Microflora of Infected Non-Vital Primary Teeth

Comparative Evaluation of Bactericidal Potential of Four Root Canal Filling Materials against Microflora of Infected Non -Vital Primary Teeth
Harini Priya M* / Sham S Bhat ** / Sundeep Hegde K ***
Background and objectives: Since complete debridement of the root canals of the primary teeth is not practically possible due to the highly variable root canal anatomy, success of the endodontic therapy depends partly on the use of antibacterial irrigating agents and root canal filling materials. Recent literature indicates that anaerobes comprise a majority of the bacteria in necrotic root canals of primary teeth. The study determined the antibacterial effectiveness of four root canal filling materials namely Calcium hydroxide, Zinc oxide eugenol, Vitapex and Metapex against microbial specimens obtained directly from necrotic root canals of primary teeth. Method: Microbial specimens were collected using sterile paper points, from 15 primary maxillary and mandibular posterior teeth of randomly selected children in the age group of 4-10 years with infected non vital primary teeth, requiring pulpectomy procedure. The microbial specimens collected were subjected to microbiological analysis and the antimicrobial potential of root canal filling materials were tested using Agar diffusion technique. Results: were statistically analyzed using one-way ANOVA. Facultative/Aerobic organisms were isolated in all the cases, anaerobic organisms were isolated in 80% of the cases, and Candida albicans was isolated in 1 case. ZOE showed superior inhibitory activity against most of the organisms isolated followed by Vitapex, Calcium hydroxide and Metapex in descending order. Conclusion: Our data may be useful as a guide for relative antimicrobial effectiveness or non-effectiveness of the materials employed. In vivo studies are required to state the specific antimicrobial activity and merits and demerits of any of the test filling material. Keywords: Zinc oxide eugenol, Vitapex, Calcium hydroxide, Metapex, micro-organisms, root canal, primary teeth. J Clin Pediatr Dent 35(1): 2330, 2010 uccessoftheendodontictherapydependspartlyontheuse s of antibacterial irrigating agents and root canal filling materials.2 Thus, an endodontic filling material with considerable antibacterial property becomes a pre requisite particularly whentreatinginfectednon-vitalprimaryteethorthosewith necroticpulp. Antimicrobialactivityofrootcanalfillingmaterialshas beenextensivelystudiedbyagardiffusiontechniquesusing pureculturesoforalbacteria.2,3 Mostoftheseinvestigations focusedonfacultativestreptococci andstaphylococci,which maynothaverepresentedthepredominantbacterialspecies foundininfectedrootcanals.Anumberofanaerobicspecies that are predominantly present in the infected root canals have not been included in agar diffusion testing of dental materials.2 Hence,ourinvestigationisanevaluationofthebactericidalpotentialoffourrootcanalfillingmaterialsagainstthe microfloraofinfectednonvitalprimaryteeth. Theobjectivesofthepresentstudywere: 1. To identify the microflora of the root canals of infectednon-vitalprimaryteeth.
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INTRODUCTION Successofendodontictherapyofprimaryteethdepends,in part, on the elimination or reduction of bacteria present in therootcanal.1 This may be accomplished by mechanical debridement anduseofantibacterialirrigatingagentsandrootcanalfilling materials.2 Since complete debridement of the root canals of the primary teeth is not practically possible,

* Harini Priya M, BDS, Post Graduate Student, Department of Pedodontics and Preventive Dentistry, Yenepoya Dental College Hospital. ** Sham S Bhat, MDS, Professor And Head Of The Department, Department of Pedodontics and Preventive Dentistry, Yenepoya Dental College Hospital. *** Sundeep Hegde K, MDS, Professor, Department of Pedodontics and Preventive Dentistry, Yenepoya Dental College Hospital. Send all correspondence to Harini Priya M, Department of Pedodontics and Preventive Dentistry, Yenepoya Dental College Hospital, Nithyananda Nagar P.O., Derelakatte, Mangalore- 575 018, Karnataka State. India. Phone + (0824) 2204668 / 2204669 Ext 227 Fax + (0824) 2204663 Email: deerpriya@rediffmail.com The Journal of Clinical Pediatric Dentistry

