Inhibitors Of Multidrug Efflux Pumps Research Articles

Efflux Pump Inhibitors in Controlling Antibiotic Resistance: Outlook under a Heavy Metal Contamination Context

Multi-drug resistance to antibiotics represents a growing challenge in treating infectious diseases. Outside the hospital, bacteria with the multi-drug resistance (MDR) phenotype have an increased prevalence in anthropized environments, thus implying that chemical stresses, such as metals, hydrocarbons, organic compounds, etc., are the source of such resistance. There is a developing hypothesis regarding the role of metal contamination in terrestrial and aquatic environments as a selective agent in the proliferation of antibiotic resistance caused by the co-selection of antibiotic and metal resistance genes carried by transmissible plasmids and/or associated with transposons. Efflux pumps are also known to be involved in either antibiotic or metal resistance. In order to deal with these situations, microorganisms use an effective strategy that includes a range of expressions based on biochemical and genetic mechanisms. The data from numerous studies suggest that heavy metal contamination could affect the dissemination of antibiotic-resistant genes. Environmental pollution caused by anthropogenic activities could lead to mutagenesis based on the synergy between antibiotic efficacy and the acquired resistance mechanism under stressors. Moreover, the acquired resistance includes plasmid-encoded specific efflux pumps. Soil microbiomes have been reported as reservoirs of resistance genes that are available for exchange with pathogenic bacteria. Importantly, metal-contaminated soil is a selective agent that proliferates antibiotic resistance through efflux pumps. Thus, the use of multi-drug efflux pump inhibitors (EPIs) originating from natural plants or synthetic compounds is a promising approach for restoring the efficacy of existing antibiotics, even though they face a lot of challenges.

In silico screening and in vitro validation of phytocompounds as multidrug efflux pump inhibitor against E. coli

Multiple drug resistance (MDR) in bacteria has increased globally in recent times. This has reduced the efficacy of antibiotics and increasing the rate of therapeutic failure. Targeting efflux pump by natural and synthetic compounds is one of the strategies to develop an ideal broad-spectrum resistance-modifying agent. Very few inhibitors of AcrB from natural sources have been reported till date. In the current study, 19 phytocompounds were screened for efflux pump inhibitory activity against AcrB protein of E. coli TG1 using molecular docking studies. The molecular dynamics simulation provided stability the protein (AcrB) and its complex with chlorogenic acid under physiological conditions. Moreover, the detailed molecular insights of the binding were also explored. The Lipinski rule of 5 and the drug-likeness prediction was determined using Swiss ADME server, while toxicity prediction was done using admetSAR and PROTOX-II webservers. Chlorogenic acid showed the highest binding affinity (−9.1 kcal mol−1) with AcrB protein among all screened phytocompounds. Consequently, all the phytocompounds that accede to Lipinski’s rule, demonstrated a high LD50 value indicating that they are non-toxic except the phytocompound reserpine. Chlorogenic acid and capsaicin are filtered out based on the synergy with tetracycline having FIC index of 0.25 and 0.28. The percentage increase of EtBr fluorescence by chlorogenic acid was 36.6% followed by piperine (24.2%). Chlorogenic acid may be a promising efflux pump inhibitor that might be employed in combination therapy with tetracycline against E. coli, based on the above relationship between in silico screening and in vitro positive efflux inhibitory activity. Communicated by Ramaswamy H. Sarma.

Inhibitor-Resistant Mutants Give Important Insights into Candida albicans ABC Transporter Cdr1 Substrate Specificity and Help Elucidate Efflux Pump Inhibition.

