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Anaerobe 18 (2012) 576e580

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Anaerobe

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Clinical microbiology

Antibiotic resistance genes in anaerobic bacteria isolated from primary dental rootcanal infections

Isabela N. Rôças, José F. Siqueira Jr. *

Department of Endodontics and Molecular Microbiology Laboratory, Faculty of Dentistry, Estácio de Sá University, Rua Alfredo Baltazar da Silveira, 580/cobertura, Recreio,Rio de Janeiro 22790-710, RJ, Brazil

a r t i c l e i n f o

Article history:Received 21 July 2012Received in revised form29 September 2012Accepted 16 October 2012Available online 26 October 2012

Keywords:Dental root canalApical periodontitisAntibiotic resistance genesBeta-lactamsTetracyclineMacrolides

* Corresponding author. Tel.: þ55 21 24978988.E-mail address: jf_siqueira@yahoo.com (J.F. Siquei

1075-9964/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.anaerobe.2012.10.001

a b s t r a c t

Fourty-one bacterial strains isolated from infected dental root canals and identified by 16S rRNA genesequence were screened for the presence of 14 genes encoding resistance to beta-lactams, tetracyclineand macrolides. Thirteen isolates (32%) were positive for at least one of the target antibiotic resistancegenes. These strains carrying at least one antibiotic resistance gene belonged to 11 of the 26 (42%)infected root canals sampled. Two of these positive cases had two strains carrying resistance genes. Sixout of 7 Fusobacterium strains harbored at least one of the target resistance genes. One Dialister invisusstrain was positive for 3 resistance genes, and 4 other strains carried two of the target genes. Of the 6antibiotic resistance genes detected in root canal strains, the most prevalent were blaTEM (17% of thestrains), tetW (10%), and ermC (10%). Some as-yet-uncharacterized Fusobacterium and Prevotella isolateswere positive for blaTEM, cfxA and tetM. Findings demonstrated that an unexpectedly large proportion ofdental root canal isolates, including as-yet-uncharacterized strains previously regarded as uncultivatedphylotypes, can carry antibiotic resistance genes.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Beta-lactams, tetracyclines and macrolides have been used indentistry to treat oral infectious conditions, including abscesses/cellulitis of endodontic origin [1]. Mechanisms of bacterial resis-tance to these antibiotics have been ascribed to resistance genesand it has been shown that the humanmicrobiota, including that ofthe oral cavity, may function as a reservoir for antibiotic resistancegenes [2]. Several antibiotic resistance genes have been identifiedin members of oral bacterial communities using molecular tech-niques, including the genes encoding resistance to beta-lactams,tetracyclines, and macrolides [3e5].

Dental root canal infections represent the primary cause ofapical periodontitis and are usually characterized by multispeciesbacterial biofilm communities conspicuously dominated by anaer-obic bacteria [6]. As with any other multispecies biofilms in nature,endodontic bacterial species are arranged in close proximity onefrom the other, which is highly conducive to the establishment ofinteractions such as food chains, quorum-sensing systems andexchange of virulence and antibiotic resistance genes [7]. Associa-tions have been reported between endodontic bacterial species and

ra).

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antibiotic resistance, including Prevotella species from dentoal-veolar abscesses [8,9] and Enterococcus faecalis from teeth withpost-treatment apical periodontitis [10]. A recent study hasattempted to detect antibiotic resistance genes directly in clinicalsamples and investigated how treatment was effective in elimi-nating detectable levels of these genes [11]. However, no study hasconsistently reported on the antibiotic resistance genes that may becarried by specific endodontic bacterial isolates.

Systemic use of antibiotics in endodontics is usually indicated foracute apical abscesses associated with systemic involvement,spreading infections, abscesses in medically compromised patientswho are at increased risk of a nonoral secondary infection follo-wing bacteremia, prophylaxis for medically compromised patientsduring routine endodontic therapy, and replantation of avulsedteeth [12]. Topic use of antibiotics in the root canal has beena recurrent theme in endodontic therapy, and recently someantibiotic-containing irrigants ormedicaments have been proposedfor use in specific clinical conditions [13,14]. Therefore, selection ofthe most effective antibiotics to be used for systemic or topical usewill depend on a better understanding of the patterns of antibioticresistance in the infected dental root canal.

