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All strains of Bifidobacterium animalis subsp. lactis described to date show medium level resistance to tetracycline. Screening of 26 strains from a variety of sources revealed the presence of tet(W) in all isolates. A transposase gene upstream of tet(W) was found in all strains, and both genes were cotranscribed in strain IPLAIC4. Mutants with increased tetracycline resistance as well as tetracycline-sensitive mutants of IPLAIC4 were isolated and genetically characterized. The native tet(W) gene was able to restore the resistance phenotype to a mutant with an alteration in tet(W) by functional complementation, indicating that tet(W) is necessary and sufficient for the tetracycline resistance seen in B. animalis subsp. lactis.
Bifidobacteria are common microorganisms in the gastrointestinal tracts of humans. The intestine of healthy newborns is quickly colonized by bifidobacteria, especially those fed with breast milk, in which they could represent the majority of the total culturable population, typically remaining dominant until weaning (7, 12). In adults, however, the intestinal bifidobacterial population is more variable, although it still constitutes one of the predominant members (29).
Different strains of the genus Bifidobacterium are considered health-promoting microorganisms for humans. Among them, Bifidobacterium animalis subsp. lactis is the most common species included in functional food products (9, 17, 27), and several beneficial effects, such as reduction of the risk of diarrhea in children and allergy relief, have been attributed to this bacterium (27).
Due to the ubiquity of B. animalis subsp. lactis in foods and dietary supplements, the presence of antibiotic resistance determinants in this bacterium is of key interest, since gut commensals possessing an antibiotic resistance gene could theoretically serve as a reservoir of resistance for intestinal pathogens via horizontal gene transfer. In this regard, resistance to tetracycline has recently been reported as the most common antibiotic resistance in bifidobacteria (1, 3, 4, 8, 18), although one incidence of erythromycin resistance related to the presence of a transposon-associated erm(X) gene has also been described previously (32). The most abundant genetic determinants responsible for the tetracycline resistance phenotype among the species of this genus are tet genes coding for ribosomal protection proteins (24). The genes tet(O), tet(M), and tet(W) and different hybrid determinants [i.e., tet(W/32/O) and tet(O/W)] were detected in bifidobacteria, tet(W) being the most commonly found (1, 4, 15, 19, 31). Remarkably, tet(W) was also found in Lactobacillus, Clostridium, and other gut bacteria, thus suggesting a wide distribution among bacteria from the mammalian gut (5, 13, 22, 23, 28). In this context, the aim of our research was to investigate the ubiquity of tet(W) among B. animalis subsp. lactis strains isolated from different origins and the influence of gastrointestinal conditions on the antibiotic resistance phenotype and to supply the basis for a correlation between genotype and phenotype.
The presence of tet(W) in 26 strains of B. animalis subsp. lactis from a variety of sources, including functional foods, probiotic supplements, dairy cultures, human feces, and culture collections, was investigated using microarray analysis and PCR. Furthermore, their tetracycline resistance levels were determined using a microdilution method. Briefly, individual colonies on agar plates were suspended in sterile saline until a density corresponding to McFarland standard of 1 was reached. The inoculated suspension was then diluted 1,000-fold in lactic acid bacterial susceptibility test medium (LSM) plus cysteine (90% Iso-Sensitest [IST] broth [Oxoid] plus 10% MRS broth [Oxoid] plus 0.3 g/liter cysteine hydrochloride [Sigma] ) containing the appropriate tetracycline concentration, and 100 μl of the diluted bacterial suspension was added to each well. Incubations were carried out at 37°C for 48 h in a Mac 500 anaerobic chamber (Don Whitley Scientific) in an atmosphere of 80% N2, 10% H2, and 10% CO2. All the strains displayed a medium level resistance to the antibiotic, close to, or above, reference breakpoints described for this genus (Table (Table1),1), suggesting the presence of specific tetracycline resistance determinants (18, 33). The microarray analysis of 24 out of the 26 strains (Table (Table1)1) was carried out as previously described (19, 30) with in-house printed DNA chips containing 223 oligonucleotides (50 to 60 nucleotides long), which represent over 430 antibiotic resistance genes, including 2 otr and 31 tet genes. The results showed, in all cases, positive hybridization signals for the oligonucleotides representing tet(W). Except for the signals with the species-specific control probes (two 60-mer oligonucleotides designed on conserved parts of the 16S rRNA [5′-gtaacggtggaatgtgtagatatcgggaagaacaccDatggcgaaggcagDtctctgggc-3′] and tuf [5′-gccgtcactcgccSttcttctcSaactaccgtccgcagttctacttccgYaccaccgacg-3′] gene sequences, respectively, of the genus Bifidobacterium (where D is A, G, or T, S is C or G, and Y is C or T), no other hybridization signals were found with any of the remaining spots, indicating the absence of other antibiotic resistance determinants tested for with this microarray. Subsequently, PCR primers were designed based on a 3,696-bp fragment of the chromosomal DNA of B. animalis subsp. lactis IPLAIC4 (GenBank accession number GU361625) (Table (Table22 and Fig. Fig.11).