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Microflora of Infected Non-Vital Primary Teeth

2. TocomparethebactericidalpotentialofrootcanalfillingmaterialsnamelyCalciumhydroxide,Zincoxide eugenol,VitapexandMetapexforprimaryteeth. MATERIALS AND METHODS The study group consisted of fifteen healthy randomly selectedpatientsofbothsexes,betweentheagegroupof48years,attendingtheout-patientblockoftheDepartmentof Pedodontics and Preventive Dentistry, Yenepoya Dental College Hospital, Derelakatte, Mangalore. Fifteen primary and mandibular posterior teeth (one from each patient), which were infected and non-vital, requiring pulpectomy procedure and with the following inclusion criteria, were includedinthestudy. Inclusion criteria 1. Healthychildrenwithoutanyknownsystemicillness. 2. Antibiotics not received by the subject four weeks priortothesampling. 3. Containedatleastonenecroticrootcanal. 4. Anabscess,sinustractorobviousradiolucencymust bepresent. 5. Did not have extensive root resorption or broken crowns.4,5 Exclusion criteria for selection 1.Subjectsknowntohaveanysystemicillness. 2.Subjectsunderantibiotictherapy.4,5 A detailed medical, dental history, history of antibiotic use was recorded before the collection of the specimen. Informed oral and written consent was obtained from the parentsoftheparticipantsbeforethecommencementofthe study. CLINICAL PROCEDURE The entire procedure was carried out under strict aseptic conditions.Noendodonticprocedurewasperformedbefore collectionofthesample,soastoavoiddisturbingtheroot canalflora. Priortothestartoftheprocedure,theinvolvedtoothand thesurroundingareawerewipedwith10%povidineiodine solutionandthetoothwasisolatedwithrubberdam. Carious dentin was removed followed by access cavity was preparation using a sterile number 4 bur followed by pulpextirpation.ThreesterileN20paperpointswereintroducedintotheaccessiblerootcanalsandleftinplaceforone minute.5, 6, 7 The three root canal samples so obtained were transferredimmediatelyinto: A)RobertsonCookedMeatMedia(R.C.M)forthecultivationofanaerobicorganisms B)Brain Heart Infusion (B.H.I) broth for cultivation of aerobicorganisms C)Sterile test tube for further smear preparation to observetheorganismsbyGramstainsrespectively. The antibacterial potential of the root canal filling 24 aterials namely: Calcium hydroxide (Deepti Dental Prodm uctsofIndia,A.P,India),Zincoxideeugenol(VishalDentocarePvtLtd.,Gujarat,India),Vitapex(NeoDentalChemical Products Co. Ltd, Tokyo, Japan), and Metapex (Meta Biomed Co. Ltd, Korea) towards the organisms isolated were assessedbyKirbeyBaucerAgarplatediffusiontechnique.8 LABORATORY PROCEDURE Smearswerepreparedonasterileglassslidefromeachof thespecimencollectedinthesteriletesttubeandsubjected to Gram staining and were observed under oil immersion lensforthepresenceofbacteria. The specimens inoculated into R.C.M and B.H.I were inoculatedat37oCovernight.Subculturesweremadefrom B.H.Ibrothintobloodagar,heatedbloodagarandMcConkeysmediaandincubatedat37oCovernight. FromR.C.M,subculturesweremadeontwobloodagar plates.OnebloodagarplatewasincubatedinMcIntoshfield jarwithGaspackforanaerobiccultureat37oCfor48hours withmetronidazoledisktoscreenoutthegrowthoffacultativeanaerobicorganisms.Thesecondbloodagarplatewas incubatedaerobicallyat37oCovernight. The development of colonies on aerobic culture and anaerobicbloodagarwerestudiedforthecolonymorphology, Gram stain and biological characterization. The standardmethodforisolationandidentificationofmicroorganisms were followed as per Cowan and Steels,9 Vette and McCarter,10 TopleyandWilson,11 MendelandBennet.12 The powder liquid ratio of all the test root canal filling materialswerestandardizedaccordingtotheformulagiven by Tchaou et al.4 An electronic balance and micro pipette wereusedtomeasuretheexactamountofpowderand liquid tobedispensed. AGAR DIFFUSION ASSAY Sensitivitytestingoftherootcanalfillingmaterialsagainst the isolates obtained was performed in Muller-Hilton agar bycup-platemethodofKirby-Bauer.8 Theagarplatesweredriedand4wellsof4mmdiameter and3mmdepthweremadeintheagarplatesusingsterile agarpuncher.Usingasterileswab,theentiresurfaceofthe agarplatewasswabbed3timestoensureevendistribution oftheinoculumandtoobtainlawnculture. Thefourrootcanalfillingmaterialsviz,Calciumhydroxide,Zincoxideeugenol,VitapexandMetapexweretestedin each plate. Each of the four filling materials was filled in eachwellintheagarplate.Theseplateswereincubatedat 37o degreesovernight.Thediameterzonesofinhibitionin millimeteraroundthefillingmaterialsweremeasuredafter 24hoursforaerobicisolatesandafter48hoursforanaerobicisolates. RESULTS In the present study, a total of 15 root canal samples were studied. Out of the total 15 samples, 12 samples (80%) exhibited polymicrobial infection, 3 samples (20%) exhibitedthepresenceoffacultative/aerobicorganisms.Candida
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G

