JOURNAL OF ORAL MICROBIOLOGY 2019, VOL. 11, 1643204 https://doi.org/10.1080/20002297.2019.1643204 ORIGINAL ARTICLE Detection and expression analysis of tet(B) in Streptococcus oralis a,b a Alexandre Arredondo , Gerard Àlvarez , José Nartc, Carolina Morc, Vanessa Blanc a 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 Tetracycline resistance can be achieved through tet genes, which code for efflux pumps, Received 21 March 2019 ribosomal protection proteins and inactivation enzymes. Some of these genes have only been Revised 28 June 2019 described in either Gram-positive or Gram-negative bacteria. This is the case of tet(B), which Accepted 3 July 2019 codes for an efflux pump and, so far, had only been found in Gram-negative bacteria. In this KEYWORDS study, tet(B) was detected in two clinical Streptococcus oralis strains isolated from the gingival Tetracycline; antibiotic sulci of two subjects. In both cases, the gene was completely sequenced, yielding 100% resistance; Streptococcus shared identity and coverage with other previously published sequences of tet(B). Moreover, oralis; tet(B); horizontal gene we studied the expression of tet(B) using RT-qPCR in the isolates grown with and without transfer; gene expression tetracycline, detecting constitutive expression in only one of the isolates, with no signs of expression in the other one. This is the first time that the presence and expression of the tet(B) gene has been confirmed in Gram-positive bacteria, which highlights the potential of the genus Streptococcus to become a reservoir and a disseminator of antibiotic resistance genes in an environment so prone to horizontal gene transfer as is the oral biofilm. Since their discovery in the 1940s, tetracyclines have most common in the oral environment is S. oralis been used for many years to treat a wide spectrum of [9]. S. oralis is a member of the Streptococcus mitis infections. However, resistance to these antibiotics group, known to be a commensal bacterium and soon appeared, and only the discovery of new deri- opportunistic pathogen [10]. S. oralis is an early vatives allowed their continued use until the 1980s, colonizer of the oral biofilm and plays an important when they were gradually replaced by other antibio- role in its establishment and homeostasis [11]. tics such as fluoroquinolones [1]. Moreover, it has been described to antagonize bac- Despite their widespread resistance, some tetracy- teria such as Streptococcus mutans, thus preventing clines are still useful in some treatments, such as in the development of dysbiotic biofilm [12]. However, acne vulgaris, periodontitis and infections caused by it is one of the most isolated bacteria in endocarditis multidrug resistant (MDR) Acinetobacter baumannii, infections [13,14] and it has been found in numerous Helicobacter pylori and methicillin-resistant Staphylo- types of infection sites [15–19]. coccus aureus [2–4]. Resistance can occur through efflux Transformation among streptococci is a common pumps, ribosomal protection or enzymatic inactivation. event, as they exhibit natural competence and easily According to http://faculty.washington.edu/marilynr/ acquire foreign DNA. Therefore, the transference of (last update May 1st, 2019), 60 tetracycline resistance antibiotic resistance genes has been largely studied in genes have been detected that code for these mechan- this genus [20,21]. Bacteria in the oral environment isms, along with 11 mosaic genes that code for ribosomal tend to grow by forming a multispecies biofilm, which protection proteins. Some of these genes, such as tet(C) increases the chances of horizontal gene transfer and and tet(32), have only been found in Gram-positive the spread of antimicrobial resistance, creating a large bacteria, while others such as tet(B) have only been reservoir of antibiotic resistance genes that could be found in Gram-negative bacteria. In a recent study by transferred to other bacteria, pathogenic or not [22]. Chander et