RND-type efflux pumps in multidrug-resistant clinical isolates of Acinetobacter baumannii: major role for AdeABC overexpression and AdeRS mutations

Abstract

Increased expression of chromosomal genes for resistance-nodulation-cell division (RND)-type efflux systems plays a major role in the multidrug resistance (MDR) of Acinetobacter baumannii. However, the relative contributions of the three most prevalent pumps, AdeABC, AdeFGH, and AdeIJK, have not been evaluated in clinical settings. We have screened 14 MDR clinical isolates shown to be distinct on the basis of multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) for the presence and overexpression of the three Ade efflux systems and analyzed the sequences of the regulators AdeRS, a two-component system, for AdeABC and AdeL, a LysR-type regulator, for AdeFGH. Gene adeB was detected in 13 of 14 isolates, and adeG and the intrinsic adeJ gene were detected in all strains. Significant overexpression of adeB was observed in 10 strains, whereas only 7 had moderately increased levels of expression of AdeFGH, and none overexpressed AdeIJK. Thirteen strains had reduced susceptibility to tigecycline, but there was no correlation between tigecycline MICs and the levels of AdeABC expression, suggesting the presence of other mechanisms for tigecycline resistance. No mutations were found in the highly conserved LysR regulator of the nine strains expressing AdeFGH. In contrast, functional mutations were found in conserved domains of AdeRS in all the strains that overexpressed AdeABC with two mutational hot spots, one in AdeS near histidine 149 suggesting convergent evolution and the other in the DNA binding domain of AdeR compatible with horizontal gene transfer. This report outlines the high incidence of AdeABC efflux pump overexpression in MDR A. baumannii as a result of a variety of single mutations in the corresponding two-component regulatory system.

Fig 1

Expression of adeB. Gene expression relative to that of rpoB was determined by qRT-PCR. Results are presented relative to susceptible strain BM4587 taken as a reference. Each isolate was tested in duplicate or triplicate in two independent experiments. The bars represent the means and the error bars the standard deviations. The adeB gene was not detected in strain BM4714.

Fig 2

Mutations in AdeS and AdeR conferring AdeABC pump overexpression were mapped on structural models generated by homology modeling with the Swiss Model Server (34). (A and E) Conserved motifs (dark gray boxes) H, N, G1, F, and G2 in AdeS and D, D, D, and K in AdeR and the amino acid positions corresponding to the sequences presented in panel E are indicated. Mutations in clinical isolates (in black), previously described mutations (in green), and catalytic residues H149 in AdeS and D63 in AdeR (in red) are indicated. (B to D) The models are based on the following templates: solution structure of the histidine kinase, adenylyl cyclase, methyl-accepting protein, and phosphatase (HAMP) linker domain of the hypothetical transmembrane receptor Af1503 from Archaeoglobus fulgidus (35, 36) (Protein Data Bank identification no. [PDB ID] 2L7H) and the catalytic core (DHp and CA) of the histidine kinase HK853 from Thermotoga maritima (PDB ID 3DGE) (32) (B); structure of the complex between HK853 and its cognate response regulator RR468 (32) (C); and the complex between the Escherichia coli PhoB output domain and its target DNA sequence (PDB ID 1GXP) (37) (D). Mutated and catalytic residues, indicated with the same color code as described above, are illustrated in stick representations. TM, transmembrane domain; DHp, dimerization and histidine-containing phosphotransfer domain; CA, catalytic and ATP-binding domain; REC, receiver domain.