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Distribution of Genes Related to Antimicrobial Resistance in Different Oral Environments: A Systematic Review
Review Article
Distribution of Genes Related to Antimicrobial Resistance in Different Oral Environments: A Systematic Review Ludmila Coutinho Moraes, DDS, MSc,* Marcus Vinıcius Reis S o, DDS, MSc, PhD,* Tatiane da Silva Dal Pizzol, PharmD, MSc, PhD,† Maria Beatriz Cardoso Ferreira, MD, MSc, PhD,‡ and Francisco Montagner, DDS, MSc, PhD* Abstract Introduction: The oral cavity is the main source of microorganisms for odontogenic infections. It is important to perform an extensive analysis regarding the reports on the presence of bacteria that carry resistance genes to antimicrobial agents. The aim of the study was to verify the reports on the distribution of genes associated with resistance to antibiotics prescribed in dentistry in different human oral sites. Methods: A systematic review was conducted in electronic databases and gray literature to analyze clinical studies that detected genes of bacterial resistance to antibiotics in saliva, supragingival biofilm, and endodontic infections. Data regarding the research group, geographic location, sample source, number of subjects, methods for sample analysis, the targeted gene groups, and the detection rates were collected. Descriptive data analysis was performed. Results: Preliminary analysis was performed in 152 titles; 50 abstracts were reviewed, and 29 full texts were obtained. Nine articles matched the inclusion criteria (saliva = 2, supragingival biofilm = 1, and endodontic infections = 6). The presence of 33 different targeted genes was evaluated. The most frequently investigated groups of genes were tetracycline and lactamics (tetM, tetQ, tetW, and cfxA). There was a wide range for the detection rates of each resistance gene among studies and for each specific gene group. Conclusions: This systematic review highlights the presence of resistance genes to antimicrobial agents in saliva, dental biofilm, and endodontic infections, especially for tetracycline and lactamics. There is a lack of reports on the presence of genes and resulting outcomes obtained through the therapeutic approaches for infection control. (J Endod 2015;-:1–8)
Key Words Dental plaque, dental pulp cavity, drug resistance, saliva
T
he human mouth harbors a wide number of microorganisms, comprising the commensal microbiota. It has an important role in maintaining oral and systemic health. The microbiota of the mouth is the source for microorganisms associated with dental caries, endodontic diseases, and periodontal diseases (1). Oral bacteria start colonizing the root canal system after pulp necrosis, remaining suspended in the root canal (planktonic state) or attached to its walls (biofilms) (2). They can reach the apical tissues causing extraradicular infections and constitute complex microbial communities associated with acute apical abscesses (3), extraradicular biofilms (4), and persistent infections (5). The composition of microbial communities associated with endodontic infections is heterogeneous and can be modulated by several factors such as the geographic location (6), the presence or absence of symptoms (7), and the clinical condition (8). Some studies also report distinct microbial profiles in paired samples from root canals and abscesses collected from the same patient (3). Few representatives of the domains Eukarya and Archea have been described; the domain Bacteria is the most predominant and diverse in endodontic infections (2). After a comprehensive review on the microbial communities associated with endodontic infections, Siqueira and R^oc¸as (2) emphasized the need for determining microbial functional roles in the community and the susceptibility to antimicrobial treatment procedures. Oral or parenteral antibiotics are generally considered adjunctive therapy for urgency endodontic treatment. Their value should not be underestimated, especially when drainage cannot be achieved or the infection shows signs of local extension or systemic involvement (9). Beta-lactam, tetracycline, and macrolide antibiotics have been prescribed in endodontics, especially for the treatment of acute apical abscesses associated with systemic involvement, spreading infections, abscesses in medically compromised patients who are at increased risk of a nonoral secondary infection after bacteremia, prophylaxis for medically compromised patients during routine endodontic therapy, and replantation of avulsed teeth (10). Local application of antimicrobial agents has been considered a possibility to improve root canal disinfection during the chemomechanical preparation (11, 12) or as an intracanal medicament (13). However, it should be emphasized that the use of antibiotics instead of biocides such as hypochlorite or chlorhexidine appears unwarranted, mainly because of the narrower spectrum of activity and resistance (14). Antibiotic resistance is a phenomenon of crucial importance in the treatment of diseases caused by pathogenic microorganisms (15). Bacterial resistance to antibiotics is multifactorial. Antibiotic resistance occurs by both intrinsic defenses (16) and genetic mutation in bacteria. Some mechanisms of bacterial resistance to antibiotics have been attributed to the resistance genes. Diverse resistance genes have been found
From the *Division of Endodontics, Department of Conservative Dentistry; †Post-Graduation Program in Epidemiology, Faculty of Medicine; and ‡Department of Pharmacology, Basic Health Science Institute, Federal University of Rio Grande do Sul, Porto Alegre, Brazil. Address requests for reprints to Dr Francisco Montagner, Universidade Federal do Rio Grande do Sul, Faculdade de Odontologia, Rua Ramiro Barcelos, 2492–Bairro Santana, Porto Alegre, RS, Brazil, 90035-003. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2014.12.018
JOE — Volume -, Number -, - 2015
Genes Related to Antimicrobial Resistance
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Review Article and characterized in human microflora, which can operate as a reservoir for antibiotic-resistant bacteria (17). The objective of this systematic review was to evaluate the distribution of genes of bacterial resistance to antibiotics in different sites of the oral cavity. A search strategy was formulated to answer a question in the PICO (Population, Intervention, Comparison, Outcome) format, defining inclusion and exclusion criteria. The question was framed as follows: In the oral sites saliva, supragingival biofilm, and endodontic infections, how are distributed genes associated with bacterial resistance to antibiotics frequently prescribed in dentistry?
Initial results for each database total: 152 articles PUBMED (135 articles), EMBASE (113 articles), SCOPUS (67 articles), ISI (36 articles), Grey literature (4 articles).
Title analysis of all selected articles = 50 abstracts
Abstract analysis of the selected articles = 29 full-text
Materials and Methods A systematic review was performed to check all clinical studies that detected genes of bacterial resistance to antibiotics in 3 sites of oral cavity: saliva, supragingival biofilm, and endodontic infections. The terms were used in various combinations in the electronic databases as follows: Medline (PubMed), Embase, Web of Science, Scopus, and OpenGrey. No language restriction was applied to the search. Figure 1 describes the search strategy adopted in the study. The search comprised articles published until January 14, 2014. The references reported on the selected articles were also reviewed. After title screening and abstract analysis, the full text of each study was obtained. The relevance of each study to the question of interest was determined through inclusion and exclusion criteria. The full texts of the articles were revised by 2 reviewers (F.M. and L.C.M) based on the following inclusion criteria: 1. Clinical studies in healthy patients or in patients with oral disease (cross-sectional or longitudinal studies) 2. Studies that detected bacterial resistance genes of antibiotics by molecular techniques 3. Studies with samples collected from oral cavity (saliva, supragingival biofilm, or root canal with primary endodontic infection) Exclusion criteria comprised the following: 1. 2. 3. 4.
Literature reviews In vitro studies Studies that analyzed mixed samples from different environments Studies that detected genes in other environments besides those cited in the inclusion criteria 5. Studies that did not use molecular methods for detection of the presence of the resistance genes 6. Studies in which the objective was not the detection of resistance genes 7. Lack of data Terms: ("mouth"[MeSH Terms] OR "mouth"[All Fields] OR ("oral"[All Fields] AND "cavity"[All Fields]) OR "oral cavity"[All Fields]) AND ("drug resistance, microbial"[MeSH Terms] OR ("drug"[All Fields] AND "resistance"[All Fields] AND "microbial"[All Fields]) OR "microbial drug resistance"[All Fields] OR ("antibiotic"[All Fields] AND "resistance"[All Fields]) OR "antibiotic resistance"[All Fields]) AND ("genes"[MeSH Terms] OR "genes"[All Fields] OR "gene"[All Fields])
Figure 1. The search strategy adopted for the study, presenting the MeSH keywords and search terms for the presence of resistance genes to antimicrobial agents in the oral environment.
2
Moraes et al.
20 excluded (Table 1)
9 selected articles
Figure 2. Results after the search strategy developed to find studies related to the presence of resistance genes to antimicrobial agents in different oral environments.
Data regarding research group, geographic location, sample source, number of subjects included in the study, methods for sample size determination, statistical analysis, the targeted genes, and their detection rates were collected from the studies. Because of the methodologic differences among the studies, it was not possible to perform a meta-analysis. Descriptive data analysis was performed.
