JOURNAL OF ORAL MICROBIOLOGY 2020, VOL. 13, 1847431 https://doi.org/10.1080/20002297.2020.1847431 Tetracycline and multidrug resistance in the oral microbiota: differences between healthy subjects and patients with periodontitis in Spain a,b a Alexandre Arredondo , Vanessa Blanc , Carolina Morc, José Nartc and Rubén León a a Department of Microbiology, Dentaid Research Center, Cerdanyola Del Vallès, Spain; bDepartament De Genètica I Microbiologia, Universitat Autònoma De Barcelona, Bellaterra, Spain; cDepartment of Periodontology, Universitat Internacional De Catalunya, Barcelona, Spain ABSTRACT ARTICLE HISTORY Introduction: Antibiotic resistance is widely found even among bacterial populations not Received 24 August 2020 having been exposed to selective pressure by antibiotics, such as tetracycline. In this study Revised 30 October 2020 we analyzed the tetracycline-resistant subgingival microbiota of healthy subjects and of Accepted 4 November 2020 patients with periodontitis, comparing the prevalence of tet genes and their multidrug KEYWORDS resistance profiles. Antibiotic resistance; Methods: Samples from 259 volunteers were analyzed, obtaining 813 tetracycline-resistant tetracycline; periodontitis; isolates. The prevalence of 12 antibiotic resistance genes was assessed, and multidrug profiles subgingival microbiota; were built. Each isolate was identified by 16S rRNA sequencing. Differences in qualitative data multidrug resistance and quantitative data were evaluated using the chi-square test and the Mann-Whitney-U test, respectively. Results: tet(M) was the most frequently detected tet gene (52.03%). We observed significant differences between the prevalence of tet(M), tet(W), tet(O), tet(32) and tet(L) in both popula­ tions studied. Multidrug resistance was largely observed, with resistance to kanamycin being the most detected (83.64%). There were significant differences between the populations in the prevalence of kanamycin, chloramphenicol, and cefotaxime resistance. Resistant isolates showed significantly different prevalence between the two studied groups. Conclusion: The high prevalence of multidrug resistance and tetracycline resistance genes found in the subgingival microbiota, highlights the importance of performing wider and more in-depth analysis of antibiotic resistance in the oral microbiota. Introduction antimicrobials in question. Such is the case of the oral Antibiotics have been used in clinical practice since environment, in which bacteria carrying tetracycline their discovery, saving countless lives. Tetracycline resistance genes have been previously detected [6,7]. was one of the first antibiotics to be discovered, in the Antibiotics have been used as adjuvants in certain den­ 1940s, and its broad-spectrum activity and relatively tal treatments such as the treatment of periodontitis, few side effects made it a widely used antibiotic [1]. Its which has been reported to benefit from the action of appealing properties led to extensive use, exerting antimicrobials [8,9]. Currently, the main antibiotics a great deal of selective pressure on bacteria, which used in periodontal treatment are amoxicillin and did not take long to become resistant [2]. Despite the metronidazole [10], however, tetracyclines have been rise of resistance, tetracyclines were and still are used in widely used in the past and there are reports of tetra­ the treatment of some human infections [3], and espe­ cycline resistance in the oral microbiota [7,11]. cially in the cattle industry as growth promoters due to Resistance to tetracycline can be expressed their anti-inflammatory effects on the gastrointestinal through efflux pumps, ribosomal protection proteins tract of the animals [4]. It has been reported that the use (RPPs) and inactivation enzymes. Tetracycline resis­ of antimicrobials in the cattle industry has an impact on tance genes code for these mechanisms and are wide­ the microbiota that lives