In IPLAIC4, tet(W) is flanked upstream by a putative transposase gene (trp). Primers were designed in a way that allowed us to amplify the trp gene together with the 5′ end of tet(W). Thus, a positive 1.47-kb amplicon implies that both genes are organized as they are in IPLAIC4 (Fig. (Fig.1).1). PCRs were carried out according to standard procedures (4). Positive Trp and Tet amplicons were obtained for the 26 strains. Afterwards, 10 Trp and 10 Tet amplicons (from strains IPLA4549, IPLAIC1, A8dOx, M6, PBT, Y1, Class11, Essen2, A12, and C64) (Table (Table1)1) were selected for sequencing (Secugen S.L., Madrid, Spain) in both directions with the primer pairs TrpF/TrpR and TetF/TetR, and they all gave homology with the corresponding sequences of IPLAIC4. This suggests that the structural organizations of trp and tet(W) are very similar, if not identical, in all our strains. Transposases catalyze the movement of DNA between different locations by recognizing insertion sequences in the DNA and could be involved in the mobilization of tet genes in bifidobacteria. In fact, one chromosomal tet(W) gene has been shown to transfer, at low frequency, from Bifidobacterium longum to Bifidobacterium adolescentis in vitro, and the site of chromosomal insertion in B. adolescentis is identical to that of the donor strain, consistent with a transposase-mediated site-site specific insertion event (13). This suggests that trp could have been involved in the acquisition of tet(W) by B. animalis and opens the question of whether trp could participate in the transfer of tet(W) to other bacteria.
The ability of IPLAIC4 to transfer tet(W) to other bacteria was tested by filter mating on glucose M17 or MRS (both supplied by Difco) plates containing cysteine (Sigma) under anaerobic conditions essentially as previously described (26). Recipient strains included Lactococcus lactis MG1614 (resistant to rifampin and streptomycin; 2 experiments), Bifidobacterium bifidum CHCC2185 (resistant to trimethoprim; 4 experiments), Enterococcus faecalis JH2-2 (resistant to rifampin, fusidic acid, and erythromycin; 1 experiment), and mutants of B. animalis subsp. animalis ATCC 25527 and ATCC 27672 resistant to both rifampin and fusidic acid (2 experiments). Donors, recipients, and transconjugants were quantitated on plates supplemented with appropriate antibiotics. No transfer of tet(W) to any recipient was detected, indicating that the frequency of transfer is less than 10−9/recipient (data not shown). The lack of transfer of tet(W) from another B. animalis subsp. lactis strain has been previously described (18). However, the experimental conditions used do not represent the complex in vivo situation in the gut, and the gastrointestinal environment most likely will offer different conditions for antibiotic resistance transfer than those provided in vitro.
