Analysis of newly detected tetracycline resistance genes and their flanking sequences in human intestinal bifidobacteria

Due to tetracycline abuse, the safe bifidobacteria in the human gastrointestinal intestinal tract (GIT) may serve as a reservoir of tetracycline resistance genes. In the present investigation of 92 bifidobacterial strains originating from the human GIT, tetracycline resistance in 29 strains was mediated by the tet(W), tet(O) or tet(S) gene, and this is the first report of tet(O)- and tet(S)-mediated tetracycline resistance in bifidobacteria. Antibiotic resistance genes harbored by bifidobacteria are transferred from other bacteria. However, the characteristics of the spread and integration of tetracycline resistance genes into the human intestinal bifidobacteria chromosome are poorly understood. Here, conserved sequences were identified in bifidobacterial strains positive for tet(W), tet(O), or tet(S), including the tet(W), tet(O), or tet(S) and their partial flanking sequences, which exhibited identity with the sequences in multiple human intestinal pathogens, and genes encoding 23 S rRNA, an ATP transporter, a Cpp protein, and a membrane-spanning protein were flanking by the 1920-bp tet(W), 1920-bp tet(O), 1800-bp tet(O) and 252-bp tet(S) in bifidobacteria, respectively. These findings suggest that tetracycline resistance genes harbored by human intestinal bifidobacteria might initially be transferred from pathogens and that each kind of tetracycline resistance gene might tend to insert in the vicinity of specific bifidobacteria genes.

There are up to 10 13 -10 14 total bacteria in the human gastrointestinal intestinal tract (GIT) 1,2 . Due to the abuse of tetracycline in the clinical and nonclinical treatment of various human infections 3 , the carriage of tetracycline resistance genes by bacteria in the human GIT has been an area of intense investigation 4 . Most studies have focused on the tetracycline resistance genes carried by clinical pathogens or opportunistic pathogens 5 and have continuously detected new tetracycline resistance genes harbored by the intestinal pathogens, such as the tet(40) gene in the human intestinal firmicute bacterium 6 . However, because bifidobacteria are ingested as probiotics in the human GIT and have acquired a "generally regarded as safe" (GRAS) status [7][8][9] , so far, only tet(W)-and tet(M)-mediated tetracycline resistance have been detected in intestinal bifidobacteria of human origin [10][11][12][13] 14 . Therefore, it remains unknown whether tetracycline resistance genes other than tet(W) and tet(M) can be detected in the bifidobacterial strains originating in the human GIT.

, and only tet(L)-, tet(O/W)-, tet(W/32/O)-, and tet(O/W/32/O/W/O)-mediated tetracycline resistance have been detected in intestinal bifidobacteria of pig origin
Antibiotic resistance (AR) genes within potentially mobile elements can spread horizontally across genera in the human GIT 15 . Comparative analysis of sequences flanking the same AR gene in one genus of bacteria can therefore further reveal the spread characteristics of the AR gene. However, although two tetracycline resistance genes [tet(W) and tet(M)] have been detected in human intestinal bifidobacteria [10][11][12][13] , only the sequences flanking the tet(W) gene in bifidobacteria have been analyzed 10,12 . Scott previously found a conserved tet(W) gene sequence of 2154 bp in 10 gut bifidobacterial strains of 5 species 12 . Ammor analyzed the flanking sequences of the tet(W) genes in another six human intestinal bifidobacteria and found an orfY gene in the downstream flanking region of the tet(W) gene in one B. thermophilum strain and one B. longum strain and a transposase gene in the downstream flanking region of the tet(W) gene in two B. longum strains 10 . Based on these results, it is not possible to determine whether the tet(W) gene inserts into common sites in the chromosome of the human intestinal bifidobacteria or whether other tetracycline resistance genes may exhibit conservation in their integration into the human intestinal bifidobacteria chromosome.
As a result of the misuse and overuse of tetracycline, the traditionally safe bifidobacteria in the human GIT may serve as a reservoir of tetracycline resistance genes and increasingly become a threat to human health. Therefore, this study was performed to assess 92 bifidobacterial strains isolated from the feces of 14 healthy individuals, one type strain and seven commercial strains via phenotypically and genotypically screening the acquired tetracycline resistance profiles and to comparatively analyze the upstream and downstream sequences flanking the tetracycline resistance genes harbored by different strains.