in soils or sediments where all spread among oral bacteria, where tet(M) is the most the wastes of such industry are left, increasing the pre­ common [1]. Other genes coding for RPPs have been valence of antimicrobial resistance genes in bacterial described in the oral environment, such as tet(Q), tet populations that had previously had little or no contact (O), tet(S) and tet(W), and genes that code for efflux with antibiotics [5]. This abuse and misuse of antimi­ pumps such as tet(B), tet(32), tet(K) and tet(L) can crobials may have repercussions on human health, also be found within the oral microbiota [1]. Several increasing people’s chances of carrying antimicrobial- genes, including tet(37) and tet(X), have been resistant bacteria without having been exposed to the described as genes coding for inactivating enzymes, CONTACT Rubén León leon@dentaid.es Department of Microbiology, Dentaid Research Center, Cerdanyola Del Vallès, Spain Supplemental data for this article can be accessed here. © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2 A. ARREDONDO ET AL. but so far only tet(37) has been found in the oral subjects had to have at least six teeth per quadrant, environment [12,13]. probing depths ≤ 3 mm and absence of moderate or Furthermore, the strong presence of Tn916/1545 severe gingivitis. Smokers of more than five cigarettes family transposons in the oral microbiota, which can per day, wearers of orthodontic appliances and preg­ carry tetracycline, macrolide and/or aminoglycoside nant or breastfeeding women were not included in resistance genes, among others [14], means that those the study. None of the volunteers took antibiotics or tetracycline-resistant microorganisms may also be nonsteroidal anti-inflammatory drugs at least three resistant to other antimicrobials. Multidrug resistance months prior to the sampling, presented any systemic is an increasingly troubling issue for the health autho­ disease or took any chronic medication. rities [15], and although multiple efforts are being Subgingival samples were taken by placing two made to provide more information, little is known sterile paper points in the deepest site of each quad­ regarding multidrug resistance in the oral rant for 20 seconds and stored in 2 ml of reduced environment. transport fluid without ethylenediaminetetraacetic It has been widely described that the subgingival acid (EDTA) [22]. Vials with the paper points were microbiota of patients with periodontitis differs from sent to the laboratory at 4°C to be processed within that of healthy subjects [16] and therefore, the pre­ the same day. Subgingival samples were dispersed by valence of antimicrobial resistance genes and the vortex for 60 seconds. Serial dilutions of each sample prevalence of multidrug resistance among these bac­ were plated on blood agar plates (Blood agar base No. terial populations could be different. 2; Oxoid Ltd, Basingstoke, UK) containing 5% horse The use of antibiotics from the past to treat infec­ blood, hemin (5 mg/L) and menadione (1 mg/L) and tions caused by antibiotic-resistant bacteria has been on blood agar plates with and without 8 μg/ml tetra­ proposed, on the grounds that due to the lack of cycline. All plates were incubated under anaerobic selective pressure, the characteristics that once con­ conditions (10% H2, 10% CO2 and 80% N2) at 37°C ferred resistance might have disappeared [17]. This for 72 h. All of the morphologically different colonies might be the case with tetracyclines, whose use has were isolated and re-plated to obtain pure cultures declined over the years. There is limited knowledge that were preserved at −80°C in a 30% glycerol regarding the distribution of tetracycline resistance in solution. the subgingival microbiota, and the few papers that address this topic are outdated or focus on either In vitro antibiotic resistance testing particular genes or specific microorganisms. Therefore, the objective of this