In vitro selection of fluoroquinolone resistance in Brucella melitensis

Moxifloxacin-resistant mutants of Brucella melitensis 16M [moxifloxacin minimum inhibitory concentration (MIC)=1mg/L] were selected in order to characterise fluoroquinolone resistance mechanisms in this species. Eight independent mutants were obtained, with moxifloxacin MICs of 16-32mg/L. The mutants displayed variable cross-resistance levels to other fluoroquinolone compounds, but no increased resistance to aminoglycosides, tetracycline, rifampicin, macrolides or co-trimoxazole. Sequencing of type II topoisomerase-encoding genes (gyrA, gyrB, parC and parE), which are natural targets for fluoroquinolones, revealed a gyrA mutation leading to the amino acid substitution Ala83Val (Escherichia coli numbering system) in five mutants with a moxifloxacin MIC of 32mg/L, whereas no mutation was found in the remaining three mutants with a MIC of 16mg/L. Phenylalanine-arginine-beta-naphthylamide dihydrochloride, an efflux pump inhibitor, reduced moxifloxacin MICs by a factor of two to eight in all resistant mutants. In B. melitensis, fluoroquinolone resistance may arise from gyrA mutation and efflux pump overexpression mechanisms.