Molecular Characterization of Resistance to Extended-Spectrum Cephalosporins in Clinical Escherichia coli Isolates from Companion Animals in the United States

Resistance to extended-spectrum cephalosporins (ESC) among members of the family Enterobacteriaceae occurs worldwide; however, little is known about ESC resistance in Escherichia coli strains from companion animals. Clinical isolates of E. coli were collected from veterinary diagnostic laboratories throughout the United States from 2008 to 2009. E. coli isolates (n = 54) with reduced susceptibility to ceftazidime or cefotaxime (MIC >= 16 mu g/ml) and extended-spectrum-beta-lactamase (ESBL) phenotypes were analyzed. PCR and sequencing were used to detect mutations in ESBL-encoding genes and the regulatory region of the chromosomal gene ampC. Conjugation experiments and plasmid identification were conducted to examine the transferability of resistance to ESCs. All isolates carried the bla(CTX-M-1)-group beta-lactamase genes in addition to one or more of the following beta-lactamase genes: bla(TEM), bla(SHV-3), bla(CMY-2), bla(CTX-M-14-like), and bla(OXA-1). Different bla(TEM) sequence variants were detected in some isolates (n = 40). Three isolates harbored a bla(TEM-181) gene with a novel mutation resulting in an Ala184Val substitution. Approximately 78% of the isolates had mutations in promoter/attenuator regions of the chromosomal gene ampC, one of which was a novel insertion of adenine between bases -28 and -29. Plasmids ranging in size from 11 to 233 kbp were detected in the isolates, with a common plasmid size of 93 kbp identified in 60% of isolates. Plasmid-mediated transfer of beta-lactamase genes increased the MICs (>= 16-fold) of ESCs for transconjugants. Replicon typing among isolates revealed the predominance of IncI and IncFIA plasmids, followed by IncFIB plasmids. This study shows the emergence of conjugative plasmid-borne ESBLs among E. coli strains from companion animals in the United States, which may compromise the effective therapeutic use of ESCs in veterinary medicine.