Results
Tetracycline susceptibility profiles. The MIC values of tetracycline in the 100 bifidobacterial strains tested are presented in Tables 1 and 2. Twenty-nine bifidobacterial strains, including the seven Bifidobacterium longum strains shown in Table 1 and two Bifidobacterium bifidum strains, six Bifidobacterium pseudocatenulatum strains, 13 Bifidobacterium lactis strains and one Bifidobacterium breve strain shown in Table 2 Detection of tetracycline resistance genes. As Tables 1 and 2 show, each of the 29 tetracycline-resistant bifidobacterial strains possessed one tetracycline resistance determinant [tet(W), or tet(O), or tet(S) gene], and none of the 13 tetracycline resistance determinants tested were detected in the 71 tetracycline-sensitive bifidobacterial strains. The occurrence of the tet(W), tet(O), and tet(S) genes among the 100 bifidobacterial strains of the seven Bifidobacterium species tested are further summarized in Table 3.
In the 6 tet(O)-positive strains including 5 B. longum subsp. longum strains and one B. pseudocatenulatum strain, tet(O) exhibited an identical DNA sequence of 1457 bp, which encoded a protein consisting of 458 amino acids that displayed 100% identity with the ribosomal protection protein tetO previously identified in Streptococcus suis BM407 (GenBank FM252032.1).
In the two tet(S)-positive strains, B. pseudocatenulatum strain F312 and B. breve strain A27, tet(S) exhibited an identical DNA sequence of 210 bp, which encoded a protein consisting of 70 amino acids that displayed 100% identity with the ribosomal protection protein tetS previously identified in Lactococcus lactis subsp. lactis strain ILIBB-JZK (GenBank KF278750.1). The

Discussion
In our previous investigation of a collection of 92 bifidobacterial strains originating from the human GIT, the macrolide, lincosamide, and streptogramin (MLS) resistance gene erm(X) was detected in 30 bifidobacterial  Table 3. Tetracycline resistance and occurrence of tetracycline resistance genes among 100 bifidobacterial strains of seven species.  strains. This study further investigated the tetracycline-resistant phenotype and genotype of these 92 strains and found that 29 bifidobacterial strains exhibited tetracycline resistance. Notably, nine bifidobacterial strains, including B. longum strains F313 and F21, B. pseudocatenulatum strains J56, H23, Z25, a39, Y1, and F312, and B. bifidum strain Y21, simultaneously exhibited MLS and tetracycline resistance. Bifidobacteria have been regarded as traditional safe probiotics in the human GIT 7,8 , and only tet(W)-and tet(M)-mediated tetracycline resistance had been reported in human intestinal bifidobacteria [10][11][12][13] . However in the present study, acquired tetracycline resistance in the 29 bifidobacterial strains was mediated by tet(W), tet(O) or tet(S), and this study provides the first report of tet(O)-and tet(S)-mediated tetracycline resistance in bifidobacteria. The finding of two new tetracycline resistance genes [tet(O) and tet(S)] in bifidobacteria suggest that the selective pressure of intensive tetracycline use has caused human intestinal bifidobacteria to acquire more tetracycline resistance genes to survive and eventually become a reservoir of tetracycline resistance genes as previously speculated by many researchers [17][18][19] . It has been generally considered that the AR resistance genes carried by bifidobacteria are transferred from other bacteria in the human GIT via a number of complex mechanisms 15,20 . Previously, it was reported that the tetracycline resistance gene tet(W) in 10 human intestinal bifidobacterial strains of 5 species had a conserved sequence of 2154 bp 10 . In the present study, the tetracycline resistance gene tet(W) in 21 human intestinal bifidobacterial strains of 4 species had a conserved sequence of 2281 bp that included the previously reported 2154 bp sequence, while the 1800-bp tet(O) gene in three human intestinal B. longum strains had a conserved sequence of 2599 bp, the 1920-bp tet(O) gene in another three human intestinal bifidobacterial strains of two species had a conserved sequence of 2719 bp, and the tet(S) gene in two human intestinal bifidobacterial strains of 2 species had a conserved sequence of 430 bp. All of these conserved sequences contained the sequence of the tetracycline resistance gene [tet(W), tet(O) or tet(S)] and its partial flanking sequence, which showed 98-100% nucleotide identity with the sequence previously identified in multiple human intestinal pathogens (Arcanobacterium, Streptococcus, Corynebacterium, Campylobacter, Listeria, etc.). Not unexpectedly, with the widespread use of tetracycline in the treatment of various human bacterial infections, pathogens are indeed more likely to harbor and retain AR genes and retain them than other bacteria in the human GIT 3,21 . Therefore, our results indicate that different tetracycline resistance genes acquired by human intestinal bifidobacteria might initially be transferred from intestinal pathogens.