study was to analyze Resistance to six other antibiotics was tested using the prevalence and distribution of 11 tet genes and blood agar plates containing 1 μg/ml of erythromycin the multidrug resistance profiles of the tetracycline- (ERY), 64 μg/ml of kanamycin (KAN), 8 μg/ml of resistant subgingival microbiota isolated from healthy chloramphenicol (CHL), 128 μg/ml of streptomycin volunteers and patients with periodontitis. (STR), 2 μg/ml of cefotaxime (CTX), and 8 μg/ml of amoxicillin (AMX) (all antimicrobials were obtained as pure powder from Sigma Aldrich, St. Louis, MO, Materials and methods USA). Antimicrobial breakpoint concentrations were set according to the recommendations of the Clinical Sample collection and culture and Laboratory Standards Institute (CLSI) [23] and The samples included in this work were part of two the European Committee on Antimicrobial previous studies [18,19], whose research protocols Susceptibility Testing (EUCAST) [24]. However, were approved by the Ethics Committee of the most of the species present in the oral environment Universitat Internacional de Catalunya (UIC), are not covered by any of these organizations. (Barcelona, Spain) with study numbers: PER-ECL Therefore, antibiotic concentrations were chosen -2011-06-NF and ODO-2014-01. Both research pro­ based on taxonomic relatedness to oral bacteria, tocols complied with the principles of the Declaration using the higher concentration of antibiotics when of Helsinki. Subgingival samples were taken from 259 in doubt (Table S1). Incubation was performed at volunteers including 129 periodontally healthy sub­ 37°C under anaerobic conditions for 72 h. jects and 130 subjects with periodontitis. Samples were obtained at the Department of Periodontology DNA extraction of the UIC. All volunteers signed an Institutional review board-approved informed consent form. DNA extraction was performed on each isolate using Patients with periodontitis were diagnosed with gen­ the QIAamp DNA Mini Kit (Qiagen, Hilden, eralized severe chronic periodontitis [20] or stage III Germany) following the manufacturer’s instructions or IV generalized grade B or C periodontitis [21]. To with some modifications. Cells were suspended in be included in the study, periodontally healthy 180 μl of a 20 mg/ml lysozyme solution (20 mM Tris- JOURNAL OF ORAL MICROBIOLOGY 3 Hcl, pH 8.0, 2 mM EDTA, 1.2% Triton X-100) and Detection of antibiotic resistance genes incubated for 30 min at 36°C. Then, 200 μl of Buffer For the detection of antibiotic resistance genes, four AL (provided in the kit), 10 μl of RNase A (20 mg/ multiplex-PCR reactions were performed using four ml) and 10 μl of proteinase K (20 mg/ml) were added sets of primers (Table 1) as previously described [6]. and incubated for 30 min at 56°C. Further steps were The amplified products were evaluated by electro­ conducted according to the manufacturer’s protocol. phoresis using a 2% agarose gel. Some of these ampli­ Lastly, DNA was re-suspended in 100 μl of buffer AE fied products were sequenced (Macrogen, Inc., (provided in the kit), visualized in a 0.5% agarose gel Amsterdam, The Netherlands), and used as positive and quantified using a Nanodrop 2000 C UV-vis controls. The GenBank accession numbers for the spectrophotometer (Nanodrop Technologies, positive controls are available in the supplementary Wilmington, DE, USA). material. Sequencing of the 16S rRNA gene Statistical analyses The 16S rRNA gene was amplified by the polymerase Qualitative data were obtained when screening for chain reaction (PCR) using universal primers 27 F genes and when testing the ability of the isolates to and 1544 R (Table 1). PCR amplification was carried grow in media containing a breakpoint antimicrobial out applying a T3000 Thermocycler (Biometra, concentration. Isolates were considered resistant to Goettingen, Germany) under the following condi­ the antimicrobial if they were able to grow in the tions: 5 min