingthatwhenPrevotellaoralis(formerly
Bacteroidesoralis) was inoculated on its own it was unable to survive, whereas when inoculated with other bacteria it
survived and dominated the established flora (11). These experiments have shown that the endodontic milieu is a
selectivehabitatthatsupportsthedevelopmentofspecificproportionsoftheanaerobicmicroflora.Oxygenandoxygen
products play an important role as ecological determinants in the development of specific proportions of the root canal
microflora (1214). The consumption of oxygen and production of carbon dioxide and hydrogen along with the
developmentofalowreductionoxidationpotentialbytheearlycolonizersfavorthegrowthofanaerobicbacteria.
Nutritionasanecologicaldriver
Thetypeandavailabilityofnutrientsisimportantinestablishingmicrobialgrowth.Nutrientsmaybederivedfromtheoral
cavity, degenerating connective tissue (13), dentinal tubule contents, or a serumlike fluid from periapical tissue (15).
Thesefactorsintherootcanalenvironmentpermitthegrowthofanaerobicbacteriacapableoffermentingaminoacids
and peptides, whereas bacteria that primarily obtain energy by fermenting carbohydrates may be restricted by lack of
available nutrients. This is the likely reason why the flora is dominated by facultatively anaerobic bacteria, such as
streptococci, in the coronal section of root canals exposed to the oral cavity, and anaerobic bacteria dominate in the
apicalsection(9,10).
The succession of strict over facultative anaerobes with time (10, 11) is most likely due to changes in available
nutrition,aswellasadecreaseinoxygenavailability.Facultativelyanaerobicbacteriagrowwellinanaerobiosishowever,
their prime energy source is carbohydrates. A decrease in availability of carbohydrates in the root canal occurs when
thereisnodirectcommunicationwiththeoralcavity,whichseverelylimitsgrowthopportunitiesforfacultativeanaerobes.
The experiments of ter Steeg and van der Hoeven (16) offer important clues about the likely dynamics of the root
canalflora.Usingserumasasubstrate,theystudiedthesuccessionofsubgingivalplaqueorganismsduringenrichment
growth.Threephasescouldbedistinguishedduringgrowth.Initially,rapidlygrowingsaccharolyticbacteriaconsumedthe
low levels of carbohydrates in serum, leading to lactic and formic acid production. In a second phase, proteins were
hydrolyzed, some amino acid fermentation took place, and there was digestion of remaining carbohydrates.
Carbohydrates were split off from the serum glycoproteins. Growth during this phase was dominated by Prevotella
intermedia, Veillonella parvula, Fusobacterium nucleatum and Eubacterium species. In a final phase, there was
progressive protein degradation. The predominant species during this phase were Peptostreptococcus micros, F.
nucleatum,andeubacteria.ThedominanceofP.microsinculturesoriginatingfromsubgingivalmicrobiota,whengrown
inserum,hasalsobeenshowninanotherstudy(17).TheecologicalnicheofP.microsmayberelatedtoitswiderange
ofpeptidaseactivities,makingaminoacidsandpeptidesavailablefromserumglycoproteins(16).Theseaminoacidscan
beusedbyP.micros,butalsobyotherbacteriathathavelittleornoproteolyticactivityinserum.
The blackpigmented anaerobic rods, Prevotella intermedia/nigrescens, Porphyromonas gingivalis and
Porphyromonasendodontalis, are proficient in degrading serum proteins and make peptides and amino acids available
forfermentation(18).ThedegradationofnativeproteinsbyPrevotellaandPorphyromonasspeciesenablesthegrowthof
bacteria that depend on the availability of peptides, such as eubacteria, fusobacteria and peptostreptococci, which
producepeptidasesbutcannothydrolyzeintactproteins(19,20).Thisisalsoofimportanceforthecapacityofrootcanal
bacteria to induce periapical abscesses. Combinations of P. micros with P. intermedia or P. endodontalis have been
implicated in the induction of periapical abscesses (21). Abscesses harboring a microflora that rapidly degrade serum
proteins have been shown to be nearly three times larger than abscesses with a microflora that lack the capacity for
breakdownofserumproteins(22).
Growthofmixedbacterialpopulationsmaydependonafoodchaininwhichthemetabolismofonespeciessupplies
essential nutrients for the growth of other members of the population (19, 2326). Blackpigmented anaerobic rods
(PrevotellaandPorphyromonasspecies)areexamplesofbacteriathathaveveryspecificnutritionalrequirements.They
are dependent on vitamin K and hemin for growth. Vitamin K can be produced by other bacteria (27). Hemin becomes
available when hemoglobin is broken down, but some bacteria may also produce hemin. Another example is
CampylobacterrectuswhichcanstimulatethegrowthofPorphyromonasspeciesbyproducingagrowthfactorrelatedto
hemin(26).C.rectusitselfderivesasourceofenergyfromthecoinhabitingmicrobialspecies.Itisstrictlydependenton
a respiratory mechanism in which only formate and hydrogen can serve as electron donors and fumarate, nitrate, or
oxygenaselectronacceptors.Thismakesthisorganismdependentonbacteriaproducingformateorhydrogen.Awide
rangeofnutritionalinteractionsisrecognizedamongoralbacteriaandthesemayalsoinfluencetheassociationsbetween
bacteriaintherootcanal(2830).
Becausethenutritionalsupplygovernsthedynamicsofthemicrobialflora,itmeansthatthebacteriapresentinthe
root canal will depend on the stage of the infection. Initially, there may be no clear associations between bacteria, but
strong positive associations develop among a restricted group of the oral flora due to the type of nutrients in the
environment(28,3133). Thus, F.nucleatum is associated with P. micros, P. endodontalis and C. rectus (28). Strong
positiveassociationsexistbetweenP.intermediaandP.micros(28,33)andP.anaerobius(28).Thereisalsoapositive
association between P. intermedia, and P. micros, P. anaerobius and the eubacteria (28). In general, species of
Eubacteria,PrevotellaandPeptostreptococcusarepositivelyassociatedwithoneanotherinendodonticsamples(28,31,
33).Propertiesthatthesebacteriahaveincommonarethattheyfermentpeptidesandaminoacidsandareanaerobic
(16),whichindicatesthatthemainsourceofnutritioninrootcanalsaretissueremnantsandaserumlikesubstrate.
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Mostofthespeciesisolatedfrominfectedrootcanalshavealsobeenidentifiedintheperiodontalpocket(2).Although
therootcanalfloraisnotascomplexasthatoftheperiodontalpocket,theyaresimilarinthatbothcontainaspecialand
limitedassortmentoftheoralflora.Itisworthnoting,however,thattherearesomedifferences.Whenthemicrobialflora
from root canals and periodontal pockets of nonvital teeth with periodontitis were compared (34), the ratio between
anaerobic and facultatively anaerobic bacteria was approximately 100 times higher in the root canal. Anaerobic
streptococci were frequent in the root canals and rarely found in the periodontal pockets. In contrast, facultative
streptococcioutnumberedanaerobicstreptococciintheperiodontalpockets.Bacteriafoundinrootcanals,butrarelyin
periodontal pockets were Peptostreptococcus species, Pseudoramibacter alactolyticus (formerly Eubacterium
alactolyticum),P.intermediaandV.parvula.Bacteriacommonlydetectedinperiodontalpockets,butnotfoundorfoundin
lowfrequencyinrootcanalswerePrevotellamelaninogenica,PrevotellacorrodensandP.gingivalis.
Methodsforisolationandcultivation
In order to identify microorganisms and study their characteristics and pathogenic potential, it is essential that accurate
methods be used for sampling and cultivation. Microbial cells must survive the accumulated stress of sampling,
dispersion,oxygenexposure,andlackofsuitablenutrientsintheculturemedia.
Anaerobicbacteriologicaltechniquesareindispensableforsamplingandcultivationofendodonticbacteria.Whenthe
Virginia Polytechnic Institute (VPI) developed and simplified (35) the methods initially developed by Hungate (36), it
resulted in a changed picture of the periodontal pocket flora and root canal flora. Application of these techniques to
endodonticsamplesrevealedthatobligateanaerobesdominatedinfectedrootcanals,comprisingasmuchas90%ofthe
flora(4,6) and confirmed the unequivocal importance of bacteria for the development of apical periodontitis in human
teeth(4).
