Anaerobe 18 (2012) 576e580

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Anaerobe
journal homepage: www.elsevier.com/locate/anaerobe

Clinical microbiology

Antibiotic resistance genes in anaerobic bacteria isolated from primary dental root
canal infections
Isabela N. Ras, Jos F. Siqueira Jr. *
Department of Endodontics and Molecular Microbiology Laboratory, Faculty of Dentistry, Estcio de S 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 a b s t r a c t

Article history: Fourty-one bacterial strains isolated from infected dental root canals and identied by 16S rRNA gene
Received 21 July 2012 sequence were screened for the presence of 14 genes encoding resistance to beta-lactams, tetracycline
Received in revised form and macrolides. Thirteen isolates (32%) were positive for at least one of the target antibiotic resistance
29 September 2012
genes. These strains carrying at least one antibiotic resistance gene belonged to 11 of the 26 (42%)
Accepted 16 October 2012
Available online 26 October 2012
infected root canals sampled. Two of these positive cases had two strains carrying resistance genes. Six
out of 7 Fusobacterium strains harbored at least one of the target resistance genes. One Dialister invisus
strain was positive for 3 resistance genes, and 4 other strains carried two of the target genes. Of the 6
Keywords:
Dental root canal
antibiotic resistance genes detected in root canal strains, the most prevalent were blaTEM (17% of the
Apical periodontitis strains), tetW (10%), and ermC (10%). Some as-yet-uncharacterized Fusobacterium and Prevotella isolates
Antibiotic resistance genes were positive for blaTEM, cfxA and tetM. Findings demonstrated that an unexpectedly large proportion of
Beta-lactams dental root canal isolates, including as-yet-uncharacterized strains previously regarded as uncultivated
Tetracycline phylotypes, can carry antibiotic resistance genes.
Macrolides 2012 Elsevier Ltd. All rights reserved.

1. Introduction antibiotic resistance, including Prevotella species from dentoal-

Beta-lactams, tetracyclines and macrolides have been used in
dentistry 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 genes
and it has been shown that the human microbiota, including that of
the oral cavity, may function as a reservoir for antibiotic resistance
genes [2]. Several antibiotic resistance genes have been identied
in 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 of
apical periodontitis and are usually characterized by multispecies
bacterial biolm communities conspicuously dominated by anaer-
obic bacteria [6]. As with any other multispecies biolms in nature,
endodontic bacterial species are arranged in close proximity one
from the other, which is highly conducive to the establishment of
interactions such as food chains, quorum-sensing systems and
exchange of virulence and antibiotic resistance genes [7]. Associa-
tions have been reported between endodontic bacterial species and
* Corresponding author. Tel.: 55 21 24978988.
E-mail address: jf_siqueira@yahoo.com (J.F. Siqueira).
veolar abscesses [8,9] and Enterococcus faecalis from teeth with
post-treatment apical periodontitis [10]. A recent study has
attempted to detect antibiotic resistance genes directly in clinical
samples and investigated how treatment was effective in elimi-
nating detectable levels of these genes [11]. However, no study has
consistently reported on the antibiotic resistance genes that may be
carried by specic endodontic bacterial isolates.
Systemic use of antibiotics in endodontics is usually indicated for
acute apical abscesses associated with systemic involvement,
spreading infections, abscesses in medically compromised patients
who are at increased risk of a nonoral secondary infection follo-
wing bacteremia, prophylaxis for medically compromised patients
during routine endodontic therapy, and replantation of avulsed
teeth [12]. Topic use of antibiotics in the root canal has been
a recurrent theme in endodontic therapy, and recently some
antibiotic-containing irrigants or medicaments have been proposed
for use in specic clinical conditions [13,14]. Therefore, selection of
the most effective antibiotics to be used for systemic or topical use
will depend on a better understanding of the patterns of antibiotic
resistance in the infected dental root canal.
