AdeR-AdeS mutations & overexpression of the AdeABC efflux system in ciprofloxacin-resistant Acinetobacter baumannii clinical isolates

Abstract

Background & objectives: : Overexpression of efflux pumps is a cause of acquired resistance to fluoroquinolones in Acinetobacter baumannii. The present study was done to investigate the presence and overexpression of AdeABC efflux system and to analyze the sequences of AdeR-AdeS regulatory system in ciprofloxacin-resistant A. baumannii isolates.

Methods: : Susceptibility of 50 clinical A. baumannii isolates to ciprofloxacin, imipenem, ceftazidime, cefepime and gentamicin antimicrobials was evaluated by agar dilution method. Isolates were screened for the evidence of active efflux pump. Isolates were also examined for adeR-adeS and adeB efflux genes by polymerase chain reaction (PCR). The adeR and adeS regulatory genes were sequenced to detect amino acid substitutions. Expression of adeB was evaluated by quantitative reverse-transcriptase PCR.

Results: : There were high rates of resistance to ciprofloxacin (88%), ceftazidime (88%), cefepime (74%) and imipenem (72%) and less resistance rate to gentamicin (64%). Phenotypic assay showed involvement of active efflux in decreased susceptibility to ciprofloxacin among 16 isolates. The 12.27-fold increase and 4.25-fold increase were found in adeB expression in ciprofloxacin-full-resistant and ciprofloxacin-intermediate-resistant isolates, respectively. Several effective mutations, including A91V, A136V, L192R, A94V, G103D and G186V, were detected in some domains of AdeR-AdeS regulators in the overexpressed ciprofloxacin-resistant isolates.

Interpretation & conclusions: The results of this study indicated that overexpression of the AdeABC efflux pump was important to reduce susceptibility to ciprofloxacin and cefepime in A. baumannii that, in turn, could be triggered by alterations in the AdeR-AdeS two-component system. However, gene expression alone does not seem adequate to explain multidrug resistance phenomenon. These results could help plan improved active efflux pump inhibitors.

Keywords: Acinetobacter baumannii - adeB - adeR - adeS - ciprofloxacin resistance.

Fig. 1

Expression of adeB mRNA transcripts as evaluated by real-time polymerase chain reaction. adeB expression in two ciprofloxacin-full resistant (R) and ciprofloxacin-intermediate (I) groups of Acinetobacter baumannii was compared relative to that in the reference ATCC 19606 strain. The error bars represent the standard deviation for the average of results from three independent experiments.

Fig. 2

Multiple-sequence alignment of AdeR (A) and AdeS (B) from Acinetobacter baumannii ATCC 17978 and clinical isolates. Sequence alignment was generated using ClustalW2 software program. The deduced amino acid sequence is designated in a single letter code. Asterisks indicate identical residues, colons indicate strongly similar residues and dots indicate weakly similar residues. Amino acid substitutions in AdeS and AdeR were highlighted in black. Some mutations leading to constitutive expression of AdeABC pump in clinical isolates such as A91V, A136V and L192R in AdeR and A94V, G103D and G186V in AdeS are indicated.