Overexpression of resistance-nodulation-cell division pump AdeFGH confers multidrug resistance in Acinetobacter baumannii

Abstract

Acinetobacter baumannii is a major nosocomial pathogen which frequently develops multidrug resistance by acquisition of antibiotic resistance genes and overexpression of intrinsic efflux systems, such as the RND efflux pumps AdeABC and AdeIJK. A third RND system was characterized by studying spontaneous mutants BM4663 and BM4664, which were selected in the presence of chloramphenicol and norfloxacin, respectively, from the AdeABC- and AdeIJK-defective derivative A. baumannii BM4652. They exhibited enhanced resistance to fluoroquinolones, tetracycline-tigecycline, chloramphenicol, clindamycin, trimethoprim, sulfamethoxazole, sodium dodecyl sulfate, and dyes such as ethidium bromide, safranin O, and acridine orange. Comparison of transcriptomes of mutants with that of their parental strain, using a microarray technology, demonstrated the overexpression of three genes that encoded an RND efflux system, named AdeFGH. Inactivation of AdeFGH in BM4664 restored an antibiotic susceptibility profile identical to that of BM4652, indicating that AdeFGH was cryptic in BM4652 and responsible for multidrug resistance in its mutants. RNA analysis demonstrated that the three genes were cotranscribed. The adeFGH operon was found in 36 out of 40 A. baumannii clinical isolates, but none of the 22 isolates tested overexpressed the pump genes. Spontaneous MDR mutant BM4684, overexpressing adeFGH, was obtained from clinical isolate BM4587, indicating that adeFGH can be overexpressed in a strain harboring adeABC-adeIJK. An open reading frame, coding a LysR-type transcriptional regulator, named adeL, was located upstream from the adeFGH operon and transcribed in the opposite direction. Mutations in adeL were found in the three adeFGH-overexpressing mutants, suggesting that they were responsible for overexpression of AdeFGH.

FIG. 1.

Schematic representation of the adeFGH operon of A. baumannii BM4664 and derivative BM4679. Open arrows indicate coding sequences and direction of transcription. The streptomycin-spectinomycin ant(3")-Ia resistance gene is hatched. Black arrows indicate the position and orientation of the primers used.

FIG. 2.

Analysis of the adeL-adeF intergenic region. Start codons of adeL and adeF genes are in bold; arrows represent transcripts and indicate direction of transcription; promoters for adeL and adeFGH, predicted by the BProm software, are indicated on each strand. +1, in bold, start of transcription; −10 and −35 regions are in italics and indicated by bold lines; the typical TTA-N₇-TAA sequence of the LTTR box is boxed.