Screening of qnr genes was carried out by multiplex PCR amplification of qnrA, qnrB and qnrS genes as described (Robicsek et al., 2006). All amplicons obtained
were purified using the Wizard® SV Gel and PCR clean-up system kit (Promega Corporations, Madison, WI). DNA sequencing of purified PCR products was performed by Macrogen (Macrogen Inc., Seoul, Korea). Nucleotide and amino acid sequences were analysed using mega-blast and psi-blast, respectively (http://www.ncbi.nlm.nih.gov). PCR-based Inc/rep typing was performed to identify the major incompatibility groups of the plasmids present in parental and transconjugant strains (Carattoli et al., 2005). Template DNA was prepared by extraction of total DNA using the see more GenElute™ Bacterial Genomic DNA commercial kit (Sigma). The PCR products obtained were then purified and sequenced as mentioned above.
To identify the relaxase MOB family of the plasmids present in parental and transconjugant strains, a PCR-based MOB amplification method was performed (Alvarado et al., 2008). The primers used to amplify the MOBP13 subfamily were MOBP13 forward (5′-AAC CCA CGC TGC AAR GAY CCV GT-3′) and MOBP13 reverse (5′-AGC GAT GTG p38 MAPK inhibitor GAT GTG AAG GTT RTC NGT RTC-3′). PCR conditions were one cycle of denaturation at 94 °C for 4 min, followed by 30 cycles at 94 °C for 30 s, 59 °C for 30 s, 72 °C for 15 s and a final extension at 72 °C for 5 min. The amplified DNA fragments were then purified and sequenced using primers MOBP13 forward and MOBP13 reverse clamp (5′-AGC GAT GTG GAT GTG AAG-3′). From each parental and transconjugant strain, plasmid profiles were visualized after DNA linearization with the S1 enzyme, followed by pulsed-field gel electrophoresis (PFGE) as described previously (Barton et al., 1995). Plasmid sizes were estimated using fingerprinting ii Montelukast Sodium informatix™ software. S1-PFGE was then transferred onto a nylon-membrane by Southern blotting. Purified DNA products obtained from the PCR of blaDHA-1,
qnrB genes and the replicon IncL/M were used as probes for hybridization of the S1-PFGE blots. These probes were labelled using the commercial kit Amersham ECL Direct Nucleic Acid Labelling and Detection Systems, as recommended by the manufacturer (GE Healthcare). An S. marcescens and an E. coli with an inducible AmpC-β-lactamase phenotype were isolated from a urine sample together with an E. coli with its natural susceptible pattern, a meticillin-resistant Staphylococcus aureus, an Enterococcus faecalis and a Morganella morganii. Primary antibiogram plates of S. marcescens and the resistant E. coli isolate showed oxyimino-β-lactams antagonism with imipenem or cefoxitin. Moreover, we observed scattered colonies located near the edge of cefoxitin, cefotaxime, ceftazidime and aztreonam. This pattern of susceptibility was compatible with the presence of a pACBL (Mirelis et al., 2006).