al., tet(B) was found in Streptococcus suis In this study we described the presence of tet(B) in isolated from pigs [5], but a lack of full sequencing of two S. oralis isolates obtained from subgingival sam- the gene prevented the confirmation of such a finding. ples of healthy volunteers. Furthermore, in order to In oral biofilm, streptococci are a great reservoir of prove the presence and transcription of the gene in tetracycline resistance genes [6–8], and one of the Gram-positive bacteria, the complete sequence of the CONTACT Rubén León leon@dentaid.es Department of Microbiology, Dentaid Research Center, Ronda Can Fatjò 10, Parc Tecnològic del Vallès, Barcelona 08290, Spain © 2019 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. gene was obtained, and its expression was evaluated DNA sequencing in both isolates. tet(B) was found in two isolates, 444.1 and 469.4, from two different subjects. In order to identify each isolate to the species level, the 16S rRNA, sodA Materials and methods and groEL genes were amplified using the primers 27- F and 1544-R, sodA-F and sodA-R, and groEL-F and Sample collection and culture groEL-R, respectively (Table 1). The PCR products Subgingival samples were collected from periodontally were purified using the E.Z.N.A.® Gel extraction Kit healthy volunteers in the dental clinic of the Universitat (Omega BIO-TEK, Norcross, GA, USA) and Internacional de Catalunya (UIC), (Barcelona, Spain), sequenced by primer walking at the Genomics and and the research protocol was approved by the Ethics Bioinformatics Service of the Autonomous University Committee of the UIC (study number: PER-ECL of Barcelona (Barcelona, Spain). Primers tetB-insert-F -2011–06-NF). Subjects had not received antibiotics and tetB-insert-R (Table 1) were used to fully 3 months prior to the study and did not show signs of sequence the tet(B) gene. These primers amplified gingivitis or periodontitis. Sterile paper points were the gene 41 bp upstream of the start codon and 72 inserted into the gingival sulci of teeth 16, 21, 36 and 41 bp downstream of the stop codon. The 1,319 bp and kept in place for 20 sec. These paper points were amplicons were obtained by PCR and were purified pooled in a vial containing 1.5 ml of sterilized reduced and sequenced by primer walking as described above. transport fluid (RTF) without EDTA [23] and sent to the The sequences obtained were analyzed using BLASTn laboratory at 4ºC for processing within 24 h. Samples software at https://blast.ncbi.nlm.nih.gov/Blast.cgi. were serially diluted and plated on blood agar (Oxoid Reference sequences for alignment were obtained Nutrient Broth No. 2; Oxoid Ltd, Basingstoke, UK) con- from the database on http://faculty.washington.edu/ taining 5% horse blood, hemin (5 mg/l), menadione marilynr/, specifically the sequences with the acces- (1 mg/ml) and 8 µg/ml of tetracycline (Sigma Aldrich, sion numbers: J01830, AF223162, V00611, AL513383, St. Louis, MO, USA) to screen for tetracycline resistant AJ277653, AF326777 and AP000342. Additionally, bacteria. Incubation was performed at 37ºC in anaerobic other sequences of tet(B) from other species were conditions, and after 72 h, all different colony morphol- used to ensure better reliability: CP015434.1, ogies were subcultured to obtain pure cultures. For each KX458222.1, CP015836.1, NG_048163.1 and selected isolate, liquid cultures with and without tetracy- LN908249.1. Alignment was performed using the cline (8 µg/ml) were done using modified brain heart Clustal Omega service at https://www.ebi.ac.uk/ infusion (brain heart infusion broth [37 g/l], yeast extract Tools/msa/clustalo/. [1 g/l], L-cysteine [0.1 g/l], sodium bicarbonate [2 g/l], and supplemented with hemin [5 mg/l] and glutamic acid [0.25%]). Cultures grew to an OD600 of 0.8–1 (≈ 108 cfu/ RNA isolation and expression of tet(B) gene ml), when DNA and RNA extractions were performed. RNA was extracted from liquid cultures with and with- out tetracycline using