ABSTRACTOverexpression of ATP-binding cassette (ABC) transporters is a major cause of drug resistance in fungal pathogens. Milbemycins, enniatin B, beauvericin, and FK506 are promising leads for broad-spectrum fungal multidrug efflux pump inhibitors. The characterization of naturally generated inhibitor-resistant mutants is a powerful tool to elucidate structure-activity relationships in ABC transporters. We isolated 20 Saccharomyces cerevisiae mutants overexpressing Candida albicans ABC pump Cdr1 variants resistant to fluconazole efflux inhibition by milbemycin α25 (8 mutants), enniatin B (8), or beauvericin (4). The 20 mutations were in just 9 residues at the centers of transmembrane segment 1 (TMS1) (6 mutations), TMS4 (4), TMS5 (4), TMS8 (1), and TMS11 (2) and in A713P (3), a previously reported FK506-resistant “hot spot 1” mutation in extracellular loop 3. Six Cdr1-G521S/C/V/R (TMS1) variants were resistant to all four inhibitors, four Cdr1-M639I (TMS4) variants were resistant to milbemycin α25 and enniatin B, and two Cdr1-V668I/D (TMS5) variants were resistant to enniatin B and beauvericin. The eight milbemycin α25-resistant mutants were altered in four amino acids as follows: G521R, M639I, A713P, and T1355N (TMS11). These four Cdr1 variants responded differently to various types of inhibitors, and each exhibited altered substrate specificity and kinetic properties. The data infer an entry gate function for Cdr1-G521 and a role for Cdr1-A713 in the constitutively high Cdr1 ATPase activity. Cdr1-M639I and -T1355N possibly cause inhibitor resistance by altering TMS contacts near the substrate/inhibitor-binding pocket. Models for the interactions of substrates and different types of inhibitors with Cdr1 at various stages of the transport cycle are presented.

Discovery of multidrug efflux pump inhibitors with a novel chemical scaffold

Multidrug efflux is a major contributor to antibiotic resistance in Gram-negative bacterial pathogens. Inhibition of multidrug efflux pumps is a promising approach for reviving the efficacy of existing antibiotics. Previously, inhibitors targeting both the efflux transporter AcrB and the membrane fusion protein AcrA in the Escherichia coli AcrAB-TolC efflux pump were identified. Here we use existing physicochemical property guidelines to generate a filtered library of compounds for computational docking. We then experimentally test the top candidate coumpounds using in vitro binding assays and in vivo potentiation assays in bacterial strains with controllable permeability barriers. We thus identify a new class of inhibitors of E. coli AcrAB-TolC. Six molecules with a shared scaffold were found to potentiate the antimicrobial activity of erythromycin and novobiocin in hyperporinated E. coli cells. Importantly, these six molecules were also active in wild-type strains of both Acinetobacter baumannii and Klebsiella pneumoniae, potentiating the activity of erythromycin and novobiocin up to 8-fold.

Efficacy of “HLE”—a multidrug efflux-pump inhibitor—as a disinfectant against surface bacteria

We evaluated the efficacy of a new disinfectant product, HLE, to inhibit multiple species of planktonic and biofilm bacterial cultures. The HLE disinfectant comprised of EDTA, lactic acid and hydrogen peroxide, and our data indicated that the disinfectant had effective antimicrobial and anti-biofilm activity even at low concentrations (0.15% to 0.4% HLE, v/v). Furthermore, the HLE disinfectant destabilized biofilm structures eradicated them due to the synergistic effect of EDTA and both antimicrobials (lactic acid and hydrogen peroxide), as revealed by confocal laser scanning microscopy. Additionally, sub-inhibitory concentrations of HLE disinfectant, with EDTA as an efflux pump inhibitor, inhibited the expression of multidrug EfrAB, NorE and MexCD efflux pumps in both planktonic and biofilm cultures. This could provide an alternative way to disinfect surfaces to avoid spreading multi-drug resistant strains in the food chain and the environment by decreasing efflux pump expression and consequently reducing the antibiotic selective pressure caused by systemic antibiotics and disinfectant use.

Boeravinone B, A Novel Dual Inhibitor of NorA Bacterial Efflux Pump of Staphylococcus aureus and Human P-Glycoprotein, Reduces the Biofilm Formation and Intracellular Invasion of Bacteria.

