Conjugation Experiments

Seven E. coli isolates harboring full-length tet(M) were used for filter mating experiments with sodium azide-resistant E. coli J53 as a recipient as previously described (Devirgiliis et al., 2009). Briefly, overnight cultures of tetracycline-resistant, sodium azide-susceptible donor (E. coli isolate) and tetracycline-susceptible, sodium azide-resistant recipient (E. coli J53), grown in Luria-Bertani (LB) broth, were seeded at a 1:50 dilution in separate flasks of fresh LB broth. Following growth to log phase (OD₆₀₀≈0.6) with shaking at the speed of 250 rpm at 37°C, 0.5 ml each of the donor and the recipient cultures were mixed and filtered through a 0.22-μm-pore-size membrane (Millipore Corp.) placed on prewarmed on MacConkey agar plates. After 24 h of incubation at 37°C, the cells were detached from filters in 5 ml of 1 × phosphate-buffered saline (pH7.4), and serial dilutions were plated on MacConkey agar plates containing 16 mg/L doxycycline and 100 mg/L sodium azide. Controls (unmixed donors and recipient cells) were treated in the same manner. Transconjugant colonies were recovered following incubation at 37°C for 24 to 72 h. Furthermore, amplification of the sequences of tet(A), tet(B), tet(C), and tet(M) in the transconjugants were performed using the primer sets in Table ​Table2.2. Template DNA was prepared as follows. Amounts of 1.5 ml of transconjugant culture were pelleted, and cells were boiled in 200 μl of H₂O for 15 min. After centrifugation, the supernatants were kept at −20°C. PCR was performed in a total volume of 50 μl, which contained 3 μl of supernatant, 50 pmol of each primer, 25 μl of PrimeSTAR® Max DNA Polymerase (TakaRa Biotechnology Co. Ltd, Japan). After an initial denaturation step of 3 min at 95°C, amplification was performed over 30 cycles, each one consisting of 30 s at 95°C, 30 s min at hybridization temperature [55°C for tet(A) and tet(C), 58°C for tet(B), and 52°C for tet(M)], and 1 min at 72°C, with a final extension step of 10 min at 72°C. The amplicons were sequenced using their corresponding primer set at commercial company (Shanghai Sangon Biological Engineering Technology and Services Co. Ltd, China). The sequences comparisons were performed using the BLASTtool available online at the National Center for Biotechnology Information of the National Library of Medicine (http://www.ncbi.nlm.nih.gov/blast/). Transconjugants carrying tet(M), but not tet(A), tet(B), or tet(C) were selected for further analysis.

Primers used in this study.