APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2009, p. 6352���6360 Vol. 75, No. 19 0099-2240/09/$08.00 0 doi:10.1128/AEM.00470-09 Copyright �� 2009, American Society for Microbiology. All Rights Reserved. Intra- and Interspecies Conjugal Transfer of Tn916-Like Elements from Lactococcus lactis In Vitro and In Vivo Joanna Boguslawska,1��� Joanna Zycka-Krzesinska,1 Andrea Wilcks,2 and Jacek Bardowski1* Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland,1 and Department of Microbiology and Risk Assessment, National Food Institute, Technical University of Denmark, M��rkh��j Bygade 19, DK-2860 S��borg, Denmark2 Received 25 February 2009/Accepted 30 July 2009 Tetracycline-resistant Lactococcus lactis strains originally isolated from Polish raw milk were analyzed for the ability to transfer their antibiotic resistance genes in vitro, using filter mating experiments, and in vivo, using germfree rats. Four of six analyzed L. lactis isolates were able to transfer tetracycline resistance determinants in vitro to L. lactis Bu2-60, at frequencies ranging from 10 5 to 10 7 transconjugants per recipient. Three of these four strains could also transfer resistance in vitro to Enterococcus faecalis JH2-2, whereas no transfer to Bacillus subtilis YBE01, Pseudomonas putida KT2442, Agrobacterium tumefaciens UBAPF2, or Escherichia coli JE2571 was observed. Rats were initially inoculated with the recipient E. faecalis strain JH2-2, and after a week, the L. lactis IBB477 and IBB487 donor strains were introduced. The first transconjugants were detected in fecal samples 3 days after introduction of the donors. A subtherapeutic concentration of tetracycline did not have any significant effect on the number of transconjugants, but transconjugants were observed earlier in animals dosed with this antibiotic. Molecular analysis of in vivo transconjugants containing the tet(M) gene showed that this gene was identical to tet(M) localized on the conjugative transposon Tn916. Primer-specific PCR confirmed that the Tn916 transposon was complete in all analyzed transconjugants and donors. This is the first study showing in vivo transfer of a Tn916-like antibiotic resistance transposon from L. lactis to E. faecalis. These data suggest that in certain cases food lactococci might be involved in the spread of antibiotic resistance genes to other lactic acid bacteria. The abuse of antibiotic use is regarded as the major cause of the accumulation and dissemination of antibiotic resistance genes in the environment (33). For several decades, studies on selection and spread of antibiotic resistance genes have fo- cused mainly on clinically relevant microbial species. Never- theless, many investigators have recently speculated that com- mensal bacteria, including lactic acid bacteria (LAB), may act as reservoirs of antibiotic resistance determinants (40). Genes conferring acquired resistance to tetracycline, erythromycin, and vancomycin have been detected and characterized for Lac- tococcus, Enterococcus, and Lactobacillus species isolated from fermented meat and milk products (13, 18, 23, 49, 50, 56). Introduction of such bacteria into humans through ingestion of commercial food products may have negative consequences by dissemination of antibiotic resistance genes via the food chain to the resident microbiota of the human gastrointestinal tract and, in the worst case, to pathogenic bacteria (4, 17, 55). Therefore, it seems important to assess the risk of antibiotic resistance gene transmission in the environment and in the guts of animals and humans and to establish the genetic basis of the detected resistance and transmission mechanisms. Dissemination of genetic information by horizontal gene trans- fer is common in the microbial world and is accomplished mainly by the following three mechanisms: natural transformation, con- jugation, and transduction (14). Many antibiotic resistance genes have been detected on mobile genetic elements, such as plasmids and conjugative transposons, and it is believed that conjugation is the main mode of horizontal dissemination of antibiotic resis- tance determinants between bacterial species. Conjugative transposons mediate their own transfer from a donor DNA molecule in one bacterial cell to a target molecule in another cell. Tn916, which spans about 18 kb and confers resistance to tetracycline via tet(M), belongs to the Tn916- Tn1545 family of conjugative transposons and was first identi- fied in Enterococcus faecalis DS16 (20). It is able to