Affiliation:
1. Division of Infectious Diseases, Department of Medicine II, University Hospital and Medical Center, Freiburg, Germany
2. Faculty of Medicine, Albert-Ludwigs University, Freiburg, Germany
Abstract
ABSTRACT
Multidrug resistance poses global challenges, particularly with regard to Gram-negative bacterial infections. In view of the lack of new antibiotics, drug enhancers, such as efflux pump inhibitors (EPIs), have increasingly come into focus. A number of chemically diverse agents have been reported to inhibit AcrB, the main multidrug transporter in
Escherichia coli
, and homologs in other Gram-negative bacteria. However, due to the often varying methodologies used for their characterization, results remain difficult to compare. In this study, using a defined selection of antibiotics known to be efflux substrates, we reevaluated 38 published compounds for their
in vitro
EPI activity. When examined in an
E. coli
strain with stable wild-type AcrB overexpression, we found 17 compounds showing at least fourfold enhancing potency with more than 2 out of 10 test drugs (belonging to eight antibiotic classes). Pyranopyridines (MBX series) were confirmed as the most potent inhibitors among agents reported so far. A new and surprising finding was that their activity, unlike that of the pyridylpiperazine EPI BDM88855, was highly susceptible to the AcrB double-mutation G141D_N282Y, which had previously been shown to diminish drug enhancing of 1-(1-naphthylmethyl)piperazine in a predominantly substrate-specific manner. Conversely, transmembrane region mutation V411A, while eliminating the drug potentiating of the BDM compound, did not decrease the activity of the MBX EPIs. Besides comparative reassessment of the potency of reported EPIs, the study demonstrated the usefulness of mutagenesis approaches providing tools for an initial discrimination of EPIs regarding their mode of function.
IMPORTANCE
Infections with difficult-to-treat multidrug-resistant bacteria pose an urgent global threat in view of the stagnating development of new antimicrobial substances. Efflux pumps in Gram-negative pathogens are known to substantially contribute to multidrug resistance making them promising targets for chemotherapeutic interventions to restore the efficacy of conventional antibiotics. In the present study, the
in vitro
activity of previously reported efflux pump inhibitors was reassessed using standardized conditions. Relevant drug sensitizing activity could be proven for almost half of the tested compounds. Further characterization of potent inhibitors was achieved by investigating the impact of specific efflux pump mutations. A double-mutation previously known to decrease the activity of the arylpiperazine 1-(1-naphthylmethyl)piperazine also impaired that of the highly efficient pyranopyridine efflux pump inhibitors. Our findings provide direct comparability of reported efflux pump inhibitors and contribute to the elucidation of their mode of action.
Funder
Open Access Publication Fund of the University of Freiburg
Publisher
American Society for Microbiology
Subject
Infectious Diseases,Cell Biology,Microbiology (medical),Genetics,General Immunology and Microbiology,Ecology,Physiology