Thesafestwaytoprotectanaerobicbacteriaistoavoidexposuretooxygenduringthevariousphasesofthesampling
and laboratory work. Oxygen is toxic because of the formation of hydrogen peroxide, superoxide radicals and hydroxyl
radicals(37).TheVPImethodwasbasedonachievingalowreductionoxidationpotentialbygassingmediawithoxygen
freegas,thusaffordingprotectionfromoxygenationduringsterilizationandsubsequenthandling.Anotherwaytoprotect
bacteria from oxygen is to use an anaerobic glove box with an atmosphere containing a mixture of nitrogen, carbon
dioxideandhydrogen.Undercertainconditions,anaerobicbacteriamaytolerateashortexposuretooxygen(14).Media
thatcontainhemolyzedbloodhaveaprotectiveeffect(14).Theprotectiveeffectofblooddependsonthepresenceofthe
enzymecatalase,whichsplitshydrogenperoxideinthemedium.
Becausethenumberofbacterialcellsinsamplesfrominfectedrootcanalsissohigh,samplesmustbedilutedand
cultivatedonsolidmediatoallowdiversespeciestogrowandbeidentifiedassinglecolonies.Thedilutionprocessisthe
most critical step with regard to oxygen exposure of the sample. Ideally, an anaerobic box and prereduced dilution
solutionsareused,butitcanalsobeperformedonthebenchbygassingjarswithoxygenfreegas.Bacteriaisolatedfrom
rootcanalshavedifferentthresholdsofsensitivitytooxygen.ThemostsensitiveareP.anaerobius,P.endodontalisand
Fusobacteriaspecies.Therateofisolationofthesespeciesisagoodindicatorofhowcarefulthemeasureshavebeento
protectthesamplefromoxygenexposureduringhandling(14).
There is a correlation between the size of the periapical lesion and the number of bacterial cells and species in the
rootcanal(4,28).Samplestakenfromteethwithlarge,longstandinglesionsharbormorebacteria(upto108cfu)than
smaller lesions. Different colony types are most easily recognized on plates with 100 or less colonies, but additional
colonytypesareoftenfoundonplateswithupto300colonies.Therefore,itisreasonabletocalculatethatthedetection
levelfordifferentspeciesis0.31%.Forsomespecies,however,thislevelmaybeloweredto0.01%byusingselective
media(38).
Thenumberofbacterialcellsisdramaticallyreducedduringrootcanalinstrumentationandirrigation(39,40),sofor
detection of bacterial cells in these samples it is necessary to inoculate solid media with undiluted samples and to use
enrichmentgrowthmedia,sincesomesamplescancontainlessthan102cells(41,42).Similarly,inrootcanalsofteeth
with posttreatment disease, the number of surviving cells can be low, and all precautions must be taken to detect the
bacteria. It is advisable to use as small amount as possible of sampling solution to avoid dilution of the sample, then
inoculate an undiluted sample on some plates and use enrichment growth (4345). The detection level for bacteria in
teethundergoingretreatmentdependsonwhatbacterialspeciesarepresentandonhowthesampleistreated,but10
102cellsmightbeexpectedinthesample.
Cultivation discloses the broadest spectrum of the endodontic flora. When all the critical steps are well performed,
cultivationallowsdetectionofsmallnumbersofcellsinasample.However,insampleswithahighcelldensitytheprocess
of serial dilution means that individual species may go undetected if a species is low in number. The methods for
detection of contamination, for example leaking rubber dam, or growth of bacteria under a restoration have all been
basedoncultivationofcontrolsamplestakenafterisolation,accesspreparationandtreatment(43,4648).
Whatisanendodonticpathogen?
Overthelastcentury,theconceptofwhatconstitutesamicrobialpathogenhaschangedwithanincreasingappreciation
ofthecomplexityofthehostpathogeninteraction.Recently,areviseddefinitionhasbeenproposedbywhichapathogen
isamicrobecapableofcausinghostdamagewherehostdamagecanresultfromeitherdirectmicrobialactionorthe
hostimmuneresponse(49).
Anendodonticpathogenisthereforedefinedasamicroorganismcapableofinducingthetissuedestructionofapical
periodontitis. This raises the question as to whether all microorganisms that inhabit the root canal space cause apical
periodontitis,orwhetherspecificorganismsofthemicrobialfloraareconsideredtocausethedisease.Teethwithapical
periodontitis almost always contain a polymicrobial infection and, whilst individual species may play different roles or
dominatevariousstagesoftheinfection,thereisnoevidencetosuggestthatparticularspeciesestablishedthereinare
notinvolvedinthepathogenesisofapicalperiodontitis.
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Limited, but valuable information is available from a classic study (11) with an eightstrain collection of species
derivedfromoneinfectedrootcanalthatwasreinoculatedinequalproportionsintoothermonkeyteeth,eitherintotoor
invariouscombinationsofseveralorseparatespecies.Attheendoftheexperimentalperiod,onespecies,Bacteroides
oralis (now Prevotella oralis), dominated in mixed infections and demonstrated a more potent capacity for tissue
destruction. In pure culture, however, B.oralis could not be reisolated from inoculated root canals (11). Such findings
illustrate the complexity of microbial interaction and that synergy between species may be essential for apical tissue
destruction.
It is clear that much more work is needed to clarify the dynamics, functions and significance, individually and
collectively,ofthespeciesconstitutingtherootcanalflora.Thehabitatselectedmicroorganismsintheinfectedrootcanal
are there because of what they share in biochemical and physiological characteristics and/or what they contribute to
others by mutual synergy. As far as we know today, all bacteria that establish in a root canal can be considered
endodonticpathogens.
The number of microorganisms in a root canal infection is of relevance to the outcome of the disease process.
Detectionofjust1050cellsofaspeciesinasamplewith106totalcellsimpliesalimitednumberofthatspeciesinthe
rootcanalandthecontribution,therefore,ofthatnumberofcellstothedevelopmentofdiseaseislikelytobelow.This
maybeareasonwhystudiesusingmolecularapproacheshavegenerallyfailedtodiscloseanassociationbetweenthe
presence of symptoms and particular species (50,51). Molecular techniques such as polymerasechainreactionbased
methods (PCR) do not give information on the abundance of microorganisms, whereas culturing and serial dilution
provides information about the number present in a sample, and therefore what contribution they can really make to
apical periodontitis. Thus, isolation and cultivation of microorganisms remain essential tools for the analysis of
pathogenicityoftherootcanalflora.
Challengesfacedbymicrobesestablishingintherootcanal
Forbacteriatoestablishsuccessfullyasendodonticpathogens,theymustovercomeaseriesofconsecutivebarrierson
and within the host tooth. It is likely that bacteria act in concert with others to penetrate hardand softtissue barriers,
establishandthensurviveintherootcanal.
TheprogressivechallengesthatmicrobesfaceinestablishingintherootcanalareshowninFig.1.Intheuntreated
canal,microbesmustbreachtoothenamelbyenteringviamicroleakage,cracksorcariesthenreachthepulpbyinvading
dentine. The pulp has its own host defense mechanism, which must be overwhelmed by the collective microbial
consortium.Oncethepulphasbeeninvaded,themicrobesmustacquiresuitablenutrientsourcesandpossiblycompete
withothersforavailablenutrition.Thenutritionavailableatthetoothsurfacemaybeverydifferentfromthatinpulporin
necroticmaterial.Finally,microbesfacepotentialkillingbythehostdefense.Ultimately,ifsuccessful,microbeswillinduce
aninflammatoryresponseattheapexresultinginapicalperiodontitis(Fig.1).
Fig.1.Challengesformicrobestoestablishintheuntreatedrootcanal.
Comparisonofculturingwithmolecularmethods
Almost 40 years ago, Mllers milestone publication (46) described how to sample the root canal in an accurate way,
withoutcontamination.Thesimpleaimwastoisolateandcultivateauthenticmicrobialinhabitantsoftherootcanal.With
few exceptions, the papers published prior to that time had not applied the careful approach necessary for accurate
isolation and cultivation. By documenting reproducible steps, recovery of microorganisms became possible without
contaminationfromadjacentsites.Theexactingprinciplesappliedtoculturemethods40yearsagoarenolessimportant
forrecoveryofmicroorganismsusingthemoleculartechnologiesoftoday.
Acquiringaccuratesamples
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Isolation of the tooth with rubber dam and decontamination of the rubber dam and tooth surface makes it possible to
sampletherootcanalwithoutcontaminationfromtheoralcavity(46).Tocheckthatthedecontaminationstepshavebeen
effective, control samples are required to ensure reliable sterility of the operative field prior to entry into the root canal
(46).Iftheresultofthecontrolsampleispositive,orintheabsenceofappropriatesterilitychecks,theveracityofthedata
fromsamplingmaybeinquestionbecauseitisunknownwhethertheisolatedbacteriaarefromwithinoroutsidetheroot
canal.
