Comparative reassessment of AcrB efflux inhibitors reveals differential impact of specific pump mutations on the activity of potent compounds.

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.IMPORTANCEInfections 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.

A Screening of Antimalarials Extends the Range of Known Escherichia coli AcrB Efflux Substrates and Reveals Two Candidates with Antimicrobial Drug-Enhancing Activity.

Efflux by resistance nodulation cell division transporters, such as AcrAB-TolC in Escherichia coli, substantially contributes to the development of Gram-negative multidrug resistance. Therefore, the finding of compounds that counteract efflux is an urgent goal in the fight against infectious diseases. Previously, an efflux inhibitory activity of the antimalarials mefloquine and artesunate was reported. In this study, we have investigated further antimalarials regarding efflux by AcrB, the pumping part of AcrAB-TolC, and their drug-enhancing potency in E. coli. We show that 10 of the 24 drugs tested are substrates of the multidrug efflux pump AcrB. Among them, tafenoquine and proguanil, when used at subinhibitory concentrations, caused an at least 4- and up to 24-fold enhancement in susceptibility to 6 and 14 antimicrobial agents, respectively. Both antimalarials are able to increase the intracellular accumulation of Hoechst 33342, with proguanil showing similar effectiveness as the efflux inhibitor 1-(1-naphthylmethyl)piperazine. In the case of proguanil, AcrB-dependent efflux inhibition could also be demonstrated in a real-time efflux assay. In addition to presenting new AcrB substrates, our study reveals two previously unknown efflux inhibitors among antimalarials. Particularly proguanil appears as a promising candidate and its chemical scaffold might be further optimized for repurposing as antimicrobial drug enhancer.

Exploring the Contribution of the AcrB Homolog MdtF to Drug Resistance and Dye Efflux in a Multidrug Resistant E. coli Isolate.

In Escherichia coli, the role of RND-type drug transporters other than the major efflux pump AcrB has largely remained undeciphered (particularly in multidrug resistant pathogens), because genetic engineering in such isolates is challenging. The present study aimed to explore the capability of the AcrB homolog MdtF to contribute to the extrusion of noxious compounds and to multidrug resistance in an E. coli clinical isolate with demonstrated expression of this efflux pump. An mdtF/acrB double-knockout was engineered, and susceptibility changes with drugs from various classes were determined in comparison to the parental strain and its acrB and tolC single-knockout mutants. The potential of MdtF to participate in the export of agents with different physicochemical properties was additionally assessed using accumulation and real-time efflux assays with several fluorescent dyes. The results show that there was limited impact to the multidrug resistant phenotype in the tested E. coli strain, while the RND-type transporter remarkably contributes to the efflux of all tested dyes. This should be considered when evaluating the efflux phenotype of clinical isolates via dye accumulation assays. Furthermore, the promiscuity of MdtF should be taken into account when developing new antibiotic agents.

Limited Multidrug Resistance Efflux Pump Overexpression among Multidrug-Resistant Escherichia coli Strains of ST131.

Gram-negative bacteria partly rely on efflux pumps to facilitate growth under stressful conditions and to increase resistance to a wide variety of commonly used drugs. In recent years, Escherichia coli sequence type 131 (ST131) has emerged as a major cause of extraintestinal infection frequently exhibiting a multidrug resistance (MDR) phenotype. The contribution of efflux to MDR in emerging E. coli MDR clones, however, is not well studied. We characterized strains from an international collection of clinical MDR E. coli isolates by MIC testing with and without the addition of the AcrAB-TolC efflux inhibitor 1-(1-naphthylmethyl)-piperazine (NMP). MIC data for 6 antimicrobial agents and their reversion by NMP were analyzed by principal-component analysis (PCA). PCA revealed a group of 17 MDR E. coli isolates (n = 34) exhibiting increased susceptibility to treatment with NMP, suggesting an enhanced contribution of efflux pumps to antimicrobial resistance in these strains (termed enhanced efflux phenotype [EEP] strains). Only 1/17 EEP strains versus 12/17 non-EEP MDR strains belonged to the ST131 clonal group. Whole-genome sequencing revealed marked differences in efflux-related genes between EEP and control strains, with the majority of notable amino acid substitutions occurring in AcrR, MarR, and SoxR. Quantitative reverse transcription-PCR (qRT-PCR) of multiple efflux-related genes showed significant overexpression of the AcrAB-TolC system in EEP strains, whereas in the remaining strains, we found enhanced expression of alternative efflux proteins. We conclude that a proportion of MDR E. coli strains exhibit an EEP, which is linked to an overexpression of the AcrAB-TolC efflux pump and a distinct array of genomic variations. Members of ST131, although highly successful, are less likely to exhibit the EEP.

New Topoisomerase Inhibitors: Evaluating the Potency of Gepotidacin and Zoliflodacin in Fluoroquinolone-Resistant Escherichia coli upon tolC Inactivation and Differentiating Their Efflux Pump Substrate Nature.