The present study was undertaken to screen a panel of bacterial
strains isolated from infected root canals and identied by 16S rRNA
gene sequence for the presence of several genes encoding resis-
tance to beta-lactams, including blaTEM, blaCMY-2, blaZ, ampC,

1075-9964/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.anaerobe.2012.10.001
 I.N. Ras, J.F. Siqueira Jr. / Anaerobe 18 (2012) 576e580
cfxA, and mecA; tetracyclines, including tetM, tetO, tetQ, tetS, and
tetW; and macrolides, including ermA, ermB, and ermC. Many of
these genes have already been detected in oral isolates or directly in
samples from the oral cavity.
2. Methods
2.1. Clinical material and sampling
The endodontic strains used in this study were isolated from
root canals of 26 patients presenting to the endodontic clinic at the
School of Dentistry, Estcio de S University, for treatment of apical
periodontitis. The teeth included in the study had intact pulp
chamber walls, necrotic pulps, and radiographic evidence of apical
periodontitis lesions. Patients who received antibiotic therapy
within 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 Ethics
Committee of the Estcio de S University.
Sampling procedures were carried out under strict asepsis
under rubber dam isolation. Prior to isolation, supragingival plaque
was removed and the tooth cleaned with pumice. Caries and/or
coronal restorations were removed and rubber dam was applied.
The operative eld, including the tooth crown, cavity, clamp, and
surroundings were cleaned and disinfected with 3% hydrogen
peroxide followed by 2.5% NaOCl solution. After completing prep-
aration of the access cavity, the operative eld, this time also
including the pulp chamber, was cleaned and disinfected once
again as above. NaOCl was neutralized with 5% sodium thio-
sulphate, and then a bacteriologic sample of the tooth surface was
obtained with sterile paper points. All teeth included in the study
yielded negative results for the bacteriological control samples
taken from the working eld.
Root canal samples were taken as follows. Three sterile paper
points were consecutively placed in the canal to a level approxi-
mately 1 mm short of the root apex and used to soak up the uid in
the canal. Paper points were then transferred aseptically to tubes
containing 500 ml of reduced transport uid (RTF) [15]. Samples
were transported to the laboratory within 15 min for microbio-
logical processing.
2.2. Culture and 16S rRNA gene identication
Samples in RTF vials were agitated in vortex for 30 s and 10-fold
serial dilutions were made in prereduced anaerobically sterilized
buffered salt solution. Aliquots of 100 ml from each dilution were
each spread onto Brucella agar plates (BBL Microbiology Systems,
Cockeysville, MD, USA) supplemented with 5% debrinated sheep
blood, hemin (5 mg/l) and menadione (1 mg/l). Plates were
immediately placed in anaerobic jars (BBL GasPak system, Becton
Dickinson Microbiology Systems, Cockeysville, MD, USA) and
incubated anaerobically at 37  C for up to 14 days. Following
incubation, one colony of each different morphology and from the
most dominant types in the sample (i.e., those presenting two or
more colonies with similar morphotype) was isolated and placed
individually in asks containing TriseEDTA buffer (10 mM Trise
HCl, 1 mM EDTA, pH 8), and stored at 20  C.
DNA was extracted from each isolate and PCR amplication of
16S rRNA genes was used for bacterial identication. The pair of
universal 16S rRNA gene primers used was 50 -GAT TAG ATA CCC TGG
TAG 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 16S
rRNA gene [16]. PCR amplication was performed in a reaction
volume of 50 ml, consisting of 0.8 mM concentration of each primer,
577
5 ml of 10 PCR buffer, 2 mM MgCl2, 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/
1 min, and then a nal step at 72  C/2 min. After the presence of the
expected PCR products was conrmed by electrophoresis in an
agarose gel, PCR products were puried using a PCR purication
system (Wizard PCR Preps, Promega, Madison, WI, USA) and then
sequenced directly on the ABI 377 automated DNA sequencer using
dye terminator chemistry (Amersham Biosciences, Little Chalfont,
Buckinghamshire, UK). Sequences were compared to the GenBank
database to identify the closest relatives by using the BLAST algo-
rithm [17] and a more than 99% similarity in the 16S rRNA gene
sequence was the criterion used to identify an isolate to the species
level. Forty-one strains that met this criterion and for which there
was no ambiguous identication were surveyed for the presence of
antibiotic resistance genes.