Results Considering all the databases, a total of 152 titles were identified after subtraction of duplicates for preliminary analysis. After title screening, 50 abstracts were revised, and full texts of 29 studies were obtained (Fig. 2). The 20 excluded articles are listed in Table 1, and the reasons for their exclusion are also shown. Nine articles matched the inclusion criteria regardless of the oral environment. There were 2 articles for saliva samples, 1 for supragingival biofilm, and 6 studies on primary endodontic infections. There was no age limit in the search, and only 1 of the selected articles reported data for children. Tables 2, 3, and 4 provide information about the selected studies. They were performed in different periods of time (from 2003–2014), and the samples were collected in Japan, the United States, Brazil, and European countries. The great majority of the authors reported ethics statements and/or conflicts of interest, except in 1 study (38). There was no description for the sample size determination methods adopted. The most frequently adopted exclusion criteria were previous exposure to antimicrobial agents, ranging from 1 to 6 months. The presence of resistance genes was assessed in clinical isolates (38–40, 43, 44, 46) and in samples from each environment (41, 42, 45). The presence of resistance genes in saliva samples was determined only from healthy subjects (38, 41). The only study that tested resistance genes in supragingival biofilm was conducted in children (39). Endodontic samples were collected from root canals (symptomatic and asymptomatic cases) and apical swelling. Descriptive statistics were reported in all studies. Inferential statistics were used to compare the prevalence of specific genes in the groups (primary vs persistent infections, preoperative vs preobturation samples, before vs after root canal preparation, and abscess vs asymptomatic apical periodontitis). JOE — Volume -, Number -, - 2015
Review Article TABLE 1. Characteristics of the Studies Excluded from the Systematic Review Authors/year
Country
Title
Andres et al, 1998 (18)
Spain
Arseze et al, 2000 (19)
Italy
Antimicrobial susceptibilities of Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens spp. isolated in Spain Detection of tetQ and ermF antibiotic resistance genes in Prevotella and Porphyromonas isolates from clinical specimens and resident microbiota of humans Distribution of the tetracycline resistance determinant tetQ gene in oral isolates of blackpigmented anaerobes in Japan Presence and antibiotic resistance of Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens in children Antibiotic resistance in oral commensal streptococci from healthy Mexicans and Cubans: resistance prevalence does not mirror antibiotic usage The mef(A) gene predominates among seven macrolide resistance genes identified in gramnegative strains representing 13 genera, isolated from healthy Portuguese children Determining the antibiotic resistance potential of the indigenous oral microbiota of humans using a metagenomic approach Antimicrobial resistance in clinical isolates of Neisseria subflava from the oral cavities of a Japanese population Genetic determinants for cfxA expression in Bacteroides strains isolated from human infections Prevalence of tetM, tetQ, nim and bla(TEM) genes in the oral cavities of Greek subjects: a pilot study Characterization of antibiotic resistance determinants in oral biofilm Molecular investigation of macrolide and tetracycline resistances in oral bacteria isolated from Tunisian children Analysis of genetic lineages and their correlation with virulence genes in Enterococcus faecalis clinical isolates from root canal and systemic infections Antimicrobial susceptibility and molecular analysis of Enterococcus faecalis originating from endodontic infections in Finland and Lithuania Genetic analysis of mobile tetQ elements in oral Prevotella species Genetic basis of erythromycin resistance in oral bacteria Prevalence of tetracycline resistance genes in oral bacteria Characterization of tet32 genes from the oral metagenome Prevalence, phenotype, and genotype of Enterococcus faecalis isolated from saliva and root canals in patients with persistent apical periodontitis Distribution of amoxicillin-resistant oral streptococci in dental plaque specimens obtained from Japanese children and adolescents at risk for infective endocarditis
Okamoto et al, 2001 (20) Sanai et al, 2002 (21) Dıaz-Mejıa et al, 2002 (22) Ojo et al, 2004 (23)
Japan Portugal Mexico Portugal
Diaz-Torres et al, 2006 (24)
UK
Furuya et al, 2007 (25)
Japan
Garcia et al, 2008 (26)
Spain
Ioannidis et al, 2009 (27)
Greece
Kim et al, 2011 (28)
US
Kouidhi et al, 2011 (29)
Tunisia
Penas et al, 2013 (30)
Brazil
Reynaud af Geijersstam et al, 2006 (31)
Sweden
Tribble et al, 2010 (32)
US
Villedieu et al, 2004 (33)
UK
Villedieu et al, 2003 (34)
UK
Warburton et al, 2009 (35)
UK
Zhu et al, 2010 (36)
China
Nemoto et al, 2013 (37)
Japan
Oral site Dental plaque
Exclusion criteria* 3, 4
Oral cavity
3
Subgingival biofilm
4
Dental plaque and buccal mucosa
3
Oral samples
6, 7
Oral and urine samples
7
Saliva and dental plaque
3
Oral cavity samples
5
Humans infections
3, 4
Dental plaque and tongue samples Dental plaque Dental caries and plaque
4 4 3, 4
Root canal
4
Root canal
4
Oral cavity samples
7
Saliva and dental plaque
3
Saliva and dental plaque
3
Oral cavity samples
5
Saliva and root canal
Supra- and subgingival plaque
4, 5
3
*Exclusion criteria were as follows: 1, literature reviews; 2, in vitro studies; 3, studies that analyzed mixed samples from different environments; 4, studies that detected genes in other environments besides those cited in the inclusion criteria; 5, studies that did not use molecular methods for detection of the presence of the resistance genes; 6, studies in which the objective was not the detection of resistance genes; 7, lack of data.
A total of 33 different genes have been evaluated in the 9 articles as follows: 1. Beta-lactam: blaTEM, blaZ, cfxA, cfxA2, blaCMY2, ampC, mecA (methicillin), pbp1A, pbp2X, and pbp2B 2. Macrolide: ermA, ermB, ermC, ermE, and ermV JOE — Volume -, Number -, - 2015
3. Tetracycline: tetA, tetB, tetC, tetD, tetK, tetL, tetM, tetO, tetQ, tetS, tetW, and tet32 4. Vancomycin: vanA, vanB, vanC1, vanC2/3, vanD, and vanE The genes for tetracycline resistance were the most frequently investigated in the different publications. The most tested genes were
Genes Related to Antimicrobial Resistance
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Review Article TABLE 2. Characteristics of the Studies Included in the Systematic Review Description of the recruitment
Authors/Year
Assessment
Participants/sample size
Nakayama and Takao, 2003 (38)
Detection of S. mitis resistant to ampicillin, cefaclor and cefotaxime Maintenance of resistant bacteria and genes that encode tetracycline resistance in children Detection of beta-lactamase resistance genes in Prevotella strains Detection of genes for resistance to tetracycline and erythromycin in oral metagenomas Detection of resistance genes in bacteria isolated from primary and persistent endodontic infections Detection of 14 genes encoding resistance to beta-lactams, tetracycline and macrolides
Healthy subjects (age: 20–30 years) N = 52 Healthy subjects (age: 4–6 years) N = 26 Patients with dentoalveolar infection (mean age: 48.1 years) N = 87 Healthy subjects (age: 21–65 years) N = 6†
Lancaster et al, 2005 (39) Iwahara et al, 2006 (40) Seville et al, 2009 (41) Jungermann et al, 2011 (42) ^ c¸as and Ro Siqueira Jr, 2012 (43) Lins et al, 2013 (44) ^ c¸as and Ro Siqueira Jr, 2013 (45) Montagner et al, 2014 (46)
Detection of resistant E. faecalis and virulence trait in primary endodontic infections Detection of genes encoding resistance to beta-lactams, tetracycline, and erythromycin in bacteria from acute or chronic endodontic infections Detection of the cfxA/cfxA2 gene and analysis of its expression in endodontic infections
Antibiotic exposure*
No
Previous 6 months
Yesjj
Previous 3 months
September 2001 and March 2005 No
Not mentioned Previous 3 months
Patients with endodontic infections (age: 19–94 years; mean: 51 years) N = 50‡ Patients with apical periodontitis (age: not specified) N = 26 Patients with primary endodontic infection (age: not specified) N = 43 Patients with asymptomatic apical periodontitis or acute apical abscess (age: 19–64 years) N = 50
No§
Previous 1 month
No§
Previous 6 months
No
Previous 6 months
No
Previous 3 months
Patients with spontaneous pain and pulp necrosis (age: nonspecified) N = 26
No
Previous 3 months
*Antibiotic exposure was considered exclusion criteria in these studies. † In this study, samples from 6 countries were analyzed, and each sample was constituted by the pool of saliva from 20 individuals/country. ‡ The final sample consisted of 45 patients with primary (n = 30) or persistent (n = 15) endodontic infections; 5 were excluded during the study. § Perhaps all patients who had endodontic infections in a nonspecified period of time. jj A total of 18 of 26 children were included in the study because they had not taken any antibiotics 3 months before sampling; 8 of 18 children had amoxicillin, ampicillin, cefalexin (cephalosporin), and ciprofloxacin in the previous 3 months; and none of them had taken tetracycline or erythromycin.
tetM for tetracycline resistance (saliva = 1 study [41], supragingival biofilm = 1 [39], endodontic infections = 4 studies [42–45]) followed by the genes tetQ/tetW (saliva = 1 study [41], supragingival biofilm = 1 [39], endodontic infections = 3 studies [42, 43, 45]). The frequency for the detection of the tetM gene ranged from 5%–100% (5%–60% in 4 root canal samples, 77.7% in the supragingival biofilms, and 100% in 1 saliva sample study). The tetracycline resistance genes tetQ and tetW were detected in 0%–10% and 20%–29% of the root canal samples, respectively; 5% of the supragingival samples had tetQ and tetW. One study (41) that evaluated the tetQ and tetW genes in saliva did not provide measurable data regarding its frequency in this specific environment. The presence of genes associated with the resistance of betalactam agents was assessed in the oral environment. The gene cfxA/cfxA2 was searched in 5 studies (40, 42, 43, 45, 46), and its detection rate ranged from 0%–40% in root canal samples. The gene blaTEM was also detected in root canals by 3 articles (42, 43, 45), with frequencies ranging from 17%–43% of the samples. The genes pbp1a, pbp2x, and pbp2b were observed in saliva samples (38). Macrolide resistance genes were detected in root canals in 3 articles (42, 43, 45), and in 1 article that evaluated saliva samples (41). The ermC gene was detected in 10%–24% of endodontic infections. Both ermA and ermB genes were not found in root canal samples. However, the ermB gene was found in saliva with high frequency. Vancomy4
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cin resistance genes were searched in root canal samples, but no positive results were observed (42, 44).
