Susceptibility tests were interpreted using the Clinical and Laboratory Ro 61-8048 Standards Institute guidelines [27]. PCR amplification DNA used as template for PCR reactions was prepared from overnight L-broth cultures incubated at 37°C. Bacterial cells were harvested by centrifugation

and re-suspended in 1 ml 10 mM Tris/HCl (pH8·0) containing 1 mM EDTA. Template DNA was obtained by boiling for 10 min and separated by centrifugation at 12,000 × g for 3 min and then stored at -20°C until analysed. PCR was carried out in 50 μl reaction volumes containing 5 μl 10× concentrated PCR buffer [100 mM Tris/HCl (pH8·3), 500 mM KCl, 15 mM MgCl2], 5 μl (10 pmol μl-1) each of primer, 4 μl dNTP mix (2·5 mM each dNTP), 0.25 μl (5 U μl-1) Taq DNA polymerase, 5 μl of template DNA and 25.75 μl sterilized distilled water. All PCR assays were performed using an automated thermal cycler (GeneAmp PCR System 9700; Applied Biosystems). PCR products were analysed by electrophoresis in

1.5% agarose gels, stained with ethidium bromide, visualized under UV light and recorded with the aid of a gel documentation system (Bio-Rad Laboratories, Hercules, Ca, USA) Conjugation experiments and SP600125 concentration PCR screening for antibiotic resistance genes The mating assays were carried using the rifampicin-resistant E. coli C600 strain as the recipient. Conjugations were carried out at 37°C for 8 hr without shaking. Transconjugants were selected on Mueller-Hinton agar plates (Oxoid Ltd; Basingstoke, Hampshire, England) containing trimethoprim (5.2 μg/ml) and rifampicin 30 μg/ml. In order to confirm that the antibiotic resistance gene markers were transferred during conjugation, the donor and transconjugants were analysed using PCR methods. Screening of the sulII gene encoding resistance to PND-1186 sulfamethoxazole, dfrA1 encoding resistance to trimethoprim and

strB encoding resistance to streptomycin was done as described previously by Ramachandran et al. [28] while detection of the floR conferring resistance to chloramphenicol and dfrA-18 gene that also confers resistance to trimethoprim was done as described previously [7, 12]. Genomic Carnitine palmitoyltransferase II DNA from V. cholerae O139 strains ATCC 51394, CO594 and VO143 were used as positive controls templates for the screening of sulII, dfr18, strB and SXT respectively and that from O1 biotype El Tor strains KO194 was used for the screening for the dfrA1 gene. Detection of mobile genetic elements All strains were further tested for the presence of the 3′-conserved sequence (3-CS) of integron class 1 using the forward primer targeting the qacEΔ1 and the reverse primer of the sulI1 gene encoding resistance to quaternary ammonium compounds (detergents) and sulphonamides, respectively. The gene cassettes flanked by the 5′-CS and the 3′-CS were amplified using a combination of primers that target the 3′-CS and the 5′-CS of the integron class 1.