2.3. Real-time PCR for antibiotic resistance genes
For improved performance of PCR in detection of antibiotic
resistance genes, DNA extracts from the root canal isolates were
subjected to multiple displacement amplication (MDA) using the
Illustra GenomiPhi V2 DNA Amplication kit (GE Healthcare, Pis-
cataway, NJ, USA) following the manufacturers instructions.
The root canal isolates were analyzed for the presence of 14
common antibiotic resistance genes that have been detected in
human-associated bacterial species (Table 1). Real-time PCR
amplication was performed with Power SYBR Green PCR Master
Mix (Applied Biosystems, Foster City, CA, USA) on an ABI 7500 Real-
time PCR instrument (Applied Biosystems) in a total reaction
volume of 20 ml. The annealing temperatures for each primer pair
were based on previous protocols established in prior studies
(Table 1). Primers in a concentration of 0.5 mM each and MDA-
amplied DNA volume of 2 ml were added to the PCR master mix
in MicroAmp Optical 96-well reaction plates. Plates were sealed,
centrifuged and then subjected to amplication. Cycling conditions
for the real-time PCR included: 95  C/10 min; 40 repeats of the
following steps: 95  C/1 min, annealing for 1 min (specic temper-
atures shown in Table 1), and 72  C/1 min. All the tests were run in
duplicate. Triplicates of appropriate negative controls containing no
template DNA were subjected to the same procedures. Positive
controls included strains or samples that yielded positive results for
these genes with results previously conrmed by amplicon
sequencing. Following amplication, melting curve analysis was
performed to determine the specicity of the amplied products.
Melting curve was obtained from 60  C to 95  C, with continuous
uorescence measurements taken at every 1% increase in temper-
ature. Data acquisition and analysis were performed using the ABI
7500 software v2.0.4 (Applied Biosystems). To conrm positive
results, the PCR products were subjected to electrophoresis in
agarose gels and representative amplicons were sequenced.
3. Results
The selected 41 bacterial strains isolated from dental root canal
infections and identied by 16S rRNA gene sequencing are shown in
Table 2. Of these, 13 (32%) isolates were positive for at least one of the
target antibiotic resistance genes. These strains carrying at least one
antibiotic resistance gene belonged to 11/26 (42%) infected root canals
sampled. Most positive cases had only one strain carrying resistance
genes, 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
578 I.N. Ras, J.F. Siqueira Jr. / Anaerobe 18 (2012) 576e580

Table 1 were positive for the presence of at least one of the target antibiotic
Primers used for detection of antibiotic resistance genes. resistance genes.
Antibiotic Primer Sequence Ta Size Reference Resistance to beta-lactam antibiotics in oral bacteria, especially
(bp) via beta-lactamase production, has been shown not to be
Beta-lactam blaCMY-2 50 -GAC AGC CTC TTT CTC 55 1014 [29] uncommon [8,9,18,19]. In line with this observation, the present
CAC A-30 study revealed that the most prevalent antibiotic resistance gene
50 -TGG ACG AAG GCT ACG
among endodontic isolates was blaTEM, which was found in 17% of
TA-30
Beta-lactam blaTEM 50 -CCA ATG CTT AAT CAG TGA 60 858 [30] the strains and 6/26 (23%) teeth. Jungermann et al. [11] previously
GG-30 reported that blaTEM was the most prevalent antibiotic resistance
50 -ATG AGT ATT CAA CAT TTC gene detected directly in 33% of the samples from primary
CG-30 endodontic infections. TEM-type beta-lactamases are reported to
Beta-lactam blaZ 50 -CAG TTC ACA TGC CAA 54 846 [31]
AGA G-30
be widespread in Gram-negative bacteria and may attack several
50 -TAC ACT CTT GGC GGT beta-lactamic antibiotics [20]. The blaTEM gene has been found
TTC-30 widely distributed among oral biolm samples in healthy and
Beta-lactam ampC 50 -TAA ACA CCA CAT ATG 50 663 [32] disease [4,5]. The present ndings may be of some concern because
TTC CG-30
beta-lactams are the main antibiotics recommended in prophylaxis
50 -ACT TAC TTC AAC TCG
CGA CG-30 or treatment in endodontics.