This study elucidated the role of boeravinone B, a NorA multidrug efflux pump inhibitor, in biofilm inhibition. The effects of boeravinone B plus ciprofloxacin, a NorA substrate, were evaluated in NorA-overexpressing, wild-type, and knocked-out Staphylococcus aureus (SA-1199B, SA-1199, and SA-K1758, respectively). The mechanism of action was confirmed using the ethidium bromide accumulation and efflux assay. The role of boeravinone B as a human P-glycoprotein (P-gp) inhibitor was examined in the LS-180 (colon cancer) cell line. Moreover, its role in the inhibition of biofilm formation and intracellular invasion of S. aureus in macrophages was studied. Boeravinone B reduced the minimum inhibitory concentration (MIC) of ciprofloxacin against S. aureus and its methicillin-resistant strains; the effect was stronger in SA-1199B. Furthermore, time–kill kinetics revealed that boeravinone B plus ciprofloxacin, at subinhibitory concentration (0.25 × MIC), is as equipotent as that at the MIC level. This combination also had a reduced mutation prevention concentration. Boeravinone B reduced the efflux of ethidium bromide and increased the accumulation, thus strengthening the role as a NorA inhibitor. Biofilm formation was reduced by four–eightfold of the minimal biofilm inhibitory concentration of ciprofloxacin, effectively preventing bacterial entry into macrophages. Boeravinone B effectively inhibited P-gp with half maximal inhibitory concentration (IC50) of 64.85 μM. The study concluded that boeravinone B not only inhibits the NorA-mediated efflux of fluoroquinolones but also considerably inhibits the biofilm formation of S. aureus. Its P-gp inhibition activity demonstrates its potential as a bioavailability and bioefficacy enhancer.

Mechanistic differences in the uptake of salicylic acid glucose conjugates by vacuolar membrane-enriched vesicles isolated from Arabidopsis thaliana.

Salicylic acid (SA) is a plant hormone involved in a number of physiological responses including both local and systemic resistance of plants to pathogens. In Arabidopsis, SA is glucosylated to form either SA 2-O-β-d-glucose (SAG) or SA glucose ester (SGE). In this study, we show that SAG accumulates in the vacuole of Arabidopsis, while the majority of SGE was located outside the vacuole. The uptake of SAG by vacuolar membrane-enriched vesicles isolated from Arabidopsis was stimulated by the addition of MgATP and was inhibited by both vanadate (ABC transporter inhibitor) and bafilomycin A1 (vacuolar H+ -ATPase inhibitor), suggesting that SAG uptake involves both an ABC transporter and H+ -antiporter. Despite its absence in the vacuole, we observed the MgATP-dependent uptake of SGE by Arabidopsis vacuolar membrane-enriched vesicles. SGE uptake was not inhibited by vanadate but was inhibited by bafilomycin A1 and gramicidin D providing evidence that uptake was dependent on an H+ -antiporter. The uptake of both SAG and SGE was also inhibited by quercetin and verapamil (two known inhibitors of multidrug efflux pumps) and salicin and arbutin. MgATP-dependent SAG and SGE uptake exhibited Michaelis-Menten-type saturation kinetics. The vacuolar enriched-membrane vesicles had a 46-fold greater affinity and a 10-fold greater transport activity with SGE than with SAG. We propose that in Arabidopsis, SAG is transported into the vacuole to serve as a long-term storage form of SA while SGE, although also transported into the vacuole, is easily hydrolyzed to release the active hormone which can then be remobilized to other cellular locations.

Detection of potential AcrAB-TolC multidrug efflux pump inhibitor in calyces extract of Hibiscus sabdariffa

Aim: The aim of this study is to investigate occurrence of potential efflux pump inhibitor (EPI) against AcrAB-TolC efflux pump in the methanol extract of H. sabdariffa. Materials and Methods: Calyces of H. sabdariffa were purchased from the local market in April 2014, methanol extract of H. sabdariffa was subjected to agar plate diffusion against Escherichia coli TG1 and its ∆acrB-∆tolC and thin layer chromatography (TLC) bioassay. The corresponding EPI fraction was eluted by methanol. The synergistic effect of antimicrobials and EPI fraction was measured by minimum inhibitory concentration (MIC) determination for E. coli and Erwinia amylovora strains, and the ability of EPI fraction to enhance EtBr accumulation was conducted. Results: E. coli TG1 was more sensitive to the methanol extracts of H. sabdariffa than E. coli ∆acrB-∆tolC, and inhibition zone corresponding to flavones on TLC bioassay plate has been formed which might be related to the fraction of potential EPI. The MIC values revealed that EPI fraction enhanced the activity of the used antimicrobials by 4 to 8 folds in E. coli TG1 and by 4 to 10 folds in E. amylovora 1189. Addition of EPI fraction in a dose-dependent manner increased the intercellular accumulation of Ethidium Bromide (EtBr) in the wild type stains of E. coli TG1 and E. amylovora 1189. Conclusion: EPI fraction behaves like a multidrug efflux pump inhibitor and further investigation should be conducted for determination of the chemical structure of EPI fraction.