Teethshouldfirstbemechanicallycleanedbypolishingwithpumice,thenchemicallycleanedanddisinfectedbefore
thesampleistaken,thedisinfectantshouldbeinactivatedsothatitwillnotinterferewithbacteriarecoveredfromtheroot
canal.Duringtheaccesspreparation,itisimportanttoincludeaseconddecontaminationstageandtakeacontrolsample
before the root canal is reached, because removal of temporary fillings, carious dentin and bacteria under fillings (52)
maycontaminatethefield(43,46,47).Forsamplingbyculture,chemicalcleansingwithhydrogenperoxideisfollowedby
disinfectionwithiodine(46).
Avoidingandmanagingcontamination
Samplingandprocessingshouldprovideatruerepresentationoftherootcanalcontents.Whenappropriateprecautions
are not applied, there is a risk that the results reported might not be authentic. The high sensitivity of molecular
techniques,especiallyPCR,meansthatstudiesusingthesemethodsmustusespecificprecautionstoeliminatemicrobial
DNA from the operation field during access preparation. When root canals are sampled by PCR, it has recently been
shown that the method for decontamination requires modification (47). Cleansing with sodium hypochlorite is more
efficientthanhydrogenperoxideandiodineatremovingdetectableDNAfromthetoothsurface13%oftoothsurfaces
werepositivefordetectableDNA,comparedwith45%,respectively(47).
Contamination may occur not only at the site of sample recovery, but potentially during handling and laboratory
procedures. The particular sensitivity of advanced culturing and molecular techniques means that many appropriate
laboratory controls are necessary to avoid contamination and manage the risk. Culturing has the advantage that
contamination during laboratory manipulation may be more readily recognized than with molecular procedures. PCR
techniqueshaveaninherentlyhighriskthatminuteamountsofcontaminatingDNAareamplifiedandreported.Thereare
manypotentialpitfallsofPCRbasedanalysis(53),soitisessentialthattheworkingenvironmentissetuptoreducetoa
minimumtheriskofcontaminationandthatitismonitoredwithblanktubes,multiplepositiveandnegativecontrols.
Therearemanyproceduralfactorsthatshouldensuretheveracityoftheresults.Someexamplesarelistedhereto
illustratethekindofdetailedattentionnecessarytodelivervaliddata:
Primerdesign.Primersshouldbespecificforthespeciesandtheamplifiedproductsufficientlylarge.Thespecificityof
theprimersandtheirsensitivityshouldbetestedanddescribed.DNAases,releasedbysomemicroorganismsatcell
death,candegradeDNAinthematerial.ThesmallerfragmentsofDNAmaypersistforalongertimethanlarger
sequences.Thus,designingtheprimerstotargetlarge,ratherthansmallPCRproductsshouldyieldfewerpositive
signalsfromdeadcells.
Crosshybridizationcontrols.ThereisariskthatthePCRprimersdesignedfromknown16SrRNAdatamaycross
hybridizewithunrelatedspecies.Therefore,controlsshouldincludecheckingbycomputerbasedBLASTsearches
andcrosshybridizationcontrolswithlivebacteriaofrelatedandunrelatedspecies.
Inhibitionreactions.Thereisthepossibilityofinhibitionreactionsfromconstituentsinthesample,whichtherefore
requirecarefulscreeninginclinicalmaterial.Collagenisoneofmanymoleculesknowntobeinvolvedininhibition
reactionsinclinicalmaterial.
OptimizingPCRprocedures.TheoptimalthermocycleparametersforPCRmaydifferbetweenspecies.PCR
conditionsthataretoo,orinsufficiently,stringentmayresultinpoorPCRreactionswithno,orwrongsequences
amplified,respectively.ThequalityandquantityofextractedDNAdependsoncelllysisconditions,whichvary
accordingtoindividualbacterialcellwallstructures.AlowDNArecoveryefficiencyforsomesamplesorsome
microorganismsmayresultinvariablePCRresults.Forthemethodtobescientificallyvalid,itisimportanttoverifythe
sensitivityandspecificityoftheprimers.
Sequencingtheamplicon.Afterconfirmingthattheampliconisthepredictedsize,sequencingofthePCRproduct
shouldsubstantiatethattheamplifiedsequencematchesthatpredictedfromtheexpectedspecies.Thesequence
matchshouldbegivenalongwiththethresholdforacceptingidentitymatchatspeciesorgenuslevels.
DetectionbyPCRandculture
An important distinction needs to be made between what is evaluated by culture and PCR assays (38). Culturing
measuresviablebacterialcellsascolonyformingunits.MolecularmethodsmeasurenucleotidesequencesandthePCR
methodallowsamplificationofveryminutequantitiesofDNAtodetectablelevels.
Whetherarootcanalsampleisevaluatedbyculturingormoleculartechniques,microorganismsareacquiredbythe
same method, usually by soaking up the fluid root canal contents with paper points. With this method, the results of
laboratory processing depend on what can be recovered from the paper point. An inherent assumption in root canal
sampling is that microorganisms acquired by paper point sampling reflect the type, number and diversity of the flora
inhabiting the root canal. However, if the paper point does not reach all microbes, there remains a possibility that the
samplemaynotaccuratelyportraytherootcanalflora.Thevalueandaccuracyofthesampleiscriticallydependenton
how carefully the tooth is prepared, how scrupulously the sample is taken and the steps to exclude contamination. A
contaminatedsampleisoflittlevalueregardlessofwhetheritislaterprocessedbymolecularmethodsorculturing.
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Culturinghastheadvantagethatitallowsallcellsinasampletogrow,besubculturedandthusbeidentified.Ithasthe
limitationthatsomespecieshaveverystringentenvironmentalandnutritionalrequirementsthatprecludecultureonsolid
media further, it is slow and time consuming. Culturing (and molecular methods) are highly technique sensitive and
withoutappropriatechecksmaybepronetocontaminationandfalseresults.Theoretically,asinglebacterialcellcanbe
detectedbyinoculatinganundilutedrootcanalsampledirectlyontothesamplingmedium,butitisrealistictocalculate
that the limit of detection is 10100 cells. There are two main reasons for this level of detection: serial dilution and cell
survival.
Serialdilutionofasampleistheprimaryreasonforthedetectionlimitof10100cells.Serialdilutionisrequiredto:
1. separatethebacteriaintorecognizablecolonyformingunits
2. reducethenumbersto50300perplateforcounting
3. distinguishdifferentcolonytypesforcharacterizingindividualspecies.
During culturing some cells may die. This depends on the nutritional demands of the bacteria, how the sample is
protected from oxygen exposure and on the inoculation time (54). The sensitivity of culturing can be improved with the
useofselectivemediaduringsubculture(38).Selectivemediacanlowerthedetectionlimitnotjustbecauseaparticular
speciesispromoted,butbecauseotherspeciesarerepressed,whichtheninvolvesfewerdilutionstodistinguishspecies.
PCRmethodsarewellsuitedtotargetknownorpreviouslyculturedandsequencedmicroorganisms.Itcanbedone
quicklywithhighspecificityandwherethetargetmaterialispresentinlowamounts.PCRanalysishastheadvantagethat
itcanamplify,withoutdilution,aparticularsequencefromtheambientbackgroundmaterial.ItisestimatedthattheDNA
from 10 cells can be detected by PCR (38). Although PCRbased methods are highly specific, a disadvantage is that
unidentifiedornontargetedspeciescannotbedetected,andcellnumberscannotbemeasuredwithconventionalPCR.
In1996,amethodthatallowsestimationofthenumberofbacteria,realtimePCR(RTPCR),wasdescribed(55),butto
ourknowledgethismethodhasnotyetbeenappliedintheanalysisofendodonticinfections.
Bacteria may be identified by PCR but not by culture (56,57), although in some cases the difference is slight (58).
Excludingthepossibilityofcontamination,thelikelyreasonsarethehighersensitivityofPCRandthatPCRidentifiesDNA
sequences,whetherlivingordead,comparedwithculturingthatdetectsonlyviablecells.Whilsteverylivingcellshouldbe
cultivable,bacteriamaybeundetectablebycultureif(i)thenumberofcellsisextremelylowor(ii)bacteriaareinjuredbut
notdead(59),(iii)thespeciesisculturedifficult(butpossible)or(iv)thespeciesisimpossibletocultureinvitro.
LimitationsofPCR
Application of molecular methods in microbiology has revolutionized the taxonomic grouping of genera and allowed
speciestobesystematicallyclassifiedaccordingtogeneticstructureratherthanphenotypicbehavior.ThePCRtechnique
in particular has allowed rapid identification of microorganisms that are difficult to culture. It is important, however, to
recognizethelimitationsofthismethodsothatinterpretationofthedataisreliableandvalid.
