Characterization of chloramphenicol and florfenicol resistance in Escherichia coli associated with bovine diarrhea

Florfenicol, a veterinary fluorinated analog of thiamphenicol, is approved for treatment of bovine respiratory pathogens in the United States. However, florfenicol resistance has recently emerged among veterinary Escherichia coli isolates incriminated in bovine diarrhea. The flo gene, which confers resistance to florfenicol and chloramphenicol, has previously been identified in Photobacterium piscicida and Salmonella enterica serovar Typhimurium DT104. The flo gene product is closely related to the CmIA protein identified in Pseudomonas aeruginosa. The cmlA gene confers nonenzymatic chloramphenicol resistance via an efflux mechanism. Forty-eight E. coli isolates recovered from calves with diarrhea, including 41 that were both chloramphenicol and florfenicol resistant, were assayed for the presence of both flo and cmlA genes. Forty-two of the 44 isolates for which florfenicol MICs were greater than or equal to 16 mug/ml were positive via PCR for the flo gene. All E. coli isolates for which florfenicol MICs were less than or equal to8 mug/ml were negative for the flo gene (n = 4). Twelve E. coli isolates were positive for cmlA, and chloramphenicol MICs for all 12 were greater than or equal to 32 mug/ml. Additionally, eight isolates were positive for both flo and cmlA, and both florfenicol and chloramphenicol MICs for these isolates were > 64 mug/ml. DNA sequence analysis of the E. coli flo gene demonstrated 98% identity to the published GenBank sequences of both serovar Typhimurium flo(St) and P. piscicida pp-flo. The flo gene was identified on high-molecular-weight plasmids of approximately 225 kb among the majority of florfenicol-resistant E. coli isolates. However, not all of the Borfenicol-resistant E. coli isolates tested contained the large flo-positive plasmids. This suggests that several of the E. coli isolates may possess a chromosomal flo gene. The E. coli flo gene specifies nonenzymatic cross-resistance to both florfenicol and chloramphenicol, and its presence among bovine E. coli isolates of diverse genetic backgrounds indicates a distribution much wider than previously thought.