Efflux-mediated resistance to florfenicol and/or chloramphenicol in Bordetella bronchiseptica: identification of a novel chloramphenicol exporter

Abstract

OBJECTIVES: Twenty florfenicol- and/or chloramphenicol-resistant Bordetella bronchiseptica isolates of porcine and feline origin were investigated for the presence of floR and cml genes and their location on plasmids. METHODS: The B. bronchiseptica isolates were investigated for their susceptibility to antimicrobial agents by broth micro- or macrodilution and for their plasmid content. Hybridization experiments and PCR assays were conducted to identify resistance genes. Transformation and conjugation studies were performed to show their transferability. Representatives of both types of genes including their flanking regions were sequenced. Moreover, inhibitor studies with the efflux pump inhibitor Phe-Arg-beta-naphthylamide (PAbetaN) were performed. RESULTS: The gene floR was found in the chromosomal DNA of 9 of the 18 florfenicol/chloramphenicol-resistant isolates. Sequence analysis revealed that the deduced FloR protein sequence differed by a single amino acid exchange from FloR of Vibrio cholerae. A chloramphenicol-resistant, but florfenicol-susceptible isolate carried a novel plasmid-borne cml gene, designated cmlB1. The CmlB1 protein revealed only 73.8-76.5% identity to known CmlA proteins. The gene cmlB1 was not part of a gene cassette. The results of inhibitor studies with PAbetaN suggested that a so-far unidentified efflux system might play a role in phenicol resistance of the remaining florfenicol- and/or chloramphenicol-resistant isolates. CONCLUSIONS: This is to the best of our knowledge the first report of a floR gene in B. bronchiseptica isolates. The identification of the first member of a new subclass of cml genes, cmlB1 from B. bronchiseptica, extends our knowledge on specific chloramphenicol exporters.