It is important to remark that a similar genetic context in B. animalis E43 was described previously (4), and an identical 3,696-bp fragment is present in the chromosomes of two recently sequenced B. animalis subsp. lactis strains (GenBank accession numbers CP001606 and CP001515). The strain AD011 also contains an almost identical DNA fragment, differing only in 1 base (GenBank accession number CP001213). The high degree of sequence conservation among our strains and the 3 currently available genome sequences indeed supports the results of Barrangou and coworkers (6) and indicates that this subspecies comprises clonal bacteria which are genomically monomorphic.
Previous studies indicated that the acquisition of a stable bile resistance phenotype or the transient exposure to bile induces an increase in tetracycline resistance in Bifidobacterium (14, 20). We tested if this phenotypic change could be correlated with the activity of tet(W). For this purpose, IPLAIC4 was incubated in MRSc broth (MRS [Difco] supplemented with 0.05% cysteine [Sigma]) containing 0.8 to 1% bile (Ox-gall; Oxoid) for 5 consecutive days (5 overnight cultures; approximately 50 generations). After that, the culture was plated on MRSc agar containing tetracycline (25, 75, or 125 μg/ml; Sigma). Hardly any colony was able to grow on 75 or 125 μg/ml, but numerous colonies were recovered on 25 μg/ml tetracycline. Interestingly, the frequency of colonies able to grow at this concentration was higher for those cultures previously grown in the presence of bile (one out of 102 to 104 CFU) than for the control culture grown in the absence of bile (one out of around 105 to 106 CFU). We analyzed 47 colonies recovered from 25-μg/ml tetracycline plates, and most of them had a transient resistance phenotype (not stable), since they revert to the original resistance level when grown in the absence of bile. This could suggest a temporal activation of tet(W) when bile is present, which is lost when bile is removed. In fact, quantitative PCR experiments indicated an induction of tet(W) (from 2- to 4-fold at bile concentrations of 0.25 and 0.75%, respectively) when IPLAIC4 was grown in the presence of bile. Also, transcriptional analysis of IPLAIC4 using dedicated genomic microarrays indicated a slight induction of tet(W) (between 1.5- and 2-fold upregulation, tested with 4 different probes) in the presence of 0.1% bile (M. Gueimonde and C. Garrigues, unpublished data). Only one of the colonies recovered from the 25-μg/ml tetracycline plates was able to grow consistently in high tetracycline concentrations. This strain was named T25 and was included in further analyses.
Mutants of IPLAIC4, sensitive to tetracycline, were isolated following mutagenesis with UV light (2 exposures of 5-min duration at 70 mJ/cm2) in the presence of ethidium bromide (20 μg/ml) and a subsequent ampicillin enrichment step. IC4-8902 and IC4-9991 were more sensitive than the original strain to tetracycline (Table (Table1).1). A mutant of IPLAIC4 with elevated resistance was isolated following subculturing in medium containing increasing concentrations of tetracycline. Mutant IC4-8658 is resistant to 100 μg/ml tetracycline.
A 3.65-kbp fragment (between primers TTF and TTR) (Fig. (Fig.1)1) containing tet(W) was sequenced in these 3 mutants and in T25 (Fig. (Fig.1),1), rendering valuable information on the potential genetic differences that lead to distinct tetracycline resistance levels. The mutant IC4-9991 contains a point mutation in tet(W) (cytosine 41 is replaced by an adenine). This introduced a different amino acid at position 14 (A14/D14) in the Walker A motif of the nucleotide binding domain (21), which could influence the activity of the protein, although in our case tet(W) seems to retain some residual activity. In the mutant IC4-8902 the thymine at position 1406 of tet(W) is missing, which yields a frameshift and a potentially shorter version of the protein (468 residues instead of 639), likely nonfunctional. On the other hand, the 3,696-bp region in the mutant IC4-8658 was 100% identical to the corresponding region in IPLAIC4, suggesting that the mutation(s) responsible for the high-resistance phenotype is located somewhere outside this region. Finally, in the mutant T25 we found a duplication of an 8-bp fragment (TGCCATAT) in the putative promoter (−35 region) of trp which could affect promoter activity.