Discussion The methods used in current studies to depict the structure and virulence factors associated with microbial communities are labor intensive and expensive. Therefore, these studies have evaluated a small number of samples obtained from a restricted population source. Systematic reviews have been suggested as a tool for a synthesis and critical analysis of the literature. They can be associated with statistical methods such as meta-analysis to obtain compiled results of studies with small sample sizes, serving as a guide for evidencebased practice (47). Thus, conducting systematic reviews may provide reliable results that can be used for clinical decision making, especially when it is possible to perform a meta-analysis. This systematic review aimed to identify, evaluate, and synthesize all clinical studies that met the prespecified eligibility criteria to answer the following research question: ‘‘How are genes of bacterial resistance to antibiotics frequently prescribed in dentistry distributed in different oral environments (saliva, supragingival biofilm, and endodontic infections)?’’ In the present review, broad topics were used in the electronic databases to reduce the possibility of overlooking studies. Four databases and gray literature were used followed by a careful hand search of JOE — Volume -, Number -, - 2015
Review Article TABLE 3. Other Characteristics of the Included Studies Authors/Year
Targeted species or bacteria
Sample source
Assessment of outcome
Nakayama and Isolates of S. mitis Takao, 2003 (38)
Saliva
PCR
Lancaster et al, 2005 (39)
Isolates from dental plaque
Dental plaque
PCR
Iwahara et al, 2006 (40)
Prevotella spp
AAA (n = 72) PCR and RC (n = 15)
Seville et al, 2009 (41)
Analysis of oral DNA metagenomes
Saliva
PCR
Jungermann et al, Bacteria and samples 2011 (42) isolated from primary and persistent endodontic infections
RC
PCR
^ c¸as and Siqueira Bacteria isolated from Ro Jr, 2012 (43) endodontic infections Lins et al, Enterococcus faecalis 2013 (44)
RC
PCR
RC
PCR
^ c¸as and Siqueira Samples from acute Ro Jr, 2013 (45) apical abscess or asymptomatic apical periodontitis
AAA (n = 25) RC (n = 25)
PCR
Montagner et al, 2014 (46)
RC (N = 26)
PCR
Bacteria isolated from acute endodontic infections
Method for resistance characterization
Comparability/ statistical analysis
Detection of bacteria Descriptive statistical with penicillin-binding analysis protein genes No comparisons between individuals with or without resistant S. mitis Detection of genes for Descriptive statistical resistance to analysis tetracycline Detection of bacteria Descriptive statistical with beta-lactamase analysis resistance genes No comparison between individuals with or without resistance genes Description of the presence of cfxA/cfxA2 in resistant-bacteria or susceptible-bacteria. Detection of genes for Descriptive statistical resistance to analysis. tetracycline and erythromycin Detection of common Descriptive and antibiotic resistance inferential statistical genes and analysis; association susceptibility to analysis (P description specific antibiotics without confidence interval); comparison of prevalence of different antibiotic resistance genes: in primary versus persistent infections; in preoperative and preobturation specimens Detection of resistance Descriptive statistical genes analysis Detection of resistant Descriptive statistical bacteria to analysis tetracycline, erythromycin, and vancomycin, and virulence trait Detection of 6 resistance Descriptive and genes to beta-lactams, inferential statistical tetracycline, and analysis; association erythromycin analysis (P description, without CI) Comparison of prevalence of the target resistance genes: in acute abscess versus asymptomatic apical periodontitis; before and after RC preparation Detection of the cfxA/ Descriptive statistical cfxA2 gene analysis
AAA, acute apical abscess; CI, confidence interval; PCR, polymerase chain reaction; RC, root canal.
references in the 29 selected articles to avoid the risk of ignoring studies. Nine articles matched the inclusion criteria: 2 articles for saliva samples, 1 for supragingival biofilm, and 6 studies on primary endodontic infections. The aim of this systematic review was to synthesize data from clinical studies with similar objectives and methodology for JOE — Volume -, Number -, - 2015
the detection of resistance genes in 3 specific oral environments. Mixed samples from different oral environments were not considered. Therefore, the present review aimed to obtain a homogeneous group of data that will provide a strict panel on the presence of resistance genes in saliva, supragingival plaque, and infected root canals. Several studies
Genes Related to Antimicrobial Resistance
5
Review Article TABLE 4. Results of the Included Studies Authors/Year
Resistance profiles
Clinical information
Nakayama and Takao, 2003 (38) Lancaster et al, 2005 (39)
All the resistant streptococci had the PBP genes
Not applied
The most prevalent gene encoding tetracycline resistance was tet(M). tet(B), tet(K), tet(O), tet(Q), tet(S), tet(W) were also found.
Iwahara et al, 2006 (40)
Presence of cfxA and cfxA2: all beta-lactamase– positive Prevotella strains; none of the betalactamase-negative Prevotella isolates Most prevalent tetracycline resistance gene: tet(M) Most abundant erythromycin resistance gene: erm(B) Common genes: tet(Q), tet(O), tet(O/32/O) tet(W) Preoperative specimens: significantly more prevalent blaTEM-1 gene in primary versus persistent infections; cfxA only present in primary infections Preobturation specimens: significant reduction of blaTEM-1 and tet(W); elimination of cfxA, blaZ, and tet(Q); no alteration of tet(M) Absence of genes vanA, vanD, and vanE (associated with vancomicin resistance) Presence of at least 1 of the target antibiotic resistance genes: 32% isolates, corresponding to 42% of the infected root canals analyzed Most prevalent genes (of the 6 antibiotic resistance genes detected): blaTEM (17% strains), tetW (10%), and ermC (10%) Presence of tetM and tetL: 60% and 20%, respectively No detection of ermA, ermB, vanA, vanB, vanC1, and vanC2/3 Initial analysis in AAA samples: 100% samples with bacteria; absence of cfxA and tetQ; 36% positive for at least 1 of 4 remained antibiotic resistance genes; blaTEM (24%), ermC (24%), tetW (12%), tetM (8%) Initial analysis in asymptomatic apical periodontitis: 100% samples with bacteria; absence of blaTEM, cfxA, and tetQ; 67% positive for at least 1 of the other antibiotic resistance genes; tetM (42%), tetW (29%), ermC (25%) blaTEM: significantly more found in acute abscess than in asymptomatic cases tetM: significantly more found in asymptomatic cases than in acute abscess cfxA/cfxA2 gene: detection in 6.9% of isolates Simultaneous detection of cfxA/cfxA2 gene and lactamase production: 1 P. buccalis strain Detection of cfxA/cfxA2 gene with no expression: 1 P. micra strain Positive lactamase production, with no detection of cfxA/cfxA2 gene: 3 strains
The results of this study have shown that tetracycline-resistant bacteria and tet(M) are maintained within the indigenous oral microbiota of children, even though they are unlikely to have been directly exposed to tetracycline No description of treatment No description of the clinical evolution of the infection Not applied
Seville et al, 2009 (41)
Jungermann et al, 2011 (42)
^ c¸as and Siqueira Jr, 2012 (43) Ro
Lins et al, 2013 (44)
^ c¸as and Siqueira Jr, 2013 (45) Ro
Montagner et al, 2014 (46)
Evaluation of the profile of antibiotic resistance genes after chemomechanical procedures No description of the clinical evolution
No description of treatment or the clinical evolution of the infection
No description of treatment or the clinical evolution of the infection Analysis of the elimination of bacteria carrying antibiotic resistance genes after chemomechanical procedures No description of the clinical evolution
No description of treatment or the clinical evolution of the infection
AAA, acute apical abscess.
were excluded because they did not provide full data in the results description. No personal correspondence was sent to the authors to obtain more detailed data. Few data are available on the presence of resistance genes in the selected oral sites. Only 1 study reported the presence of tetracycline genes in bacteria isolated from supragingival biofilm in children (39). Of the 6 selected articles that evaluated the presence of resistance genes in endodontic infections, 2 examined directly pooled samples of 6
Moraes et al.
acute endodontic infections (42, 45), and 4 tested bacterial strains isolates from those infections (40, 43, 44, 46). The critical evaluation of the selected studies showed few factors connecting them. It was especially noticed the diversity of patient characteristics. The sample size was variable, ranging from 26 to 87. No sample size determination methods were described. There are different outcomes that can limit the definition of the parameter to be the basis of the sample size determination. The presence of strict inclusion criteria JOE — Volume -, Number -, - 2015
Review Article and the labor-intensive characteristics for root canal sampling may restrict the number of subjects who were enrolled in the studies. It may also require a long period of time to obtain all the volunteers who would join the study. Furthermore, it should be considered that root canal sampling is labor intensive, and strict procedures should be used to avoid external contaminants. Therefore, there are intrinsic limitations such as the complexity of the sampling process and the wide range of variables associated with the patient that may introduce bias to the results. During the literature search, it was observed that few data about the inclusion criteria of the patients were reported. Both healthy and diseased patients with endodontic infections were included in the samples, and saliva samples were collected. Supragingival biofilm samples were collected from healthy children (39). However, details on their recruitment, sex, and clinical conditions were not described. Prior exposure to antibiotics was used as an exclusion criterion. There was no consensus on the literature regarding the previous period that the patient should not take the medication (ranging from 1– 6 months). Stark et al (48) reported that a previous intake of amoxicillin can change the microbial composition inside the oral cavity, with a slight increase in the number of resistant strains. Baglie et al (49) reported a decrease in colony-forming units counting 60 minutes after oral amoxicillin intake. In a study by Feres et al (50), the consumption of amoxicillin and metronidazole for 14 days determined a transient modification in the percentage of resistant strains in subgingival biofilm although a large proportion remained susceptible during this period. The prevalence of bacteria resistant to amoxicillin only returned to the baseline levels in 90 days. These data suggest that short periods of suspension of antibiotic treatment (ie, 1 or 2 months) may not be sufficient for the microbiota to return to baseline levels of susceptibility. Thus, the information obtained may still reflect the influence of prior use of the drug. Another factor that must be considered is the geographic location. It can interfere in the composition of the microbial communities associated with endodontic infections (51). Seville et al (41) compared the presence of resistance genes in different European countries without limiting the evaluation to a specific geographic area. Nakayama and Takao (38) investigated the percentage of betalactam resistance genes in isolates of Streptococcus spp. Jungermann et al (42) also reported the presence of resistance genes in samples collected after chemomechanical preparation; however, only the results obtained before the treatment were considered in the present study. Regarding the selected articles that evaluated root canal samples, 4 were conducted in Brazil, 1 in Japan, and 1 in the United States. It is known that the profile of resistance depends on the geographic region because it is related to the pattern of antibiotic prescription by professionals and the profile of use by the patients (52). Therefore, studies performed in different countries are affected by the different pattern of use of antibiotics. According to the present literature review, the genes blaTEM, cfxA, tetW, tetM, and ermC were frequently detected in oral samples. Although the detection of resistance genes for specific antibiotics does not necessarily mean antibiotic resistance of the specimens, it indicates the potential for the expression of the encoded protein. Montagner et al (46) detected the cfxA/cfxA2 gene in 2 of 26 isolates from acute endodontic infections, but only 1 strain had expressed this gene as determined through phenotypic methods. Furthermore, it is also important to evaluate the clinical response of patients who harbor antibiotic resistance genes. Some studies (40, 42, 46) evaluated the bacterial susceptibility to antibiotics and the presence of resistance genes in samples collected from patients with dentoalveolar infections. However, no one described the clinical outcome after the JOE — Volume -, Number -, - 2015
treatment or determined the relation between the presence of resistance genes and the clinical evolution of patients. Thus, this systematic review highlights the presence of resistance genes to antimicrobial agents in the oral cavity, especially for tetracycline and beta-lactam agents in saliva, supragingival biofilm, and acute endodontic infections. It also showed the lack of reports about the presence of these genes and resulting clinical outcomes, considering the therapeutic approaches used for infection control.