at 95°C, followed by 35 cycles at 95°C medium containing antibiotic and susceptible if they for 60 sec, 57°C for 60 sec and 72°C for 60 sec; were not. To compare the prevalence of antibiotic followed by another 10 min at 72°C. Sequencing of resistance and of antibiotic resistance genes in both the gene 16S rRNA was performed in Macrogen, Inc. healthy subjects and subjects with periodontitis, data (Amsterdam, The Netherlands). The sequences were analyzed using the chi-square test. To compare obtained were aligned to form a single contig and the bacterial loads resistant to tetracycline, the non- were identified by comparison with those available at parametric Mann–Whitney U test was used. the National Center for Biotechnology Information A nominal significance level of 5% (p < 0.05) was database (http://www.ncbi.nlm.nih.gov) using BLAST applied for both tests. software (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Only sequences with > 99% of similarity were Results accepted to identify isolates at species level and the 16S rRNA sequences were deposited in GenBank One hundred and twenty-nine subgingival samples (accession numbers MT807114-MT807900). from periodontally healthy subjects (SPHS) and 130 Table 1. List of primers used in this study. Primers Sequence 5ʹ-3’ Size (bp) Tm Tm Multiplex Multiplex group Ref. tetMF GCG TAC AAG CAC AGA CTC GT 1142 61 64 1 [6] tetMR AGC CAT AGC GTA TCC CCT CC tetWF GAG AGC CTG CTA TAT GCC AGC 168 64 [57] tetWR GGG CGT ATC CAC AAT GTT AAC intF GGC TAC AGA CCG AGT ACC AGC 684 61 [6] intR GGA ACT TGA CGT TCG CCA CT ermBF GGT AAA GGG CAT TTA ACG AC 494 55 60 2 [58] ermBR CGA TAT TCT CGA TTG ACC CA tetQF AGA ATC TGC TGT TTG CCA GTG 167 50 [57] tetQR CGG AGT GTC AAT GAT ATT GCA tet32F GAA CCA GAT GCT GCT CTT 620 57 [59] tet32R CAT AGC CAC GCC CAC ATG AT tetLF TCG TTA GCG TGC TGT CAT TC 267 55 [60] tetLR GTA TCC CAC CAA TGT AGC CG tetOF AAC TTA GGC ATT CTG GCT CAC 515 55 58 3 [61] tetOR TCC CAC TGT TCC ATA TCG TCA tetSF GAA AGC TTA CTA TAC AGT AGC 168 50 [57] tetSR AGG AGT ATC TAC AAT ATT TAC tet31F CAA TCA CGC CCA AAA GAA 564 53 [62] tet31R TGT GCC ATC CCA GTT TGT tetBF AAT AGC CAC TAA ATG GGG CG 243 58 56.5 4 [6] tetBR ATA ACA CCG GTT GCA TTG GT tetKF TCG ATA GGA ACA GCA GTA 169 52.8 [60] tetKR CAG CAG ATC CTA CTC CTT tet37F ATG GTT CGC TAT TAC TCT AAC 170 50 [6] tet37R ATC AGT CTC ATA TTT CGA CA 27 F GAG TTT GAT CCT GGC TCA G approx. 1500 57 - - [63] 1544 R AGA AAG GAG GTG ATC CAG CC 4 A. ARREDONDO ET AL. subgingival samples from subjects with periodontitis more isolates. Significant differences were also (SSP) were collected. SPHS were from patients between observed between the isolates for susceptibility to all the ages of 19–24 (mean of 21.5 ± 3.3) years and SSP the antimicrobials, where SPHS isolates showed two were from patients 24 to 82 (mean of 51.25 ± 11.97) times higher susceptibility. Moreover, we found that years of age. 91% of all the isolates were resistant to other anti­ SSP grown on blood agar showed a mean bacterial biotics besides tetracycline (Table 3). Resistance to load of 6.54 log10 colony-forming units per milliliter KAN was the most frequently observed (83.64%), (cfu/ml) (± 0.91), while SPHS showed a mean bacter­ followed by ERY (67.16%), STR (20.66%), CTX ial load of 5.64 log10 cfu/ml (± 0.8). When grown on (18.08%), AMX (15.50%), and CHL (14.64%). media with tetracycline, SSP showed a mean of 4.87 Prevalence and significant differences of the studied log10 cfu/ml (± 2.09), 2.14% of the total bacterial load, genes and resistances between the two groups are versus the 3.66 log10 cfu/ml (±1.63) for SPHS, 1.05% shown in Figure 1. of the total bacterial load, which did not represent All the identified species and their prevalence of a significant difference (p = 0.76). Eighty-six-point the antibiotic resistance genes screened and the resis­ eighty-two percent (86.82%) (n = 112) of SPHS and tance to six antibiotics are presented in Table S2. 