Occurrence and characterization of resistance to extended-spectrum cephalosporins mediated by beta-lactamase CMY-2 in Salmonella isolated from food-producing animals in Canada

Abstract

Resistance of Salmonella to extended-spectrum cephalosporins (ESCs) is being reported with increasing frequency. In humans, infections with Salmonella resistant to ESCs threaten the efficacy of ceftriaxone, the drug of choice for treating salmonellosis in children. To determine the occurrence of resistance to ESCs, we examined 8426 strains isolated from food-producing animals in Canada in 1994-99 for reduced susceptibility or resistance to ceftriaxone. Of the 8 such strains identified (7 from turkeys and 1 from cattle), 5 had reduced susceptibility, and 3 were resistant; 2 were isolated in 1995, 1 was isolated in each of 1996 and 1997, and 4 were isolated in 1999. Isoelectric focusing showed that all 8 isolates produced a beta-lactamase with a pI > or = 9. The strains were resistant to cefoxitin and not inhibited by clavulanic acid. Primers specific for the Citrobacter freundii blaAmpC gene produced the expected product in the polymerase chain reaction. DNA sequencing showed that all isolates possessed the blaCMY-2 gene. Plasmid DNA from all 8 isolates transformed Escherichia coli DH10B, whereas only 1 isolate transferred blaCMY-2 conjugally. All transformants and the transconjugant were resistant to ampicillin, cefoxitin, ceftiofur, cephalothin, streptomycin, sulfisoxazole, and tetracycline. Southern blots of plasmids from the isolates, the transformants, and the transconjugant showed that blaCMY-2 was located on similar-sized plasmids (60 or 90 MDa) in the transformants and the transconjugant. In the S. Typhimurium DT104 and S. Ohio isolates, the floSt gene was found on the same plasmid. Class 1 integrons with the aadB gene cassette were detected in the S. Bredeney isolates but not in their transformants or the transconjugant. Pulsed-field gel electrophoresis and plasmid profiles indicated that both clonal dispersion and horizontal transfer of blaCMY-2 may have caused dissemination of the resistance determinant.

Figure 1. Southern blot hybridization of the Salmonella Bredeney SA993178 donor and the Escherichia coli C600N transconjugant using a digoxigenin-labeled bla_CMY-2 probe. Lane 1 shows the S. Bredeney donor, lane 2 the cefoxitin-resistant E. coli transconjugant, and lane 3 the non-conjugated E. coli.

Figure 2. Pulsed-field gel electrophoresis patterns of Salmonella isolates possessing the bla_CMY-2 gene. Lanes 1, 5, and 9 are S. Newport am01144 digested with XbaI; lanes 2, 3, 4, and 6 are S. Bredeney SA993178, SA992542, SA992721, and SA992306, respectively; lane 7 is S. Typhimurium DT104 (SA972816); and lane 8 is the S. Typhimurium var. Copenhagen strain provided by Dr. Paul Fey. All isolates were digested with BlnI (AvrII).