AUTHOR=Migliaccio Antonella , Esposito Eliana Pia , Bagattini Maria , Berisio Rita , Triassi Maria , De Gregorio Eliana , Zarrilli Raffaele 

TITLE=Inhibition of AdeB, AceI, and AmvA Efflux Pumps Restores Chlorhexidine and Benzalkonium Susceptibility in Acinetobacter baumannii ATCC 19606

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.790263

DOI=10.3389/fmicb.2021.790263

ISSN=1664-302X

ABSTRACT=<p>The management of infections caused by <italic>Acinetobacter baumannii</italic> is hindered by its intrinsic tolerance to a wide variety of biocides. The aim of the study was to analyze the role of different <italic>A. baumannii</italic> 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 <italic>A. baumannii</italic>. <italic>A. baumannii</italic> 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 <italic>adeB</italic> and <italic>adeJ</italic> (RND superfamily), <italic>aceI</italic> (PACE family) and <italic>amvA</italic> (MFS superfamily) EP genes. The values of CHX MIC and MBC decreased by eightfold in Δ<italic>adeB</italic> and twofold in Δ<italic>amvA</italic> or Δ<italic>aceI</italic> mutants, respectively, while not affected in Δ<italic>adeJ</italic> 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 <italic>adeB</italic> gene. BZK MIC was decreased by fourfold in Δ<italic>adeB</italic> mutant, and twofold in Δ<italic>amvA</italic> and Δ<italic>aceI</italic> mutants, respectively; EPs double and triple deletion mutants showed an additive effect on BZK MIC. BZK susceptibility was recovered in Δ<italic>adeB</italic> Δ<italic>aceI</italic> Δ<italic>adeJ</italic> and Δ<italic>amvA</italic> Δ<italic>adeB</italic> Δ<italic>adeJ</italic> 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 <italic>A. baumannii</italic> ATCC 19606 and in Δ<italic>adeJ</italic>, Δ<italic>aceI</italic>, or Δ<italic>amvA</italic> mutants, but not in Δ<italic>adeB</italic> mutant. Either piperine (PIP) or resveratrol (RV) at non-toxic concentrations inhibited CHX MIC in <italic>A. baumannii</italic> 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 <italic>A. baumannii</italic> ATCC 19606 parental strain and EPs mutants. These results demonstrate that tolerance to CHX and BZK in <italic>A. baumannii</italic> 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 <italic>A. baumannii.</italic></p>