Beta-lactam cfxA 50 -GCG CAA ATC CTC CTT 60 802 [8] In spite of being widespread in many species [21], especially
TAA CAA-30 Gram-negative bacteria, we are not aware of previous studies
50 -ACC GCC ACA CCA ATT reporting on the detection of the blaTEM gene in strains of Fuso-
TCG-30
Beta-lactam mecA 50 -AAA ATC GAT GGT AAA 54 533 [33]
bacterium, D. invisus and Campylobacter curvus. Except for a study that
(methicillin) GGT TGG C-30 detected blaTEM in E. faecalis [22], there are not previous reports of
50 -AGT TCT GCA GTA CCG this gene in Gram-positive bacteria, but one Propionibacterium pro-
GAT TTG C-30 pionicum strain, a species that may be involved in apical actinomy-
Macrolide ermA 50 -GTT CAA GAA CAA TCA 52 421 [34]
cosis, was positive for this gene. A previous report claimed that PCR
ATA CAG AG-30
50 -GGA TCA GGA AAA GGA reagents may be contaminated with the blaTEM gene [23]. In the
CAT TTT AC-30 present study, negative controls were run for each batch of samples
Macrolide ermB 50 -CCG TTT ACG AAA TTG 55 359 [34] analyzed to rule out the possibility of reagent contamination.
GAA CAG GTA AAG GGC-30 Noteworthy was that 6 of the 7 Fusobacterium strains tested e 4
50 -GAA TCG AGA CTT GAG
TGT GC-30
Fusobacterium nucleatum strains and 2 uncharacterized strains,
Macrolide ermC 50 -AAT CGG CTC AGG AAA 54 562 [31] were positive for at least one resistance gene. In addition to
AGG-30 carrying the blaTEM gene, Fusobacterium strains were also positive
50 -ATC GTC AAT TCC TGC for genes encoding resistance to macrolides and tetracyclines.
ATG-30
Some strains concomitantly carried resistance genes to two classes
Tetracycline tetM 50 -GTG GAC AAA GGT ACA 55 406 [35]
ACG AG-30 of antibiotics. These ndings are of some concern and need to be
50 -CGG TAA AGT TCG TCA further explored as Fusobacterium species are ubiquitous in diverse
CAC AC-30 sites of the human body and may be involved with different
Tetracycline tetO 50 -AAC TTA GGC ATT CTG 55 515 [35] conditions requiring antibiotic therapy [24e26].
GCT CAC-30
50 -TCC CAC TGT TCC ATA
The cfxA gene has also been frequently detected in oral biolms
TCG TCA-30 associated with periodontal disease [4]. A study detected this gene
Tetracycline tetS 50 -CAT AGA CAA GCC GTT 55 667 [35] in 11% of root canal samples [11]. It has been demonstrated that cfxA
GAC C-30 in oral bacterial communities mainly originates from Prevotella
50 -ATG TTT TTG GAA CGC
species [8,19]. Indeed, the only strain positive for cfxA in this study
CAG AG-30
Tetracycline tetQ 50 -TTA TAC TTC CTC CGG 55 904 [35] was an uncharacterized strain of Prevotella.
CAT CG-30 Three of the tetracycline resistance genes targeted in this study
50 -ATC GGT TCG AGA ATG were found in endodontic bacteria: tetW, tetM and tetS. Resistance
TCC AC-30 to tetracyclines may be related to ribosomal protection, efux
Tetracycline tetW 50 -GAG AGC CTG CTA TAT 64 168 [27]
pumps and enzymatic inactivation [27]. The tetW gene, which
GCC AGC-30
50 -GGG CGT ATC CAC AAT encodes the former, has been detected in the oral cavity in both
GTT AAC-30 pathogenic and non-pathogenic species [28]. Along with ermC, it
was the second most prevalent resistance gene found in this study;
4 strains from 2 species carried this gene: F. nucleatum (3 strains)
strains, 5%), and cfxa and tetS (both in 1 strain each, 2%). No strain and Parvimonas micra. The tetM gene, also encoding ribosomal
was positive for the 8 other resistance genes targeted in this study. protection, was found in 2 strains (D. invisus and a Fusobacterium
Six out of 7 Fusobacterium strains showed at least one of the strain). This gene has been very prevalent in the oral cavity of adults
target resistance genes. Some as-yet-uncharacterized Fusobacte- and children [3e5]. In addition, one Pseudoramibacter alactolyticus
rium and Prevotella isolates were positive for blaTEM, cfxA and tetM. strain was positive for tetS, while tetQ, which has been highly
Five strains were positive for more than one of the target genes: one prevalent in the oral cavity [4,5], was not detected in this study.