86.15% (n = 112) of SSP harbored tetracycline- resistant bacteria, from which we obtained a total of Discussion 813 isolates, belonging 448 to SPHS and 365 to SSP. Streptococcus sp. were the most frequently isolated The present study investigated the tetracycline- species, representing 75.62% in SSP and 75.89% in resistant bacteria in subgingival samples of 129 peri­ SPHS. In both groups, Streptococcus oralis was the odontally healthy subjects and 130 patients with per­ most frequently isolated species (n = 83), followed by iodontitis, finding high loads of tetracycline-resistant other streptococcal species such as Streptococcus mitis bacteria, and a high prevalence of tet genes and (n = 81), Streptococcus intermedius (n = 79) and multidrug resistance in both populations. Streptococcus constellatus (n = 58). The most fre­ Bacterial counts, both total and that of resistant quently isolated non-streptococcal species was bacteria, were 10 times higher in SSP than in SPHS. Prevotella intermedia (n = 31) followed by Prevotella This agrees with previous studies [25,26], in which nigrescens (n = 25). the bacterial load in the periodontal pocket of When comparing the prevalence of certain species patients with periodontitis was higher than that between the two groups of subjects, significant differ­ observed from periodontally healthy subjects. ences were observed: Gemella haemolysans (5-fold, A higher percentage of tetracycline-resistant bacteria p = 0.016), S. oralis (3-fold, p < 0.01), Streptococcus was also observed in SSP, probably due to the higher pneumoniae (4-fold, p < 0.01) and Streptococcus sangui­ bacterial load of those samples. nis (4-fold, p < 0.01) were more prevalent in SPHS, Although the percentages of total tetracycline- while Streptococcus anginosus (2-fold, p = 0.036), resistant streptococci were similar in both groups of S. constellatus (6-fold, p < 0.01), Streptococcus gordonii subjects, differences were observed at species level (2-fold, p = 0.022), Streptococcus parasanguinis (3-fold, (Table S2). S. sanguinis, S. oralis and S. pneumoniae, p = 0.017) and Streptococcus tigurinus (7-fold, p = 0.049) which in our study were significantly more prevalent were more prevalent in SSP (Table S2). in SPHS, have been previously associated to com­ A list of the identified species of the tetracycline- mensal biofilms [27,28], while S. anginosus, resistant microorganisms isolated in this study, the S. constellatus, S. gordonii, S. tigurinus and prevalence of the antibiotic resistance genes screened S. parasanguinis, which in our study were signifi­ and the multidrug resistance profile for the six anti­ cantly more prevalent in SSP, have been related to biotics tested is displayed in Table 2. The genes tet a periodontitis-associated microbiota [29–33]. (M) (52.03%), tet(32) (8.24%) and tet(O) (7.75%) However, the role of these species in periodontitis is were the most frequently detected tet genes. not yet clear [34,35]. Significant differences were also observed in the pre­ The prevalence of G. haemolysans was significantly valence of tet(M) (p < 0.01), tet(W) (p < 0.01), and tet higher in SPHS. This species has been described by (O) (p < 0.01), which were higher in SPHS, and tet metagenomic studies as an early colonizer of the oral (32) (p < 0.01) and tet(L) (p < 0.01), which were biofilm [36,37] and therefore as part of the commen­ higher in SSP. The intTn gene, which codes for an sal microbiota. However, it has also been linked to integrase located in transposons of the family Tn916/ opportunistic infections [38]. 