The present study was undertaken to screen a panel of bacterialstrains isolated from infected root canals and identified by 16S rRNAgene sequence for the presence of several genes encoding resis-tance to beta-lactams, including blaTEM, blaCMY-2, blaZ, ampC,

I.N. Rôças, J.F. Siqueira Jr. / Anaerobe 18 (2012) 576e580 577

cfxA, and mecA; tetracyclines, including tetM, tetO, tetQ, tetS, andtetW; and macrolides, including ermA, ermB, and ermC. Many ofthese genes have already been detected in oral isolates or directly insamples from the oral cavity.

2. Methods

2.1. Clinical material and sampling

The endodontic strains used in this study were isolated fromroot canals of 26 patients presenting to the endodontic clinic at theSchool of Dentistry, Estácio de Sá University, for treatment of apicalperiodontitis. The teeth included in the study had intact pulpchamber walls, necrotic pulps, and radiographic evidence of apicalperiodontitis lesions. Patients who received antibiotic therapywithin the previous three months were not included in the study.Selected teeth showed no periodontal pockets deeper than 4 mm.Approval for the study protocol was obtained from the EthicsCommittee of the Estácio de Sá University.

Sampling procedures were carried out under strict asepsisunder rubber dam isolation. Prior to isolation, supragingival plaquewas removed and the tooth cleaned with pumice. Caries and/orcoronal restorations were removed and rubber dam was applied.The operative field, including the tooth crown, cavity, clamp, andsurroundings were cleaned and disinfected with 3% hydrogenperoxide followed by 2.5% NaOCl solution. After completing prep-aration of the access cavity, the operative field, this time alsoincluding the pulp chamber, was cleaned and disinfected onceagain as above. NaOCl was neutralized with 5% sodium thio-sulphate, and then a bacteriologic sample of the tooth surface wasobtained with sterile paper points. All teeth included in the studyyielded negative results for the bacteriological control samplestaken from the working field.

Root canal samples were taken as follows. Three sterile paperpoints were consecutively placed in the canal to a level approxi-mately 1 mm short of the root apex and used to soak up the fluid inthe canal. Paper points were then transferred aseptically to tubescontaining 500 ml of reduced transport fluid (RTF) [15]. Sampleswere transported to the laboratory within 15 min for microbio-logical processing.

2.2. Culture and 16S rRNA gene identification

Samples in RTF vials were agitated in vortex for 30 s and 10-foldserial dilutions were made in prereduced anaerobically sterilizedbuffered salt solution. Aliquots of 100 ml from each dilution wereeach spread onto Brucella agar plates (BBL Microbiology Systems,Cockeysville, MD, USA) supplemented with 5% defibrinated sheepblood, hemin (5 mg/l) and menadione (1 mg/l). Plates wereimmediately placed in anaerobic jars (BBL GasPak system, BectonDickinson Microbiology Systems, Cockeysville, MD, USA) andincubated anaerobically at 37 �C for up to 14 days. Followingincubation, one colony of each different morphology and from themost dominant types in the sample (i.e., those presenting two ormore colonies with similar morphotype) was isolated and placedindividually in flasks containing TriseEDTA buffer (10 mM TriseHCl, 1 mM EDTA, pH 8), and stored at �20 �C.

DNA was extracted from each isolate and PCR amplification of16S rRNA genes was used for bacterial identification. The pair ofuniversal 16S rRNA gene primers usedwas 50-GAT TAGATACCC TGGTAG TCC AC-30 and 50-CCC GGG AAC GTA TTC ACC G-30, corre-sponding to base positions 786e808 and 1369e1387, respectively,and spanning the variable regions V5eV8 of the Escherichia coli 16SrRNA gene [16]. PCR amplification was performed in a reactionvolume of 50 ml, consisting of 0.8 mM concentration of each primer,

5 ml of 10� PCR buffer, 2 mMMgCl2, 1.25 U of Taq DNA polymerase,0.2 mM concentration of each deoxyribonucleoside triphosphate.Cycling parameters included an initial denaturation step at 95 �C/2 min, followed by 36 cycles of 95 �C/30 s, 60 �C/1 min, and 72 �C/1min, and then a final step at 72 �C/2 min. After the presence of theexpected PCR products was confirmed by electrophoresis in anagarose gel, PCR products were purified using a PCR purificationsystem (Wizard PCR Preps, Promega, Madison, WI, USA) and thensequenced directly on the ABI 377 automated DNA sequencer usingdye terminator chemistry (Amersham Biosciences, Little Chalfont,Buckinghamshire, UK). Sequences were compared to the GenBankdatabase to identify the closest relatives by using the BLAST algo-rithm [17] and a more than 99% similarity in the 16S rRNA genesequence was the criterion used to identify an isolate to the specieslevel. Forty-one strains that met this criterion and for which therewas no ambiguous identification were surveyed for the presence ofantibiotic resistance genes.

