A plasmid-mediated qnrB19 marker was detected in four isolates and this gene was completely characterized. A collection of 93 Salmonella spp. isolates recovered between 2002 and 2009 from a variety of food products and animals
in Colombia was obtained from the University of Cordoba (Colombia). Isolates were streaked on XLD medium (Oxoid, Basingstoke, UK) to check for purity, and were confirmed as Salmonella using a Salmonella latex test (Oxoid). Susceptibilities to 15 drugs were determined by disc diffusion and interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines (2007). The following antimicrobial compounds were used: amoxicillin–clavulanic acid 20/10 μg (AMC), ampicillin 10 μg (AMP), cefpirome 30 μg (CFP), cefpodoxime 10 μg (CPD), ceftiofur 30 μg (CFR), cephalothin 30 μg (KF), see more chloramphenicol 30 μg (C), ciprofloxacin 5 μg (CIP),
gentamicin 10 μg (GM), kanamycin 30 μg (KAN), nalidixic acid 30 μg (NA), neomycin 30 μg (NEO), streptomycin 10 μg (S), trimethoprim/sulfamethoxazole 25 μg (SXT), and tetracycline 30 μg (TE). Discs were purchased from Oxoid. Escherichia coli ATCC® 25922 was included as a control. MICs for nalidixic acid (Sigma-Aldrich, Ireland) and ciprofloxacin (Sigma-Aldrich) were determined by the broth microdilution method (CLSI, 2007), in the absence and presence of 40 μg mL−1 phe-arg-β-naphthylamide (PAβN) (Sigma–Aldrich). Genomic DNA extraction, PCR Selleck Bcl2 inhibitor purification and sequencing were performed as described previously (O’Regan et al., 2009). Table 1 provides the details of all primer sequences, annealing temperatures and amplicon sizes. Positive controls for the detection of PMQR genes
were included: E. coli Lo qnrA1+, K. pneumoniae B1 qnrB1+, E. coli S7 qnrS1+, E. coli TOP10+pCR2.1W qepA and E. coli Meloxicam 78-01 aac(6′)-Ib-cr+. Nalidixic acid-resistant isolates were assessed for all known PMQR markers using previously published primers (Table 1). Plasmids were purified from nalidixic acid-resistant isolates using the PureYield™ Plasmid Midiprep System (Promega, Madison, WI) and their profiles were determined in a 0.9% agarose gel SeaKem®LE Agarose (Lonza, Wokingham, UK) after electrophoresis in 1 × Tris-HCl (pH 8)–boric acid–EDTA buffer containing 0.1 μg mL−1 ethidium bromide (Sigma-Aldrich). Using a PCR-based method developed previously by Pallecchi et al. (2010), the ColE-like plasmid carrying qnrB19 genetic determinant was amplified and the sequence was determined (Qiagen, Hilden, Germany). Complete amplified plasmid products were subjected to restriction fragment length polymorphism (RFLP) analysis with MboII enzyme (New England Biolabs, Ipswich, MA) to identify any sequence-based polymorphisms. The complete sequence of these plasmids was determined (Qiagen) and analysed using blast (http://www.ncbi.nlm.nih.gov/BLAST/), clustalw (http://www.ebi.ac.uk/clustalw/) and dnastar (DNAStar Inc., Madison, WI) programs.