1545 was widely detected (79.58%), being more pre­ Isolates of the genus Prevotella were significantly valent in SPHS (81.92%) than in SSP (76.71%). more prevalent in SSP (2-fold). The increased rich­ When assessing resistance to six different antimi­ ness of Prevotella species in SSP was responsible for crobials, a significant difference was found between this, increasing the total number of Prevotella isolates the groups, where the SSP group showed three times in SSP. However, P. intermedia and P. nigrescens, two Table 2. Species identified from SPHS and SSP and the number of isolates carrying each antimicrobial resistance gene and their phenotypical resistance. The asterisk indicates significant differences between SPHS and SSP isolates. AMX: amoxicillin, CTX: cefotaxime, CHL: chloramphenicol, STR: streptomycin, ERY: erythromycin, KAN: kanamycin. Antimicrobial resistance genes Antimicrobial resistance Species N intTn tet(W) tet(O) tet(M) tet [31] tet [32] tet(B) tet(L) tet(S) tet(Q) tet [37] tet(K) erm(B) AMX CTX CHL STR ERY KAN SSP Actinomyces sp. 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 1 Bacteroides sp. 2 0 0 0 0 0 0 0 0 0 2 0 0 0 0 2 0 2 2 2 Gemella sp. * 2 2 0 0 2 0 0 0 0 0 0 0 0 0 1 0 0 1 2 2 Granulicatella sp. 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Lachnoanaerobaculum sp. 3 2 0 0 0 0 0 0 0 0 0 0 0 2 1 2 0 2 2 2 Mogibacterium sp. 1 0 1 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 1 1 Neisseria sp. 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 2 0 Peptoniphilus sp. 2 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Peptostreptococcus sp. 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Prevotella sp. * 48 16 0 0 3 0 18 7 0 0 27 0 2 3 28 36 2 15 24 42 Proteus sp. 1 1 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 Pseudomonas sp. 2 0 0 0 0 0 0 0 0 0 0 0 0 0 2 2 2 2 2 2 Rothia sp. 2 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 2 2 Slackia sp. 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 Staphylococcus sp. 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 Streptococcus sp. 276 248 0 18 116 0 31 0 12 0 3 0 0 128 20 56 29 48 188 271 Veillonella sp. 6 6 0 0 5 0 0 0 0 0 0 0 0 2 1 4 0 4 5 2 Not identified 10 2 0 0 2 0 1 1 0 0 1 0 0 1 1 2 1 1 3 4 SPHS Abiotrophia sp. 5 5 0 0 2 0 0 0 0 0 0 0 0 4 0 0 1 0 5 2 Bacilus sp. 1 1 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 1 1 Butyrivibrio sp. 4 1 0 2 1 0 1 0 0 0 0 0 0 1 2 0 0 2 1 4 Eubacterium sp. 3 2 1 0 3 0 1 1 0 0 0 0 0 2 0 0 0 1 3 3 Gemella sp. * 13 13 1 0 8 0 0 1 0 0 1 0 0 0 1 1 4 0 5 4 Granulicatella sp. 3 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 Haemophilus sp. 4 2 1 0 2 0 1 2 0 0 0 0 0 3 0 0 0 0 4 1 Neisseria sp. 2 0 0 0 2 0 0 0 0 0 0 0 0 2 0 0 0 1 2 0 Prevotella sp. * 25 0 0 0 0 0 1 3 0 0 16 0 1 0 16 15 4 13 18 22 Rothia sp. 4 1 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 2 Staphylococcus sp. 5 1 0 0 0 0 0 0 0 0 0 0 3 4 3 3 2 3 4 0 Streptococcus sp. 340 305 44 42 250 0 12 2 0 1 5 0 0 218 39 12 68 59 227 283 Veillonella sp. 12 11 5 1 9 0 0 0 0 0 0 0 0 5 3 5 2 4 12 6 Not identified 27 24 3 0 17 0 0 0 0 0 3 0 0 20 7 0 3 7 27 20 JOURNAL OF ORAL MICROBIOLOGY 5 6 A. ARREDONDO ET AL. Table 3. Number of isolates resistant to other antimicrobials. In this study, we found the genes tet(W), tet(O), The asterisk indicates significant differences between samples tet(32), tet(B), tet(Q) and tet(K), distributed among from periodontally healthy subjects (SPHS) and the samples the genera Eubacterium, Gemella, Haemophilus, from subjects with periodontitis (SSP) isolates. In parenth­ eses, the percentage of isolates over the total number of Veillonella, Butyrivibrio and Prevotella which, accord­ isolates from each population. Define meaning of asterisk. ing to the tetracycline resistance genes database avail­ Number of other antimicrobials SPHS isolates (%) SSP isolates (%) able at http://faculty.washington.edu/marilynr/, had 0* 48 (10.71) 21 (5.75) not been previously reported (Table 2). However, 1 82 (18.30) 68 (18.63) further studies are needed to confirm these results. 