Improved Potency of Indole-Based NorA Efflux Pump Inhibitors: From Serendipity toward Rational Design and Development.

The NorA efflux pump is a potential drug target for reversal of resistance to selected antibacterial agents, and recently we described indole-based inhibitor candidates. Herein we report a second class of inhibitors derived from them but with significant differences in shape and size. In particular, compounds 13 and 14 are very potent inhibitors in that they demonstrated the lowest IC values (2 μM) ever observed among all indole-based compounds we have evaluated.

Indole Based Weapons to Fight Antibiotic Resistance: A Structure-Activity Relationship Study.

Antibiotic resistance represents a worldwide concern, especially regarding the outbreak of methicillin-resistant Staphylococcus aureus, a common cause for serious skin and soft tissues infections. A major contributor to Staphylococcus aureus antibiotic resistance is the NorA efflux pump, which is able to extrude selected antibacterial drugs and biocides from the membrane, lowering their effective concentrations. Thus, the inhibition of NorA represents a promising and challenging strategy that would allow recycling of substrate antimicrobial agents.

Mutations within the mepA operator affect binding of the MepR regulatory protein and its induction by MepA substrates in Staphylococcus aureus.

The expression of mepA, encoding the Staphylococcus aureus MepA multidrug efflux protein, is repressed by the MarR homologue MepR. Repression occurs through binding of two MepR dimers to an operator with two homologous and closely approximated pseudopalindromic binding sites (site 1 [S1] and site 2 [S2]). MepR binding is impeded in the presence of pentamidine, a MepA substrate.

Clonal relatedness is a predictor of spontaneous multidrug efflux pump gene overexpression in Staphylococcus aureus.

Increased expression of genes encoding multidrug resistance efflux pumps (MDR-EPs) contributes to antimicrobial agent and biocide resistance in Staphylococcus aureus. Previously identified associations between norA overexpression and spa type t002 meticillin-resistant S. aureus (MRSA), and a similar yet weaker association between mepA overexpression and type t008 meticillin-susceptible S.

Structural mechanism of transcription regulation of the Staphylococcus aureus multidrug efflux operon mepRA by the MarR family repressor MepR.

The multidrug efflux pump MepA is a major contributor to multidrug resistance in Staphylococcus aureus. MepR, a member of the multiple antibiotic resistance regulator (MarR) family, represses mepA and its own gene. Here, we report the structure of a MepR-mepR operator complex.

A new plant-derived antibacterial is an inhibitor of efflux pumps in Staphylococcus aureus.

An in-depth evaluation was undertaken of a new antibacterial natural product (1) recently isolated and characterised from the plant Hypericum olympicum L. cf. uniflorum.

The molecular mechanisms of allosteric mutations impairing MepR repressor function in multidrug-resistant strains of Staphylococcus aureus.

Unlabelled: Overexpression of the Staphylococcus aureus multidrug efflux pump MepA confers resistance to a wide variety of antimicrobials. mepA expression is controlled by MarR family member MepR, which represses mepA and autorepresses its own production. Mutations in mepR are a primary cause of mepA overexpression in clinical isolates of multidrug-resistant S.

Functional consequences of substitution mutations in MepR, a repressor of the Staphylococcus aureus MepA multidrug efflux pump gene.

The expression of mepA, encoding the Staphylococcus aureus MepA multidrug efflux protein, is repressed by the MarR homologue MepR. MepR dimers bind differently to operators upstream of mepR and mepA, with affinity being greatest at the mepA operator. MepR substitution mutations may result in mepA overexpression, with A103V most common in clinical strains.

Mutagenesis and modeling to predict structural and functional characteristics of the Staphylococcus aureus MepA multidrug efflux pump.

MepA is a multidrug and toxin extrusion (MATE) family protein and the only MATE protein encoded within the Staphylococcus aureus genome. Structural data for MATE proteins are limited to a single high-resolution example, NorM of Vibrio cholerae. Substitution mutations were created in MepA using gradient plates containing both a substrate and reserpine as an efflux pump inhibitor.

Expression of multidrug resistance efflux pump genes in clinical and environmental isolates of Staphylococcus aureus.

Increased expression of multidrug resistance efflux pump (MDR-EP) genes in clinical isolates of Staphylococcus aureus occurs frequently, but its temporal and geographic variability is unknown. Such strains may contaminate the hospital environment, posing an infection control problem. Nearly 700 clinical isolates from different geographic locales as well as 91 environmental isolates recovered from two Detroit hospitals were studied.

Mechanisms of in-vitro-selected daptomycin-non-susceptibility in Staphylococcus aureus.

Daptomycin is highly active against Staphylococcus aureus, including multidrug-resistant strains and those with reduced susceptibility to vancomycin. However, daptomycin-non-susceptible (Dap(NS)) strains [minimum inhibitory concentration (MIC) >1mg/L] have been derived clinically and in vitro. The mechanism(s) by which this occurs is incompletely understood, but existing data indicate that it is multifactorial.

Ethidium bromide MIC screening for enhanced efflux pump gene expression or efflux activity in Staphylococcus aureus.

Multidrug resistance efflux pumps contribute to antimicrobial and biocide resistance in Staphylococcus aureus. The detection of strains capable of efflux is time-consuming and labor-intensive using currently available techniques. A simple and inexpensive method to identify such strains is needed.

In silico genetic correlations of multidrug efflux pump gene expression in Staphylococcus aureus.

Regulatory mechanisms for chromosomal genes encoding multidrug resistance (MDR) efflux pumps (EPs) in Staphylococcus aureus are poorly defined. Microbiological, quantitative gene expression, mRNA half-life and genome data for 11 strains of S. aureus combined with bioinformatic analyses were used to identify correlates of increased MDR EP gene expression.