2.3. Real-time PCR for antibiotic resistance genes

For improved performance of PCR in detection of antibioticresistance genes, DNA extracts from the root canal isolates weresubjected to multiple displacement amplification (MDA) using theIllustra GenomiPhi V2 DNA Amplification kit (GE Healthcare, Pis-cataway, NJ, USA) following the manufacturer’s instructions.

The root canal isolates were analyzed for the presence of 14common antibiotic resistance genes that have been detected inhuman-associated bacterial species (Table 1). Real-time PCRamplification was performed with Power SYBR Green PCR MasterMix (Applied Biosystems, Foster City, CA, USA) on an ABI 7500 Real-time PCR instrument (Applied Biosystems) in a total reactionvolume of 20 ml. The annealing temperatures for each primer pairwere based on previous protocols established in prior studies(Table 1). Primers in a concentration of 0.5 mM each and MDA-amplified DNA volume of 2 ml were added to the PCR master mixin MicroAmp Optical 96-well reaction plates. Plates were sealed,centrifuged and then subjected to amplification. Cycling conditionsfor the real-time PCR included: 95 �C/10 min; 40 repeats of thefollowing steps: 95 �C/1 min, annealing for 1 min (specific temper-atures shown in Table 1), and 72 �C/1 min. All the tests were run induplicate. Triplicates of appropriate negative controls containing notemplate DNA were subjected to the same procedures. Positivecontrols included strains or samples that yielded positive results forthese genes with results previously confirmed by ampliconsequencing. Following amplification, melting curve analysis wasperformed to determine the specificity of the amplified products.Melting curve was obtained from 60 �C to 95 �C, with continuousfluorescence measurements taken at every 1% increase in temper-ature. Data acquisition and analysis were performed using the ABI7500 software v2.0.4 (Applied Biosystems). To confirm positiveresults, the PCR products were subjected to electrophoresis inagarose gels and representative amplicons were sequenced.

3. Results

The selected 41 bacterial strains isolated from dental root canalinfections and identified by 16S rRNA gene sequencing are shown inTable 2. Of these,13 (32%) isolates were positive for at least one of thetarget antibiotic resistance genes. These strains carrying at least oneantibiotic resistancegenebelonged to11/26 (42%) infectedroot canalssampled. Most positive cases had only one strain carrying resistancegenes, except for 2 cases that harbored 2 positive strains each.

Of the 6 antibiotic resistance genes detected in root canal strains,the most prevalent were blaTEM, which occurred in 7 strains (17%),followed by tetW (4 strains, 10%), ermC (4 strains, 10%), tetM (2

Table 1Primers used for detection of antibiotic resistance genes.

Antibiotic Primer Sequence Ta Size(bp)

Reference

Beta-lactam blaCMY-2 50-GAC AGC CTC TTT CTCCAC A-30

50-TGG ACG AAG GCT ACGTA-30

55 1014 [29]

Beta-lactam blaTEM 50-CCA ATG CTT AAT CAG TGAGG-30

50-ATG AGT ATT CAA CAT TTCCG-30

60 858 [30]

Beta-lactam blaZ 50-CAG TTC ACA TGC CAAAGA G-30

50-TAC ACT CTT GGC GGTTTC-30

54 846 [31]

Beta-lactam ampC 50-TAA ACA CCA CAT ATGTTC CG-30

50-ACT TAC TTC AAC TCGCGA CG-30

50 663 [32]

Beta-lactam cfxA 50-GCG CAA ATC CTC CTTTAA CAA-30

50-ACC GCC ACA CCA ATTTCG-30

60 802 [8]

