Characteristics and diversity of mutations in regulatory genes of resistance-nodulation-cell division efflux pumps in association with drug-resistant clinical isolates of Acinetobacter baumannii

Abstract

Background This study was aimed to characterize the genetic diversity and expression of three putative resistance-nodulation-cell division (RND)-type efflux systems and their contribution to multidrug efflux in clinical isolates of Acinetobacter baumannii. Methods Antimicrobial susceptibility testing of 95 A. baumannii isolates was determined by Kirby-Bauer disk diffusion for 18 antibiotics and minimum inhibitory concentration (MIC) of colistin was determined by the broth microdilution method. Moreover, the MIC of five classes of antibiotics was assessed using E-test strips in the presence and absence of phenylalanine-arginine beta-naphthylamide (PAβN). Regulatory genes of the RND efflux pumps (adeRS, adeL, adeN and baeSR) were subjected to sequencing. The relative expression of adeB, adeG and adeJ genes was determined by quantitative real-time PCR (qRT-PCR). Results Overall, the majority of isolates (94%) were extensively drug-resistant (XDR). In the phenotypic assay, efflux pump activity was observed in 40% of the isolates against multiple antibiotics mainly tigecycline. However, we found no efflux activity against imipenem. Several amino acid substitutions were detected in the products of regulatory genes; except in AdeN. Of note, G186V mutation in AdeS was found to be associated with overexpression of its efflux pump. No insertion sequences were detected. Conclusions Our findings outlined the role of RND efflux pumps in resistance of A. baumannii to multiple antibiotics particularly tigecycline, and pointed out the importance of a variety of single mutations in the corresponding regulatory systems. Further studies are required to decipher the precise role of RND efflux pumps in multidrug-resistant clinical isolates of A. baumannii.

Emergence of Ceftazidime/Avibactam and Tigecycline Resistance in Carbapenem-Resistant Klebsiella pneumoniae Due to In-Host Microevolution

“…The MICs of carbapenem and fosfomycin (with supplementation of glucose-6-phosphate in the agar) were determined by the agar dilution method, and the others were determined by the broth microdilution method with fresh cation-adjusted Mueller-Hinton broth. The MICs of TGC were also determined in the presence of Phe-Arg-β-naphthylamide (PAβN) at a concentration of 50 μg/mL to verify the function of the efflux pump in TGC resistance ( Salehi et al., 2021 ).…”

Relationship between the Biofilm-Forming Capacity and Antimicrobial Resistance in Clinical Acinetobacter baumannii Isolates: Results from a Laboratory-Based In Vitro Study

“…To establish the effects of the overexpression of resistance-nodulation-division (RND) efflux pumps on the susceptibility of tested antimicrobials, a phenylalanine-arginine βnaphthylamide (PAβN) plate-based assay was performed, as described by Salehi et al [30].…”

The Impact of Omega-3 Fatty Acids on the Evolution of Acinetobacter baumannii Drug Resistance

“…The development of resistance in A. baumannii is driven largely by its existence in the hospital environment, where the use of a diverse array of antimicrobial compounds has been associated with constitutive upregulation of genes encoding RND efflux systems, particularly adeABC and adeIJK , via regulatory mutations ( 11 , 14 , 15 ). Specifically, selective pressure exerted by antimicrobials selects for direct mutations within the efflux systems or their corresponding regulatory components, including adeRS ( adeABC ) and adeN ( adeIJK ) ( 11 , 14 , 16 , 17 ).…”