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Novel Piperazine Arylideneimidazolones Inhibit the AcrAB-TolC Pump in Escherichia coli and Simultaneously Act as Fluorescent Membrane Probes in a Combined Real-Time Influx and Efflux Assay.

ABSTRACT: In this study, we tested five compounds belonging to a novel series of piperazine arylideneimidazolones for the ability to inhibit the AcrAB-TolC efflux pump. The biphenylmethylene derivative (BM-19) and the fluorenylmethylene derivative (BM-38) were found to possess the strongest efflux pump inhibitor (EPI) activities in the AcrAB-TolC-overproducingEscherichia colistrain 3-AG100, whereas BM-9, BM-27, and BM-36 had no activity at concentrations of up to 50 ?M in a Nile red efflux assay. MIC microdilution assays demonstrated that BM-19 at 1/4 MIC (intrinsic MIC, 200 ?M) was able to reduce the MICs of levofloxacin, oxacillin, linezolid, and clarithromycin 8-fold. BM-38 at 1/4 MIC (intrinsic MIC, 100 ?M) was able to reduce only the MICs of oxacillin and linezolid (2-fold). Both compounds markedly reduced the MIC of rifampin (BM-19, 32-fold; and BM-38, 4-fold), which is suggestive of permeabilization of the outer membrane as an additional mechanism of action. Nitrocefin hydrolysis assays demonstrated that in addition to their EPI activity, both compounds were in fact weak permeabilizers of the outer membrane. Moreover, it was found that BM-19, BM-27, BM-36, and BM-38 acted as near-infrared-emitting fluorescent membrane probes, which allowed for their use in a combined influx and efflux assay and thus for tracking of the transport of an EPI across the outer membrane by an efflux pump in real time. The EPIs BM-38 and BM-19 displayed the most rapid influx of all compounds, whereas BM-27, which did not act as an EPI, showed the slowest influx.

SUBMITTER: Bohnert JA

PROVIDER: S-EPMC4808168 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Novel Piperazine Arylideneimidazolones Inhibit the AcrAB-TolC Pump in Escherichia coli and Simultaneously Act as Fluorescent Membrane Probes in a Combined Real-Time Influx and Efflux Assay.

Bohnert Jürgen A JA Schuster Sabine S Kern Winfried V WV Karcz Tadeusz T Olejarz Agnieszka A Kaczor Aneta A Handzlik Jadwiga J Kieć-Kononowicz Katarzyna K

Antimicrobial agents and chemotherapy 20160325 4

In this study, we tested five compounds belonging to a novel series of piperazine arylideneimidazolones for the ability to inhibit the AcrAB-TolC efflux pump. The biphenylmethylene derivative (BM-19) and the fluorenylmethylene derivative (BM-38) were found to possess the strongest efflux pump inhibitor (EPI) activities in the AcrAB-TolC-overproducingEscherichia colistrain 3-AG100, whereas BM-9, BM-27, and BM-36 had no activity at concentrations of up to 50 μM in a Nile red efflux assay. MIC micr ...[more]

PMID: 26824939

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Project description:Antibacterial therapy is of paramount importance in treatment of several acute and chronic infectious diseases caused by pathogens. Over the years extensive use and misuse of antimicrobial agents has led to emergence of multidrug resistant (MDR) and extensive drug resistant (XDR) pathogens. This drastic escalation in resistant phenotype has limited the efficacy of available therapeutic options. Thus, the need of the hour is to look for alternative therapeutic approaches to mitigate healthcare concerns caused due to MDR bacterial infections. Nanoparticles have gathered much attention as potential candidates for antibacterial therapy. Equipped with advantages of, wide spectrum bactericidal activity at very low dosage, inhibitor of biofilm formation and ease of permeability, nanoparticles have been considered as leading therapeutic candidates to curtail infections resulting from MDR bacteria. However, substrate non-specificity of efflux pumps, particularly those belonging to resistance nodulation division super family, have been reported to reduce efficacy of many potent antibacterial therapeutic drugs. Previously, we had reported antibacterial activity of polysaccharide-capped silver nanoparticles (AgNPs) toward MDR bacteria. We showed that AgNPs inhibits biofilm formation and alters expression of cytoskeletal proteins FtsZ and FtsA, with minimal cytotoxicity toward mammalian cells. In the present study, we report no reduction in antibacterial efficacy of silver nanoparticles in presence of AcrAB-TolC efflux pump proteins. Antibacterial tests were performed according to CLSI macrobroth dilution method, which revealed that both silver nanoparticles exhibited bactericidal activity at very low concentrations. Further, immunoblotting results indicated that both the nanoparticles modulate the transporter AcrB protein expression. However, expression of the membrane fusion protein AcrA did show a significant increase after exposure to AgNPs. Our results indicate that both silver nanoparticles are effective in eliminating MDR Enterobacter cloacae isolates and their action was not inhibited by AcrAB-TolC efflux protein expression. As such, the above nanoparticles have strong potential to be used as effective and alternate therapeutic candidates to combat MDR gram-negative Enterobacterial pathogens.

Dataset Information

Novel Piperazine Arylideneimidazolones Inhibit the AcrAB-TolC Pump in Escherichia coli and Simultaneously Act as Fluorescent Membrane Probes in a Combined Real-Time Influx and Efflux Assay.

Publications

Novel Piperazine Arylideneimidazolones Inhibit the AcrAB-TolC Pump in Escherichia coli and Simultaneously Act as Fluorescent Membrane Probes in a Combined Real-Time Influx and Efflux Assay.

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