Beta-lactam(methicillin)

mecA 50-AAA ATC GAT GGT AAAGGT TGG C-30

50-AGT TCT GCA GTA CCGGAT TTG C-30

54 533 [33]

Macrolide ermA 50-GTT CAA GAA CAA TCAATA CAG AG-30

50-GGA TCA GGA AAA GGACAT TTT AC-30

52 421 [34]

Macrolide ermB 50-CCG TTT ACG AAA TTGGAA CAG GTA AAG GGC-30

50-GAA TCG AGA CTT GAGTGT GC-30

55 359 [34]

Macrolide ermC 50-AAT CGG CTC AGG AAAAGG-30

50-ATC GTC AAT TCC TGCATG-30

54 562 [31]

Tetracycline tetM 50-GTG GAC AAA GGT ACAACG AG-30

50-CGG TAA AGT TCG TCACAC AC-30

55 406 [35]

Tetracycline tetO 50-AAC TTA GGC ATT CTGGCT CAC-30

50-TCC CAC TGT TCC ATATCG TCA-30

55 515 [35]

Tetracycline tetS 50-CAT AGA CAA GCC GTTGAC C-30

50-ATG TTT TTG GAA CGCCAG AG-30

55 667 [35]

Tetracycline tetQ 50-TTA TAC TTC CTC CGGCAT CG-30

50-ATC GGT TCG AGA ATGTCC AC-30

55 904 [35]

Tetracycline tetW 50-GAG AGC CTG CTA TATGCC AGC-30

50-GGG CGT ATC CAC AATGTT AAC-30

64 168 [27]

I.N. Rôças, J.F. Siqueira Jr. / Anaerobe 18 (2012) 576e580578

strains, 5%), and cfxa and tetS (both in 1 strain each, 2%). No strainwas positive for the 8 other resistance genes targeted in this study.

Six out of 7 Fusobacterium strains showed at least one of thetarget resistance genes. Some as-yet-uncharacterized Fusobacte-rium and Prevotella isolates were positive for blaTEM, cfxA and tetM.Five strains were positive formore than one of the target genes: oneDialister invisus strain was positive for 3 resistance genes, while 4other strains yielded positive results for 2 genes.

4. Discussion

The present study demonstrated that 32% of the root canalisolates tested, including some as-yet-uncharacterized strains,

were positive for the presence of at least one of the target antibioticresistance genes.

Resistance to beta-lactam antibiotics in oral bacteria, especiallyvia beta-lactamase production, has been shown not to beuncommon [8,9,18,19]. In line with this observation, the presentstudy revealed that the most prevalent antibiotic resistance geneamong endodontic isolates was blaTEM, which was found in 17% ofthe strains and 6/26 (23%) teeth. Jungermann et al. [11] previouslyreported that blaTEM was the most prevalent antibiotic resistancegene detected directly in 33% of the samples from primaryendodontic infections. TEM-type beta-lactamases are reported tobe widespread in Gram-negative bacteria and may attack severalbeta-lactamic antibiotics [20]. The blaTEM gene has been foundwidely distributed among oral biofilm samples in healthy anddisease [4,5]. The present findings may be of some concern becausebeta-lactams are the main antibiotics recommended in prophylaxisor treatment in endodontics.

In spite of being widespread in many species [21], especiallyGram-negative bacteria, we are not aware of previous studiesreporting on the detection of the blaTEM gene in strains of Fuso-bacterium,D. invisus andCampylobacter curvus. Except for a study thatdetected blaTEM in E. faecalis [22], there are not previous reports ofthis gene in Gram-positive bacteria, but one Propionibacterium pro-pionicum strain, a species that may be involved in apical actinomy-cosis, was positive for this gene. A previous report claimed that PCRreagents may be contaminated with the blaTEM gene [23]. In thepresent study, negative controls were run for each batch of samplesanalyzed to rule out the possibility of reagent contamination.

Noteworthy was that 6 of the 7 Fusobacterium strains tested e 4Fusobacterium nucleatum strains and 2 uncharacterized strains,were positive for at least one resistance gene. In addition tocarrying the blaTEM gene, Fusobacterium strains were also positivefor genes encoding resistance to macrolides and tetracyclines.Some strains concomitantly carried resistance genes to two classesof antibiotics. These findings are of some concern and need to befurther explored as Fusobacterium species are ubiquitous in diversesites of the human body and may be involved with differentconditions requiring antibiotic therapy [24e26].

