Abstract
The management of infections caused by Acinetobacter baumannii is hindered by its intrinsic tolerance to a wide variety of biocides. The aim of the study was to analyze the role of different A. baumannii efflux pumps (EPs) in tolerance to chlorhexidine (CHX) and benzalkonium (BZK) and identify non-toxic compounds, which can restore susceptibility to CHX and BZK in A. baumannii. A. baumannii ATCC 19606 strain was tolerant to both CHX and BZK with MIC and MBC value of 32 mg/L. CHX subMIC concentrations increased the expression of adeB and adeJ (RND superfamily), aceI (PACE family) and amvA (MFS superfamily) EP genes. The values of CHX MIC and MBC decreased by eightfold in ΔadeB and twofold in ΔamvA or ΔaceI mutants, respectively, while not affected in ΔadeJ mutant; EPs double and triple deletion mutants showed an additive effect on CHX MIC. CHX susceptibility was restored in double and triple deletion mutants with inactivation of adeB gene. BZK MIC was decreased by fourfold in ΔadeB mutant, and twofold in ΔamvA and ΔaceI mutants, respectively; EPs double and triple deletion mutants showed an additive effect on BZK MIC. BZK susceptibility was recovered in ΔadeB ΔaceI ΔadeJ and ΔamvA ΔadeB ΔadeJ triple mutants. The structural comparison of AdeB and AdeJ protomers showed a more negatively charged entrance binding site and F-loop in AdeB, which may favor the transport of CHX. The carbonyl cyanide m-chlorophenylhydrazine protonophore (CCCP) EP inhibitor reduced dose-dependently CHX MIC in A. baumannii ATCC 19606 and in ΔadeJ, ΔaceI, or ΔamvA mutants, but not in ΔadeB mutant. Either piperine (PIP) or resveratrol (RV) at non-toxic concentrations inhibited CHX MIC in A. baumannii ATCC 19606 parental strain and EPs gene deletion mutants, and CHX-induced EP gene expression. Also, RV inhibited BZK MIC and EP genes expression in A. baumannii ATCC 19606 parental strain and EPs mutants. These results demonstrate that tolerance to CHX and BZK in A. baumannii is mediated by the activation of AdeB, AceI and AmvA EPs, AdeB playing a major role. Importantly, inhibition of EP genes expression by RV restores CHX and BZK susceptibility in A. baumannii.
Highlights
Bacteria belonging to the genus Acinetobacter are glucose nonfermentative Gram-negative coccobacilli that are a frequent cause of health-care associated infections and hospital outbreaks
Because it has been demonstrated that CHX increased the expression of aceI efflux pump (EP) gene in A. baumannii ATCC 17978 (Hassan et al, 2013), we asked if CHX was able to regulate the expression of EPs genes in ATCC 19606
Preliminary data showed that basal level of expression of adeB, adeG, adeJ, belonging to resistance– nodulation–cell division (RND) superfamily, amvA and craA belonging to major facilitator superfamily (MFS) superfamily, aceI, belonging to proteobacterial antimicrobial compound efflux (PACE) superfamily, and abeS and abeM, belonging to the SMR superfamily were different in A. baumannii ATCC 19606
Summary
Bacteria belonging to the genus Acinetobacter are glucose nonfermentative Gram-negative coccobacilli that are a frequent cause of health-care associated infections and hospital outbreaks. The most successful Acinetobacter clones show resistance to a broad range of antimicrobials and tolerance to disinfectants and share virulence features such as biofilm formation on biotic and abiotic surfaces, resistance to desiccation and adherence to epithelial cells (Giannouli et al, 2013; Wong et al, 2017; Harding et al, 2018). A. baumannii persistence in the contaminated hospital environment is contributed by reduced susceptibility of the bacteria to a broad range of biocides used as antiseptics or disinfectants, such as the bisphenol triclosan (TRI), the quaternary ammonium compounds benzalkonium chloride (BZK), dequalinium chloride (DQ), and cetrimide (CT), and the biguanide chlorhexidine (CHX) (McDonnell and Russell, 1999). Activation of AdeB and AdeJ resistance– nodulation–cell division (RND) efflux systems (Rajamohan et al, 2010a; Tucker et al, 2014), AmvA and CraA major facilitator superfamily (MFS) efflux systems (Rajamohan et al, 2010b; Foong et al, 2019) have been shown to induce tolerance to CHX and other disinfectants in clinical A. baumannii isolates
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