Because bifidobacteria rarely harbor plasmids, it is generally believed that the acquired AR genes tend to be integrated into the chromosome of bifidobacteria 22,23 (O) or tet(S) in the bifidobacterial strains in this study only exhibited 98-100% nucleotide identity with these sequences previously identified in Bifidobacterium. Hence, our results provide evidence for revealing the insertion regularity of different tetracycline resistance genes into the chromosome of human intestinal bifidobacteria, and we speculate that each kind of acquired tetracycline resistance gene might tend to insert into the vicinity of specific genes in bifidobacteria. In Gram-positive anaerobes other than bifidobacteria, a few researchers had also investigated the integration characteristic of the acquired tetracycline resistance genes tet(W) and tet(S). However, no similar genes was found flanking the tetracycline resistance genes tet(W) in the two Lactobacillus reuteri strains 24 and no similar genes were found flanking the tetracycline resistance genes tet(S) in the six Streptococcus dysgalactiae subsp. equisimilis strains 25 . Thus, unlike in bifidobacteria, the tetracycline resistance genes tet(W) and tet(S) in the other Gram-positive anaerobes might exhibit random insertion sites, which remains to be further studied.
Commercially used bifidobacterial strains are commonly screened from the healthy human GIT 26,27 . However, it had been verified that one B. longum strain F8 isolated from the healthy human GIT could transfer the tetracycline resistance gene tet(W) to Butyrivibrio adolescentis strain L2-3229 12 . Thus, considering that the AR genes harbored by bifidobacterial strains could have the potential risk of transfer to pathogenic bacteria in the human GIT and become a treat to human healthy 28,29 , the EFSA recommended that bacterial strains for commercial use should not harbor any transferable AR genes 16 . Over the past few years, only tet(W)-and tet(M)-mediated tetracycline resistance had been detected in human intestinal bifidobacteria [10][11][12][13] ; thus, human intestinal bifidobacterial strains lacking the tet(W) and tet(M) genes would be considered as relatively safe. However, this study detected two new tetracycline resistance genes, tet(O) and tet(S), in human intestinal bifidobacteria in addition to tet(W) and further investigated the potential transferability of tet(W), tet(O) and tet(S) in bifidobacteria via filter mating experiments. Although no transfer of tet(W), tet(O) or tet(S) was observed via filter mating, this does not confirm that the tet(W), tet(O) or tet(S) in these bifidobacterial strains could not be transferred in the human GIT, since the actual transfer process of AR genes that occurs in the GIT usually occurs over a much longer period of time 15 . Therefore, the presence of the tetracycline resistance genes tet(O) and tet(S) should also be considered in the safety assessment of human intestinal bifidobacterial strains prior to commercial use.
In summary, this study has provided additional genetic knowledge regarding acquired tetracycline resistance in bifidobacteria isolated from the healthy human GIT. The detection of two new tetracycline resistance genes [tet(O) and tet(S)] in human bifidobacteria indicates that human intestinal bifidobacteria have begun to harbor more AR genes, and that the screening of bifidobacterial strains from the healthy human GIT for commercial use faces additional challenges. Bacterial strains and growth conditions. One hundred individual bifidobacterial strains belonging to seven species were investigated in the present study: of these, one was a type strain, seven were commercial strains, and 92 were isolated from the feces of 14 healthy individuals (Tables 1 and 2). The first letter in the names of the 92 strains, "J", "L", "F", "W", "N", "Y", "A", "Z", "D", "X", "H", "a", "B", or "S", indicates the origin among the 14 individuals. The number of strains of each species in the 100 tested strains was as follows: Bifidobacterium longum, 45; Bifidobacterium breve, 18; Bifidobacterium lactis 19; Bifidobacterium pseudocatenulatum, 12; Bifidobacterium bifidum, 3; Bifidobacterium adolescentis, 2; Bifidobacterium infantis, 1.

Antimicrobial susceptibility.
