In the present study, a representative sample of 45 isolates was chosen to characterize their IncA/C plasmids. The code labels of the strains were designed to include relevant information about their isolation. The first two letters indicate the state: YU, Yucatán; SL, San Luis Potosí; MI, eFT-508 molecular weight Michoacán; and SO, Sonora. The third and fourth letters indicate the isolation source: HS, human;
PUS, pork meat; RES, beef meat; POLS, chicken meat; RAPUS, pork intestine; and RARES, beef intestine. The first two numbers indicate the year of isolation (from 2002-2007), and the last numbers are the isolate numbers. Plasmid DNA extraction and plasmid profiles Plasmid profiles were obtained by a modified alkaline lysis procedure [29] and were visualized by electrophoresis in 0.7% agarose gels subjected to 60 V for 8 hours. Plasmid profiles of E. coli V157 [30], E. coli E2348/69 [31] and E. coli AR060302 [6] were used as molecular markers for large plasmids, and supercoiled DNA ladders (Invitrogen) were used for smaller plasmids. To resolve plasmids larger than 50 kb, we performed S1 restriction PFGE. Briefly, total DNA was embedded in agarose plugs, and slices were treated with 8 U of nuclease S1 (Promega) at 37°C for 45 min. The PFGE running conditions were 6 V/Cm at 14°C for 15 hours, and switching times
ranged from 1 sec to 25 sec. A-769662 cell line The Low Range PFG Marker was used as the reference standard (New England Biolabs). Plasmid transformation and antimicrobial susceptibility testing Plasmid DNA was introduced into E. coli DH5α and TOP10 through electroporation. Transformants were selected on Luria-Bertani (LB) agar containing either 2-μg/ml ceftriaxone for the CMY+ isolates or 15-μg/ml chloramphenicol AZD9291 ic50 for the CMY- isolates. Susceptibility testing was performed by disk diffusion according to Clinical and Laboratory Standards Institute (CLSI) recommendations [32]. The following commercially purchased disks (Becton, Dickinson and Company, Sparks, MD, USA) were used: ampicillin (A), 10 μg; chloramphenicol (C), 30 μg; streptomycin (S), 10
μg; sulfonamides (Su), 250 μg; tetracycline (T), 30 μg; ceftriaxone (Ax), 30 μg; gentamicin (G), 10 μg; CYC202 mouse trimethoprim-sulfamethoxazole (Sxt), 1.25/23.75 μg; kanamycin (K), 10 μg; nalidixic acid (N), 30 μg. Resistance to ceftriaxone was confirmed by agar dilution using a breakpoint of ≥4 μg/ml. Plasmid Pst I restriction and Southern hybridization Plasmid restriction analysis with Pst I has been used for the classification of CMY+ plasmids according to Giles types [12, 20]. Giles type A has been correlated with IncA/C plasmids carrying a single bla CMY-2 copy, type B with IncI1 plasmids, and type C with IncA/C plasmids carrying two bla CMY-2 copies [6, 19]. Plasmid DNA was treated with 15 U of Pst I (Invitrogen) at 37°C for 6 hours and was electrophoresed in 0.7% agarose for 3 hours at 100 V.