The cfxA gene has also been frequently detected in oral biofilmsassociated with periodontal disease [4]. A study detected this genein 11% of root canal samples [11]. It has been demonstrated that cfxAin oral bacterial communities mainly originates from Prevotellaspecies [8,19]. Indeed, the only strain positive for cfxA in this studywas an uncharacterized strain of Prevotella.

Three of the tetracycline resistance genes targeted in this studywere found in endodontic bacteria: tetW, tetM and tetS. Resistanceto tetracyclines may be related to ribosomal protection, effluxpumps and enzymatic inactivation [27]. The tetW gene, whichencodes the former, has been detected in the oral cavity in bothpathogenic and non-pathogenic species [28]. Along with ermC, itwas the second most prevalent resistance gene found in this study;4 strains from 2 species carried this gene: F. nucleatum (3 strains)and Parvimonas micra. The tetM gene, also encoding ribosomalprotection, was found in 2 strains (D. invisus and a Fusobacteriumstrain). This gene has been very prevalent in the oral cavity of adultsand children [3e5]. In addition, one Pseudoramibacter alactolyticusstrain was positive for tetS, while tetQ, which has been highlyprevalent in the oral cavity [4,5], was not detected in this study.These findings are in agreement with a previous study ofendodontic infections [11], in which tetM and tetW were morecommon than tetQ. The clinical relevance of tetracycline resistancein endodontics is mostly related to the use of tetracycline-containing irrigation solutions.

Resistance to erythromycin is most commonly due to theacquisition of erm genes which encodes for rRNAmethylases. In the

Table 2Antibiotic resistance genes detected in bacterial strains isolated from primarily infected dental root canals.

Species Case blaTEM blaCMY-2 blaZ ampC cfxA mecA ermA ermB ermC tetM tetO tetS tetQ tetW

Actinomyces odontolyticus 1Sb e e e e e e e e e e e e e e

Actinomyces sp. oral clone GU009 8Ta e e e e e e e e e e e e e e

Actinomyces sp. oral clone GU009 14Sa e e e e e e e e e e e e e e

Campylobacter curvus 10Sa Positive e e e e e e e e e e e e e

Campylobacter gracilis 4Sa e e e e e e e e e e e e e e

Campylobacter rectus 12Ta e e e e e e e e e e e e e e

Dialister invisus 8Sa Positive e e e e e e e Positive Positive e e e e

Enterococcus faecalis MB 35a e e e e e e e e e e e e e e

Fusobacterium nucleatum 1Sa e e e e e e e e e e e e e PositiveF. nucleatum 2Ta e e e e e e e e Positive e e e e PositiveF. nucleatum 4Ta e e e e e e e e e e e e e e

F. nucleatum 6Ka Positive e e e e e e e e e e e e e

F. nucleatum 16Sa e e e e e e e e Positive e e e e PositiveFusobacterium sp. clone BS019 10Ka Positive e e e e e e e e e e e e e