Dialister invisus strain was positive for 3 resistance genes, while 4 These ndings are in agreement with a previous study of
other strains yielded positive results for 2 genes. endodontic infections [11], in which tetM and tetW were more
common than tetQ. The clinical relevance of tetracycline resistance
4. Discussion in endodontics is mostly related to the use of tetracycline-
containing irrigation solutions.
The present study demonstrated that 32% of the root canal Resistance to erythromycin is most commonly due to the
isolates tested, including some as-yet-uncharacterized strains, acquisition of erm genes which encodes for rRNA methylases. In the
 I.N. Ras, J.F. Siqueira Jr. / Anaerobe 18 (2012) 576e580 579

Table 2
Antibiotic 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 Positive
F. nucleatum 2Ta e e e e e e e e Positive e e e e Positive
F. 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 Positive
Fusobacterium 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 Positive
Porphyromonas 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

present study, 4 strains were positive for ermC: F. nucleatum (2
strains), D. invisus and P. alactolyticus. No endodontic strain was
positive for ermA or ermB, even though the latter has been regarded
as one of the most abundant erm genes in the oral microbiota [3].
Endodontic isolates were identied by 16S rRNA gene in order to
provide a more accurate identication. This resulted in identica-
tion and testing not only of valid well-known or newly named
species, but also of 6 strains from 5 species-level taxa that had not
been previously cultivated. These taxa were rst disclosed by
culture-independent molecular methods and previously regarded
as uncultivable phylotypes, i.e., species known only by a 16S rRNA
gene sequence. These ndings indicate that some of the so-called
uncultivable phylotypes are in fact cultivable bacteria that were
not previously detected by culture because of either insufcient
sampling coverage or difculties in phenotypic identication. The
methodology used in this study allowed for the screening of these
still-to-be-characterized oral strains for the presence of antibiotic
resistance genes and is probably the rst study in this regard. Of
these 6 uncharacterized strains, 3 (two Fusobacterium and one
Prevotella) were positive for one or two of the resistance genes. All
three carried the blaTEM gene, the Prevotella strain was also posi-
tive for cfxA and one Fusobacterium strain for the tetM gene. These
ndings 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 harbored
at least one strain positive for the target antibiotic resistance genes.
Even though this gure may be considered high, it is still highly
likely to be an underestimate. This is because the design of the
present study included only the most dominant isolates for iden-
tication and analysis, and excluded uncultivable bacteria. There-
fore, the contribution of low-dominance species and uncultivable
bacteria to resistance genes was not evaluated. Direct screening of
clinical samples for the presence of resistance genes would be more
appropriate for this purpose. Moreover, traditional root canal
sampling with paper points fail to sample bacteria located deep
into biolms and distributed along irregularities and ramications
of 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-
tied the presence of several antibiotic resistance genes, this
information 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 clinician
should be aware of this information when deciding on local or
580 I.N. Ras, J.F. Siqueira Jr. / Anaerobe 18 (2012) 576e580
systemic antibiotic therapy. For instance, because of the risks
of bacteremia during treatment of infected canals, antibiotic
prophylaxis is indicated for patients at risk of developing bacterial
endocarditiis. Acute infections (abscesses) are also usually resultant
of exacerbations of chronic infections. The knowledge of the
potential of antibiotic resistance pattern in members of the
endodontic microbiota may inuence the decision-making process
of antibiotic prescription in endodontics.