70 60 50
Percentage

40 30 20 10 0
S. cc vi i r i S. d sa ans l S. iva py riu s Ps o e u gen do u m s on A Kle a s . bs Pe str e p iell pt os to a c tr ep oc to ci co V cc F u eill i s o ion b a el c t la Po er rp iu m h y Pre vo ro t m e C an o n l l a as di da al s p bi ca ns re au te En h ro co us

St

ap

Organisms
Graph 1. Microorganisms isolated

albicans was isolated from 1 specimen. The organisms solatedaregiveninGraph1. i A total of 14 species so isolated were employed in the experimental procedure. The isolated organisms were dividedinto5groupsbasedonbacteriaorfungi,aerobicor anaerobicbacteria,grampositiveorgramnegativebacteria. Themeanzonesofinhibitionofthefourrootcanalfilling materials against the 14 organisms isolated are given in
Table 1. Mean Zones Of Inhibition (Mm) Of 4 Filling Materials Against 14 Organisms Organisms Staphylococcus aureus Enterococci Streptococcus viridans Streptococcus salivarius Streptococcus pyogenus Pseudomonas Klebsiella Anaerobic streptococci Peptostreptococci Veillionella Fusobacterium Prevotella Porphyromonas Candida albicans Ca(OH)2 12.5 12.75 13.2 13.5 13 12 14 13.5 13 14 12 11.6 13.33 4 ZOE 16.5 16.75 18.1 18 20 15 20 20 18 20 20 18.8 18 8 Vitapex 14 14.75 13.3 10.5 6 12 18 14.5 13 17 14 12.6 13 4 Metapex 12.5 13.5 11.9 7.5 2 12 13 14 13 15 12 11.2 12.66 1

Table1. Measurements of the inhibitory zones are ranked arbitrarily into the following three categories according to the proportionaldistributionofthedataset:4,5 1. No/Weakinhibition(NW) 2. Mediuminhibition(M) 3. Stronginhibition(S). Zonesizecategoriesandproportionsofdatarepresented ineachcategoryarerepresentedinTable2.
Table 2. Ranking Scheme For Microbial Inhibition Rank RANK RANGE OF ZONE DIAMETERS % OF DATA SET (mm) REPRESENTED 12.5 46.42 41.07 FREQUENCY (N= 56) 7 26 23