Inactivating tolC in multidrug-resistant Escherichia coli with differing sequence types and quinolone resistance-determining mutations reveals remarkably potentiated activity of the first-in-class topoisomerase inhibitors gepotidacin and zoliflodacin. Differences between both structurally unrelated compounds in comparison to fluoroquinolones regarding the selectivity of E. coli RND (resistance-nodulation-cell division)-type transporters, efflux inhibitors, and AcrB porter domain mutations were demonstrated. The findings should reinforce efforts to develop efflux-bypassing drugs and provide AcrB targets with critical relevance for this purpose.

Detection of High-Level Rifaximin Resistance in Enteric Bacteria by Agar Screen.

Objectives: This study aimed at determining the prevalence of rifaximin resistance in a large collection of Enterobacterales resistant to third-generation cephalosporins. A simple agar screen was developed to detect high-level resistance. Methods: A total of 401 isolates nonsusceptible to third-generation cephalosporins (including 342 Escherichia coli and 39 Klebsiella spp. and 20 Enterobacter spp.) were tested by microdilution for their MICs of rifaximin and rifampicin. Isolates with a confirmed rifaximin minimal inhibitory concentration (MIC) of >64 mg/L and a number of high-level resistant, and susceptible control isolates were tested for growth on Mueller-Hinton agar supplemented with rifaximin or rifampicin at a concentration of 256 mg/L. Amino acid mutations in rpoB and the presence of rifaximin resistance-associated genes arabidopsis response regulator (arr) 2/3 were investigated. Results: Microdilution assays identified rifaximin resistance in nine E. coli and three Klebsiella spp. isolates with complete cross-resistance to rifampicin (MICs of both >64 mg/L). The rifaximin agar screen correctly identified 9/9 clinical E. coli isolates, 2/2 E. coli controls, and 3/3 Klebsiella spp. with high-level rifaximin resistance, and was negative in 45 control clinical isolates with rifaximin MICs ranging between 2 and 32 mg/L according to broth microdilution. All nine high-level rifaximin agar screen-positive E. coli clinical isolates (vs. none of the tested controls) had rpoB mutations or carried arr2/3. Conclusions: Our agar screen test has the potential to detect high-level rifaximin-resistant Enterobacterales. Such strains remain rare among extended spectrum beta-lactamase (ESBL)-positive enteric bacteria, but may emerge among patients receiving rifaximin for prevention of hepatic encephalopathy and spontaneous bacterial peritonitis or among patients receiving rifaximin for other indications.

Efflux-Mediated Resistance to New Oxazolidinones and Pleuromutilin Derivatives in Escherichia coli with Class Specificities in the Resistance-Nodulation-Cell Division-Type Drug Transport Pathways.

A major contribution of the resistance-nodulation-cell division (RND)-transporter AcrB to resistance to oxazolidinones and pleuromutilin derivatives in Escherichia coli was confirmed. However, we discovered significant differences in efflux inhibitor activities, specificities of the homologous pump YhiV (MdtF), and the impact of AcrB pathway mutations. Particularly, entrance channel double-mutation I38F I671T and distal binding pocket mutation F615A revealed class-specific transport routes of oxazolidinones and pleuromutilin derivatives. The findings could contribute to the understanding of the RND-type multidrug transport pathways.

Proof of an Outer Membrane Target of the Efflux Inhibitor Phe-Arg-ß-Naphthylamide from Random Mutagenesis.

Phe-Arg-ß-naphthylamide (PAßN) has been characterized as an efflux pump inhibitor (EPI) acting on the major multidrug resistance efflux transporters of Gram-negative bacteria, such as AcrB in Eschericha coli. In the present study, in vitro random mutagenesis was used to evolve resistance to the sensitizing activity of PAßN with the aim of elucidating its mechanism of action. A strain was obtained that was phenotypically similar to a previously reported mutant from a serial selection approach that had no efflux-associated mutations. We could confirm that acrB mutations in the new mutant were unrelated to PAßN resistance. The next-generation sequencing of the two mutants revealed loss-of-function mutations in lpxM. An engineered lpxM knockout strain showed up to 16-fold decreased PAßN activity with large lipophilic drugs, while its efflux capacity, as well as the efficacy of other EPIs, remained unchanged. LpxM is responsible for the last acylation step in lipopolysaccharide (LPS) synthesis, and lpxM deficiency has been shown to result in penta-acylated instead of hexa-acylated lipid A. Modeling the two lipid A types revealed steric conformational changes due to underacylation. The findings provide evidence of a target site of PAßN in the LPS layer, and prove membrane activity contributing to its drug-sensitizing potency.

Mycobacteria exploit nitric oxide-induced transformation of macrophages into permissive giant cells.