Acknowledgments Supported by a grant from the Fundac¸~ao de Apoio a Pesquisa do Estado do Rio Grande do Sul (ARD-FAPERGS 140112-0) to Dr Montagner. The authors deny any conflicts of interest related to this study.
References 1. Wade WG. The oral microbiome in health and disease. Pharmacol Res 2013;69: 137–43. 2. Siqueira JF Jr, R^oc¸as IN. Community as the unit of pathogenicity: an emerging concept as to the microbial pathogenesis of apical periodontitis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;107:870–8. 3. Montagner F, Gomes BP, Kumar PS. Molecular fingerprinting reveals the presence of unique communities associated with paired samples of root canals and acute apical abscesses. J Endod 2010;36:1475–9. 4. Subramanian K, Mickel AK. Molecular analysis of persistent periradicular lesions and root ends reveals a diverse microbial profile. J Endod 2009;35:950–7. 5. Signoretti FG, Gomes BP, Montagner F, Jacinto RC. Investigation of cultivable bacteria isolated from longstanding retreatment-resistant lesions of teeth with apical periodontitis. J Endod 2013;39:1240–4. 6. Baumgartner JC, Siqueira JF Jr, Xia T, R^oc¸as IN. Geographical differences in bacteria detected in endodontic infections using polymerase chain reaction. J Endod 2004; 30:141–4. 7. Santos AL, Siqueira JF Jr, R^oc¸as IN, et al. Comparing the bacterial diversity of acute and chronic dental root canal infections. PLoS One 2011;6:e28088. 8. Gomes BP, Pinheiro ET, Gad^e-Neto CR, et al. Microbiological examination of infected dental root canals. Oral Microbiol Immunol 2004;19:71–6. 9. Poeschl PW, Crepaz V, Russmueller G, et al. Endodontic pathogens causing deep neck space infections: clinical impact of different sampling techniques and antibiotic susceptibility. J Endod 2011;37:1201–5. 10. Siqueira JF Jr. Systemic antibiotics in endodontics. In: Siqueira JF Jr, ed. Treatment of Endodontic Infections. London: Quintessence Publishing; 2011:381–96. 11. Tong Z, Ling J, Lin Z, et al. The effect of MTADN on 10 Enterococcus faecalis isolates and biofilm: an in vitro study. J Endod 2013;39:674–8. 12. Torabinejad M, Shabahang S, Aprecio RM, Kettering JD. The antimicrobial effect of MTAD: an in vitro investigation. J Endod 2003;29:400–3. 13. Diogenes AR, Ruparel NB, Teixeira FB, Hargreaves KM. Translational science in disinfection for regenerative endodontics. J Endod 2014;40:S52–7. 14. Zehnder M. Root canal irrigants. J Endod 2006;32:389–98. 15. Vester B, Long KS. Antibiotic resistance in bacteria caused by modified nucleosides in 23S ribosomal RNA. In: Grosjean H, ed. DNA and RNA Modification Enzymes: Structure, Mechanism, Function and Evolution. Austin: Landes Bioscience; 2009. 16. Wise R, Hart T, Cars O, et al. Antimicrobial resistance is a major threat to public health. Br Med J 1998;317:609–10. 17. Sommer MO, Church GM, Dantas G. The human microbiome harbors a diverse reservoir of antibiotic resistance genes. Virulence 2010;1:299–303. 18. Andres MT, Chung WO, Roberts MC, Fierro JF. Antimicrobial susceptibilities of Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens spp. isolated in Spain. Antimicrob Agents Chemother 1998;42:3022–3. 19. Arzese AR, Tomasetig L, Botta GA. Detection of tetQ and ermF antibiotic resistance genes in Prevotella and Porphyromonas isolates from clinical specimens and resident microbiota of humans. J Antimicrob Chemother 2000;45:577–82. 20. Okamoto M, Takano K, Maeda N. Distribution of the tetracycline resistance determinant tetQ gene in oral isolates of black-pigmented anaerobes in Japan. Oral Microbiol Immunol 2001;16:224–8. 21. Sanai Y, Persson GR, Starr JR, et al. Presence and antibiotic resistance of Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens in children. J Clin Periodontol 2002;29:929–34. 22. Dıaz-Mejıa JJ, Carbajal-Saucedo A, Amabile-Cuevas CF. Antibiotic resistance in oral commensal streptococci from healthy Mexicans and Cubans: resistance prevalence does not mirror antibiotic usage. FEMS Microbiol Lett 2002;217:173–6.
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Review Article 23. Ojo KK, Ulep C, Van Kirk N, et al. The mef(A) gene predominates among seven macrolide resistance genes identified in gram-negative strains representing 13 genera, isolated from healthy Portuguese children. Antimicrob Agents Chemother 2004;48:3451–6. 24. Diaz-Torres ML, Villedieu A, Hunt N, et al. Determining the antibiotic resistance potential of the indigenous oral microbiota of humans using a metagenomic approach. FEMS Microbiol Lett 2006;258:257–62. 25. Furuya R, Onoye Y, Kanayama A, et al. Antimicrobial resistance in clinical isolates of Neisseria subflava from the oral cavities of a Japanese population. J Infect Chemother 2007;13:302–4. 26. Garcıa N, Gutierrez G, Lorenzo M, et al. Genetic determinants for cfxA expression in Bacteroides strains isolated from human infections. J Antimicrob Chemother 2008; 62:942–7. 27. Ioannidis I, Sakellari D, Spala A, et al. Prevalence of tetM, tetQ, nim and bla(TEM) genes in the oral cavities of Greek subjects: a pilot study. J Clin Periodontol 2009;36: 569–74. 28. Kim SM, Kim HC, Lee SW. Characterization of antibiotic resistance determinants in oral biofilms. J Microbiol 2011;49:595–602. 29. Kouidhi B, Zmantar T, Hentati H, et al. Molecular investigation of macrolide and tetracycline resistances in oral bacteria isolated from Tunisian children. Arch Oral Biol 2011;56:127–35. 30. Penas PP, Mayer MP, Gomes BP, et al. Analysis of genetic lineages and their correlation with virulence genes in Enterococcus faecalis clinical isolates from root canal and systemic infections. J Endod 2013;39:858–64. 31. Reynaud af Geijersstam AH, Ellington MJ, Warner M, et al. Antimicrobial susceptibility and molecular analysis of Enterococcus faecalis originating from endodontic infections in Finland and Lithuania. Oral Microbiol Immunol 2006;21:164–8. 32. Tribble GD, Garza JJ, Yeung VL, et al. Genetic analysis of mobile tetQ elements in oral Prevotella species. Anaerobe 2010;16:604–9. 33. Villedieu A, Diaz-Torres ML, Roberts AP, et al. Genetic basis of erythromycin resistance in oral bacteria. Antimicrob Agents Chemother 2004;48:2298–301. 34. Villedieu A, Diaz-Torres ML, Hunt N, et al. Prevalence of tetracycline resistance genes in oral bacteria. Antimicrob Agents Chemother 2003;47:878–82. 35. Warburton P, Roberts AP, Allan E, et al. Characterization of tet(32) genes from the oral metagenome. Antimicrob Agents Chemother 2009;53:273–6. 36. Zhu X, Wang Q, Zhang C, et al. Prevalence, phenotype, and genotype of Enterococcus faecalis isolated from saliva and root canals in patients with persistent apical periodontitis. J Endod 2010;36:1950–5. 37. Nemoto H, Nomura R, Ooshima T, Nakano K. Distribution of amoxicillin-resistant oral streptococci in dental plaque specimens obtained from Japanese children and adolescents at risk for infective endocarditis. J Cardiol 2013;62:296–300.
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38. Nakayama A, Takao A. Beta-Lactam resistance in Streptococcus mitis isolated from saliva of healthy subjects. J Infect Chemother 2003;9:321–7. 39. Lancaster H, Bedi R, Wilson M, Mullany P. The maintenance in the oral cavity of children of tetracycline-resistant bacteria and the genes encoding such resistance. J Antimicrob Chemother 2005;56:524–31. 40. Iwahara K, Kuriyama T, Shimura S, 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;44:172–6. 41. Seville LA, Patterson AJ, Scott KP, et al. Distribution of tetracycline and erythromycin resistance genes among human oral and fecal metagenomic DNA. Microb Drug Resist 2009;15:159–66. 42. Jungermann GB, Burns K, Nandakumar R, et al. Antibiotic resistance in primary and persistent endodontic infections. J Endod 2011;37:1337–44. 43. R^ocas IN, Siqueira JF Jr. Antibiotic resistance genes in anaerobic bacteria isolated from primary dental root canal infections. Anaerobe 2012;18:576–80. 44. Lins RX, de Oliveira Andrade A, Hirata Junior R, et al. Antimicrobial resistance and virulence traits of Enterococcus faecalis from primary endodontic infections. J Dent 2013;41:779–86. 45. R^oc¸as IN, Siqueira JF Jr. Detection of antibiotic resistance genes in samples from acute and chronic endodontic infections and after treatment. Arch Oral Biol 2013;58:1123–8. 46. Montagner F, Jacinto RC, Signoretti FG, et al. Beta-lactamic resistance profiles in Porphyromonas, Prevotella and Parvimonas species isolated from acute endodontic infections. J Endod 2014;40:339–44. 47. Spangberg LS. Systematic reviews in endodontics—examples of GIGO? Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103:723–4. 48. Stark CA, Adamsson I, Edlund C, et al. Effects of omeprazole and amoxycillin on the human oral and gastrointestinal microflora in patients with Helicobacter pylori infection. J Antimicrob Chemother 1996;38:927–39. 49. Baglie S, Del Ruenis AP, Motta RH, et al. Plasma and salivary amoxicillin concentrations and effect against oral microorganisms. Int J Clin Pharmacol Ther 2007;45: 556–62. 50. Feres M, Haffajee AD, Allard K, et al. Antibiotic resistance of subgingival species during and after antibiotic therapy. J Clin Periodontol 2002;29:724–35. 51. R^oc¸as IN, Baumgartner JC, Xia T, Siqueira JF Jr. Prevalence of selected bacterial named species and uncultivated phylotypes in endodontic abscesses from two geographic locations. J Endod 2006;32:1135–8. 52. van Winkelhoff AJ, Herrera Gonzales D, Winkel EG, et al. Antimicrobial resistance in the subgingival microflora in patients with adult periodontitis. A comparison between The Netherlands and Spain. J Clin Periodontol 2000;27:79–86.