NO/WEAK 1-8 MEDIUM STRONG 8.1-13.5 >13.5

Statistical Analysis Statistical analysis was carried out by one-way ANOVA usingSPSSversion11.5withpost-hocteststocomparethe statistical difference of antimicrobial effects between the materials tested with each of the four bacterial groups (AerobicGrampositive,AerobicGram-negative,Anaerobic Gram-positiveandAnaerobicGramnegative). 25

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18 16 14 12 Mean of zones of 10 inhibition 8 6 4 2 0
Ca(OH)2 ZOE Vitapex Metapex

17.65

12.31

12.62 10.59

Filling materials
Graph 2. Mean of zones of inhibition of Filling materials

Statisticaldifferenceoftheantimicrobialeffectsbetween the materials against the fifth group (fungi) could not be doneasCandidaalbicanswasobtainedinonlyonecase. ZOE exhibited the maximum inhibitory effect followed by Vitapex, Calcium hydroxide and Metapex. The mean zones of inhibition of the filling materials are given in Graph2. Statsticallytherewasveryhighsignificantdifferencein theinhibitoryactivitybetweenthematerials.(p=0.001) The statistical difference in the inhibitory activities between ZOE and Vitapex were highly significant (p=0.008). ThedifferencesintheinhibitoryactivitiesbetweenZOE and Metapex were statistically very highly significant (p=0.001). The statistical difference in the inhibitory activities between ZOE and Ca (OH) 2 were highly significant (p=0.004). DISCUSSION Thereareveryfewstudiesconcerningrootcanalmicroflora of the primary teeth. Marsh and Largent13 reported alpha hemolytic streptococci as the predominant microorganism whereasotherstudies14,15 reportedthatthemostpredominant microorganisminrootcanalsofprimaryteethwithnecrotic pulp and periapical lesions were Streptococcus salivarius. Anaerobic organisms represented over 70% of the microflora of the primary molars that had been treated unsuccessfullyandwerealsothemostprevalentbacteriain teethindicatedforextraction. In the present study, aerobic and anaerobic bacteria, black-pigmented bacilli, streptococci and Gram-negative aerobic rods were found. This is in agreement with Toyoshimaet al16 whoreportedthatinrootcanalsofprimary 26

