Studies on the mechanisms of P-lactam resistance in Bordetella bronchiseptica

Objectives: Little is currently known about beta-lactam resistance in Bordetella bronchiseptica. So far, only a single beta-lactamase gene, bla(BOR-1), has been identified. In a previous study, high MICs of ampicillin, cefalotin and ceftiofur were determined among 349 porcine B. bronchiseptica isolates. The aim of this study was to identify genes associated with elevated MICs of beta-lactams and their transferability. Methods: Selected isolates were investigated by PCR for commonly found bla genes and class 1 integrons; selected amplicons were sequenced. Plasmid location of resistance genes was confirmed by conjugation. beta-Lactamases were characterized by SDS-PAGE and isoelectric focusing. The genomic relatedness of the isolates was investigated by XbalI macrorestriction analysis. Inhibition studies with efflux pump inhibitors were conducted. The permeability of cephalosporins into intact cells was measured exemplarily for one isolate. Results: Of the 349 B. bronchiseptica isolates, eight isolates carried a class 1 integron with a bla(OXA-2) cassette on a conjugative plasmid of ca. 50 kb. In addition, one plasmid-free isolate also carried this class 1 integron. Besides blaBOR-1, no other beta-lactamase gene was detected in the remaining isolates with high MICs of ampicillin of >= 32 mg/L. Inhibition experiments suggested that efflux does not play a role in beta-lactam resistance. Instead, membrane permeability for cephalosporins was reduced as shown for B. bronchiseptica isolate B543. Conclusions: This is to the best of our knowledge the first report of a mobile bla gene in B. bronchiseptica. Reduced membrane permeability of B. bronchiseptica seems to decrease susceptibility against cephalosporins.