Acknowledgments
This study was supported by grants from Fundao Carlos
Chagas Filho de Amparo Pesquisa do Estado do Rio de Janeiro
(FAPERJ) and Conselho Nacional de Desenvolvimento Cientco e
Tecnolgico (CNPq), Brazilian Governmental Institutions.
The authors deny any conicts of interest.
References
[1] Pallasch TJ. Pharmacology of anxiety, pain and infection. In: Ingle JI,
Bakland LK, editors. Endodontics. 4th ed. Baltimore: Williams & Wilkins; 1994.
p. 641e79.
[2] Sommer MO, Dantas G, Church GM. Functional characterization of the
antibiotic resistance reservoir in the human microora. Science 2009;325:
1128e31.
[3] Seville LA, Patterson AJ, Scott KP, Mullany P, Quail MA, Parkhill J, et al.
Distribution of tetracycline and erythromycin resistance genes among human
oral and fecal metagenomic DNA. Microb Drug Resist 2009;15:159e66.
[4] Kim SM, Kim HC, Lee SW. Characterization of antibiotic resistance determi-
nants in oral biolms. J Microbiol 2011;49:595e602.
[5] Ioannidis I, Sakellari D, Spala A, Arsenakis M, Konstantinidis A. Prevalence of
tetM, tetQ, nim and bla(TEM) genes in the oral cavities of Greek subjects:
a pilot study. J Clin Periodontol 2009;36:569e74.
[6] Siqueira Jr JF, Ras IN. Distinctive features of the microbiota associated with
different forms of apical periodontitis. J Oral Microbiol 2009;1. http://dx.doi.
org/10.3402/jom.v1i0.2009.
[7] Marsh PD. Dental plaque: biological signicance of a biolm and community
life-style. J Clin Periodontol 2005;32(Suppl. 6):7e15.
[8] Iwahara K, Kuriyama T, Shimura S, Williams D, Yanagisawa M, Nakagawa K,
et al. Detection of cfxA and cfxA2, the beta-lactamase genes of Prevotella spp.,
in clinical samples from dentoalveolar infection by real-time PCR. J Clin
Microbiol 2006;44:172e6.
[9] Kuriyama T, Williams DW, Yanagisawa M, Iwahara K, Shimizu C, Nakagawa K, et al.
Antimicrobial susceptibility of 800 anaerobic isolates from patients with den-
toalveolar infection to 13 oral antibiotics. Oral Microbiol Immunol 2007;22:285e8.
[10] Pinheiro ET, Gomes BP, Drucker DB, Zaia AA, Ferraz CC, Souza-Filho FJ. Anti-
microbial susceptibility of Enterococcus faecalis isolated from canals of root
lled teeth with periapical lesions. Int Endod J 2004;37:756e63.
[11] Jungermann GB, Burns K, Nandakumar R, Tolba M, Venezia RA, Fouad AF.
Antibiotic resistance in primary and persistent endodontic infections. J Endod
2011;37:1337e44.
[12] Siqueira Jr JF. Systemic antibiotics in endodontics. In: Siqueira Jr JF, editor.
Treatment of endodontic infections. London: Quintessence Publishing; 2011.
p. 381e6.
[13] Windley 3rd W, Teixeira F, Levin L, Sigurdsson A, Trope M. Disinfection of
immature teeth with a triple antibiotic paste. J Endod 2005;31:439e43.
[14] Torabinejad M, Shabahang S, Aprecio RM, Kettering JD. The antimicrobial
effect of MTAD: an in vitro investigation. J Endod 2003;29:400e3.
[15] Syed SA, Loesche WJ. Survival of human dental plaque ora in various
transport media. Appl Microbiol 1972;24:638e44.
[16] Ashimoto A, Chen C, Bakker I, Slots J. Polymerase chain reaction detection of 8
putative periodontal pathogens in subgingival plaque of gingivitis and
advanced periodontitis lesions. Oral Microbiol Immunol 1996;11:266e73.
[17] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment
search tool. J Mol Biol 1990;215:403e10.