Inhibitors of multidrug efflux pumps of Pseudomonas aeruginosa from natural sources: An in silico high-throughput virtual screening and in vitro validation

Pseudomonas aeruginosa is resistant to a wide range of antibiotics, thus making troublesome the infection treatment. Efflux systems are the main mechanisms involved; among these, MexAB-OprM is a tripartite efflux pump responsible for resistance to ciprofloxacin, aztreonam, gentamicin, tetracycline and tobramycin. In an attempt to contrast antibiotic efflux, databases of natural compounds were tested for their ability to bind MexB, the inner membrane channel, using a high-throughput virtual screening approach. The comparison of their common pharmacophoric features was the basis for inhibitor identification and selection process. In silico screening against the MexB protein was performed by Autodock/Vina and further refined using a minimization/focused docking protocol on the obtained complexes. The compounds showing the best docking and resulting potentially active at nanomolar concentration have been selected and used in combination with antibiotics usually exported by MexAB-OprM in antimicrobial in vitro synergy tests (checkerboard and time kill assays) against multidrug-resistant P. aeruginosa clinical isolates. The combinations morelloflavone and pregnan-20-one-derivative/ciprofloxacin showed a four-fold MIC decrease and 100-fold increase of the bacterial killing compared to the antibiotic alone. The two chosen hits were validated by ethidium bromide accumulation assay for their efflux inhibition potency. These compounds showed the ability to increase the accumulation of ethidium bromide inside the bacterial cells as evidenced by the increase of its fluorescence in the presence of the each of them. Finally, their toxicity has been preliminary tested through hemolysis assay. The observed good correlation between in silico docking and in vitro synergy tests, indicates these two compounds as promising drugs to be used in combination therapy against MDR and MexAB-OprM overexpressing P. aeruginosa.

Antibacterial activity of epigallocatechin-3-gallate (EGCG) and its synergism with β-lactam antibiotics sensitizing carbapenem-associated multidrug resistant clinical isolates of Acinetobacter baumannii

BackgroundInfections caused by Acinetobacter baumannii were responsive to conventional antibiotic therapy. However, recently, carbapenem-associated multidrug resistant isolates have been reported worldwide and present a major therapeutic challenge. Epigallocatechin-3-Gallate (EGCG) extracted from green tea exhibits antibacterial activity. PurposeWe evaluated the antibacterial activity of EGCG and possible synergism with antibiotics in carbapenem-associated multidrug resistant A. baumannii. A potential mechanism for synergism was also explored. Materials and methodsSeventy clinical isolates of A. baumannii collected from geographically different areas were analyzed by minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of EGCG. Checkerboard and time-killing assays were performed to exam the synergism between EGCG and antibiotics. The effects of EGCG on a multidrug efflux pump inhibitor (1-[1-naphthylmethyl] piperazine; NMP) and β-lactamase production were also examined in A. baumannii. ResultsSixty-three of 70 clinical isolates of A. baumannii carried carbapenemase-encoding genes with carbapenem-associated multidrug resistance. Levels of MIC and MBC of EGCG ranged from 64 to 512µg/ml and from 128 to ≥1024µg/ml, respectively among the clinical isolates. MIC90 and MBC86 levels were 256µg/ml and 512µg/ml of EGCG, respectively. Subinhibitory concentration of EGCG in combination with all antibiotics tested, including carbapenem, sensitized (MICs fall≤1.0µg/ml) all carbapenem-associated multidrug resistant isolates. Checkerboard and time-killing assays showed synergism between EGCG and meropenem (or carbenicillin) counted as fractional inhibitory concentration of < 0.5 and cell numbers’ decrease per ml of >2log10 within 12h, respectively. EGCG significantly increased the effect of NMP but was unrelated to β-lactamase production in A. baumannii, suggesting EGCG may be associated with inhibition of efflux pumps. ConclusionOverall we suggest that EGCG-antibiotic combinations might provide an alternative approach to treat infections with A. baumannii regardless of antibiotic resistance.

Reviving Antibiotics: Efflux Pump Inhibitors That Interact with AcrA, a Membrane Fusion Protein of the AcrAB-TolC Multidrug Efflux Pump.

