Individual rectal swabs from wild pigs, samples of intestinal content from small terrestrial mammals or samples of faeces were placed overnight in buffered peptone water (BPW) at 37°C, then cultivated for E. coli and tested for susceptibility to 12 antimicrobial agents as previously described (Literak et al. 2007). Briefly, one colony of each plate was tested for susceptibility to antimicrobial agents in accordance with CLSI (The Clinical and Laboratory Standards Institute, Wayne, PA, USA). Antibiotic susceptibility was tested by disk diffusion method on Mueller–Hinton agar (CM337; Oxoid, UK) using antibacterial substances: amoxicillin–clavulanic acid (30 μg), ampicillin (10 μg), cephalothin (30 μg), ceftazidime (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), gentamicin (10 μg), nalidixic acid (30 μg), streptomycin (10 μg), trimethoprim–sulfamethoxazole (25 μg), sulfonamides compounds (300 μg) and tetracycline (30 μg) (Oxoid). In E. coli isolates found to be resistant to one or more of the antibiotics listed above, polymerase chain reaction (PCR) was used to detect specific antibiotic-resistant genes, integrase genes int1 and int2, and gene cassettes within class 1 and 2 integrons (Dolejska et al. 2007; Literak et al. 2007). The presence of genes tetA, tetB, tetC, tetD, tetE, tetG, blaTEM, blaSHV, blaOXA-2-like, blaOXA-1/-30, cat, cmlA, floR, sul1, sul2, sul3, strA, int1, int2, variable region of class 1 integron, variable region of class 2 integron, dhfr1, dhfr12, dhfr17, aadA1, aadA2, aadA5, estX and sat1/2 was tested with primers and under the conditions listed in Table 1. The control strains E. coli F134, H195, HR17, M2, M44, M66, M148, OP1, R128, Aeromonas sp. A233, Salmonella enterica serovar Typhimurium DT104S1 and Klebsiella pneumoniae ATTC700603 were used. All E. coli strains were identified by the API 10S test (BioMerieux, France).
Antimicrobial susceptibility of all the ESBL-positive isolates was tested quantitatively by broth microdilution with cation-adjusted Mueller–Hinton broth, according to CLSI guidelines (CLSI 2008b). Microtitre trays were used with dehydrated dilution ranges of custom-made panels of antibiotics (Trek Diagnostic System, East Grinstead, UK). The following antimicrobial agents were included in the panel: amoxicillin–clavulanic acid, ampicillin, apramycin, cefpodoxime, ceftiofur, cephalotin, chloramphenicol, ciprofloxacin, colistin, florfenicol, gentamicin, nalidixic acid, neomycin, spectinomycin, streptomycin, sulfamethoxazole, tetracycline and trimethoprim with the test ranges and Subcommittee on Veterinary Antimicrobial Susceptibility Testing (CLSI/VAST) breakpoints as described previously (CLSI 2008b). Identification of E. coli phylogenetic groups was performed using a multiplex PCR assay (Clermont et al. 2000) in all the ESBL isolates. By this method, E. coli isolates can be devided into four main phylogenetic groups (A, B1, B2 and D) according to the presence of chuA and yjaA genes and DNA fragment TSPE4.C2. The isolates were typed by XbaI-pulsed field gel electrophoresis (PFGE) (CDCP 2004). The samples with no band differences were designed as indistinguishable and possibly epidemiologically linked, and the samples with the different PFGE patterns (more than three band changes) were epidemiologically unrelated (Tenover et al. 1995).
Transferability of bla genes was tested by conjugation. Plate mating experiments were performed using plasmid-free, rifampicin-resistant and nalidixic acid-resistant E. coli MT102RN and Salm. Typhimurium SL5325 as recipients (Caroff et al. 1999; Olesen et al. 2004). The strains were grown to exponential phase, mixed (1 : 1), and 500 μl of the donor and recipient mixture was incubated using a bacteriological filter on the surface of blood agar at 37°C overnight. Transconjugants were selected on brain heart infusion (BHI) medium supplemented with 25 mg l−1 rifampicin, 32 mg l−1 nalidixic acid and 2 mg l−1 cefotaxime.
The following abbreviations are used for resistance phenotype in this study: ampicillin (A), amoxicillin–clavulanic acid (Ac), chloramphenicol (C), cephalotin (Cf), ceftazidime (Cfz), ciprofloxacin (Cip), gentamicin (Gn), nalidixic acid (Na), streptomycin (S), sulfonamides cp. (Su), trimethoprim–sulfamethoxazole (Sxt) and tetracycline (T).