ThePCRmethodisahighlysensitiveassayforDNAdetection,butviableordeadcellsareindistinguishablebytheir
nucleotide sequence. Thus, microbial detection by PCR reveals current inhabitants of the root canal, but it may also
representahistoricalrecordofthosemicroorganismsthathaveentered,buthavenothadthecapacitytoestablishand
survive. How long DNA from dead microorganisms may persist in the root canal is unknown. PCR methods have been
successfully used to amplify Mycobacterium tuberculosis from several hundred to 1000yearold animal and human
remains(6063).
The special binding affinity of hydroxyapatite for DNA has long been known (64) and this complicates recovery and
analysisoftheauthenticendodonticmicrobialflora.OnceDNAisadsorbedbydentine,itmayprovedifficulttoextract(47,
65), and this problem raises several questions. For example, DNA from dead bacterial cells could be bound to coronal
dentine, which could potentially contaminate the root canal sample. What is the fate of DNA from bacteria that have
enteredandnotsurvivedintherootcanal?Todate,theseissueshavenotbeenadequatelyaddressedinstudiesusing
molecular techniques, which leave open the question as to whether the reported species truly represent the living
microbialfloraoftherootcanalatthetimeofsampling.
Therearefewreportsintheendodonticliteraturecomparingresultswhenalternativemolecularmethodshavebeen
applied to the same clinical material. In one study (66), checkerboard DNADNA hybridization was compared with 16S
rDNAbasedPCR.Thetwomethodsproduceddisparateresultsforthenumberofteethpositiveforindividualspeciesand
dissimilar matching between positive results. For example, Treponema denticola was detected by PCR in 23 of 50
samples and by checkerboard in five of the same 50 samples, with four samples positive by both methods. Tanerella
forsythensiswaspositivein11ofthe39samplesbyPCRandin15samplesbycheckerboard,andonlyeightsamples
hadmatchingpositiveresults(66).Severalfactors,suchasoligonucleotidedesignandpreparation,andtime/temperature
protocolsforhybridizationandamplificationcouldaccountforthedisparateresults.
The lack of studies with calibration of molecular methods suggests uncertainty about the authenticity and validity of
reported results. Molecular methods offer sensitivity and precision, but a rigorous scientific approach with appropriate
controlsisessentialfortheretobeconfidenceinthevalidityofthedata.
Florainuntreatedrootcanals
Therootcanalinfectionisadynamicprocessandvariousbacterialspeciesdominateatdifferentstagesofthisprocess.
Inalongstandinginfection,thereisashifttowardsdominanceofthecommunitybyselectedspecies.Themostimportant
factorsdrivingthisdevelopmentareavailabilityofnutrition,oxygenlevel(redoxpotential)andthelocalpHwithintheroot
canal.
Exogenousnutrients,suchasfermentablecarbohydrates,canaffectthemicrobialecologyofthecoronalpartsofan
exposed root canal, but endogenous proteins and glycoproteins are the principal nutrients in the main body of the root
canal system. It might appear that the source of proteins in the root canal is restricted because of the progressive
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degradationofthesmallvolumeofpulpaltissue,butthebacteriainduceaperiapicalinflammationthatleadstoinfluxofa
serumlike exudate into the canal. This fluid is a sustainable nutrient source containing proteins and glycoproteins for
thosebacteriathathaveaproteolyticcapacity.Thebacteriathatdominatethisstageoftheinfectionarelikelytobethose
that either have a proteolytic capacity, or maintain a cooperative synergy with those that can utilize this substrate for
bacterial metabolism. Bacterial metabolism of the serumlike fluid also causes reduction of the redox potential and a
concomitantriseinthepH(8).
Thespeciescommonlyrecoveredbyculturefromrootcanalsofteethwithapicalperiodontitishavebeenpreviously
reviewed(5).Applicationofmolecularmethodsformicrobialdetectionhasmeantthatseveraladditionalspeciescanbe
includedastypicalofthemicrobialfloraoftheinfectedrootcanal,whicharedescribedbelow.
Developmentswithmoleculartechniques
During the last decade, molecular techniques have increased our ability to differentiate bacteria and led to the
establishmentofnewgeneraandspecies.Toagreatextentthesearesplitofffrompreviouslyestablishedgeneraand
species. An example of cultured bacteria that are subdivided and reclassified, are the fastidious, asaccharolytic, strict
anaerobic,slowgrowing,small,GrampositiverodsbelongingtothegenusEubacterium. When they were first reported
fromrootcanalsamples,thestrainshydrolyzingargininewerecharacterizedasEubacteriumlentum,andstrainsnegative
inthisaspectweredesignatedasEubacteriumgroup4(4).Later,strainsbelongingtothelattergroupwereclassifiedas
a new species Eubacteriumtimidum(67). On the basis of phenotypic characteristics, DNADNA hybridization data and
phylogenetic analysis with 16S rRNA gene sequence data, new species have been established from these two
eubacteria.OralstrainsofE.lentumhavebeenreclassifiedasEubacteriumexiguum,laterSlackiaexigua(68,69).The
newgeneraandspeciesMogibacterium timidum, Mogibacterium pumilum, Mogibacterium vescum (70), Mogibacterium
neglectum(71) and Cryptobacteriumcurtum(72) have been established from root canal and periodontal isolates of E.
timidum(68).
Whilstmolecularmethodshavehelpedtodifferentiatethesespecies,theisolateswereoriginallyobtainedbyculturing
from infected canals. A comparison between advanced culturing and molecular methods is possible for the species E.
lentumandS.exigua,sinceS.exiguaistheonlyspeciesderivedfromE.lentum(68).WithaspeciesspecificPCRprimer,
and a detection limit of 10 organisms, S.exigua was found in41% of teeth undergoing root canal treatment (73). This
correspondswellwithculturingwhereE.lentumhasbeenfoundin31%ofteethwithnecroticpulpsandperiapicallesions
(28). The material may not be fully comparable, but the differences in detection limits (approximately 0.5% for culture)
mayindicatethatwhenthisbacteriumoccurs,itisinnumbersabovethislevel.
Recently,studiesusingPCRhavereportedthespeciesDialisterpneumosintes(formerlyBacteroides pneumosintes)
and Filifactor alocis (formerly Fusobacterium alocis) to occur in 66% and 46% of root canals of teeth with apical
periodontitis,respectively(74,75).Becausethesespeciesarenonfermentative,strictlyanaerobic,slowgrowing,forming
diminutive colonies even after long incubation and unreactive in most biochemical tests, there are few reports of
identificationincultureisolates.AlthoughD.pneumosinteswasonlydescribedasaspeciesin1994(76),theyhavebeen
isolatedbyculturing(28,77),butinlowerfrequencies(3035%).ThesensitivityofPCRbasedscreeningisoneobvious
reasonforthehigherfrequencyofdetection,buttheproblemsinvolvedingrowth,isolationandidentificationarefurther
possiblereasonsforinfrequentidentificationbyculturemethods.
UsingthePCRmethod,severalbacterialspecieshavebeenfoundtobemoreprevalentinrootcanalsthanpreviously
reportedbyculturebasedmethodsandanumberofselectedspeciesareshowninTable1.
Blackpigmentedbacteria
Blackpigmented bacteria (BPB) have frequently been isolated from infected root canals and, due to their proteolytic
activity,areimplicatedinapicalabscessformation(18,20,21,78,79).P.intermedia(formerlyBacteroidesintermedius)
hasbeenthemostcommonlyisolatedspecies.In1992,isolatespreviouslyclassifiedasP.intermediawerereclassified
asanewspecies,P.nigrescens(80).P.intermediaandP.nigrescenscannotbedifferentiatedusingroutinephenotypic
methods. Using sodium dodecyl sulfatepolyacrylamide gel electrophoresis and PCR, it has since been shown that P.
nigrescensismorecommoninendodonticinfectionsthanP.intermedia(81).P.intermediaandP.nigrescenshavebeen
culturedfrom2640%ofrootcanalsofteethwithapicalperiodontitis(28,32,82),althoughinonePCRbasedstudy,P.
intermedia/nigrescenswasdetectedinonly13%ofinfectedrootcanals(51).
Table1.Comparisonofculture(28)andmolecularmethodsselectedspeciesfromuntreatedinfectedroot
canals
Prevalence(%)
Study
P.
F.
Streptococcus P.
P.
A.
P.
intermedia/ P.
P.
C.
F.
Enterococcus
nucleatum sp.*
micros propionicum israelii alactolyticus P.
gingivalis endodontalis rectus alocis sp.
nigrescens
Fouadet
al.(50)
82
41
50
36
18
14
Siqueiraet
al.(102)
10
Siqueira
etal.(51)
13
28
43
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Siqueiraet
al.(120)
23
Siqueira
&Ras
(209)
23
56
Siqueira&
Ras
(210)
Siqueira
&Ras
(198)
36
Siqueira&
Ras(74)
46
48
35
34
11
34
34
25
Sundqvist
(28)
*Includes
facultative
andstricdly
anaerobic
species.