teeth with necrotic pulp and periapical lesions there is a polymicrobialinfection,similartomicrobiotaofpermanent teeth. Thepresentstudyalsoreportedthepresenceof Candida in1case;thereareconsiderablyfewreportsintheliterature regardingthepresenceofCandida intherootcanal.6 Facultative / aerobic organisms were present in all 15 cases (100%). However, this finding differs from that of Silva et al 17 who reported aerobic organisms in 60% of necroticrootcanalsofprimaryteeth.Streptococciwerepresent in 86.65% of the cases, which is consistent with the findingsofSilva et al 17 whoreported85%prevalenceand also comparable with the findings of Marsh and Largent13 whoreported82%prevalence. In our study, Streptococcus viridans was the organism waspresentinmajorityofthecases(66.66%),whichiscomparabletothatofthefindingsofMarshandLargent13 who reported Streptococcus viridans (alpha hemolytic streptococci)asthepredominantorganism. Streptococcus salivarius werepresentin13.33%andStreptococcuspyogenusin 6.66%ofthecaseswhichdiffersfromthatofCohen et al 14 whoisolated Streptococcus salivarius in70%ofthecases. However,inthestudybyCohen et al 14 theorganismswere isolated from open, infected primary teeth whereas in our study only those teeth which did not have broken crowns weretakentoavoidthepossiblecontaminationoftheroot canalmicroflorawiththatofthesalivaryorganisms. Enterococci werefoundin26.66%ofthecaseswhichis consistentwiththefindingsofRocas et al 18 whoreported 33%. Staphylococcus aureus wereisolatedin5%ofthecases byCohenet al 14 while13%inourstudy. In the present study, aerobic Gram-negative organisms werefoundin20%ofthecases[Pseudomonas (13.33%)and
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Klebsiella (6.66%)],whichiscomparablewiththefindings of Silva et al who isolated Gram-negative aerobic rods in 15%ofnecroticprimarymolars. Anaerobicbacteriawerepresentin12canals(80%)inthe presentstudy. Veillonella wasfoundin6.66%whichiscomparablewith thefindingsofPeterset al19whoisolatedVeillonella species in4%ofinfectedrootcanals. Amongtheanaerobicorganisms,black-pigmentedbacilli havefrequentlybeenisolatedfromrootcanalsofpermanent teeth with necrotic pulp.17 Sundqvist et al 20 reported their presence in 30% of the cases, Toyoshima et al16 isolated black pigmented bacilli in 44.4% of retreatment cases. While another study,reported their presence in 30% of the cases.17 In the present study, black-pigmented bacilli (Prevotella and Porphyromonas)werepresentin53.33%ofthe caseswhichmaybecomparablewiththefindingsofaprevious investigation that found 49%. Peptostreptococcus wereisolatedin6.66%ofthecases,whichisconsistentwith thefindingsofChuet al 21 [10%]andAdibet al 22 [6.7%]of thecasesanddiffersfromthatofGomeset al 23whoisolated theirpresencein35%ofinfecteddentalrootcanals. Anaerobicstreptococciwerefoundin13.33%inthepresentstudy,whichiscomparablewiththefindingsofAdibet al 22 who reported 17.5%. Fusobacterium were isolated in 6%ofthecasesinourstudy. Reported rates of yeast incidence in root canals vary widely(3to55%),dependinguponthepopulationsampled andthesensitivityofthesamplingtechnique.6 Candida albicans wereisolatedin6.66%ofthecasesinourstudy,which may be comparable with a previous study byAdib et al 22 whoisolated2.4%of Candida species. Most of the previous studies that tested antibacterial activityoffillingmaterialsagainstpureculturesusedGrampositive cocci, Gram-negative rods, or other bacteria that maynothaverepresentedthepredominantbacterialspecies found in infected root canals, i.e. Peptostreptococci, Prevotella speciesandobligateandfacultative streptococci.2 Whileourobjectivewastotestbacteriathatwererepresentativeofendodonticmicrobiota,comparingourdatawith previous