2 179 (39.96) 140 (38.36) 3 93 (20.76) 77 (21.10) For instance, the detection of the gene tet(B) in 4 31 (6.92) 37 (10.14) streptococci, as recently described [43], or new tet 5 12 (2.68) 13 (3.56) 6* 3 (0.67) 9 (2.47) genes being described in the oral environment [44] show that there is still much to be unveiled regarding tetracycline resistance genes. The prevalence of the gene tet(M) among the SPHS in our study was similar Prevotella species that were present in both popula­ (65.85%) to what has been previously described tions, did not show significantly different prevalences. [7,45]. Nevertheless, the prevalence of this gene in The genus Prevotella has been linked to the progres­ SSP was much lower (35.07%) than what other sion of periodontitis, and therefore, a higher preva­ authors found in Greek and US subjects with period­ lence of this genus was expected in SSP. However, the ontitis [45,46], but similar to the percentages found increased richness of tetracycline-resistant Prevotella in Dominican patients [6]. These differences might be species observed in SSP is worth mentioning, since explained by the geographical constraints of the most reports have focused their attention on populations involved in the studies or due to their P. intermedia and P. nigrescens [39,40], and little is methodological approaches, such as pooling the sam­ known about the prevalence of tetracycline resistance ples, the DNA or different selection criteria of the in other oral Prevotella species. isolates. As previously discussed, the streptococcal The prevalence of Gram-negative anaerobes was species were significantly different between SPHS low, and species usually isolated from patients with and SSP, which might be the cause for the differences periodontitis, such as Porphyromonas gingivalis or in prevalence of tet(M) between the two groups stu­ Fusobacterium nucleatum, were not detected in this died. While 73.53% of the streptococci isolated from study. This might be due to the high susceptibility SPHS showed tet(M), only 42.02% of the streptococci that these organisms have to tetracycline, in most isolated from SSP did, indicating that those strepto­ cases showing minimum inhibition concentrations cocci associated to a healthy biofilm might be more lower than the 8 μg/ml tetracycline breakpoint con­ susceptible to carry tet(M). In our study, the preva­ centration used in this study [41,42], which makes lence of tet [32] was higher in SSP, as previously them unlikely to be isolated in such conditions, as described [6], which might be due to the increased seen in previous studies [7]. prevalence of this gene in periodontitis-associated Figure 1. Prevalence (%) of tet, int and erm(B) genes and the prevalence (%) of subgingival isolates resistant to different antimicrobials (KAN: kanamycin, ERY: erythromycin, STR: streptomycin, CHL: Chloramphenicol, AMX: Amoxicillin, CTX: cefotax­ ime. The asterisk indicates significant differences between the isolates obtained from samples from periodontally healthy subjects and from samples from subjects with periodontitis. JOURNAL OF ORAL MICROBIOLOGY 7 species such as S. constellatus and P. intermedia¸ lactams and KAN [56], was twice as prevalent in SSP although other authors have also detected this gene compared to SPHS. On the other hand, the differences in in some commensal bacteria [47]. The gene tet(W) CHL resistance might be due to the unequal distribution can be frequently detected in the DNA from pooled of CHL resistance among streptococcal species, resulting saliva and plaque samples [7,48,49], but its distribu­ in different percentages of this resistance between both tion within the subgingival biofilm has only been groups (Table S2). The highest ratio of P. intermedia and studied by Collins et al. [6]. In that study, tet(W) P. nigrescens and the high prevalence of S. constellatus was, on average, twice as prevalent in SSP than in CTX-resistant in SSP were determinant for the higher SPHS. In our study, tet(W) was more ubiquitous in prevalence of CTX resistance in SSP. Although the genus SPHS, since S. intermedius and S. oralis, which were Prevotella is already known for its β-lactam resistance, more prevalent in SPHS, were some