29
ThespeciesPrevotellatanneraehasalsobeenincludedintheBPBgroupsincestrainsofthisspeciesmayproduce
tan to black pigment when grown on rabbit blood agar (83). Root canal isolates initially characterized as P.
intermedia/nigrescens(84)havethenbeenshowntobeP.tannerae by 16S rRNA gene sequencing (85). Interestingly,
PCRusingprimersspecificforP.tanneraerevealedthat60%ofclinicalisolatesfromrootcanalsandabscesses/cellulitis
of endodontic origin were positive for P.tannerae. Thus, P.tannerae is an example of a species that was detected in
earlierculturingstudies,butunderaformername.Thehigherfrequencyreportedinmolecularcomparedwithculturing
studiesispartlyexplainedbytheinconsistencyofthisspeciestoformpigmentedcolonies(83),sothatisolatesmayhave
beenclassifiedasnonpigmentingsaccharolyticPrevotellaspecies.
OfotherBPB,P.endodontalisandP.gingivalishavebeenreported,inculturestudies,tooccurinfrequencieslower
than 10% (28, 82). In contrast, PCR assays have detected P. endodontalis and P. gingivalis in 43% and 28%,
respectively, of samples from necrotic pulps (51). The sensitivity of the PCR method probably accounts for the higher
reportedprevalenceofPorphyromonasspecies.
Identificationofculturedifficultspecieswithmoleculartechniques
Spirochetes are the group of organisms for which PCRbased identification has brought about the greatest revision of
reportedprevalenceinendodonticinfections.Morethan100yearsago,someofMillersdrawings(9)clearlyindicatedthe
presenceofspirochetes.Spirocheteshavethenbeenfoundinnecroticrootcanalsusingmicrobiologicalmethods(7,86,
87), darkfield microscopy (8890) and transmission electron microscopy (91). Together, these publications suggested
thatspirocheteswereonlyoccasionallyfoundandwhentheyoccurred,madeupasmallproportionoftheflora.
Recently,evidencehasemergedfromPCRbasedanalysisthatinfectedrootcanalscontainarangeofspirochetesin
muchhigherprevalencethanwaspreviouslythought(Table2).Themostpredominantspirochetesininfectedrootcanals
are T. denticola (9294) and Treponema socranskii (92, 93). The species Treponema lecithinolyticum (95) and
Treponema maltophilum (92, 95, 96) are moderately prevalent, and Treponema amylovorum (92, 95), Treponema
medium (95), Treponema pectinovorum (92, 93) and Treponema vincentii (92, 93) are infrequent inhabitants of the
infectedrootcanal(Table2).
Thesestudies(Table2)showconsiderablediversityofvaluesbetweenresearchgroups,forexampleprevalenceofT.
denticola has been reported at 13% and 78% (50, 93). Even the results derived from the same research group and
clinical material reveal dissimilar prevalence values, for example T.denticola is reported at 43% and 78% (93, 94). An
issueyettoberesolvediswhetherornotarelationshipexistsbetweenthepresenceofspirochetesandsymptoms.Some
findings support such a relationship (92), whereas other data suggest no association (93). Despite some progress in
understandingspirocheteecologyandpathogenicity(97),moreinformationisneededtoexplaintheirroleinendodontic
infections.
Tanerella forsythensis (formerly Bacteroides forsythus) is an example of a bacterium that is extremely difficult to
culture.ThisGramnegativeanaerobeisdependentonotherbacteriaforgrowthandwillnotgrowindependentlyinvitro,
ifthemediumisnotsupplementedwithNacetylmuramicacid(98).T.forsythensisisimplicatedinmarginalperiodontitis,
but to our knowledge has not been cultured from root canals. This organism was first described in infected root canals
whenPCRbasedtestsrevealedevidenceofitin18%ofsampledrootcanals(99).Usingsimilarmethods,othergroups
havefoundevidencethatT.forsythensisisarelativelyfrequentinhabitant(Table3),withreportedprevalencevaluesof
1655%(50,66,96,100103).
Moleculartechniquesdifferentiationornewspecies?
Molecularmethodshavegreatlyfacilitatedidentificationofculturedifficultspeciesandexpeditedtaxonomicgroupingwith
easeandenhancedprecision.However,theimpactofmolecularmethodsonunderstandingthediversityoftherootcanal
microbiotahasnotbeenasdramaticasitmightseem.WiththeexceptionofspirochetesandthespeciesT.forsythensis,
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whichareprevalentininfectedrootcanalsyetdifficulttocultivate,molecularmethodshaveidentifiedspeciesthathave
beenpreviouslydetectedbyconventionalculture.
Table2.Spirochetesininfectedrootcanals
Prevalence
(%)
Study
No.of
cases
(n)
T.
T.
T.
T.
T.
T.
T.
T.
Other
amylovorum denticota lecithinolyticum malthophilum medium pectinovorum socranskii vincentii Treponemes
Baumgartneret
al.(92)
138
29
30
14
45
Fouadetal.(50)
24
13
Jungetal.(96)
26
Rasetal.(93)
32
78
41
16
Siqueira&Ras
(95)
31
26
39
13
Siqueiraetal.(94)
54
43
Table3.Tanerellaforsythensis(formerlyBacteroidesforsythus)ininfectedrootcanals
Study
Numberofcases(n)
Prevalence
(%)
Fouadetal.(50)
24
17
Gonalves&Mouton(100)
11
55
Jungetal.(96)
73
16
Rasetal.(101)
50
26
Siqueiraetal.(102)
80
20
Siqueiraetal.(66)
39
28
Siqueira&Ras(103)
50
52
Florainrootfilledcanals
It is generally acknowledged that persistence of disease is most commonly due to difficulties that occur during initial
endodontictreatment.Inadequateasepticcontrol,pooraccesscavitydesign,missedcanals,inadequateinstrumentation,
andleakingtemporaryorpermanentrestorationsareexamplesofproceduralpitfallsthatmayresultinendodonticpost
treatmentdisease(104).
The reasons for disease persistence in welltreated rootfilled teeth have been poorly characterized until a series of
studies published during the 1990s. Using block biopsy material from nonhealed periapical tissues including apices of
therootfilledteeth,analysisbycorrelativelightandelectronmicroscopyhasshownthattherearefivefactorsthatmay
contributetopersistenceofaperiapicalradiolucencyaftertreatment.Thefactorsare:(i)intraradicularinfection(105)(ii)
extraradicularinfectionbybacteriaofthespeciesActinomycesisraeliiandPropionibacteriumpropionicum(106108)(iii)
foreign body reaction (109,110) (iv) cysts, especially those containing cholesterol crystals (111) and (v) fibrous scar
tissuehealing(112).Ofallthesefactors,itisgenerallybelievedthatthemajorcauseofpersistentdiseaseafterrootcanal
treatmentisthepersistenceofmicroorganismsintheapicalpartofrootfilledteeth.
Endodontic posttreatment disease, or apical periodontitis associated with a rootfilled tooth, can continue for many
yearsandmaybecomeapparentonlywhenatoothrequiresanewrestoration.Thefactthatsomemicroorganismsare
capable of survival under harsh, nutrientlimited conditions of the rootfilled canal for so long is remarkable. Yet, little
information was known about the microorganisms involved in persistent intracanal infection after root filling until 1998,
whentwostudiesrevealedthatthemicrobialfloraassociatedwithendodonticposttreatmentdiseaseisquiteunlikethat
foundinotheroralinfections,orthatoftheuntreatedrootcanal(44,45).
Microbiologyofcanalswithpersistentinfection
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Usually one or just a few species are recovered from canals of teeth with posttreatment disease. These are
predominantlyGrampositivemicroorganismsandthereisanequaldistributionoffacultativeandobligateanaerobes(44,
45).Thismicrobialfloraisdistinctlydifferentfrominfectionsinuntreatedrootcanals,wherethelattertypicallyconsistsof
a polymicrobial mix with approximately equal proportions of Grampositive and Gramnegative species, dominated by
obligateanaerobes.
There is some diversity of species isolated from rootfilled teeth with persistent periapical disease, but there is a
consensusamongstmoststudiesthatthereisahighprevalenceofenterococciandstreptococci(4446,113117).Other
speciesfoundinhigherproportionsinindividualstudiesarelactobacilli(44),Actinomycesspeciesandpeptostreptococci
(116) and P. alactolyticus, P. propionicum, D. pneumosintes, and F. alocis (113). Some bacteriological findings from
studiesofrootfilledteethwithpersistentdiseaseareshowninTable4.