studies is difficult because of the different test strains,mediaandcultureconditionsinvolved. Thebacterialspecimensinthisstudywerecollectedfrom therootcanalsofinfectednon-vitalprimaryteethwhichdid nothavebrokencrowntopreventcontaminationoftheroot canal microflora by the organisms from saliva and carious lesionoftheteeth. An in vitro study cannot stimulate perfectly an in vivo studybutitcancontrolfactorsthatan in vivo studycannot, suchasquantitativeevaluationofantibacterialactivitybya variety of filling materials. As the in vitro method also requiredthefillingmaterialstodiffuseintotheagar,thenet inhibitory effect was a combination of diffusion potential andanti-bacterialactivity.Theabilitytodiffuseintodentinal tubulesisadesiredcharacteristicofanantibacterialagent.4 Hobson found that microorganisms penetrated into the tubulesofdentinalwallsinrootcanalsin70%ofextracted teethwithnecrotictissue.24 Evenifseveralfillingmaterialshavebeenusedthrough the years, the most common ones are Zinc oxide eugenol, Iodoform and Calcium hydroxide. Generally after a good filling and irrigation, the final outcome of the pulpectomy procedure depends on the quality of the root canal filling materials, which can neutralize any remaining pulp tissue andmicroorganisms. In this study, ZOE exhibited strong inhibitory action against all the four groups of bacteria except against Candida albicans. Broisman et al,25, Cox et al,3 Grossman,26 Pupo et al ,27 Rahmat,28 Canalda and Pumarola,29 and Pumarola et al 30 agree that the sealers with ZOE base are thosethathavegreaterinhibitoryeffectagainstthemicroorganismsfoundinrootcanals.Theantimicrobialactivityof ZOEisattributedtotheeugenolcontentofthematerial.Cox et al 3 demonstratedthatzincoxidehadnoinhibitoryeffect andtheadditionofeugenoltozincoxideretardedthegrowth ofonlytheGram-positiveorganisms.Theinclusionofzinc acetateasasettingacceleratorinhibitedbothGram-positive andGram-positivebacteria. Calciumhydroxideshowedmediumormoderateinhibition against all the four bacterial groups and showed least inhibitionagainstCandida albicans inourstudy. ThisisinagreementwiththeresultsofTchaouet al 4 who foundthatcalciumhydroxideproducedmediumormediumstronginhibitionagainst P.intermedia strains.Theresultof ourstudycontradictswiththatofapreviousstudy5 inwhich calcium hydroxide exhibited weak antimicrobial activity against facultative/aerobic Gram-positive, facultative/aerobicGram-negativeorganismsbutfailedtoinhibitanaerobic bacteria.Theresultofourstudyalsodiffersfromotherstudies by Difore et al,31 Abdulkader et al,32 Siqueira and Gonclaves.33 They demonstrated that calcium hydroxide associatedwithaninertsubstance(distilledwater,salineor glycerine) was ineffective against several obligatory and facultativeanaerobicbacteria.Theweakinhibitoryeffectof calciumhydroxideinagardiffusionassaycanbeexplained by the fact that blood or buffer present in the agar media might have neutralized calcium hydroxide, a phenomenon thatmayalsooccurinvivowherebloodandbufferingsystemsarepresent. Vitapexshowedstronginhibitoryactionagainstfacultative/aerobic Gram-negative bacteria and anaerobic Gramnegative organisms, moderate inhibition against facultative/aerobic Gram-positive and anaerobic Gram-positive organismsbutshowedweakinhibitoryactionagainst Candida albicans.ThisfindingdiffersfromtheresultsofPabla et al 34 andTchaouet al 4 accordingtowhosestudies,Vitapex showedtheleastornoantibacterialactivity. However, Nurko and Garcia-Godoy35 studied the effectiveness of Vitapex in the root canal treatment of primary teeth.Thetreatmentwasdeemedsuccessfulif,clinically,the tooth was painless, without pathological mobility, and the gingivawashealthywithoutasinusorfistula.Theauthors 27