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Distribution of Genes Related to Antimicrobial Resistance in Different Oral Environments: A Systematic Review Ludmila Coutinho Moraes, DDS, MSc,* Marcus Vinıcius Reis S o, DDS, MSc, PhD,* Tatiane da Silva Dal Pizzol, PharmD, MSc, PhD,† Maria Beatriz Cardoso Ferreira, MD, MSc, PhD,‡ and Francisco Montagner, DDS, MSc, PhD* Abstract Introduction: The oral cavity is the main source of microorganisms for odontogenic infections. It is important to perform an extensive analysis regarding the reports on the presence of bacteria that carry resistance genes to antimicrobial agents. The aim of the study was to verify the reports on the distribution of genes associated with resistance to antibiotics prescribed in dentistry in different human oral sites. Methods: A systematic review was conducted in electronic databases and gray literature to analyze clinical studies that detected genes of bacterial resistance to antibiotics in saliva, supragingival biofilm, and endodontic infections. Data regarding the research group, geographic location, sample source, number of subjects, methods for sample analysis, the targeted gene groups, and the detection rates were collected. Descriptive data analysis was performed. Results: Preliminary analysis was performed in 152 titles; 50 abstracts were reviewed, and 29 full texts were obtained. Nine articles matched the inclusion criteria (saliva = 2, supragingival biofilm = 1, and endodontic infections = 6). The presence of 33 different targeted genes was evaluated. The most frequently investigated groups of genes were tetracycline and lactamics (tetM, tetQ, tetW, and cfxA). There was a wide range for the detection rates of each resistance gene among studies and for each specific gene group. Conclusions: This systematic review highlights the presence of resistance genes to antimicrobial agents in saliva, dental biofilm, and endodontic infections, especially for tetracycline and lactamics. There is a lack of reports on the presence of genes and resulting outcomes obtained through the therapeutic approaches for infection control. (J Endod 2015;-:1–8)
Key Words Dental plaque, dental pulp cavity, drug resistance, saliva
T
he human mouth harbors a wide number of microorganisms, comprising the commensal microbiota. It has an important role in maintaining oral and systemic health. The microbiota of the mouth is the source for microorganisms associated with dental caries, endodontic diseases, and periodontal diseases (1). Oral bacteria start colonizing the root canal system after pulp necrosis, remaining suspended in the root canal (planktonic state) or attached to its walls (biofilms) (2). They can reach the apical tissues causing extraradicular infections and constitute complex microbial communities associated with acute apical abscesses (3), extraradicular biofilms (4), and persistent infections (5). The composition of microbial communities associated with endodontic infections is heterogeneous and can be modulated by several factors such as the geographic location (6), the presence or absence of symptoms (7), and the clinical condition (8). Some studies also report distinct microbial profiles in paired samples from root canals and abscesses collected from the same patient (3). Few representatives of the domains Eukarya and Archea have been described; the domain Bacteria is the most predominant and diverse in endodontic infections (2). After a comprehensive review on the microbial communities associated with endodontic infections, Siqueira and R^oc¸as (2) emphasized the need for determining microbial functional roles in the community and the susceptibility to antimicrobial treatment procedures. Oral or parenteral antibiotics are generally considered adjunctive therapy for urgency endodontic treatment. Their value should not be underestimated, especially when drainage cannot be achieved or the infection shows signs of local extension or systemic involvement (9). Beta-lactam, tetracycline, and macrolide antibiotics have been prescribed in endodontics, especially for the treatment of acute apical abscesses associated with systemic involvement, spreading infections, abscesses in medically compromised patients who are at increased risk of a nonoral secondary infection after bacteremia, prophylaxis for medically compromised patients during routine endodontic therapy, and replantation of avulsed teeth (10). Local application of antimicrobial agents has been considered a possibility to improve root canal disinfection during the chemomechanical preparation (11, 12) or as an intracanal medicament (13). However, it should be emphasized that the use of antibiotics instead of biocides such as hypochlorite or chlorhexidine appears unwarranted, mainly because of the narrower spectrum of activity and resistance (14). Antibiotic resistance is a phenomenon of crucial importance in the treatment of diseases caused by pathogenic microorganisms (15). Bacterial resistance to antibiotics is multifactorial. Antibiotic resistance occurs by both intrinsic defenses (16) and genetic mutation in bacteria. Some mechanisms of bacterial resistance to antibiotics have been attributed to the resistance genes. Diverse resistance genes have been found
From the *Division of Endodontics, Department of Conservative Dentistry; †Post-Graduation Program in Epidemiology, Faculty of Medicine; and ‡Department of Pharmacology, Basic Health Science Institute, Federal University of Rio Grande do Sul, Porto Alegre, Brazil. Address requests for reprints to Dr Francisco Montagner, Universidade Federal do Rio Grande do Sul, Faculdade de Odontologia, Rua Ramiro Barcelos, 2492–Bairro Santana, Porto Alegre, RS, Brazil, 90035-003. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2014.12.018
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Review Article and characterized in human microflora, which can operate as a reservoir for antibiotic-resistant bacteria (17). The objective of this systematic review was to evaluate the distribution of genes of bacterial resistance to antibiotics in different sites of the oral cavity. A search strategy was formulated to answer a question in the PICO (Population, Intervention, Comparison, Outcome) format, defining inclusion and exclusion criteria. The question was framed as follows: In the oral sites saliva, supragingival biofilm, and endodontic infections, how are distributed genes associated with bacterial resistance to antibiotics frequently prescribed in dentistry?
Initial results for each database total: 152 articles PUBMED (135 articles), EMBASE (113 articles), SCOPUS (67 articles), ISI (36 articles), Grey literature (4 articles).
Title analysis of all selected articles = 50 abstracts
Abstract analysis of the selected articles = 29 full-text
Materials and Methods A systematic review was performed to check all clinical studies that detected genes of bacterial resistance to antibiotics in 3 sites of oral cavity: saliva, supragingival biofilm, and endodontic infections. The terms were used in various combinations in the electronic databases as follows: Medline (PubMed), Embase, Web of Science, Scopus, and OpenGrey. No language restriction was applied to the search. Figure 1 describes the search strategy adopted in the study. The search comprised articles published until January 14, 2014. The references reported on the selected articles were also reviewed. After title screening and abstract analysis, the full text of each study was obtained. The relevance of each study to the question of interest was determined through inclusion and exclusion criteria. The full texts of the articles were revised by 2 reviewers (F.M. and L.C.M) based on the following inclusion criteria: 1. Clinical studies in healthy patients or in patients with oral disease (cross-sectional or longitudinal studies) 2. Studies that detected bacterial resistance genes of antibiotics by molecular techniques 3. Studies with samples collected from oral cavity (saliva, supragingival biofilm, or root canal with primary endodontic infection) Exclusion criteria comprised the following: 1. 2. 3. 4.
Literature reviews In vitro studies Studies that analyzed mixed samples from different environments Studies that detected genes in other environments besides those cited in the inclusion criteria 5. Studies that did not use molecular methods for detection of the presence of the resistance genes 6. Studies in which the objective was not the detection of resistance genes 7. Lack of data Terms: ("mouth"[MeSH Terms] OR "mouth"[All Fields] OR ("oral"[All Fields] AND "cavity"[All Fields]) OR "oral cavity"[All Fields]) AND ("drug resistance, microbial"[MeSH Terms] OR ("drug"[All Fields] AND "resistance"[All Fields] AND "microbial"[All Fields]) OR "microbial drug resistance"[All Fields] OR ("antibiotic"[All Fields] AND "resistance"[All Fields]) OR "antibiotic resistance"[All Fields]) AND ("genes"[MeSH Terms] OR "genes"[All Fields] OR "gene"[All Fields])
Figure 1. The search strategy adopted for the study, presenting the MeSH keywords and search terms for the presence of resistance genes to antimicrobial agents in the oral environment.
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20 excluded (Table 1)
9 selected articles
Figure 2. Results after the search strategy developed to find studies related to the presence of resistance genes to antimicrobial agents in different oral environments.
Data regarding research group, geographic location, sample source, number of subjects included in the study, methods for sample size determination, statistical analysis, the targeted genes, and their detection rates were collected from the studies. Because of the methodologic differences among the studies, it was not possible to perform a meta-analysis. Descriptive data analysis was performed.