Thereisadifferenceinthemicrobialflorabetweenpoorlytreatedandwelltreatedteethwhenthecanalsaresampled
atretreatment.Althoughonlyonepoorlyrootfilledtoothwasreported,thepolymicrobialflorawasfoundtobesimilarto
thatseeninuntreatedrootcanals(45).Thisobservationhasrecentlybeenconfirmedinastudy(117)wherecomparison
of the isolates in 38 poorly filled canals with 22 wellfilled canals revealed a significant association of the former with
polymicrobial infections. When teeth are poorly treated, it is not surprising that the flora after root canal filling should
approximate that of the untreated canal, especially if it is also poorly restored and there is microleakage from the oral
cavitythatallowsaninfluxofcarbohydratesandpossiblynewbacteria.
Table4.Bacteriologicalfindingsinrootfilledteethwithpersistentperiapicallesions
Study
Speciesperrootcanalwithbacteria
Enterococcussp.*
Streptococcussp.*
Candidasp.*
Actinomycessp.*
Mller(46)
1.6
29
16
ND
Molanderetal.(44)
1.7
47
20
Sundqvistetal.(45)
1.3
38
25
13
Hancocketal.(116)
1.7
32
21
27
Peciulieneetal.(115)
1.6
64
18
Cheung&Ho(118)
2.6(1.8)
ND
50
17
ND
Pinheiroetal.(117)
2.1(1.8)
55
33
20
Siqueira&Ras(113)
4.1
77
23
*Percentprevalence,incanalswithmicroorganisms.
IdentificationbyPCR.Allotherstudiesbyculture.
Excludingpoorlyfilledrootcanals.
ND,notdetected.
Theprevalenceofenterococcihasbeenaconspicuousfindinginallstudiesthathaveinvestigatedtheflorainroot
filled teeth (4446, 113117), with one exception (118), and implicates Enterococcus faecalis as an opportunistic
pathogeninpersistentapicalperiodontitis.Streptococciarealsocommonlyisolatedfromrootfilledcanalswithpersistent
lesions(Table4).Othermicroorganismsofinterestbecauseoftheirassociationwithendodonticposttreatmentdisease
arespeciesofActinomycesandCandida.Somepropertiesofthesespeciesaredescribedinmoredetailbelow.
Enterococci
Studies that have recovered microbes from filled root canals with persistent periapical disease have shown a high
proportionofenterococci,rangingfrom29%to77%(4446,113,115117).Thiscontrastswitharatherlowproportion
ofenterococci,around5%orless,recoveredfromuntreatedinfectedrootcanals(57,119,120)andraisesthequestion
ofhowandwhenenterococciestablishintherootcanal.Althoughmoreresearchisneededtoaddressthisissue,there
areseveralpossibleexplanations.
OnepossibilityisthatE.faecaliscouldbepresentinuntreatedcanals,butinsuchlownumbersthatitisnotrecovered,
orisoutcompetedbyothermicroorganismsinthebacterialconsortium.Whenenvironmentalconditionsimprove,itmay
growtohigherandrecoverableproportions.Inanimalexperiments(11),afterinoculationofaneightstraincollectionin
equal (12.5%) proportions, E.faecalis was reisolated at about 1% of the total microbial flora, which was similar to its
proportion when originally recovered from an infected tooth. Whilst this might account for some cases, it is unlikely to
explainallcasessinceevenwithsensitivemolecularmethods,E.faecaliswasonlydetectedin7.5%ofinfectedrootcanal
samples(120)comparedwithtentimesthatprevalenceincanalsassociatedwithposttreatmentdisease(113).
TheremustbeanotherexplanationforthehighprevalenceofE.faecalisinrootfilledcanalsassociatedwithdisease
and the most likely one is that E. faecalis enters the canal in the process of treatment, during or between treatment
sessions.E.faecalishasbeenfoundinahigherproportionofcanalsthatwereinadequatelysealedforaperiodoftime
duringthetreatment,orweretreatedover10ormoresessions(121).Althoughitisunlikelytooccurwhenthetoothhas
beenwellrestored,itisconceivablethatE.faecaliscouldenterafterrootfilling,asithasbeenshownthatpoorlyrestored
teethhaveahigherrateofendodonticposttreatmentdisease(122).
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Enterococciarepartofastablehostadaptedbacterialcommunityinhabitingthelargebowelofmostadulthumansin
numbers as high as 108 cfu/g of feces (123). They have a commensal relationship with the host, but under favorable
circumstancesmaytakeadvantageoftemporaryweaknessesinthehostdefensetoestablishinfection.ThespeciesE.
faecalis has some intrinsic characteristics that allow it to survive in conditions that are commonly lethal for many other
microorganisms.Thesepropertiesincludeanabilitytogrowinhighsaltconcentrations(6.5%NaCl),awidetemperature
range(1060C),40%bile,abroadpHrange,aswellaspersistinthepresenceofdetergents(124129).
E. faecalis and Enterococcus faecium are significant human pathogens particularly in nosocomial and antibiotic
resistant infections, yet their virulence factors are just beginning to be understood (130135). Some virulence factors
identified to date (123) are: (i) secreted factors such as a cytolysin and gelatinase (136) (ii) adhesins such as
aggregation substance, enterococcal surface protein (Esp), collagen adhesin (Ace) (137 141) and (iii) surface
structures such as capsular polysaccharide (142). A notable cause for concern has been the special capacity of E.
faecalisforacquiringantibioticresistancegenesfromotherorganismsorbyspontaneousmutation,makingitparticularly
difficulttocontrolanestablishedenterococcalinfection(143).
Thecharacteristicsrequiredforpersistentinfectionintherootcanalareunlikelytobethesameasthoseseeninsoft
tissueinfectioninotherpartsofthebody.Onepathogenicpropertyisaspecialcapacityforinvasionofdentinaltubules
(144146), particularly in the presence of serum (15) and in the absence of immunoglobulin G (147). Adhesion of E.
faecalis to dentine could be another factor of relevance for pathogenesis. A recent study has shown that the serine
proteaseandacollagenbindingprotein(Ace)areinvolvedinbindingE.faecalistodentine(148).
TheintrinsiccapacityofE.faecalistowithstandawidepHrangerepresentsaproblemforclinicalantibacterialcontrol.
Calcium hydroxide, which is generally a highly potent antimicrobial dressing (40, 149, 150), is ineffective because E.
faecaliscanendureahighalkalinityuptoaroundpH11.5(40,145,146,151154).Thenaturalbufferingeffectofdentine
(155158) affords further protection to alkalineresistant organisms since levels in dentine do not reach higher than pH
10.8 in cervical and pH 9.7 in apical dentine (156). The mechanisms of alkaline tolerance in E. faecalis have been
essentially unknown until recently when it was shown that a functioning cellwallassociated proton pump, which drives
protonsintothecelltoacidifythecytoplasm,isimportantforsurvivalofE.faecalisinahighlyalkalineenvironment(151).
WhilsttheabilityofE.faecalistoresisttheantimicrobialeffectofcalciumhydroxideremainsasignificantclinicalchallenge
inendodonticretreatment,itmaynotbeacriticalfactorforitsinvolvementinposttreatmentdisease.Arecentstudyof
retreatedteethinaNorthAmericanpopulation,wherecalciumhydroxideisinfrequentlyusedasarootcanaldressing,
showed that E. faecalis was recovered in similarly high proportions (116), which suggests that resistance to calcium
hydroxidemaynotbetheexplanationforselectionofthismicroorganism.
Anotherinherentcharacteristicofenterococciisanabilitytoadapttofluctuatinglevelsofnutrientsupplyandlimitation,
anditisthistraitthatmayfacilitatethepersistenceofE.faecalisinthecanallongafterrootfilling.Recently,thisproperty
wasexploredinaseriesoflongtermstarvationassays(159).E.faecalissurvivedinwaterformorethan4months,which
demonstratedthecapacityofE.faecalistoendurelongtermstarvation.Attheonsetofstarvationtherewasarapidfallin
viablecellnumbers,leavingaresidualsmallpopulationofstarvedcells(159).Thesestarvationstatecellswereshownto
beinaminimalmetabolicstate,sinceadditionofcellwallandDNAsynthesisinhibitorstoE.faecalis starvation cultures
resultedinlimitedchangeintherateoflossofviablecellnumbers.
