Efflux-mediated aminoglycoside and macrolide resistance in Burkholderia pseudomallei

Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsic ally resistant to a wide range of antimicrobial agents including beta-lacta ms, aminoglycosides, macrolides, and polymyxins. We used Tn5-OT182 to mutag enize B. pseudomallei to identify the genes involved in aminoglycoside resi stance. We report here on the identification of AmrAB-OprA, a multidrug eff lux system in B. pseudomallei which is specific for both aminoglycoside and macrolide antibiotics. We isolated two transposon mutants, RM101 and RM102 , which had 8- to 128-fold increases in their susceptibilities to the amino glycosides streptomycin, gentamicin, neomycin, tobramycin, kanamycin, and s pectinomycin. In addition, both mutants, in contrast to the parent, were su sceptible to the macrolides erythromycin and clarithromycin but not to the lincosamide clindamycin. Sequencing of the DNA flanking the transposon inse rtions revealed a putative operon consisting of a resistance, nodulation, d ivision-type transporter, a membrane fusion protein, an outer membrane prot ein, and a divergently transcribed regulatorprotein. Consistent with the pr esence of an effluxystem,both mutants accumulated [H-3]dihydrostreptomycin, whereas the parent strain did not. We constructed an amr deletion strain, B. pseudomallei DD503, which was hypersusceptible to aminoglycosides and ma crolides and which was used successfully in allelic exchange experiments. T hese results suggest that an efflux system is a major contributor to the in herent high-level aminoglycoside and macrolide resistance found in B. pseud omallei.