Structural and biochemical characterization of MepR, a multidrug binding transcription regulator of the Staphylococcus aureus multidrug efflux pump MepA.

MepR is a multidrug binding transcription regulator that represses expression of the Staphylococcus aureus multidrug efflux pump gene, mepA, as well as its own gene. MepR is induced by multiple cationic toxins, which are also substrates of MepA. In order to understand the gene regulatory and drug-binding mechanisms of MepR, we carried out biochemical, in vivo and structural studies.

Inability of a reserpine-based screen to identify strains overexpressing efflux pump genes in clinical isolates of Staphylococcus aureus.

Overexpression of efflux pump genes conferring multidrug resistance (MDR) in Staphylococcus aureus results in reduced susceptibility to select biocides, dyes and fluoroquinolones. Reserpine is commonly used as an inhibitor of MDR efflux pumps and previous work from our laboratory using a reserpine-based screen to identify clinical isolates with an efflux phenotype revealed that nearly one-half overexpressed norA-B-C, mepA or mdeA. The accuracy of reserpine in predicting efflux pump gene overexpression in clinical strains was examined in detail.

Multidrug efflux pump overexpression in Staphylococcus aureus after single and multiple in vitro exposures to biocides and dyes.

Biocides and dyes are commonly employed in hospital and laboratory settings. Many of these agents are substrates for multiple-drug resistance (MDR)-conferring efflux pumps of both Gram-positive and Gram-negative organisms. Several such pumps have been identified in Staphylococcus aureus, and mutants overexpressing the NorA and MepA MDR pumps following exposure to fluoroquinolones have been identified.

The phenolic diterpene totarol inhibits multidrug efflux pump activity in Staphylococcus aureus.

The phenolic diterpene totarol had good antimicrobial activity against effluxing strains of Staphylococcus aureus. Subinhibitory concentrations reduced the MICs of selected antibiotics, suggesting that it may also be an efflux pump inhibitor (EPI). A totarol-resistant mutant that overexpressed norA was created to separate antimicrobial from efflux inhibitory activity.

Efflux-related resistance to norfloxacin, dyes, and biocides in bloodstream isolates of Staphylococcus aureus.

Efflux is an important resistance mechanism in Staphylococcus aureus, but its frequency in patients with bacteremia is unknown. Nonreplicate bloodstream isolates were collected over an 8-month period, and MICs of four common efflux pump substrates, with and without the broad-spectrum efflux pump inhibitor reserpine, were determined (n = 232). A reserpine-associated fourfold decrease in MIC was considered indicative of efflux.

Mechanisms of daptomycin resistance in Staphylococcus aureus.

Daptomycin resistance in Staphylococcus aureus emerged during therapy of tricuspid endocarditis. Susceptibility to daptomycin of the parent strain (SA-675), other daptomycin-susceptible strains and the non-susceptible mutant (SA-684) was heterogeneous; however, subpopulations growing at concentrations above the minimum inhibitory concentration (MIC) were not stably resistant. Stable resistance was produced only by serial passage on daptomycin-containing media.

MepR, a repressor of the Staphylococcus aureus MATE family multidrug efflux pump MepA, is a substrate-responsive regulatory protein.

The mepRAB gene cluster of Staphylococcus aureus encodes a MarR family repressor (MepR; known to repress mepA expression), a MATE family multidrug efflux pump (MepA), and a protein of unknown function (MepB). In this report, we show that MepR also is autoregulatory, repressing the expression of its own gene. Exposure of strains containing a mepR::lacZ fusion with mepR provided in trans under the control of an inducible promoter, or a mepA::lacZ fusion alone, to subinhibitory concentrations of MepA substrates resulted in variably increased expression mainly of mepA.

Multidrug resistance in Staphylococcus aureus due to overexpression of a novel multidrug and toxin extrusion (MATE) transport protein.

Efflux is an important mechanism of multidrug resistance (MDR) in bacteria. The multidrug and toxin extrusion (MATE) family is the most recently described group of MDR efflux proteins, none of which have previously been identified in Staphylococcus aureus. Two independently derived S.

Effect of promoter region mutations and mgrA overexpression on transcription of norA, which encodes a Staphylococcus aureus multidrug efflux transporter.

NorA is a Staphylococcus aureus multidrug transporter that confers resistance to structurally distinct compounds. The MgrA global regulatory protein is reported to augment norA expression when mgrA is overexpressed from an undefined plasmid-based promoter. Further details about norA regulatory mechanisms are scant.

Effect of substrate exposure and other growth condition manipulations on norA expression.

Objectives: Multidrug efflux is a resistance mechanism that simultaneously affects susceptibility to many structurally unrelated compounds. The regulation of norA expression, which encodes the Staphylococcus aureus NorA multidrug efflux pump, is not well understood but the MgrA global regulator and the arlRS locus are involved. The expression of genes encoding proteins related to NorA, such as QacA of S.

Phenylpiperidine selective serotonin reuptake inhibitors interfere with multidrug efflux pump activity in Staphylococcus aureus.

Structural variants of phenylpiperidine selective serotonin reuptake inhibitors (P-SSRIs) inhibited the function of two unique Staphylococcus aureus multidrug efflux pumps. The most active compound was the paroxetine isomer NNC 20-7052, which had an IC(50) for ethidium, acriflavine, and pyronin Y efflux of 9, 53, and 18% of its MIC, respectively, against the NorA pump. The unbalanced effect of NNC 20-7052 on the efflux of different substrates suggests the possibility that P-SSRIs function by a physical interaction with NorA.