The MIC values of tetracycline in these 100 bifidobacterial strains were determined using Etest strips (bioMérieux, Marcy-l'Étoile, France), according to the manufacturer's recommendations. Prior to the assay, the strains were anaerobically cultured in MRSC medium at 37 °C for 24 h. An inoculum was then suspended in MRSC broth to achieve the turbidity of a 1.0 McFarland standard (3 × 10 8 cells/ml) and was subsequently uniformly applied to an agar plate with a sterile cotton swab in three directions. After drying for 20 or 30 min, tetracycline Etest strips with antimicrobial gradients ranging from 0.016 to 256 μg/ml were placed on the agar plates. The MIC values were visually defined as the lowest tetracycline concentration at which no growth was observed with the Etest strip after aerobic incubation at 37 °C for 48 h. The interpretation of the tetracycline susceptibility status of these strains was based on the tetracycline breakpoint for Bifidobacterium (MIC = 8 μg/ ml) defined by the EFSA 16 . Each assay was repeated three times in duplicate.
PCR amplification and sequencing. Genomic DNA from the 100 bifidobacterial strains was extracted according to the method of Ausubel and colleagues 30 Table 4. The primers used to detect tet(M), tet(T), tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(K), and tet(L) were chosen as previously described [31][32][33] , while three sets of primers (tetW_F and tetW_R, tetO_F and tetO_R, and tetS_F and tetS_R) were designed to detect the tet(W), tet(O), and tet(S) genes based on the tet(W) sequence of Bifidobacterium animalis subsp. lactis CNCM I-2494 (GenBank CP002915.1), the tet(O) sequence of Streptococcus suis BM407 (GenBank FM252032.1), and the tet(S) sequence of Lactococcus lactis subsp. lactis strain ILIBB-JZK (GenBank KF278750.1), respectively. PCR assay was performed with TaKaRa Ex Taq DNA polymerase using the component concentration recommended by the provider (TaKaRa, Dalian, China). PCR products were separated by electrophoresis on a 1.0% agarose gel and visualized by ethidium bromide staining. All positive amplicons were purified by a PCR purification spin kit (Qiagen, Germany) and subsequently sequenced by the BGI Company (Shanghai, China). The obtained sequences were compared with those in GenBank.
Genome walking. Nested PCR was conducted to amplify the flanking sequences of the tet(W) genes in 21 bifidobacterial strains, the tet(O) genes in 6 bifidobacterial strains, and the tet(S) genes in two bifidobacterial strains using a Genome Walking Kit (TaKaRa, Dalian, China), following the manufacturer's recommendations. The nested PCR assays were performed in three steps using the same AP primer and three reverse SP primers (SP1, SP2, and SP3) designed under the conditions suggested by the kit instructions. The SP primers groups (SP1, SP2, and SP3) are listed in Table 3 and were designed to amplify the upstream and downstream sequences flanking the tet(W), tet(S), and tet(O) genes. In particular, two groups of SP primers were designed to amplify the downstream flanking sequences of the 1457-bp and 1800-bp tet(O) genes. All positive amplicons obtained in ScienTific RepORts | 7: 6267 | DOI:10.1038/s41598-017-06595-0 Filter mating experiments. The potential transferability of the tet(W) genes from 21 bifidobacterial strains, the tet(O) genes from 6 bifidobacterial strains, and the tet(S) genes from two bifidobacterial strains (donors) to Enterococcus faecalis StF-EFM (recipient) was investigated by filter mating experiments, following the method of Gevers and colleagues 34 . Briefly, the donor and recipient cells were grown to mid-exponential phase in MRSC medium prior to assay, and 1 ml of donor and 1 ml of recipient culture were mixed. Subsequently, the mixture (2 ml) was dispensed onto a sterile filter (0.45 μm; MF-Millipore membrane filter, HAWP 02500, Millipore) that was then anaerobically incubated on non-selective BHI agar (Oxoid) at 37 °C for 24 h. The cells were collected from the filters by centrifugation and resuspended in 1 ml of PBS. The transconjugants were aerobically detected on Pfizer Enterococcus Selective (PSE) agar supplemented with tetracycline (16 μg/ml), since only Enterococcus faecalis StF-EFM (recipient) can grow on PSE agar under aerobic conditions. Transfer frequencies were defined as the number of transconjugant colonies per recipient colony formed after the mating period. Data Availability. The datasets generated during the current study are included in this article and are available from the corresponding author on reasonable request.