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recommendedtheuseofVitapexasarootcanalfillingmaterialastheyobservedvariousadvantagesofthismaterial. Ontheotherhand,Estrelaet al 36 verifiedtheinfluenceof iodoformontheantimicrobialpotentialofcalciumhydroxide on Staphylococcus aureus, Enterococci faecalis, P. aeruginosa, B. subtilis and C.albicans by direct exposure test and agar diffusion test.The results showed significant antimicrobial effectiveness for calcium hydroxide paste or iodoformplussaline. Metapexshowedthelowestantimicrobialactivitywhen comparedtotheotherthreerootcanalfillingmaterialstested inthisstudy.However,itshowedmoderateinhibitoryactivity against facultative/aerobic Gram-negative bacteria, anaerobicGram-positivebacteriaandanaerobicGram-negative bacteria but showed weak inhibitory activity against facultative/aerobic Gram-positive organisms and failed to inhibitCandida albicans. Theweakinhibitoryactivitymaybeexplainedbythefact that calcium hydroxide an ingredient of Metapex has been demonstrated to interfere with the antiseptic capacity of dyadiccombinationsofendodonticmedicaments. Most of the studies related to antimicrobial activity of rootcanalfillingmaterialshavebeendoneusingstandardized bacterial strains (ATCC-AmericanType Culture Collection).Veryfewstudieswerereportedinthedentalliterature exclusively on bacterial strains isolated from infected primaryteeth.4,5,34 Themeanzoneofinhibitionofthematerialsinthisstudy cannot be compared with previous studies because of the variabilitys bacterial strains, culture media, culture conditionsandpowderandliquidratioofthetestmaterials. Basedontheresultsofthisstudy,ZOEshowedsuperior antimicrobial activity against most of the organisms isolated,followedbyVitapex,CalciumhydroxideandMetapex indescendingorder. CONCLUSION The following conclusions were drawn from the present study: 1.Therootcanalsofinfectedprimaryteetharepolymicrobialinnature,withanaerobicandfacultative/aerobicorganismspredominatelypresent. 2.Allthetestfillingmaterialsshowedvariedantimicrobialactivityagainstthemicroorganismstested. 3.ZOEshowedsuperiorinhibitoryactivityagainstmost of the organisms isolated followed by Vitapex, CalciumhydroxideandMetapexindescendingorder. Itisdifficulttodrawconclusionsbasedoninvitroevaluation of antimicrobial activity with isolated bacteria. It is wellknownthatendodonticinfectionsaremixedwithcomplexfloralinteractions.Theeffectofthetestfillingmaterials against a single strain may not be effective against a mixedvarietyofinfection.Theuseofartificialmediaalso plays an important role in determining the experimental results.Itispossiblethatdifferentresultsmighthavebeen obtained if other methods of testing antimicrobial activity i.e. Agar dilution method, Direct contact test etc. were employed. Microbial interactions in the oral cavity should be consideredbeforeconcludingtheidealtestresults.Invivostudies are required to state the specific antimicrobial activity andmeritsanddemeritsofanyofthetestfillingmaterial. The clinical relevance of the findings from this study, however,canonlybedeterminedinclinicaltrials.Ourdata maybeusefulasaguideforrelativeantimicrobialeffectivenessornon-effectivenessofthematerialsemployed. REFERENCES
1. TronstadL.Recentdevelopmentinendodonticresearch.ScandJDent Res,100:529.1992. 2. TchaouWS,TurngBF,MinahGE,CollJA.Inhibitionofpurecultures of oral bacteria by root canal filling materials. Pediatr Dent, 18(7): 44449,1996. 3. CoxST,HembreeJH,McknightJPetal.Thebactericidalpotentialof various endodontic materials for primary teeth. Oral Surg, 45(6): 94754,1978. 4. TchaouWS,TurngBF,MinahGE,CollJA.Invitroinhibitionofbacteria from root canals of primary teeth by various dental materials. PediatrDent,17(5):35155,1995. 5. ReddyS,RamakrishnaY.Evaluationofantimicrobialefficacyofvariousrootcanalfillingmaterialsusedinprimaryteeth:Amicrobiologicalstudy.JClinPediatrDent,31(3):19599,2007. 6. AkdenizBG,KoparalE,SenBH,AtesM,DenizciAA.Prevalenceof candida albicans in oral cavities and root canals of children. J Dent Child,28992,2002. 7. Pazelli LC, Freitas AC, Ito IY, Souza-Gugelmin MC, Medeiros AS, Nelson-FilhoP.Prevalenceofmicroorganismsinrootcanalsofhuman deciduous teeth with necrotic pulp and chronic periapical lesions. PesquiOdontolBras,17(4):36771,2003. 8. BauerA,W,KirbyW,SherrisJCandTurchM.Antibioticsusceptibilitytestingbystandardizedsinglediscmethod.AmerJClinPathol,45: 493,1966. 9. Cowan and Steel. Manual for identification of Medical bacteria. 3rd edition.Cambridgeuniversitypress.11214. 10. Y.Vette S, McCater. Oral and Maxillofacial infection. Topazian, 4th edition;chapter3:4361. 11. TopleyandWilson.Microbiologyandmicrobialinfections.9thedition: vol3,22527. 12. Mendel and Bennet. Principle and practice of infectious disease. 5th edition,28689. 13. MarshSJ,LargentMD.Abacteriologicalstudyofthepulpcanalsof infectedprimarymolars.JDentChild,34:460470,1967. 14. Cohen MM, Joress SM, Calisti LP. Bacteriologic study of infected deciduousmolars.OralSurgOralMedOralPathol,13(11):138286, 1960. 15. Tomic-KarovicK,JelinekE.Comparativestudyofthebacteriological flora in the surroundings, the root canals and sockets of deciduous molars.IntDentJ,21:37588,1971. 16. ToyoshimoY, Fukusima H, Inoue JI, SasakiY,Yamamoto. Bacteriologicalstudyoftheperiapicalpathosisondeciduousteeth.JPNDentJ, 26:44958,1988. 17. SilvaLAB,Nelson-FilhoP,FariaGetal.Bacterialprofileinprimary teeth with necrotic pulp and periapical lesions. Braz Dent J, 17(2): 14448,2006. 18. RocasIN,SiqueiraJF,SantosKRN.AssociationofEnterococcusfaecaliswithdifferentformsofperiradiculardiseases.JEndodon,30(5): 31520,2004. 19. PetersLB,WesselinkPR,vanWinkelhoffAJ.Combinationsofbacterialspeciesinendodonticinfections.IntEndodJ,35(8):698702,2002.