Fusobacterium sp. clone CZ006 8Sa Positive e e e e e e e e Positive e e e e

Lactococcus garvieae 8Tc e e e e e e e e e e e e e e

Mogibacterium neglectum 13Sa e e e e e e e e e e e e e e

Parvimonas micra 8Ka e e e e e e e e e e e e e e

P. micra 1Kb e e e e e e e e e e e e e PositivePorphyromonas endodontalis 1SdqA e e e e e e e e e e e e e e

Porphyromonas gingivalis 16Sa e e e e e e e e e e e e e e

P. gingivalis 10BBc e e e e e e e e e e e e e e

Prevotella marshii 9Ta e e e e e e e e e e e e e e

Prevotella oralis 16Sa e e e e e e e e e e e e e e

Prevotella sp. oral clone FM005 1Sb Positive e e e Positive e e e e e e e e e

Prevotella sp. oral clone GU027 9Sa e e e e e e e e e e e e e e

Propionibacterium acnes 12Kc e e e e e e e e e e e e e e

Propionibacterium propionicum 7Ka e e e e e e e e e e e e e

P. propionicum 16Sa e e e e e e e e e e e e e e

P. propionicum 2SdqA Positive e e e e e e e e e e e e e

Pseudoramibacter alactolyticus 6Sa e e e e e e e e e e e e e e

P. alactolyticus 12Ka e e e e e e e e e e e Positive e e

P. alactolyticus 11Sa e e e e e e e e Positive e e e e e

Streptococcus anginosus 1Kb e e e e e e e e e e e e e e

S. anginosus 10Ta e e e e e e e e e e e e e e

S. anginosus 9Ta e e e e e e e e e e e e e e

Streptococcus constellatus/intermedius 9Ka e e e e e e e e e e e e e e

Streptococcus infantis 2Sc e e e e e e e e e e e e e e

Streptococcus mitis 1Sa e e e e e e e e e e e e e e

S. mitis 10Sb e e e e e e e e e e e e e e

Streptococcus parasanguinis 8Ta e e e e e e e e e e e e e e

Total 7 0 0 0 1 0 0 0 4 2 0 1 0 4% 17 0 0 0 2 0 0 0 10 5 0 2 0 10

I.N. Rôças, J.F. Siqueira Jr. / Anaerobe 18 (2012) 576e580 579

present study, 4 strains were positive for ermC: F. nucleatum (2strains), D. invisus and P. alactolyticus. No endodontic strain waspositive for ermA or ermB, even though the latter has been regardedas one of the most abundant erm genes in the oral microbiota [3].

Endodontic isolates were identified by 16S rRNA gene in order toprovide a more accurate identification. This resulted in identifica-tion and testing not only of valid well-known or newly namedspecies, but also of 6 strains from 5 species-level taxa that had notbeen previously cultivated. These taxa were first disclosed byculture-independent molecular methods and previously regardedas uncultivable phylotypes, i.e., species known only by a 16S rRNAgene sequence. These findings indicate that some of the so-called“uncultivable” phylotypes are in fact cultivable bacteria that werenot previously detected by culture because of either insufficientsampling coverage or difficulties in phenotypic identification. Themethodology used in this study allowed for the screening of thesestill-to-be-characterized oral strains for the presence of antibioticresistance genes and is probably the first study in this regard. Ofthese 6 uncharacterized strains, 3 (two Fusobacterium and onePrevotella) were positive for one or two of the resistance genes. Allthree carried the blaTEM gene, the Prevotella strain was also posi-tive for cfxA and one Fusobacterium strain for the tetM gene. Thesefindings reinforce the need for improved efforts in cultivating and

characterizing oral bacterial species previously regarded as uncul-tivable so as to determine species names and unravel their patho-genic and antibiotic resistance patterns.

Of interest was the fact that 42% of the infected teeth harboredat least one strain positive for the target antibiotic resistance genes.Even though this figure may be considered high, it is still highlylikely to be an underestimate. This is because the design of thepresent study included only the most dominant isolates for iden-tification and analysis, and excluded uncultivable bacteria. There-fore, the contribution of low-dominance species and uncultivablebacteria to resistance genes was not evaluated. Direct screening ofclinical samples for the presence of resistance genes would bemoreappropriate for this purpose. Moreover, traditional root canalsampling with paper points fail to sample bacteria located deepinto biofilms and distributed along irregularities and ramificationsof the root canal system and may also have contributed to under-estimating the prevalence of cases with resistance genes.

It is salient to point out that, although the present study iden-tified the presence of several antibiotic resistance genes, thisinformation does not necessarily translate into functional resis-tance, since phenotypic resistance tests were not performed [11].However, they indicate a potential for resistance and the clinicianshould be aware of this information when deciding on local or

I.N. Rôças, J.F. Siqueira Jr. / Anaerobe 18 (2012) 576e580580

systemic antibiotic therapy. For instance, because of the risksof bacteremia during treatment of infected canals, antibioticprophylaxis is indicated for patients at risk of developing bacterialendocarditiis. Acute infections (abscesses) are also usually resultantof exacerbations of chronic infections. The knowledge of thepotential of antibiotic resistance pattern in members of theendodontic microbiota may influence the decision-making processof antibiotic prescription in endodontics.

Acknowledgments

This study was supported by grants from Fundação CarlosChagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro(FAPERJ) and Conselho Nacional de Desenvolvimento Científico eTecnológico (CNPq), Brazilian Governmental Institutions.

The authors deny any conflicts of interest.

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