Results Considering all the databases, a total of 152 titles were identified after subtraction of duplicates for preliminary analysis. After title screening, 50 abstracts were revised, and full texts of 29 studies were obtained (Fig. 2). The 20 excluded articles are listed in Table 1, and the reasons for their exclusion are also shown. Nine articles matched the inclusion criteria regardless of the oral environment. There were 2 articles for saliva samples, 1 for supragingival biofilm, and 6 studies on primary endodontic infections. There was no age limit in the search, and only 1 of the selected articles reported data for children. Tables 2, 3, and 4 provide information about the selected studies. They were performed in different periods of time (from 2003–2014), and the samples were collected in Japan, the United States, Brazil, and European countries. The great majority of the authors reported ethics statements and/or conflicts of interest, except in 1 study (38). There was no description for the sample size determination methods adopted. The most frequently adopted exclusion criteria were previous exposure to antimicrobial agents, ranging from 1 to 6 months. The presence of resistance genes was assessed in clinical isolates (38–40, 43, 44, 46) and in samples from each environment (41, 42, 45). The presence of resistance genes in saliva samples was determined only from healthy subjects (38, 41). The only study that tested resistance genes in supragingival biofilm was conducted in children (39). Endodontic samples were collected from root canals (symptomatic and asymptomatic cases) and apical swelling. Descriptive statistics were reported in all studies. Inferential statistics were used to compare the prevalence of specific genes in the groups (primary vs persistent infections, preoperative vs preobturation samples, before vs after root canal preparation, and abscess vs asymptomatic apical periodontitis). JOE — Volume -, Number -, - 2015
Review Article TABLE 1. Characteristics of the Studies Excluded from the Systematic Review Authors/year
Country
Title
Andres et al, 1998 (18)
Spain
Arseze et al, 2000 (19)
Italy
Antimicrobial susceptibilities of Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens spp. isolated in Spain Detection of tetQ and ermF antibiotic resistance genes in Prevotella and Porphyromonas isolates from clinical specimens and resident microbiota of humans Distribution of the tetracycline resistance determinant tetQ gene in oral isolates of blackpigmented anaerobes in Japan Presence and antibiotic resistance of Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens in children Antibiotic resistance in oral commensal streptococci from healthy Mexicans and Cubans: resistance prevalence does not mirror antibiotic usage The mef(A) gene predominates among seven macrolide resistance genes identified in gramnegative strains representing 13 genera, isolated from healthy Portuguese children Determining the antibiotic resistance potential of the indigenous oral microbiota of humans using a metagenomic approach Antimicrobial resistance in clinical isolates of Neisseria subflava from the oral cavities of a Japanese population Genetic determinants for cfxA expression in Bacteroides strains isolated from human infections Prevalence of tetM, tetQ, nim and bla(TEM) genes in the oral cavities of Greek subjects: a pilot study Characterization of antibiotic resistance determinants in oral biofilm Molecular investigation of macrolide and tetracycline resistances in oral bacteria isolated from Tunisian children Analysis of genetic lineages and their correlation with virulence genes in Enterococcus faecalis clinical isolates from root canal and systemic infections Antimicrobial susceptibility and molecular analysis of Enterococcus faecalis originating from endodontic infections in Finland and Lithuania Genetic analysis of mobile tetQ elements in oral Prevotella species Genetic basis of erythromycin resistance in oral bacteria Prevalence of tetracycline resistance genes in oral bacteria Characterization of tet32 genes from the oral metagenome Prevalence, phenotype, and genotype of Enterococcus faecalis isolated from saliva and root canals in patients with persistent apical periodontitis Distribution of amoxicillin-resistant oral streptococci in dental plaque specimens obtained from Japanese children and adolescents at risk for infective endocarditis
Okamoto et al, 2001 (20) Sanai et al, 2002 (21) Dıaz-Mejıa et al, 2002 (22) Ojo et al, 2004 (23)
Japan Portugal Mexico Portugal
Diaz-Torres et al, 2006 (24)
UK
Furuya et al, 2007 (25)
Japan
Garcia et al, 2008 (26)
Spain
Ioannidis et al, 2009 (27)
Greece
Kim et al, 2011 (28)
US
Kouidhi et al, 2011 (29)
Tunisia
Penas et al, 2013 (30)
Brazil
Reynaud af Geijersstam et al, 2006 (31)
Sweden
Tribble et al, 2010 (32)
US
Villedieu et al, 2004 (33)
UK
Villedieu et al, 2003 (34)
UK
Warburton et al, 2009 (35)
UK
Zhu et al, 2010 (36)
China
Nemoto et al, 2013 (37)
Japan
Oral site Dental plaque
Exclusion criteria* 3, 4
Oral cavity
3
Subgingival biofilm
4
Dental plaque and buccal mucosa
3
Oral samples
6, 7
Oral and urine samples
7
Saliva and dental plaque
3
Oral cavity samples
5
Humans infections
3, 4
Dental plaque and tongue samples Dental plaque Dental caries and plaque
4 4 3, 4
Root canal
4
Root canal
4
Oral cavity samples
7
Saliva and dental plaque
3
Saliva and dental plaque
3
Oral cavity samples
5
Saliva and root canal
Supra- and subgingival plaque
4, 5
3
*Exclusion criteria were as follows: 1, literature reviews; 2, in vitro studies; 3, studies that analyzed mixed samples from different environments; 4, studies that detected genes in other environments besides those cited in the inclusion criteria; 5, studies that did not use molecular methods for detection of the presence of the resistance genes; 6, studies in which the objective was not the detection of resistance genes; 7, lack of data.
A total of 33 different genes have been evaluated in the 9 articles as follows: 1. Beta-lactam: blaTEM, blaZ, cfxA, cfxA2, blaCMY2, ampC, mecA (methicillin), pbp1A, pbp2X, and pbp2B 2. Macrolide: ermA, ermB, ermC, ermE, and ermV JOE — Volume -, Number -, - 2015
3. Tetracycline: tetA, tetB, tetC, tetD, tetK, tetL, tetM, tetO, tetQ, tetS, tetW, and tet32 4. Vancomycin: vanA, vanB, vanC1, vanC2/3, vanD, and vanE The genes for tetracycline resistance were the most frequently investigated in the different publications. The most tested genes were
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Review Article TABLE 2. Characteristics of the Studies Included in the Systematic Review Description of the recruitment
Authors/Year
Assessment
Participants/sample size
Nakayama and Takao, 2003 (38)
Detection of S. mitis resistant to ampicillin, cefaclor and cefotaxime Maintenance of resistant bacteria and genes that encode tetracycline resistance in children Detection of beta-lactamase resistance genes in Prevotella strains Detection of genes for resistance to tetracycline and erythromycin in oral metagenomas Detection of resistance genes in bacteria isolated from primary and persistent endodontic infections Detection of 14 genes encoding resistance to beta-lactams, tetracycline and macrolides
Healthy subjects (age: 20–30 years) N = 52 Healthy subjects (age: 4–6 years) N = 26 Patients with dentoalveolar infection (mean age: 48.1 years) N = 87 Healthy subjects (age: 21–65 years) N = 6†
Lancaster et al, 2005 (39) Iwahara et al, 2006 (40) Seville et al, 2009 (41) Jungermann et al, 2011 (42) ^ c¸as and Ro Siqueira Jr, 2012 (43) Lins et al, 2013 (44) ^ c¸as and Ro Siqueira Jr, 2013 (45) Montagner et al, 2014 (46)
Detection of resistant E. faecalis and virulence trait in primary endodontic infections Detection of genes encoding resistance to beta-lactams, tetracycline, and erythromycin in bacteria from acute or chronic endodontic infections Detection of the cfxA/cfxA2 gene and analysis of its expression in endodontic infections
Antibiotic exposure*
No
Previous 6 months
Yesjj
Previous 3 months
September 2001 and March 2005 No
Not mentioned Previous 3 months
Patients with endodontic infections (age: 19–94 years; mean: 51 years) N = 50‡ Patients with apical periodontitis (age: not specified) N = 26 Patients with primary endodontic infection (age: not specified) N = 43 Patients with asymptomatic apical periodontitis or acute apical abscess (age: 19–64 years) N = 50
No§
Previous 1 month
No§
Previous 6 months
No
Previous 6 months
No
Previous 3 months
Patients with spontaneous pain and pulp necrosis (age: nonspecified) N = 26
No
Previous 3 months
*Antibiotic exposure was considered exclusion criteria in these studies. † In this study, samples from 6 countries were analyzed, and each sample was constituted by the pool of saliva from 20 individuals/country. ‡ The final sample consisted of 45 patients with primary (n = 30) or persistent (n = 15) endodontic infections; 5 were excluded during the study. § Perhaps all patients who had endodontic infections in a nonspecified period of time. jj A total of 18 of 26 children were included in the study because they had not taken any antibiotics 3 months before sampling; 8 of 18 children had amoxicillin, ampicillin, cefalexin (cephalosporin), and ciprofloxacin in the previous 3 months; and none of them had taken tetracycline or erythromycin.
tetM for tetracycline resistance (saliva = 1 study [41], supragingival biofilm = 1 [39], endodontic infections = 4 studies [42–45]) followed by the genes tetQ/tetW (saliva = 1 study [41], supragingival biofilm = 1 [39], endodontic infections = 3 studies [42, 43, 45]). The frequency for the detection of the tetM gene ranged from 5%–100% (5%–60% in 4 root canal samples, 77.7% in the supragingival biofilms, and 100% in 1 saliva sample study). The tetracycline resistance genes tetQ and tetW were detected in 0%–10% and 20%–29% of the root canal samples, respectively; 5% of the supragingival samples had tetQ and tetW. One study (41) that evaluated the tetQ and tetW genes in saliva did not provide measurable data regarding its frequency in this specific environment. The presence of genes associated with the resistance of betalactam agents was assessed in the oral environment. The gene cfxA/cfxA2 was searched in 5 studies (40, 42, 43, 45, 46), and its detection rate ranged from 0%–40% in root canal samples. The gene blaTEM was also detected in root canals by 3 articles (42, 43, 45), with frequencies ranging from 17%–43% of the samples. The genes pbp1a, pbp2x, and pbp2b were observed in saliva samples (38). Macrolide resistance genes were detected in root canals in 3 articles (42, 43, 45), and in 1 article that evaluated saliva samples (41). The ermC gene was detected in 10%–24% of endodontic infections. Both ermA and ermB genes were not found in root canal samples. However, the ermB gene was found in saliva with high frequency. Vancomy4
Moraes et al.
cin resistance genes were searched in root canal samples, but no positive results were observed (42, 44).