Although there is little known about the source and type of nutrition available at the apex of a rootfilled canal, the
microbial flora may be sustained by a periapical tissue transudate. This is likely to be a serumderived fluid from
surrounding tissue (15, 160). Growth of E. faecalis in serum is possible (15, 161, 162). Longterm experiments with
culturesofE.faecalisinhumanserumshowedahighnumberofcellswerestillviableafter4months(159).Cellsalready
inastarvationstatewereshowntobecapableofrecoveryuponadditionofserum(159).ItislikelythatE.faecalismay
encounterperiodsofstarvationintherootfilledcanal,brokenbyopportunitiestoaccessserumorserumlikefluid.Under
suchconditions,evenasmallnumberofcellscangainthenutritionalsupportrequiredforsurvivalandwouldtherefore
havethepotentialtomaintainaperiapicaldiseaseprocess.
A more detailed review of enterococci and their role in posttreatment apical periodontitis appears elsewhere in this
issue.
Streptococci
Streptococcicomprisearelativelyhighproportion,approximately20%(range1650%,Table4),ofthemicroorganisms
recoveredfromthecanalsofteethwithposttreatmentdisease(44,45,115118).However,therecoveryofstreptococci
islessremarkablewhenitistakeninthecontextofitshighprevalenceinuntreatedinfectedcanals(5,32).
The genus Streptococcus contains a diverse range of species of which oral streptococci fall into four broad groups
(163,164). Analysis of the Streptococcus species isolated from teeth with endodontic posttreatment disease indicates
thatnoparticularspeciesorgrouphaveahigherprevalence.Whatstreptococcihaveincommonisapreferentialcapacity
forinvasionofdentinaltubules(165167),whichshouldfavortheirabilitytoenterandestablishintherootcanalsystem.
Streptococcal surface adhesins mediate binding to dentin as well as facilitating dentin invasion (166, 167) and
streptococcalinvasionofdentinmayalsofacilitatecoinvasionofotherspecies(168).
The ability of streptococci to penetrate or hide in dentinal tubules may be attributable to their pattern of growth in
chains,aphenotypiccharacteristicsharedwithenterococci.Thisabilitymayalsoaccountforthefindingofstreptococciin
approximatelythesameprevalenceininitialandposttreatmentrootcanalinfections.
Thereissomeevidencesuggestingthatstreptococciaredifficulttoeradicateduringtreatmentoftherootcanal.Ina
studythatevaluatedbacteriabeforeandafterinstrumentationoftherootcanals,Streptococcusspecieswererepeatedly
isolatedatuptothreesessionsoftreatment(32).Interestingly,inthesamestudy,Candidaspecieswerealsodifficultto
eradicate,whichdemonstratesthechallengesfacedinantimicrobialcontrol.
Candida
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Candidaalbicanshasbeenperiodicallyreportedinteethwithpersistentposttreatmentapicalperiodontitis(44,45,113,
115118)andyeastshavealsobeenobservedbyelectronmicroscopyinsuchteeth(105). Yeasts are seldom seen in
untreated root canals, unless canals have been open to the oral cavity (169) or there has been a history of protracted
treatment(170).Inonestudy,theprevalenceofC.albicansininfectedrootcanalswasreportedtobehigher,although
thetypeofclinicalmaterialwasnotstated(171).
Yeastshaveseveralpropertiesincommonwithenterococci.Yeastshavethecapacitytosurviveasamonoinfection
(170, 172) and several studies have shown a capacity for growth and invasion of dentine (173175), although in
comparisonwithE.faecalis,thispropertyisweak(175).Notsurprisingly,sodiumhypochloriteisapotentagentinkilling
Candidaspecies(176178)andEDTAisalsoreportedtobeeffective(179).Severalinvitro studies have reported that
Candida species resist the antimicrobial action of calcium hydroxide (176,180), which may be a factor for selection of
Candidainpersistentrootcanalinfections.
ThesecharacteristicssuggestthatbothCandidaandenterococcishareseveralpropertiesnecessarytoestablishand
survive in the harsh environment of the rootfilled canal. The properties include resistance to antimicrobials used in
endodontic treatment, an ability to grow in monoinfections, survival in conditions of nutrient limitation and an ability to
evadethehostresponsebysequestrationwithintherootcanalsystem.
Actinomyces
A.israeliiisofinterestbecauseitisaknownandrepeatedculpritintherapyresistantcases(107,181183)andisbyfar
themostcommonspeciesinvolvedinactinomycosis(184).ThelikelysiteofA.israelii infection is the periapical tissues
whereitisknowntobeinvolvedinperiapicalactinomycosishowever,itisinterestingthatithasbeenrecoveredfromthe
root canals of retreated teeth (45,116,117). The presence of A.israeliiin the root canal suggests the possibility of a
communication between the periapical tissues and the canal, where some protection may be afforded from the host
defense.
HowA.israeliiestablishesintheperiapicaltissuesisunknown.Itmaygrowoutasaclumpfromtherootcanalintothe
periapical tissues, or it may be forced from the root canal during instrumentation, thus inoculating the periapical tissue.
StudiesofexperimentalinfectionwithA.israeliiinanimalshaveshowncharacteristiclesionsofacohesivebacterialmass
ofbranchingfilamentssurroundedbyhostleukocytes(185188).
Identification of Actinomyces species has been hampered by problems with traditional biochemical methods of
characterization.AlthoughsomestudieshaveappliedDNAhybridizationmethods(120,189191),thesearenotreadily
applicable and reproducible from one lab to another. The partial characterization of the 16S rRNA gene (192) has
facilitatedthedevelopmentofprobessuitedtowidespreadapplication(193195).
A. israelii is the most prevalent Actinomyces species isolated from human abscesses however, Actinomyces
gerencseriae(formerlyA.israeliiserotypeII)isalsoprevalentandtheyarefoundin56%and25%ofhumanabscesses,
respectively(184).UsingcheckerboardDNADNAhybridizationanalysisofrootcanalsamplesfromteethdiagnosedwith
periapical abscesses, A. israelii and A. gerencseriae have been reported in 14.8% and 7.4% of samples, respectively
(120)however,theroleofA.gerencseriaeinpersistentinfectionafterrootfillingisunknown.
Recently, a new Actinomyces species, Actinomyces radicidentis (196), was found to be involved in posttreatment
disease(197).UsingPCRbaseddetection,ithasbeenshowntobepresentinuntreatedrootcanalinfectionsandroot
filledteethwithchronicapicalperiodontitis(198),althoughitsprevalenceinbothtypesofinfectionwaslow.
Actinomycesspeciessharesomepropertieswithenterococci,streptococciandCandidaincludingagrowthpatternof
cohesive filaments or chains, resistance to antimicrobials used in endodontic treatment, an ability to grow in
monoinfectionsandtoevadethehostresponse.
AmoredetailedreviewofActinomycesspeciesandtheirroleinposttreatmentapicalperiodontitisappearselsewhere
inthisissue.
Ecologicaldifferencesbetweenuntreatedandrootfilledrootcanals
The untreated infected root canal is an environment that provides microorganisms with nutritional diversity in a shifting
patternovertime.Thespeciesthatestablishhavetypicallyinvadedbycaries,cracksormicroleakagearoundfillingsand
theyseekshelter,nutritionandafavorablehabitat.Initially,theremaybeaninfluxofcarbohydratesfacilitatinggrowthof
facultative anaerobes, but as the infection matures, the available nutrients are mainly peptides and amino acids, which
favoranaerobicproteolyticspecies.
Whilstthemicrobialflorainanuntreatedinfectedrootcanalmayexperiencefeast,inthewellfilledrootcanalthereis
predominantlyfamine.Mostoralloftheoriginalnecroticpulpwillhavebeeneliminatedleavingdry,barrenconditionsfor
survivingmicrobialcells.Thesemicrobeswouldexperienceastaticenvironmentandstarvation,butwithsomeluckmay
encounteraserumlikefluidtransudatefromtheperiapicaltissue.Thespeciesthatpersistherearethosethathaveeither
survived the antimicrobial treatment and are the last ones remaining, or have entered during treatment and found it
possibletoestablishwhereotherscannotdoso.
Propertiesofspeciesassociatedwithendodonticposttreatmentdisease
With the exception of Actinomyces, which is primarily involved in extraradicular infection, other species associated with
persistent intraradicular infection described here, i.e. Candida, streptococci and enterococci, can be viewed as
opportunisticpathogens.Abehaviorincommonistoleavetheirnormalhabitatoftheoralcavityandestablishelsewhere,
intherootcanal,wheretheytakeadvantageofthelocalecologicalchangeintheenvironmentandwheretherehasbeen
eliminationofmicrobialcompetitors.
