AdeIJK, a resistance-nodulation-cell division pump effluxing multiple antibiotics in Acinetobacter baumannii

Abstract

We have identified a second resistance-nodulation-cell division (RND)-type efflux pump, AdeIJK, in clinical isolate Acinetobacter baumannii BM4454. The adeI, adeJ, and adeK genes encode, respectively, the membrane fusion, RND, and outer membrane components of the pump. AdeJ belongs to the AcrB protein family (57% identity with AcrB from Escherichia coli). mRNA analysis by Northern blotting and reverse transcription-PCR indicated that the genes were cotranscribed. Overexpression of the cloned adeIJK operon was toxic in both E. coli and Acinetobacter. The adeIJK genes were detected in all of the 60 strains of A. baumannii tested. The two latter observations suggest that the AdeIJK complex might contribute to intrinsic but not to acquired antibiotic resistance in Acinetobacter. To characterize the substrate specificity of the pump, we have constructed derivatives of BM4454 in which adeIJK (strain BM4579), adeABC (strain BM4561), or both groups of genes (strain BM4652) were inactivated by deletion-insertion. Determination of the antibiotic susceptibility of these strains and of BM4652 and BM4579, in which the adeIJK operon was provided in trans, indicated that the AdeIJK pump contributes to resistance to beta-lactams, chloramphenicol, tetracycline, erythromycin, lincosamides, fluoroquinolones, fusidic acid, novobiocin, rifampin, trimethoprim, acridine, safranin, pyronine, and sodium dodecyl sulfate. The chemical structure of these molecules suggests that amphiphilic compounds are the preferred substrates. The AdeABC and AdeIJK efflux systems contributed in a more than additive fashion to tigecycline resistance.

FIG. 1.

Schematic representation of the adeIJK and adeABC operons from A. baumannii BM4454 and derivatives BM4579 and BM4651. Open arrows represent coding sequences and indicate the sense of transcription. The regions corresponding to the kanamycin and apramycin resistance genes (Km^R and Ap^R, respectively) are hatched. Black arrows indicate the positions and orientations of the primers used. The probes are represented by thick black horizontal lines.

FIG. 2.

Analysis of the transcription of the adeIJK genes by Northern hybridization. Total RNAs from BM4454 (lane 1), CIP 70-10 (lane 2), CIP 70-21 (lane 3), and BM4579 (lane 4) were hybridized with the probes indicated at the bottom. The sizes of the transcripts relative to RNA molecular weight marker I (Roche) are indicated at the left.