We determined the expression levels of tet(W) in the different mutants grown in MRSc in the presence of tetracycline (antibiotic concentrations were selected on the basis of similar levels of growth inhibition among the different strains: 0.4 μg/ml for IC4-8902, 1 μg/ml for IC4-9991, 38 μg/ml for T25 and IC4-8658, and 12 μg/ml for IPLAIC4. Quantitative PCR was carried out as previously described (10) using the primers qRT-Tet1F and qRT-Tet1R (Table (Table22 and Fig. Fig.2).2). Expression levels were determined by relative quantification using the ΔΔCT method, in which the expression level in the control culture (IPLAIC4) is arbitrarily set to 1 and the expression levels in the samples are calculated relative to that control. Experiments were carried out in duplicate, each measured in duplicate in two independent PCR runs. This experiment indeed suggested that the increased tetracycline resistance in mutants T25 and IC4-8658 is due to an increased expression of tet(W).
A quantitative reverse transcription-PCR (RT-PCR) analysis was carried out in order to ascertain if trp and tet(W) were expressed independently or in the same transcript. Primers were designed in order to amplify internal fragments of the 2 genes (qRT-TrpF/qRT-TrpR and qRT-Tet1F/qRT-Tet1R), the intergenic region (qRT-IntF/qRT-IntR), the trp 3′ end together with the 5′ end of the intergenic region (qRT-TrpIntF/qRT-TrpIntR), and the 5′ end of tet(W) together with the 3′ end of the intergenic region (qRT-IntTetF/qRT-IntTetR) (Fig. (Fig.11 and Table Table2).2). The analysis was carried out with strains IPLAIC4, T25, and IC4-8902. All primers produced amplification products for the 5 amplicons in the three strains. In addition, the five amplified fragments were found to be present in equal amounts, indicating that trp and tet(W) are transcribed together as part of a bicistronic operon.
Finally, in order to unequivocally demonstrate that the tetracycline resistance phenotype in B. animalis subsp. lactis is conferred by tet(W) and not by other genes present in its genome, whose function could have passed unnoticed by us, we cloned the 3.65-kb fragment from IPLAIC4 containing trp and its upstream region and the native tet(W) gene in the mutant IC4-8902, in which a truncated, nonfunctional version of tet(W) is present. The fragment was amplified using Pfx DNA polymerase (Invitrogen) and the primers TTF and TTR according to the manufacturer's instructions. The PCR product was digested with XbaI and cloned in the Escherichia coli-Bifidobacterium shuttle vector pAM1 (2), digested with the same enzyme, to generate the plasmid pAM-TB. Cloning and transformation procedures were carried out as previously described (2). The IC4-8902 strain containing the empty vector (pAM1) displayed a tetracycline MIC of 1 μg/ml, similar to that for IC4-8902. However, IC4-8902 harboring pAM-TB showed a MIC of 32 μg/ml, even higher than that for IPLAIC4; this difference is probably due to the presence of multiple copies of the tet(W) gene. This result indicates that tetracycline resistance in B. animalis subsp. lactis is conferred by tet(W) and suggests that no other genetic determinants participate in this process.
In summary, this work shows the wide distribution of tet(W) among B. animalis subsp. lactis strains and its involvement in the tetracycline resistance phenotype of this bacterium. Bile exposure slightly induces tet(W) expression, which could influence resistance levels in certain environments. Upstream of tet(W) is a transposase gene, which is cotranscribed in tandem. Transposases have been found to be involved in the horizontal gene transfer of genetic elements among bacteria, but to date there is no evidence that tet(W) in B. animalis subsp. lactis is transmissible.
This work was financed by European Union FEDER funds and the Spanish Plan Nacional de I+D (project AGL2007-61805) and by the Consejo Superior de Investigaciones Científicas (CSIC PIE 200870I049).
María Fernández is especially thanked for her excellent assistance, and Baltasar Mayo and Pablo Álvarez-Martín are acknowledged for the generous gift of the plasmid pAM1. Christel Garrigues and Morten Danielsen are kindly acknowledged for sharing unpublished data, and Eric Johansen is thanked for fruitful discussions.
Published ahead of print on 26 March 2010.