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Formicrobestomaintainapicalperiodontitisandcauseposttreatmentdisease,theymustdomorethanjustsurvivein
therootfilledcanaltheymustalsopossessthepathogenicpropertiesnecessarytoperpetuateinflammationexternalto
therootcanalsystem.Ingeneral,microorganismsinvolvedinpersistentinfectionsimplementoneofthreestrategiesto
evadetheimmuneresponsesequestration,cellularorhumoralevasion(199).Sequestrationinvolvesaphysicalbarrier
betweenthemicrobeandthehost.Cellularevasionmeansthatmicroorganismsavoidleukocytedependentantibacterial
mechanisms.Humoralevasionmeansthatextracellularbacteriaavoidthehostsantibodiesandcomplement.
At least two of the three strategies are deployed by microorganisms involved in endodontic posttreatment disease
(200).A.israeliiisanexampleofanendodonticpathogenthatdisplayscellularevasionbyavoidingphagocytosisbyPMN
leukocytesinvivo(185,187,188) primarily through a mechanism of collective cohesion (188).E.faecalis and Candida
speciesarerepresentativeofmicrobesthatareabletoremainsequesteredwithintherootcanalsystem.
The properties necessary for microorganisms to persist in the rootfilled canal are outlined in Fig. 2. Some of the
physiologicaltraitsrequiredforentryandinitialestablishmentmaybesimilartothatofmicrobesinhabitinganecroticpulp
in an untreated canal, such as an ability to find nutrients, compete with other microorganisms and evade initial host
defenses.
For species to survive endodontic treatment (Fig. 2, phase 2), there must be an ability to withstand biomechanical
cleaning and antimicrobial dressing. There are numerous reports confirming the bactericidal efficacy of sodium
hypochloriteagainstseveralspeciesinvolvedinpersistentinfectionsuchasA.israelii(201),E.faecalis(151, 202, 203)
andCandida(176178). It therefore seems reasonable to assume that these species may have the capacity to shelter
from the main root canal in weblike areas, or in dentinal tubules where some level of protection or buffering of the
antimicrobial agent is possible (157, 204). Although most root canal bacteria are sensitive to the high pH of calcium
hydroxide (40), several species involved in persistent infection are now known to have a capacity to resist the
antimicrobialeffectofthiscommonlyusedagent(40,145,146,151,152,180,201).
Fig.2.Challengesformicrobesinvolvedinpersistentinfection.
Howbacteriaendurerootfillingisunknown,butstudiesthathavesampledtherootcanalpriortorootfillingandthen
followedthetreatmentoutcomeofinfectedteethhaveshownthatsomelesionsheal(41,45,205,206),implyingthatthe
bacteria did not survive or were not in a position to inflame the periapical tissue. Whether or not bacteria survive root
canal filling may depend on whether they are entombed, or blocked from acquiring nutrition (104). It is possible, even
likely,thatbacteriamayundergoaperiodofstarvation.Here,theabilityofE.faecalistowithstandperiodsofstarvation
(159,207,208),isatraitthatmaybecrucialforsurvival.
Apicalperiodontitisisadynamicprocessinvolvinganinteractionbetweenhostandlivingbacteria,andthemicrobes
needtofindsubstratesforgrowth(Fig.2,phase3).Inawellinstrumentedrootcanalwherenecroticpulptissuehasbeen
removedandthereisnocommunicationwithexogenousnutrientsfromtheoralcavity,nutritionislikelytocomefroma
periapicalfluidtransudate,whichisprobablyserumlikeinnature(15).Anabilitytoutilizecollagenwithindentinemayalso
beusefulandthereareindicationsthatE.faecalismayhavethisproperty(15,148).Theprocessofacquiringsubstrates
forgrowthprobablyinvolvesenzymaticbreakdownofserumandtissuemolecules,andthispropertyincombinationwith
anabilitytoavoidthehostdefenseinduceaninflammatoryresponseintheperiapicaltissue.
Concurrentconditionsforpersistentinfection
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In a study that examined the influence of infection at the time of root filling on the outcome of treatment (41), 68% of
teeth, which were infected at root filling, healed after the treatment. Similar results have also been reported in other
studies(45,205,206).Whilstinfectionatthetimeofrootcanalfillingwilladverselyaffecttheoutcomeoftreatment,the
presenceofapersistentpathogen,alone,isnotsufficientforpersistenceofdisease.Theremustbeaset of conditions
thatoccurincombinationtoresultinpersistenceofendodonticdisease.TheseconditionsareshownconceptuallyinFig.
3.Asetofmicrobialcharacteristics,coincidingwithasetoflocationparameterspermitsaninteractionwiththehostthat
willdeterminewhethertherewillbepersistentapicalperiodontitis.
Thesetofconditionsformicroorganismsincludeanabilitytoevadetheantimicrobialstagesoftreatment,persistence
characteristicssuchasastarvationsurvivalpotential,andacapacitytoinflamehosttissue(Fig.3).Locationparameters
arealsoimportant.Providedthatmicrobescanenterandreachtheapicalarea,theymustbesituatedneartheapical(or
accessory)foramenandhaveanopencommunicationforthefreeexchangeoffluid,moleculesandorganismsinorderto
inflameperiapicaltissue.Theintersectionofalltheseconditionswiththehostdefenseresultsinpersistenceofdisease.
Fateofbacteriathathaveenteredtherootcanalbutdonotsurvive
Allbacteriahavethetheoreticalcapacitytoenterandestablishintherootcanal,butfewdoso.Somemayenterdentine,
butdonotreachtherootcanal.Othersmayreachtherootcanal,butdonotsurvive.Thefateofthosebacteriathatenter
andreachtherootcanalbutcannotestablishorsurviveisunknownhowever,theircellcontentspresumablydisintegrate
oraredegradedbyothermicroorganisms.
ThefateofDNAfromdeadspeciesisalsouncertain.Thereremainsapossibilitythatafterlysis,theDNAfragments
fromthesecellsmightlingerinthecanalorbeboundtodentineandifso,suchminuteamountswouldconceivablybe
detectedandamplifiedbyPCR.ThepresenceofintrinsicorexogenousDNAaseswouldalsoinfluencehowlongtheDNA
wouldpersist.
Intheonlyexperimentalstudyknowntousthathasexaminedtheroleandfateofaknownmicrobialcollection(11),
variousknowncombinationsofaneightstraincollectionofindigenousoralbacteriawereinoculatedintomonkeyteeth.At
theendoftheexperimentalperiod,Bacteroidesoralis(nowPrevotellaoralis)dominatedinmixedinfections,yetcouldnot
bereisolatedwhentheyhadbeeninoculatedinpureculture.Thefateofthebacteriathatwereinoculatedinitially,but
werenotdetectedattheendoftheexperimentalperiodisamatterofspeculation.Whilstthespeciespresumablydied,it
isalsopossiblethatsomecellssurvivedbutinsuchlownumbersthatwerenotdetectablebyculture.
Fig.3.Persistentinfectionrequiresnotone,butaseriesofcoupledcharacteristics.Bacteriamustpossessan
abilitytosurvivethestagesoftreatment,persistencecharacteristicsandanultimateabilitytoinflamehost
tissue.Thelocationofbacteriaiscriticalforthemtosourcenutrientsandinflametissue.Theconcomitant
interactionofthesecharacteristicswiththehostdefenseresultsinfailuretoheal.
Inanotherstudy,subgingivalplaquewasgrowninseruminachemostat(16).Oneofthemembersofthemicrobial
consortium, P. intermedia, was not detected initially, but after repeated serum enrichment it dominated the flora. This
informationshowsthatsomebacteriacanbepresentinlownumbers,belowthedetectionlimitofthecultivationmethod.
Summary
Infection of the root canal is not a random event. The type and mix of the microbial flora develop in response to the
surrounding environment. Factors that influence whether species die or survive are the particular ecological niche,
nutrition,anaerobiosis,pHandcompetitionorcooperationwithothermicroorganisms.Speciesthatestablishapersistent
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rootcanalinfectionareselectedbythephenotypictraitsthattheyshareincommonandthataresuitedtothemodified
environment.Someofthesesharedcharacteristicsincludethecapacitytopenetrateandinvadedentine,agrowthpattern
ofchainsorcohesivefilaments,resistancetoantimicrobialsusedinendodontictreatment,aswellasanabilitytogrowin
monoinfections, to survive periods of starvation and to evade the host response. Microorganisms that establish in the
untreatedrootcanalwouldexperienceanenvironmentofnutritionaldiversitythatchangeswithtime.Incontrast,thewell
filledrootcanaloffersthemicrobialfloralittlemorethanshelterfromthehostandmicrobialcompetitors,butinasmall,
dry,nutritionallylimitedspace.Inallcases,itistheenvironmentthatselectsformicroorganismsthatpossesstraitssuited
toestablishingandsustainingthediseaseprocess.
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