of the species high prevalence of CTX resistance among oral strepto­ that carried tet(W) most often. cocci has not been previously reported; thus, it might Transposons of the Tn916/1545 family are frequently pose a serious health issue, even more when considering found in the oral microbiota [50]. These transposons their multidrug resistance capabilities. usually carry tetracycline resistance genes, and in some While the differences found in this study were based cases, genes that confer resistance to macrolides and/or on the periodontal diagnoses of the subjects, more cer­ aminoglycosides [51,52]. In order to estimate the pre­ tainty about the causes of the differences observed would sence of these transposons among the isolates, we used most likely be achieved by including either the age or the PCR to screen the intTn gene, which codes for an inte­ sex of the subjects as variables of interest. Nonetheless, grase located at the 3ʹ- ends of these transposons [14], the this study has shown high loads of bacteria exhibiting tet(M) gene, and the erm(B) gene, which confers resis­ multidrug resistance and a variety of tetracycline resis­ tance to macrolides and is often found in these conjuga­ tance genes to be present in Spanish SSPs and SPHSs. tive elements [53]. The results showed a high prevalence However, significant differences were detected between of both genes in both groups, although they were signifi­ the two groups in terms of i) bacterial species resistant to cantly more prevalent in SPHS. The previously discussed tetracycline; ii) prevalence of the screened genes and iii) differing prevalence of streptococcal species when com­ multidrug resistance profiles. Moreover, the presence of paring SPHS and SSP might be the reason for these some tet genes was detected in certain bacterial genera, differences, given that the streptococci of this study which had not been previously described. Overall, we showed different profiles of antibiotic resistance genes. found that in Spanish subjects, although the use of tetra­ These results suggest a high prevalence of transposons of cycline has been declining for many years, resistance to the Tn916/1545 family in the tetracycline-resistant oral this antibiotic is still present in subgingival bacteria, microbiota of healthy subjects and patients with which are a reservoir of tetracycline resistance genes periodontitis. and multidrug resistance, which, coupled with the high Most of the tetracycline-resistant isolates obtained in prevalence of conjugative transposons in the oral envir­ this study showed resistance to other antibiotics. onment, might foster further increased spread of antimi­ Resistance to two antimicrobials besides tetracycline crobial resistance. was the most common pattern, and a large part of this multidrug resistance was to KAN and ERY, which could be linked to the presence of the previously mentioned Acknowledgments transposons [51]. Levels of multidrug resistance were We would like to express our gratitude to all the volunteers high and similar in both populations, showing that the that participated in the study and would also like to thank tetracycline-resistant subgingival microbiota is an impor­ Ann Bangle for editing the manuscript. tant reservoir of antimicrobial resistance, which might be specially striking in subjects who have received little or no antibiotic therapy. The high prevalence of isolates resis­ Disclosure statement tant to antibiotics not commonly used, and therefore not No potential conflict of interest was reported by the exposed to selective pressure, might be due to the low authors. fitness costs associated with the acquisition and mainte­ nance of some mobile genetic elements that carry anti­ biotic resistance genes [54,55]. References Significant differences were found when testing KAN and CHL resistance between both groups. The former [1] Chopra I, Roberts M. 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