Discussion The methods used in current studies to depict the structure and virulence factors associated with microbial communities are labor intensive and expensive. Therefore, these studies have evaluated a small number of samples obtained from a restricted population source. Systematic reviews have been suggested as a tool for a synthesis and critical analysis of the literature. They can be associated with statistical methods such as meta-analysis to obtain compiled results of studies with small sample sizes, serving as a guide for evidencebased practice (47). Thus, conducting systematic reviews may provide reliable results that can be used for clinical decision making, especially when it is possible to perform a meta-analysis. This systematic review aimed to identify, evaluate, and synthesize all clinical studies that met the prespecified eligibility criteria to answer the following research question: ‘‘How are genes of bacterial resistance to antibiotics frequently prescribed in dentistry distributed in different oral environments (saliva, supragingival biofilm, and endodontic infections)?’’ In the present review, broad topics were used in the electronic databases to reduce the possibility of overlooking studies. Four databases and gray literature were used followed by a careful hand search of JOE — Volume -, Number -, - 2015
Review Article TABLE 3. Other Characteristics of the Included Studies Authors/Year
Targeted species or bacteria
Sample source
Assessment of outcome
Nakayama and Isolates of S. mitis Takao, 2003 (38)
Saliva
PCR
Lancaster et al, 2005 (39)
Isolates from dental plaque
Dental plaque
PCR
Iwahara et al, 2006 (40)
Prevotella spp
AAA (n = 72) PCR and RC (n = 15)
Seville et al, 2009 (41)
Analysis of oral DNA metagenomes
Saliva
PCR
Jungermann et al, Bacteria and samples 2011 (42) isolated from primary and persistent endodontic infections
RC
PCR
^ c¸as and Siqueira Bacteria isolated from Ro Jr, 2012 (43) endodontic infections Lins et al, Enterococcus faecalis 2013 (44)
RC
PCR
RC
PCR
^ c¸as and Siqueira Samples from acute Ro Jr, 2013 (45) apical abscess or asymptomatic apical periodontitis
AAA (n = 25) RC (n = 25)
PCR
Montagner et al, 2014 (46)
RC (N = 26)
PCR
Bacteria isolated from acute endodontic infections
Method for resistance characterization
Comparability/ statistical analysis
Detection of bacteria Descriptive statistical with penicillin-binding analysis protein genes No comparisons between individuals with or without resistant S. mitis Detection of genes for Descriptive statistical resistance to analysis tetracycline Detection of bacteria Descriptive statistical with beta-lactamase analysis resistance genes No comparison between individuals with or without resistance genes Description of the presence of cfxA/cfxA2 in resistant-bacteria or susceptible-bacteria. Detection of genes for Descriptive statistical resistance to analysis. tetracycline and erythromycin Detection of common Descriptive and antibiotic resistance inferential statistical genes and analysis; association susceptibility to analysis (P description specific antibiotics without confidence interval); comparison of prevalence of different antibiotic resistance genes: in primary versus persistent infections; in preoperative and preobturation specimens Detection of resistance Descriptive statistical genes analysis Detection of resistant Descriptive statistical bacteria to analysis tetracycline, erythromycin, and vancomycin, and virulence trait Detection of 6 resistance Descriptive and genes to beta-lactams, inferential statistical tetracycline, and analysis; association erythromycin analysis (P description, without CI) Comparison of prevalence of the target resistance genes: in acute abscess versus asymptomatic apical periodontitis; before and after RC preparation Detection of the cfxA/ Descriptive statistical cfxA2 gene analysis
AAA, acute apical abscess; CI, confidence interval; PCR, polymerase chain reaction; RC, root canal.
references in the 29 selected articles to avoid the risk of ignoring studies. Nine articles matched the inclusion criteria: 2 articles for saliva samples, 1 for supragingival biofilm, and 6 studies on primary endodontic infections. The aim of this systematic review was to synthesize data from clinical studies with similar objectives and methodology for JOE — Volume -, Number -, - 2015
the detection of resistance genes in 3 specific oral environments. Mixed samples from different oral environments were not considered. Therefore, the present review aimed to obtain a homogeneous group of data that will provide a strict panel on the presence of resistance genes in saliva, supragingival plaque, and infected root canals. Several studies
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Review Article TABLE 4. Results of the Included Studies Authors/Year
Resistance profiles
Clinical information
Nakayama and Takao, 2003 (38) Lancaster et al, 2005 (39)
All the resistant streptococci had the PBP genes
Not applied
The most prevalent gene encoding tetracycline resistance was tet(M). tet(B), tet(K), tet(O), tet(Q), tet(S), tet(W) were also found.
Iwahara et al, 2006 (40)
Presence of cfxA and cfxA2: all beta-lactamase– positive Prevotella strains; none of the betalactamase-negative Prevotella isolates Most prevalent tetracycline resistance gene: tet(M) Most abundant erythromycin resistance gene: erm(B) Common genes: tet(Q), tet(O), tet(O/32/O) tet(W) Preoperative specimens: significantly more prevalent blaTEM-1 gene in primary versus persistent infections; cfxA only present in primary infections Preobturation specimens: significant reduction of blaTEM-1 and tet(W); elimination of cfxA, blaZ, and tet(Q); no alteration of tet(M) Absence of genes vanA, vanD, and vanE (associated with vancomicin resistance) Presence of at least 1 of the target antibiotic resistance genes: 32% isolates, corresponding to 42% of the infected root canals analyzed Most prevalent genes (of the 6 antibiotic resistance genes detected): blaTEM (17% strains), tetW (10%), and ermC (10%) Presence of tetM and tetL: 60% and 20%, respectively No detection of ermA, ermB, vanA, vanB, vanC1, and vanC2/3 Initial analysis in AAA samples: 100% samples with bacteria; absence of cfxA and tetQ; 36% positive for at least 1 of 4 remained antibiotic resistance genes; blaTEM (24%), ermC (24%), tetW (12%), tetM (8%) Initial analysis in asymptomatic apical periodontitis: 100% samples with bacteria; absence of blaTEM, cfxA, and tetQ; 67% positive for at least 1 of the other antibiotic resistance genes; tetM (42%), tetW (29%), ermC (25%) blaTEM: significantly more found in acute abscess than in asymptomatic cases tetM: significantly more found in asymptomatic cases than in acute abscess cfxA/cfxA2 gene: detection in 6.9% of isolates Simultaneous detection of cfxA/cfxA2 gene and lactamase production: 1 P. buccalis strain Detection of cfxA/cfxA2 gene with no expression: 1 P. micra strain Positive lactamase production, with no detection of cfxA/cfxA2 gene: 3 strains
The results of this study have shown that tetracycline-resistant bacteria and tet(M) are maintained within the indigenous oral microbiota of children, even though they are unlikely to have been directly exposed to tetracycline No description of treatment No description of the clinical evolution of the infection Not applied
Seville et al, 2009 (41)
Jungermann et al, 2011 (42)
^ c¸as and Siqueira Jr, 2012 (43) Ro
Lins et al, 2013 (44)
^ c¸as and Siqueira Jr, 2013 (45) Ro
Montagner et al, 2014 (46)
Evaluation of the profile of antibiotic resistance genes after chemomechanical procedures No description of the clinical evolution
No description of treatment or the clinical evolution of the infection
No description of treatment or the clinical evolution of the infection Analysis of the elimination of bacteria carrying antibiotic resistance genes after chemomechanical procedures No description of the clinical evolution
No description of treatment or the clinical evolution of the infection
AAA, acute apical abscess.
were excluded because they did not provide full data in the results description. No personal correspondence was sent to the authors to obtain more detailed data. Few data are available on the presence of resistance genes in the selected oral sites. Only 1 study reported the presence of tetracycline genes in bacteria isolated from supragingival biofilm in children (39). Of the 6 selected articles that evaluated the presence of resistance genes in endodontic infections, 2 examined directly pooled samples of 6
Moraes et al.
acute endodontic infections (42, 45), and 4 tested bacterial strains isolates from those infections (40, 43, 44, 46). The critical evaluation of the selected studies showed few factors connecting them. It was especially noticed the diversity of patient characteristics. The sample size was variable, ranging from 26 to 87. No sample size determination methods were described. There are different outcomes that can limit the definition of the parameter to be the basis of the sample size determination. The presence of strict inclusion criteria JOE — Volume -, Number -, - 2015
Review Article and the labor-intensive characteristics for root canal sampling may restrict the number of subjects who were enrolled in the studies. It may also require a long period of time to obtain all the volunteers who would join the study. Furthermore, it should be considered that root canal sampling is labor intensive, and strict procedures should be used to avoid external contaminants. Therefore, there are intrinsic limitations such as the complexity of the sampling process and the wide range of variables associated with the patient that may introduce bias to the results. During the literature search, it was observed that few data about the inclusion criteria of the patients were reported. Both healthy and diseased patients with endodontic infections were included in the samples, and saliva samples were collected. Supragingival biofilm samples were collected from healthy children (39). However, details on their recruitment, sex, and clinical conditions were not described. Prior exposure to antibiotics was used as an exclusion criterion. There was no consensus on the literature regarding the previous period that the patient should not take the medication (ranging from 1– 6 months). Stark et al (48) reported that a previous intake of amoxicillin can change the microbial composition inside the oral cavity, with a slight increase in the number of resistant strains. Baglie et al (49) reported a decrease in colony-forming units counting 60 minutes after oral amoxicillin intake. In a study by Feres et al (50), the consumption of amoxicillin and metronidazole for 14 days determined a transient modification in the percentage of resistant strains in subgingival biofilm although a large proportion remained susceptible during this period. The prevalence of bacteria resistant to amoxicillin only returned to the baseline levels in 90 days. These data suggest that short periods of suspension of antibiotic treatment (ie, 1 or 2 months) may not be sufficient for the microbiota to return to baseline levels of susceptibility. Thus, the information obtained may still reflect the influence of prior use of the drug. Another factor that must be considered is the geographic location. It can interfere in the composition of the microbial communities associated with endodontic infections (51). Seville et al (41) compared the presence of resistance genes in different European countries without limiting the evaluation to a specific geographic area. Nakayama and Takao (38) investigated the percentage of betalactam resistance genes in isolates of Streptococcus spp. Jungermann et al (42) also reported the presence of resistance genes in samples collected after chemomechanical preparation; however, only the results obtained before the treatment were considered in the present study. Regarding the selected articles that evaluated root canal samples, 4 were conducted in Brazil, 1 in Japan, and 1 in the United States. It is known that the profile of resistance depends on the geographic region because it is related to the pattern of antibiotic prescription by professionals and the profile of use by the patients (52). Therefore, studies performed in different countries are affected by the different pattern of use of antibiotics. According to the present literature review, the genes blaTEM, cfxA, tetW, tetM, and ermC were frequently detected in oral samples. Although the detection of resistance genes for specific antibiotics does not necessarily mean antibiotic resistance of the specimens, it indicates the potential for the expression of the encoded protein. Montagner et al (46) detected the cfxA/cfxA2 gene in 2 of 26 isolates from acute endodontic infections, but only 1 strain had expressed this gene as determined through phenotypic methods. Furthermore, it is also important to evaluate the clinical response of patients who harbor antibiotic resistance genes. Some studies (40, 42, 46) evaluated the bacterial susceptibility to antibiotics and the presence of resistance genes in samples collected from patients with dentoalveolar infections. However, no one described the clinical outcome after the JOE — Volume -, Number -, - 2015
treatment or determined the relation between the presence of resistance genes and the clinical evolution of patients. Thus, this systematic review highlights the presence of resistance genes to antimicrobial agents in the oral cavity, especially for tetracycline and beta-lactam agents in saliva, supragingival biofilm, and acute endodontic infections. It also showed the lack of reports about the presence of these genes and resulting clinical outcomes, considering the therapeutic approaches used for infection control.
Acknowledgments Supported by a grant from the Fundac¸~ao de Apoio a Pesquisa do Estado do Rio Grande do Sul (ARD-FAPERGS 140112-0) to Dr Montagner. The authors deny any conflicts of interest related to this study.