be main- tained in a wide range of clinically important gram-positive and gram-negative species (12, 44). Excision of Tn916 from the donor molecule is required for conjugative transposition and results in a covalently closed circu- lar transposon molecule that is an intermediate in conjugal trans- fer (10). A single strand of the covalently closed circular transpo- son is transferred to the recipient cell, where the complementary strand is synthesized to recreate a double-stranded circular trans- poson, which inserts into a target site (48). Lactococcus lactis strains are used worldwide as starter or- ganisms in the dairy industry and for the manufacturing of many fermented products. Conjugation has been described widely for lactococci, although mainly for exploitation of this process for development of improved starter strains (22, 38, 39, 51, 53). The objective of the present study was to establish the ability of wild-type L. lactis isolates to transfer tetracycline resistance determinants to gram-positive bacteria, namely, L. lactis Bu2- 60, E. faecalis JH2-2, and Bacillus subtilis YBE01, and to gram- negative bacteria, namely, Pseudomonas putida KT2442, Agro- * Corresponding author. Mailing address: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland. Phone: 48 22/659 70 72. Fax: 48 22/592 21 90. E-mail: jacek@ibb.waw.pl. ��� Present address: Department of Biochemistry and Molecular Bi- ology, The Medical Centre of Postgraduate Education, Marymoncka 99, 01-813 Warsaw, Poland. Published ahead of print on 7 August 2009. 6352

bacterium tumefaciens UBAPF2, and Escherichia coli JE2571, by using the filter mating approach. In order to confirm whether these donor strains were able to transfer the tetracy- cline resistance genes to E. faecalis JH2-2 in vivo in the gas- trointestinal tract, we also used germfree rats. MATERIALS AND METHODS Bacterial strains and growth conditions. The strains used in this study and their growth conditions are listed in Table 1. The L. lactis tetracycline-resistant strains IBB28, IBB160, IBB161, IBB224, IBB477, and IBB487, used as donor strains for mating experiments, were originally isolated from samples of Polish artisanal dairy products or raw milk (J. Zycka-Krzesinska, J. Boguslawska, and J. Bardowski, unpublished data) and were grown on GM17 plates containing tet- racycline at 30��C for 24 to 48 h. The following strains were used as recipients in conjugal transfer experiments: E. faecalis JH2-2, E. faecalis JH2-SS, L. lactis subsp. lactis bv. diacetylactis Bu2-60, B. subtilis YBE01, P. putida KT2442, A. tumefaciens UBAPF2, and E. coli JE257. Transconjugants were grown on recip- ient-specific agar plates supplemented with tetracycline. Antibiotics (Sigma, St. Louis, MO) were used at the following concentrations: rifampin (rifampicin), 50 g ml 1 streptomycin, 500 g ml 1 tetracycline, 10 g ml 1 fusidic acid, 25 g ml 1 spectinomycin, 500 g ml 1 chloramphenicol, 25 g ml 1 and kanamy- cin, 50 g ml 1. In vitro mating experiments. The ability of the lactococcal strains to transfer the tetracycline resistance genes to E. faecalis JH2-2, E. faecalis JH2-SS, L. lactis Bu2-60, and B. subtilis YBE01 was examined by the filter mating approach. The mating procedure was performed as described earlier (32). In short, exponen- tially growing donor and recipient strains were mixed in a total volume of 2 ml and poured onto a sterile membrane filter (HAWP04700 Millipore, Bedford, MA), which was incubated right side up for 18 to 20 h at 30��C, 37��C, or 42��C (depending on the recipient) on nonselective recipient-specific agar plates. Con- trol cultures of recipient and donor strains alone were treated in the same manner. After incubation, the bacteria were washed from the filters by rigorous shaking in 2 ml of PPS (8.5 g/l NaCl [Merck, Darmstadt, Germany] containing 1 g/liter neutralized bacteriological peptone [Oxoid, Hampshire, England]). Dilu- tions were spread onto donor-, recipient-, and transconjugant-selective agar plates. After growth at 30��C for 1 to 2 days, the numbers of transconjugant, donor, and recipient colonies were determined. Potential transfer of tetracycline resistance to P. putida KT2442, A. tumefaciens UBAPF2, and E. coli JE2571 was examined by the filter mating approach de- scribed above, with various modifications, as follows. Exponentially growing donor and recipient strains were mixed at a 20:1 ratio (41). Aliquots of 2 ml of mating mixture were filtered by sterile membrane filters, which were then placed