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20. SundqvistG,JohanssonE,SjogrenU.PrevalenceofBlack-pigmented Bacteroidesspeciesinrootcanalinfections.JEndodon,15(1):1319, 1989. 21. ChuFCS,TsangCSP,ChowTW,SamaranayakeLP.Identificationof cultivable microorganisms from primary endodontic infections with exposedandunexposedpulpspace.JEndodon,31(6):42429,2005. 22. Adib V, Spratt D, Ng YL, Gulabivala K. Cultivable microbial flora associatedwithpersistentperiapicaldiseaseandcoronalleakageafter rootcanaltreatment:apreliminarystudy.IntEndodJ,37(8):54251, 2004. 23. GomesBP,PinheiroET,Gade-NetoCRetal.Microbiologicalexaminationofinfecteddentalrootcanals.OralMicrobiolImmunol,19(2): 716,2004. 24. HobsonP.Pulptreatmentofdeciduousteeth.BrDentJ,128:2328, 27582,1970. 25. Broisman H, Van Houte J, Gron P and Krakow A.A. Antimicrobial effectsofN2invitro.OralSurg,OralMed,OralPathol,45:116122, 1978. 26. GrossmanL.I.Antimicrobialeffectofrootcanalcements.JofEndod, 6:594507,1980. 27. Pupo J, Biral R.R, Benatti O, Abe A and Valrighi L. Antimicrobial effectsofendodonticfillingmaterialsonmicroorganismsfoundinroot canal.OralSurg,74:216220,1992. 28. RahmatAB.Evaluationofantimicrobialactivityof10rootcanalfillingmaterialsonStreptococcussanguisandStreptococcusmutans.Oral Surg,OralMed,OralPathol,68:99102,1989. 29. CanaldaC,PumarolaJ.Bacterialgrowthinhibitionproducedbyroot canal sealer cements with calcium hydroxide base. Oral Surg, Oral Med,OralPathol,68:99102,1989. 30. PumarolaJ,EstebanBandCanaldaC.Antimicrobialactivityof7root canalsealers.OralSurg,OralMed,OralPathol,74:216220,1992. 31. DiFiore PM, Peters DD, Setterstrom JA, Lortan L. The antibacterial effectsofcalciumhydroxideapexificationpastesonStreptococcussanguis.OralSur,g55(1):9194,1983. 32. AbdulkaderA,DuguidRandSaundersE.M.Theantimicrobialactivity of endodontic sealers to anaerobic bacteria. Int Endod J, 29: 280283,1996. 33. SiqueriaJ.FandGonclavesR.B.Antibacterialactivitiesofrootcanal againstselectedanaerobicbacteria.JEndod,22:890,1996. 34. PablaT,GulatiMS,MohanU.Evaluationofantimicrobialefficacyof variousrootcanalfillingmaterialsforprimaryteeth.JIndSocPedod PrevDent,15(4):13440,1997. 35. Nurko C, Garcia-Godoy F. Evaluation of a calcium hydroxide / iodoformpaste(vitapex)inrootcanaltherapyforprimaryteeth.JClinPed Dent,23:28994,1999. 36. Estrela C, Estrela CRA, Hollanda ACB, Decurcio DA, Pecora JD. Influenceiodoformonantimicrobialpotentialofcalciumhydroxide.J ApplOralSci,14:3337,2006.

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