the High Pure RNA Isolation kit (Roche Diagnostics, Mannheim, Germany) according Detection of tet(B) to the instructions provided by the manufacturer. An extra step to remove any remaining DNA was done DNA was extracted from all the isolates using the using the TURBO DNA-freeTM Kit (Thermo Fisher QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) Scientific, Vilnius, Lithuania). The absence of DNA in according to the manufacturer’s instructions, quantified the samples was verified by PCR using the sets of using a Nanodrop 2000 UV-vis spectrophotometer primers tetB-RT-F/tetB-RT-R, and 16S-So-F/16S-So-R (Nanodrop Technologies, Wilmington, DE, USA) and (Table 1), visualizing the results in a 3% agarose gel. The visualized in a 0.5% agarose gel stained with ethidium remaining RNA was quantified in a Qubit 4 bromide. Fluorometer using the Qubit RNA XR Assay Kit All the isolates were screened for a battery of tet genes (Thermo Fisher Scientific, Eugene, OR, USA), and its (data not shown). To detect tet(B) the primers tetB-F and integrity was verified on a 2% agarose gel. tetB-R were used (Table 1) with the following PCR con- Expression of tet(B) was measured by Quantitative ditions: an initial denaturation of 5 min at 95ºC, 30 cycles Reverse Transcription PCR (RT-qPCR). Reactions of 30 sec of denaturation at 95ºC, 30 sec of annealing at were performed with a LightCycler® 480 II 56.5ºC and 45 sec of extension at 72ºC, and a final Instrument using the LightCycler® 480 RNA Master extension of 10 min at 72ºC. The result of the PCR was Hydrolysis Probes kit (Roche Diagnostics, Mannheim visualized in a 3% agarose gel stained with ethidium Germany), following the instructions provided by the bromide, where a band of 242 bp could indicate the manufacturer. The sets of primers tetB-RT and 16S- presence of tet(B). So (Table 1) were used together with probes 45 and JOURNAL OF ORAL MICROBIOLOGY 3 Table 1. List of primers used in this study. Primers Sequence 5ʹ – 3’ Product size (bp) Annealing Temp (°C) Reference 27-F AGA GTT TGA TCC TGG CTC AG 1,533 57 [37] 1544-R AGA AAG GAG GTG ATC CAG CC sodA-F CCI TAY ICI TAY GAY GCI YTI GAR CC 500 37 [38] sodA-R ARR TAR TAI GCR TGY TCC CAI ACR TC groEL-F GAH GTN GTI GAA GGI ATG CA 800 52 [39] groEL-R ATT TGR CGI AYW GGY TCT TC tetB-F TTG GTT AGG GGC AAG TTT TG 242 55 [40] tetB-R GTA ATG GGC CAA TAA CAC CG tetB-insert-F ACC AAA GCT TAG TTA TTC TAC CAC TCC CTA TCA GT 1,319 53 This study tetB-insert-R CTT CTT CGA ATG CCC TCT TGG GTT ATC AAG tetB-RT-F TTC AAG TGC GCT TTG GAT GC 111 60 This study tetB-RT-R CGT TGA GAA GCT GAG GTG GT 16S-So-F CGC TCG GGA CCT ACG TAT TA 59 60 This study 16S-So-R TAC CAG AAA GGG ACG GCT AA M1-F GAT TTT TTA GCA GAA GTA 629 55 This study M1-NdeI-R CCG ATA CCA TAT GCC GAT ATT CTA ACC GAA T M2-HindIIIF CCG ATA CAA GCT TAG TGA CCC GCT TCT GCG A 782 55 This study M2-R GAC TAT TTG GAC GAC GGG ermB-NdeI-F GCG TTA GCA TAT GTA CGT TAG ATT AAT TCC TAC CAG 887 55 This study ermB-HindIII-R CCG ATA CAA GCT TTT ATT TCC TCC CGT TAA ATA 66 from the Roche Diagnostics’ Universal Diagnostics, Mannheim, Germany) according to ProbeLibrary for the genes tet(B) and 16S rRNA, the manufacturer’s instructions. The construct was respectively. The RT-qPCR conditions were as fol- then purified using the E.Z.N.A.® Gel extraction Kit lows: i) 75 ng of RNA were used in each reaction and transformed into 469.4 by electroporation using for the synthesis of cDNA, which was performed at the Gene Pulser Xcell (BioRad, Hercules, CA, USA). 