on brain heart infusion agar containing DNase I (100 U/ml) to hamper transfer by transformation. After 18 h of incubation at 37��C, the cells were washed out of the filters and plated on agar containing appropriate antibiotics to select for the donor, recipient, and transconjugants. To study whether subinhibitory (sub-MIC) concentrations of tetracycline (which had no inhibitory effect on bacterial growth) would have any influence on conjugal transfer frequency, conjugation experiments between donors L. lactis IBB477 and IBB487 and recipients L. lactis Bu2-60 and E. faecalis JH2-2 were conducted as described above, with the following modifications: sterile membrane filters were incubated on nonselective recipient agar plates and on recipient agar plates contain- ing tetracycline at the following concentrations: 0.1, 0.15, and 0.2 g/ml. Animal management and experimental design. Seventeen male germfree Sprague-Dawley rats, originally supplied by Taconic (Germantown, NY), were bred at the National Food Institute, Technical University of Denmark. The rats were 3 months old at the beginning of the experiment and were housed and fed as previously described (57). The germfree status of the animals was verified by testing fecal samples for aerobic and anaerobic growth of bacteria and yeasts. The rats were caged in two isolators and grouped in the following way: group A, one rat receiving only the E. faecalis JH2-2 recipient strain, used as a control group B, four animals dosed with the donor strain L. lactis IBB477 and with E. faecalis JH2-2 group C, four animals dosed with L. lactis IBB477, E. faecalis JH2-2, and drinking water containing tetracycline at 50 g/ml group D, one rat receiving only the E. faecalis JH2-2 recipient strain, used as a control group E, four animals dosed with the donor strain L. lactis IBB487 and with E. faecalis JH2-2 and group F, three animals dosed with L. lactis IBB487, E. faecalis JH2-2, and drinking water containing tetracycline at 50 g/ml. Isolator 1 contained groups A to C, whereas isolator 2 contained groups D to F. At day 0, all rats received a 1-ml dose containing about 1010 CFU of the recipient strain E. faecalis JH2-2. The recipient strain was allowed to establish itself in the rats for 1 week, after which the donor strains were introduced. Each day from days 8 to 10, all rats in isolator 1 (except the control rat) received a 1-ml dose containing 108 CFU L. lactis IBB477 and all rats in isolator 2 (except the control rat) were dosed with 1 ml containing 108 CFU L. lactis IBB487. Tetra- cycline was added to the dosing cultures of donors and received by animals in groups C and F during the same period. Overnight cultures of all strains were washed and resuspended in phosphate-buffered saline (Oxoid), and 1 ml was given to each rat by oral gavage. Fresh fecal samples (100 to 300 mg) were obtained directly from the rats each working day by gently pressing the abdomens of the animals. Intestinal samples from the duodenum, ileum, cecum, and colon were taken at sacrifice. The samples were initially diluted 10-fold (wt/vol) in phosphate-buffered saline, thor- oughly homogenized, further diluted, and plated on the appropriate selective agar plates for enumeration of donors, recipients, and transconjugants. DNA preparation and manipulations. Total DNA was isolated from L. lactis cells by the following method. Cells from 2 ml of overnight culture were harvested, TABLE 1. Bacterial strains used in this study and their cultivation conditionsa Strain Relevant properties Cultivation conditions Reference or origin Gram-positive bacteria E. faecalis strains BHI, 42��C JH2-2 Rifr Fusr 27 JH2-SS StrrSpr 51 L. lactis strains IBB28 Tetr tet(S) gene GM17, 30��C Zycka-Krzesinska et al., unpublished data IBB160 Tetr tet(S) gene IBB161 Tetr tet(M) gene IBB224 Tetr tet(S) and tet(M) genes IBB477 Tetr tet(S) and tet(M) genes IBB487 Tetr tet(M) gene 35 Bu2-60 Strr Rifr B. subtilis YBE01 Cmr LB, 37��C 20 Gram-negative bacteria P. putida KT2442 Cmr Kmr Rifr LB, 37��C 10 A. tumefaciens UBAPF2 Strr Kmr Rifr LB, 30��C 25 E. coli JE2571 Rifr LB, 37��C 7 a Abbreviations: Rifr, rifampin resistance Fusr, fusidic acid resistance Strr, streptomycin resistance Spr, spectinomycin resistance Tetr, tetracycline resistance Cmr, chloramphenicol resistance Kmr, kanamycin resistance BHI, brain heart infusion medium (Oxoid) GM17, M17 medium supplemented with 0.5% glucose (Difco) LB, Luria-Bertani medium (Difco). 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