Immunity to mycobacteria involves the formation of granulomas, characterized by a unique macrophage (MΦ) species, so-called multinucleated giant cells (MGC). It remains unresolved whether MGC are beneficial to the host, that is, by prevention of bacterial spread, or whether they promote mycobacterial persistence. Here, we show that the prototypical antimycobacterial molecule nitric oxide (NO), which is produced by MGC in excessive amounts, is a double-edged sword. Next to its antibacterial capacity, NO propagates the transformation of MΦ into MGC, which are relatively permissive for mycobacterial persistence. The mechanism underlying MGC formation involves NO-induced DNA damage and impairment of p53 function. Moreover, MGC have an unsurpassed potential to engulf mycobacteria-infected apoptotic cells, which adds a further burden to their antimycobacterial capacity. Accordingly, mycobacteria take paradoxical advantage of antimicrobial cellular efforts by driving effector MΦ into a permissive MGC state.

Contribution of AcrAB-TolC to multidrug resistance in an Escherichia coli sequence type 131 isolate.

Drug efflux by resistance-nodulation-cell division (RND)-type transporters, such as AcrAB-TolC of Escherichia coli, is an important resistance mechanism in Gram-negative bacteria; however, its contribution to multidrug resistance (MDR) in clinical isolates is poorly defined. We inactivated acrB of a sequence type 131 E. coli human isolate that showed high-level MDR, but had no mutations within the known efflux-associated local or global regulators. The resistance profile of the acrB deletion mutant revealed significantly increased susceptibility to drugs from seven antibiotic classes, including agents usually inactive against Gram-negative bacteria, notably the new oxazolidinone, tedizolid (512-fold enhanced susceptibility). AcrB deficiency reduced, but did not abolish, the efflux of dyes, which indicates the activity of at least one more efflux transporter. The findings demonstrate the efficacy of AcrAB-TolC-mediated broad-spectrum drug efflux, including agents primarily developed for Gram-positive pathogens, in a clinical isolate representative of a globally-emerging lineage. The results illustrate the need to develop molecules modified to impede their transport by AcrAB-TolC and its homologues and new efflux inhibitors.

Evidence of a Substrate-Discriminating Entrance Channel in the Lower Porter Domain of the Multidrug Resistance Efflux Pump AcrB.

Efflux pumps of the resistance nodulation cell division (RND) transporter family, such as AcrB of Escherichia coli, play an important role in the development of multidrug resistance, but the molecular basis for their substrate promiscuity is not yet completely understood. From a collection of highly clarithromycin-resistant AcrB periplasmic domain mutants derived from in vitro random mutagenesis, we identified variants with an unusually altered drug resistance pattern characterized by increased susceptibility to many drugs of lower molecular weight, including fluoroquinolones, tetracyclines, and oxazolidinones, but unchanged or increased resistance to drugs of higher molecular weight, including macrolides. Sequencing of 14 such "divergent resistance" phenotype mutants and 15 control mutants showed that this unusual phenotype was associated with mutations at residues I38 and I671 predominantly to phenylalanine and threonine, respectively, both conferring a similar susceptibility pattern. Reconstructed I38F and I671T single mutants as well as an engineered I38F I671T double mutant with proved efflux competence revealed an equivalent phenotype with enhanced or unchanged resistance to many large AcrB substrates but increased susceptibility to several lower-molecular-weight drugs known to bind within the distal binding pocket. The two isoleucines located in close vicinity to each other in the lower porter domain of AcrB beneath the bottom of the proximal binding pocket may be part of a preferential small-drug entrance pathway that is compromised by the mutations. This finding supports recent indications of distinct entrance channels used by compounds with different physicochemical properties, of which molecular size appears to play a prominent role.

Thioridazine and chlorpromazine inhibition of ethidium bromide efflux in Mycobacterium avium and Mycobacterium smegmatis.

OBJECTIVES: Therapy of AIDS patients infected with Mycobacterium avium is problematic due to its intrinsic resistance to antibiotics. We have characterized the efflux pump activity of M. avium wild-type strain through an automated fluorometric method and correlated it with intrinsic resistance to antibiotics. METHODS: M. avium ATCC 25291(T) and Mycobacterium smegmatis mc(2)155 were evaluated for accumulation and efflux of ethidium bromide in the presence or absence of the efflux pump inhibitors (EPIs) thioridazine, chlorpromazine, verapamil and the proton uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). For this purpose, a new automated fluorometric method was used that separately assesses accumulation and extrusion of ethidium bromide. RESULTS: The automated fluorometric method described in this paper allowed the detection and quantification of ethidium bromide transport across M. avium and M. smegmatis cell walls. Accumulation of ethidium bromide was found to be temperature-dependent and significantly increased by EPIs thioridazine, chlorpromazine, verapamil and CCCP in a concentration-dependent manner. Efflux of ethidium bromide under optimum conditions of temperature and glucose is inhibited by the above agents. At half their intrinsic MICs, both thioridazine and chlorpromazine, similarly to verapamil and CCCP, significantly increased the susceptibility of M. avium to erythromycin, suggesting an effect upon an efflux pump with ethidium bromide and erythromycin as substrates. A similar effect was observed for M. smegmatis with verapamil only. CONCLUSIONS: M. avium and M. smegmatis intrinsic resistance is affected by EPIs such as thioridazine or chlorpromazine, an effect that might be important in research and development of new, more effective antimycobacterial therapies.