[18] Khemaleelakul S, Baumgartner JC, Pruksakorn S. Identication of bacteria in
acute endodontic infections and their antimicrobial susceptibility. Oral Surg
Oral Med Oral Pathol Oral Radiol Endod 2002;94:746e55.
[19] Handal T, Olsen I, Walker CB, Caugant DA. Detection and characterization of
beta-lactamase genes in subgingival bacteria from patients with refractory
periodontitis. FEMS Microbiol Lett 2005;242:319e24.
[20] Livermore DM. beta-Lactamases in laboratory and clinical resistance. Clin
Microbiol Rev 1995;8:557e84.
[21] Paterson DL, Bonomo RA. Extended-spectrum beta-lactamases: a clinical
update. Clin Microbiol Rev 2005;18:657e86.
[22] Chouchani C, El Salabi A, Marrakchi R, Ferchichi L, Walsh TR. First report of
mefA and msrA/msrB multidrug efux pumps associated with blaTEM-1 beta-
lactamase in Enterococcus faecalis. Int J Infect Dis 2012;16:e104e9.
[23] Koncan R, Valverde A, Morosini MI, Garcia-Castillo M, Canton R, Cornaglia G,
et al. Learning from mistakes: Taq polymerase contaminated with beta-
lactamase sequences results in false emergence of Streptococcus pneumo-
niae containing TEM. J Antimicrob Chemother 2007;60:702e3.
[24] Siqueira Jr JF, Ras IN. The microbiota of acute apical abscesses. J Dent Res
2009;88:61e5.
[25] Manseld JM, Campbell JH, Bhandari AR, Jesionowski AM, Vickerman MM.
Molecular analysis of 16S rRNA genes identies potentially periodontal
pathogenic bacteria and archaea in the plaque of partially erupted third
molars. J Oral Maxillofac Surg 2012;70:1507e14. e1506.
[26] Brook I. Microbiology of intracranial abscesses associated with sinusitis of
odontogenic origin. Ann Otol Rhinol Laryngol 2006;115:917e20.
[27] Aminov RI, Garrigues-Jeanjean N, Mackie RI. Molecular ecology of tetracycline
resistance: development and validation of primers for detection of tetracy-
cline resistance genes encoding ribosomal protection proteins. Appl Environ
Microbiol 2001;67:22e32.
[28] Villedieu A, Diaz-Torres ML, Hunt N, McNab R, Spratt DA, Wilson M, et al.
Prevalence of tetracycline resistance genes in oral bacteria. Antimicrob Agents
Chemother 2003;47:878e82.
[29] Frye JG, Jesse T, Long F, Rondeau G, Porwollik S, McClelland M, et al. DNA
microarray detection of antimicrobial resistance genes in diverse bacteria. Int
J Antimicrob Agents 2006;27:138e51.
[30] Call DR, Bakko MK, Krug MJ, Roberts MC. Identifying antimicrobial resistance
genes with DNA microarrays. Antimicrob Agents Chemother 2003;47:3290e5.
[31] Perreten V, Vorlet-Fawer L, Slickers P, Ehricht R, Kuhnert P, Frey J. Microarray-
based detection of 90 antibiotic resistance genes of gram-positive bacteria.
J Clin Microbiol 2005;43:2291e302.
[32] Bou G, Martinez-Beltran J. Cloning, nucleotide sequencing, and analysis of the
gene encoding an AmpC beta-lactamase in Acinetobacter baumannii. Anti-
microb Agents Chemother 2000;44:428e32.
[33] Louie L, Goodfellow J, Mathieu P, Glatt A, Louie M, Simor AE. Rapid detection
of methicillin-resistant staphylococci from blood culture bottles by using
a multiplex PCR assay. J Clin Microbiol 2002;40:2786e90.
[34] Lina G, Quaglia A, Reverdy ME, Leclercq R, Vandenesch F, Etienne J. Distri-
bution of genes encoding resistance to macrolides, lincosamides, and strep-
togramins among staphylococci. Antimicrob Agents Chemother 1999;43:
1062e6.
[35] Ng LK, Martin I, Alfa M, Mulvey M. Multiplex PCR for the detection of tetra-
cycline resistant genes. Mol Cell Probes 2001;15:209e15.