63ºC for 3 min; ii) cycling conditions were 95ºC for Electroporated cells were plated on blood agar plates 30 sec, followed by 45 cycles of 15 sec at 95ºC, 40 sec containing 5 µg/ml of erythromycin (Sigma Aldrich, at 60ºC and 1 sec at 72ºC. Data were analyzed using St. Louis, MO, USA) and incubated in anaerobic the LightCycler® 480 Software 1.5. The 16S rRNA conditions for 24–48 h. The recombination event gene was used as an endogenous control, and the was confirmed by PCR using primers M1-F and formula 2ΔCptet(B)/2ΔCp16S rRNA was used to evaluate M2-R. differences in the expression of tet(B) in the presence and absence of tetracycline. Antibiotic susceptibility testing Construction of tet(M) defective mutants Susceptibility to tetracycline was tested in 444.1, 469.4 and 469.4Δtet(M)::erm(B)(EryR) using E-test strips Isolate 469.4 was found to harbor the gene tet(M). (Sigma Aldrich, St. Louis, MO, USA) according to Therefore, in order to determine the contribution of manufacturer’s instructions. Streptococcus pneumo- tet(B) to the tetracycline resistance of this isolate, niae ATCC 49619 was used as a quality control strain. disruption of the gene tet(M) was performed by introducing the gene erm(B), which confers resis- tance to erythromycin, between the positions 853 Results and 1,110 of the tet(M) gene. To this end, we Species identification and tet(B) sequences made a construct consisting of the gene erm(B) surrounded by two sequences homologous to tet From a library of 448 tetracycline resistant isolates (data (M), which we named M1 (the 5ʹ region) and M2 not shown), two isolates obtained from two different (the 3ʹ region). erm(B) was extracted by PCR using subjects were found to carry the tet(B) gene. Sequencing the primers ermB-NdeI-F and ermB-HindIII-R from of the 16S rRNA gene revealed that both isolates were a S. oralis clinical strain resistant to erythromycin S. oralis and shared the same sequence, which showed pertaining to the Dentaid Research Center 100% identity with the 16S rRNA sequences from (Cerdanyola del Vallès, Spain) strain collection. M1 S. oralis ATCC 700233, DSMZ 20,066 and CIP and M2 sequences were obtained by PCR from the 103216. To further corroborate this result, sequences isolate 469.4 using the primers M1-F and M1-NdeI of the genes sodA and groEL were also aligned to strep- -R for the M1 sequence, and M2-HindIII-F and M2- tococcal sequences, observing the highest homology R for the M2 sequence (Table 1). PCR products with S. oralis sequences. To ensure the presence of tet were purified using the E.Z.N.A.® Gel extraction (B), full sequencing of the gene and further alignment Kit and digested with NdeI and HindIII (New against reference sequences was performed, showing an England Biolabs Inc., Ipswich, MA, USA). Digested identity of 99.99–100% of the tet(B) isolated in this fragments were ligated using T4 ligase (Roche study. Sequence J01830 was used as a reference, being 4 A. ARREDONDO ET AL. the first published sequence of tet(B) [24]. Point muta- A transcriptional analysis of the gene tet(B) was tions were found in all of the sequences used compared performed using RT-qPCR. tet(B) mRNA was detected to J01830 (Supplementary Figure 1). tet(B) sequences of in isolate 469.4 but not in isolate 444.1, despite showing isolates 444.1 and 469.4, as well as sequences AF223162, very similar levels of 16S rRNA expression (Figure 1). AF326777, AL513383, CP015836.1, KX458222.1, As for isolate 469.4, no differences were observed in the LN908249.1 and NG_048163.1, presented five point expression of tet(B) when applying tetracycline to the mutations in positions 463, 794, 854, 940 and 1012. medium. The gene expression value, normalized with Sequence CP015434.1 exhibited an additional point the 16S rRNA gene, was 0.06 (log2). mutation in position 1,140, and sequence AP000342 According to previous screening for tet genes, had four of these point mutations in positions 794, isolate 469.4 contained a tet(M) gene in addition 854, 940 and 1,012. All these mutations resulted in to tet(B). In order to determine the influence of tet amino acid substitutions with the exception of the (B) on the tetracycline resistance of the isolate mutations in position 1,140 (Table 2 and 469.4, the mutant 469.4Δtet(M)::erm(B)(EryR) was Supplementary Figure 2). constructed. Sequences obtained in this study were submitted to GenBank (https://www.ncbi.nlm.nih.gov/genbank/), and the following accession numbers were retrieved: Antibiotic susceptibility MK611935 and MK611934 for the 16S rRNA gene of Susceptibility to tetracycline was tested in isolates 444.1 and 469.4, respectively, and MK611936 and 444.1 and 469.4 using E-test strips, observing MK611937 for the tet(B) gene of 444.1 and 469.4, a minimum inhibitory concentration (MIC) of 32 µg/ respectively. ml to tetracycline. In order to assess whether tetracy- cline resistance was being conferred by the gene tet(M) as opposed to tet(B), susceptibility to tetracycline in the mutant strain 469.4Δtet(M)::erm(B)(EryR) was also Expression of tet(B) tested, observing a MIC of 32 µg/ml. The level of expression of tet(B) in isolates 444.1 and 469.4 was determined comparing log-phase liquid cultures with and without tetracycline of each isolate. Discussion The exposure to tetracycline did not decrease the Resistance to tetracyclines is widespread, and although growth rate of the bacteria recovered from the culture tetracyclines are still useful in some treatments, only the and yielded similar amounts of RNA. most recent versions are being used to treat multidrug Table 2. Positions of nucleotide substitutions in the tet(B) sequences used as comparison. Between parentheses, the positions and amino acid substitutions corresponding to each nucleotide substitution. NC: no changes. Nucleotide substitution Sequences 463A>G (Thr155Ala) 794A>G (Glu265Gly) 854A>T (Asp285Val) 940G>C (Glu314Gln) 1012A>G (Thr338Ala) 1140A>G (NC) 469.4 + + + + + 444.1 + + + + + AF223162 + + + + + AF326777 + + + + + AL513383 + + + + + CP015836.1 + + + + + KX458222.1 + + + + + LN908249.1 + + + + + NG_048163.1 + + + + + CP015434.1 + + + + + + AP000342 + + + + 20 Relative fluorescence units 444 16S w TC 15 444 16S w/o TC 469 16S w TC 469 16S w/o TC 10 444 tet(B) w TC 444 tet(B) w/o TC 469 tet(B) w TC 5 469 tet(B) w/o TC 0 1 11 21 31 41 Cycles Figure 1. Amplification curves of the 16S rRNA and tet(B) genes obtained by RT-qPCR from RNA of 444.1 and 469.4. JOURNAL OF ORAL MICROBIOLOGY 5 resistant bacteria [25]. Nevertheless, a new formulation constitutive, since its transcription did not depend of minocycline, a second generation tetracycline, has on the addition of tetracycline, as it was observed been proven effective against MDR A. baumannii infec- once normalized with the expression values of the tions, allowing an old antibiotic to be used again [4,25]. 16S rRNA gene. Both isolates carried tet(M) as Unfortunately, resistance to these antibiotics is possible well, which might have conferred resistance to tet- through different mechanisms such as efflux pumps like racycline, as opposed to tet(B). We disabled the tet the one coded by tet(B), which can recognize different (M) gene in the 469.4 isolate through homologous tetracyclines as substrates, including minocycline, dox- recombination to determine its role in the level of ycycline and even glycylcyclines through punctual tetracycline resistance, and we found no changes in mutations [26]. tet(B) is the most distributed tetracy- the level of susceptibility, indicating that tet(B) cline resistance gene among Gram-negatives [27] and could be responsible for tetracycline resistance in so far it has not been described in Gram-positives. isolate 469.4, although it cannot be ruled out that However, a recent study [5] reported to have found tet resistance to tetracycline could be provided by (B) in S. suis, being the first time to describe the pre- another gene or mechanism. sence of this gene in a Gram-positive bacteria. The presence of tet(B) in Gram-positive bacteria Nonetheless, and although sequencing was performed is another step in the dissemination of antibiotic and 100% identity was obtained, only 54.55% of the resistance genes. Although tet(B) seemed to be gene was covered, raising doubts of whether the gene expressed in only one of the isolates, the presence might or might not be tet(B). According to Levy et al. of this tetracycline resistance gene had never been [28], tetracycline