JAC Advance Access published online on January 29, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkl515
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Studies on the mechanisms of ß-lactam resistance in Bordetella bronchiseptica
1 Institut für Tierzucht, Bundesforschungsanstalt für Landwirtschaft (FAL), Höltystr. 10, 31535 Neustadt-Mariensee, Germany 2 Institut für Medizinische Mikrobiologie, Immunologie und Parasitologie, Pharmazeutische Mikrobiologie, Universität Bonn, Meckenheimer Allee 168, 53115 Bonn, Germany
Received 5 October 2006; returned 17 November 2006; revised 18 November 2006; accepted 20 November 2006
* Corresponding author. Tel: +49-5034-871-241; Fax: +49-5034-871-246; E-mail: stefan.schwarz{at}fal.de
OBJECTIVES: Little is currently known about ß-lactam resistance in Bordetella bronchiseptica. So far, only a single ß-lactamase gene, blaBOR-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 ß-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. ß-Lactamases were characterized by SDS–PAGE and isoelectric focusing. The genomic relatedness of the isolates was investigated by XbaI 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 blaOXA-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 ß-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 ß-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.
Key Words: class D ß-lactamases , blaOXA-2 , class 1 integrons , membrane permeability , efflux , cephalosporins