References 1. Wade WG. The oral microbiome in health and disease. Pharmacol Res 2013;69: 137–43. 2. Siqueira JF Jr, R^oc¸as IN. Community as the unit of pathogenicity: an emerging concept as to the microbial pathogenesis of apical periodontitis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;107:870–8. 3. Montagner F, Gomes BP, Kumar PS. Molecular fingerprinting reveals the presence of unique communities associated with paired samples of root canals and acute apical abscesses. J Endod 2010;36:1475–9. 4. Subramanian K, Mickel AK. Molecular analysis of persistent periradicular lesions and root ends reveals a diverse microbial profile. J Endod 2009;35:950–7. 5. Signoretti FG, Gomes BP, Montagner F, Jacinto RC. Investigation of cultivable bacteria isolated from longstanding retreatment-resistant lesions of teeth with apical periodontitis. J Endod 2013;39:1240–4. 6. Baumgartner JC, Siqueira JF Jr, Xia T, R^oc¸as IN. Geographical differences in bacteria detected in endodontic infections using polymerase chain reaction. J Endod 2004; 30:141–4. 7. Santos AL, Siqueira JF Jr, R^oc¸as IN, et al. Comparing the bacterial diversity of acute and chronic dental root canal infections. PLoS One 2011;6:e28088. 8. Gomes BP, Pinheiro ET, Gad^e-Neto CR, et al. Microbiological examination of infected dental root canals. Oral Microbiol Immunol 2004;19:71–6. 9. Poeschl PW, Crepaz V, Russmueller G, et al. Endodontic pathogens causing deep neck space infections: clinical impact of different sampling techniques and antibiotic susceptibility. J Endod 2011;37:1201–5. 10. Siqueira JF Jr. Systemic antibiotics in endodontics. In: Siqueira JF Jr, ed. Treatment of Endodontic Infections. London: Quintessence Publishing; 2011:381–96. 11. Tong Z, Ling J, Lin Z, et al. The effect of MTADN on 10 Enterococcus faecalis isolates and biofilm: an in vitro study. J Endod 2013;39:674–8. 12. Torabinejad M, Shabahang S, Aprecio RM, Kettering JD. The antimicrobial effect of MTAD: an in vitro investigation. J Endod 2003;29:400–3. 13. Diogenes AR, Ruparel NB, Teixeira FB, Hargreaves KM. Translational science in disinfection for regenerative endodontics. J Endod 2014;40:S52–7. 14. Zehnder M. Root canal irrigants. J Endod 2006;32:389–98. 15. Vester B, Long KS. Antibiotic resistance in bacteria caused by modified nucleosides in 23S ribosomal RNA. In: Grosjean H, ed. DNA and RNA Modification Enzymes: Structure, Mechanism, Function and Evolution. Austin: Landes Bioscience; 2009. 16. Wise R, Hart T, Cars O, et al. Antimicrobial resistance is a major threat to public health. Br Med J 1998;317:609–10. 17. Sommer MO, Church GM, Dantas G. The human microbiome harbors a diverse reservoir of antibiotic resistance genes. Virulence 2010;1:299–303. 18. Andres MT, Chung WO, Roberts MC, Fierro JF. Antimicrobial susceptibilities of Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens spp. isolated in Spain. Antimicrob Agents Chemother 1998;42:3022–3. 19. Arzese AR, Tomasetig L, Botta GA. Detection of tetQ and ermF antibiotic resistance genes in Prevotella and Porphyromonas isolates from clinical specimens and resident microbiota of humans. J Antimicrob Chemother 2000;45:577–82. 20. Okamoto M, Takano K, Maeda N. Distribution of the tetracycline resistance determinant tetQ gene in oral isolates of black-pigmented anaerobes in Japan. Oral Microbiol Immunol 2001;16:224–8. 21. Sanai Y, Persson GR, Starr JR, et al. Presence and antibiotic resistance of Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens in children. J Clin Periodontol 2002;29:929–34. 22. Dıaz-Mejıa JJ, Carbajal-Saucedo A, Amabile-Cuevas CF. Antibiotic resistance in oral commensal streptococci from healthy Mexicans and Cubans: resistance prevalence does not mirror antibiotic usage. FEMS Microbiol Lett 2002;217:173–6.
Genes Related to Antimicrobial Resistance
7
Review Article 23. Ojo KK, Ulep C, Van Kirk N, et al. The mef(A) gene predominates among seven macrolide resistance genes identified in gram-negative strains representing 13 genera, isolated from healthy Portuguese children. Antimicrob Agents Chemother 2004;48:3451–6. 24. Diaz-Torres ML, Villedieu A, Hunt N, et al. Determining the antibiotic resistance potential of the indigenous oral microbiota of humans using a metagenomic approach. FEMS Microbiol Lett 2006;258:257–62. 25. Furuya R, Onoye Y, Kanayama A, et al. Antimicrobial resistance in clinical isolates of Neisseria subflava from the oral cavities of a Japanese population. J Infect Chemother 2007;13:302–4. 26. Garcıa N, Gutierrez G, Lorenzo M, et al. Genetic determinants for cfxA expression in Bacteroides strains isolated from human infections. J Antimicrob Chemother 2008; 62:942–7. 27. Ioannidis I, Sakellari D, Spala A, et al. Prevalence of tetM, tetQ, nim and bla(TEM) genes in the oral cavities of Greek subjects: a pilot study. J Clin Periodontol 2009;36: 569–74. 28. Kim SM, Kim HC, Lee SW. Characterization of antibiotic resistance determinants in oral biofilms. J Microbiol 2011;49:595–602. 29. Kouidhi B, Zmantar T, Hentati H, et al. Molecular investigation of macrolide and tetracycline resistances in oral bacteria isolated from Tunisian children. Arch Oral Biol 2011;56:127–35. 30. Penas PP, Mayer MP, Gomes BP, et al. Analysis of genetic lineages and their correlation with virulence genes in Enterococcus faecalis clinical isolates from root canal and systemic infections. J Endod 2013;39:858–64. 31. Reynaud af Geijersstam AH, Ellington MJ, Warner M, et al. Antimicrobial susceptibility and molecular analysis of Enterococcus faecalis originating from endodontic infections in Finland and Lithuania. Oral Microbiol Immunol 2006;21:164–8. 32. Tribble GD, Garza JJ, Yeung VL, et al. Genetic analysis of mobile tetQ elements in oral Prevotella species. Anaerobe 2010;16:604–9. 33. Villedieu A, Diaz-Torres ML, Roberts AP, et al. Genetic basis of erythromycin resistance in oral bacteria. Antimicrob Agents Chemother 2004;48:2298–301. 34. Villedieu A, Diaz-Torres ML, Hunt N, et al. Prevalence of tetracycline resistance genes in oral bacteria. Antimicrob Agents Chemother 2003;47:878–82. 35. Warburton P, Roberts AP, Allan E, et al. Characterization of tet(32) genes from the oral metagenome. Antimicrob Agents Chemother 2009;53:273–6. 36. Zhu X, Wang Q, Zhang C, et al. Prevalence, phenotype, and genotype of Enterococcus faecalis isolated from saliva and root canals in patients with persistent apical periodontitis. J Endod 2010;36:1950–5. 37. Nemoto H, Nomura R, Ooshima T, Nakano K. Distribution of amoxicillin-resistant oral streptococci in dental plaque specimens obtained from Japanese children and adolescents at risk for infective endocarditis. J Cardiol 2013;62:296–300.
8
Moraes et al.
38. Nakayama A, Takao A. Beta-Lactam resistance in Streptococcus mitis isolated from saliva of healthy subjects. J Infect Chemother 2003;9:321–7. 39. Lancaster H, Bedi R, Wilson M, Mullany P. The maintenance in the oral cavity of children of tetracycline-resistant bacteria and the genes encoding such resistance. J Antimicrob Chemother 2005;56:524–31. 40. Iwahara K, Kuriyama T, Shimura S, 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;44:172–6. 41. Seville LA, Patterson AJ, Scott KP, et al. Distribution of tetracycline and erythromycin resistance genes among human oral and fecal metagenomic DNA. Microb Drug Resist 2009;15:159–66. 42. Jungermann GB, Burns K, Nandakumar R, et al. Antibiotic resistance in primary and persistent endodontic infections. J Endod 2011;37:1337–44. 43. R^ocas IN, Siqueira JF Jr. Antibiotic resistance genes in anaerobic bacteria isolated from primary dental root canal infections. Anaerobe 2012;18:576–80. 44. Lins RX, de Oliveira Andrade A, Hirata Junior R, et al. Antimicrobial resistance and virulence traits of Enterococcus faecalis from primary endodontic infections. J Dent 2013;41:779–86. 45. R^oc¸as IN, Siqueira JF Jr. Detection of antibiotic resistance genes in samples from acute and chronic endodontic infections and after treatment. Arch Oral Biol 2013;58:1123–8. 46. Montagner F, Jacinto RC, Signoretti FG, et al. Beta-lactamic resistance profiles in Porphyromonas, Prevotella and Parvimonas species isolated from acute endodontic infections. J Endod 2014;40:339–44. 47. Spangberg LS. Systematic reviews in endodontics—examples of GIGO? Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103:723–4. 48. Stark CA, Adamsson I, Edlund C, et al. Effects of omeprazole and amoxycillin on the human oral and gastrointestinal microflora in patients with Helicobacter pylori infection. J Antimicrob Chemother 1996;38:927–39. 49. Baglie S, Del Ruenis AP, Motta RH, et al. Plasma and salivary amoxicillin concentrations and effect against oral microorganisms. Int J Clin Pharmacol Ther 2007;45: 556–62. 50. Feres M, Haffajee AD, Allard K, et al. Antibiotic resistance of subgingival species during and after antibiotic therapy. J Clin Periodontol 2002;29:724–35. 51. R^oc¸as IN, Baumgartner JC, Xia T, Siqueira JF Jr. Prevalence of selected bacterial named species and uncultivated phylotypes in endodontic abscesses from two geographic locations. J Endod 2006;32:1135–8. 52. van Winkelhoff AJ, Herrera Gonzales D, Winkel EG, et al. Antimicrobial resistance in the subgingival microflora in patients with adult periodontitis. A comparison between The Netherlands and Spain. J Clin Periodontol 2000;27:79–86.
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