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

Abstract

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 CmlA 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 > or =16 microg/ml were positive via PCR for the flo gene. All E. coli isolates for which florfenicol MICs were < or =8 microg/ml were negative for the flo gene (n = 4) Twelve E. coli isolates were positive for cmlA, and chloramphenicol MICs for all 12 were > or =32 microg/ml. Additionally, eight isolates were positive for both flo and cmlA, and both florfenicol and chloramphenicol MICs for these isolates were > or =64 microg/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 florfenicol-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.

FIG. 1

Phylogenetic tree of florfenicol-resistant bovine E. coli. Total DNA was digested with XbaI and separated by PFGE. Molecular weight markers were S. cerevisiae strain YPH 755 chromosomes. Similarities among E. coli PFGE patterns were identified by cluster analysis using the neighbor-joining method to draw a phylogenetic tree (21). Phylogenetic analysis identified 44 PFGE patterns in the 45 florfenicol-resistant bovine E. coli isolates assayed.

FIG. 2

Comparison of the location of the flo gene in serovar Typhimurium DT104, P. piscicida, and E. coli. PCR primers were designed to amplify a portion of flo and genes mapping downstream (tetR) within the Salmonella MDR locus or to amplify flo and the RSF1010 replicon identified in P. piscicida. Primers flomap2F and pp-floMapR anneal to nucleotide positions 1953 and 2723 of the P. piscicida plasmid-mediated flo sequence, resulting in a 771-bp amplicon. Primers flomap2F and flomap2R anneal at nucleotide positions 5330 of flo and 6073 of tetR within the serovar Typhimurium DT104 MDR locus, resulting in a 744-bp amplicon.

FIG. 3

Location of the flo resistance gene in bovine E. coli genomes by PFGE and Southern analysis. Total E. coli genomic DNA, plasmid and chromosome, was cut with XbaI, separated by PFGE, and probed with labeled flo. Lanes 2 to 15, bovine E. coli isolates. Avian E. coli strain 5790 served as the positive control for flo (lane 1).