resistance determinants should have ≤ confirmed in Gram positives. Whether tet(B) is 80% of amino acid identity in order to be considered being expressed or not, it seems clear that the different genes, which makes it impossible for the pre- reservoir for this gene has expanded to the genus viously discussed analysis to determine if the gene stu- Streptococcus, an enormous bacterial group, died was tet(B). increasing the chances for its dissemination. The In our study we found two isolates of S. oralis transfer of antibiotic resistance genes between susceptible of carrying tet(B). In both cases, we Gram-positive and Gram-negative bacteria has performed full sequencing of the gene, resulting been previously reported [31-33], and the oral in 100% coverage, and compared the sequences to environment offers a great opportunity for these those tet(B) sequences previously published, show- transfers to occur. The characteristics of the oral ing some minor differences involving up to six biofilm make it prone to genetic exchange, allowing nucleotides in a sequence of 1,206 base pairs, the bacteria that live in such environment to which led to up to five amino acid substitutions acquire and share antibiotic resistance genes, in a sequence of 401 amino acids (100% – 98.75% among others [22,34-36]. Furthermore, the oral identity). Most of the polymorphisms observed in environment is a transit area for many bacteria the tet(B) sequences obtained from isolates 444.1 that might end up in other niches of the human and 469.4 were shared by all the published body, thus creating the opportunity for these bac- sequences used in this study with the exception of teria to acquire genetic determinants present in the V00611 and AJ277653, which were identical to the oral microbiome. Given that the tet(B) sequence reference sequence J01830 (Table 2). These three analyzed in this study was identical to other sequences were obtained from Escherichia coli, sequences of tet(B) observed in Gram-negatives, although CP015836.1, another E. coli sequence the functionality of this gene, being transferred to used in this study, presented five point mutations Gram-negatives, would be expected. In this study compared to J01830, implying that these mutations we confirmed the presence and expression of the might have occurred at some point during replica- gene tet(B) in Gram-positive bacteria. However, tion or transference and were maintained in certain further studies should be conducted in order to strains. establish the function of the gene tet(B) in isolates tet(B) expression was studied to understand the 444.1 and 469.4. Moreover, a full characterization functionality of the gene in the two S. oralis isolates of the surroundings of the gene and its ability to obtained. It is known that, in the presence of tetra- transfer among other streptococci might help to cycline, the transcription of tetracycline resistance understand the actual potential of the tet(B) gene genes is induced [29,30]. For this reason, cultures in Gram-positive bacteria. were made with and without tetracycline and RNA was extracted to quantify the expression of the gene. tet(B) mRNA could only be found in isolate Disclosure statement 469.4, meaning that the gene was not being expressed under those conditions in isolate 444.1. No potential conflict of interest was reported by the Furthermore, expression in 469.4 seemed to be authors. 6 A. ARREDONDO ET AL. ORCID bite in a 2-month-old infant. Paediatr Int Child Health. 2016;36(2):160–162. Alexandre Arredondo http://orcid.org/0000-0003-4459- [18] Chung JK, Lee SJ. Streptococcus mitis/oralis 1300 endophthalmitis management without phakic Gerard Àlvarez http://orcid.org/0000-0002-0022-1669 intraocular lens removal in patient with iris-fixated Vanessa Blanc http://orcid.org/0000-0002-2740-6559 phakic intraocular lens implantation. BMC Rubén León http://orcid.org/0000-0001-6400-4906 Ophthalmol. 2014;14:92. 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