Antibiotic resistance is a major threat to human welfare. Inhibitors of multidrug efflux pumps (EPIs) are promising alternative therapeutics that could revive activities of antibiotics and reduce bacterial virulence. Identification of new druggable sites for inhibition is critical for the development of effective EPIs, especially in light of constantly emerging resistance. Here, we describe EPIs that interact with periplasmic membrane fusion proteins, critical components of efflux pumps that are responsible for the activation of the transporter and the recruitment of the outer-membrane channel. The discovered EPIs bind to AcrA, a component of the prototypical AcrAB-TolC pump, change its structure in vivo, inhibit efflux of fluorescent probes, and potentiate the activities of antibiotics in Escherichia coli and other Gram-negative bacteria. Our findings expand the chemical and mechanistic diversity of EPIs, suggest the mechanism for regulation of the efflux pump assembly and activity, and provide a promising path for reviving the activities of antibiotics in resistant bacteria.

Bacterial Multidrug Efflux Pumps: Much More Than Antibiotic Resistance Determinants.

Bacterial multidrug efflux pumps are antibiotic resistance determinants present in all microorganisms. With few exceptions, they are chromosomally encoded and present a conserved organization both at the genetic and at the protein levels. In addition, most, if not all, strains of a given bacterial species present the same chromosomally-encoded efflux pumps. Altogether this indicates that multidrug efflux pumps are ancient elements encoded in bacterial genomes long before the recent use of antibiotics for human and animal therapy. In this regard, it is worth mentioning that efflux pumps can extrude a wide range of substrates that include, besides antibiotics, heavy metals, organic pollutants, plant-produced compounds, quorum sensing signals or bacterial metabolites, among others. In the current review, we present information on the different functions that multidrug efflux pumps may have for the bacterial behaviour in different habitats as well as on their regulation by specific signals. Since, in addition to their function in non-clinical ecosystems, multidrug efflux pumps contribute to intrinsic, acquired, and phenotypic resistance of bacterial pathogens, the review also presents information on the search for inhibitors of multidrug efflux pumps, which are currently under development, in the aim of increasing the susceptibility of bacterial pathogens to antibiotics.

Therapeutic options and emerging alternatives for multidrug resistant staphylococcal infections.

Methicillin-resistant Staphylococcus aureus (MRSA) remains the single biggest challenge in infectious disease in the civilized world. Moreover, vancomycin resistance is also spreading, leading to fears of untreatable infections as were common in ancient times. Molecular microbiology and bioinformatics have revealed many of the mechanisms involved in resistance development. Mobile genetic elements, up-regulated virulence factors and multi-drug efflux pumps have been implicated. A range of approved antibiotics from the glycopeptide, lipopeptide, pleuromutilin, macrolide, oxazolidinone, lincosamide, aminoglycoside, tetracycline, steptogramin, and cephalosporin classes has been employed to treat MRSA infections. The upcoming pipeline of drugs for MRSA includes some new compounds from the above classes, together with fluoroquinolones, antibacterial peptide mimetics, aminomethylciclines, porphyrins, peptide deformylase inhibitors, oxadiazoles, and diaminopyrimidines. A range of non-drug alternative approaches has emerged for MRSA treatment. Bacteriophage-therapy including purified lysins has made a comeback after being discovered in the 1930s. Quorum-sensing inhibitors are under investigation. Small molecule inhibitors of multi-drug efflux pumps may potentiate existing antibiotics. The relative failure of staphylococcal vaccines is being revisited by efforts with multi-valent vaccines and improved adjuvants. Photodynamic therapy uses non-toxic photosensitizers and harmless visible light to produce reactive oxygen species that can nonspecifically destroy bacteria while preserving host cells. Preparation of nanoparticles can kill bacteria themselves, as well as improve the delivery of anti-bacterial drugs. Anti-MRSA drug discovery remains an exciting field with great promise for the future.

Evaluation of the Efficiency of Synthesized Efflux Pump Inhibitors on Salmonella enterica ser. Typhimurium Cells

Multidrug efflux pump inhibitors have a great potential as pharmacological agents that increase the drug susceptibility of bacterial pathogens. Our study was focused on the synthesis and evaluation of the efficiency of resistance-nodulation-division (RND) family efflux pump inhibitors. The efficiency of these inhibitors was investigated on Salmonella enterica ser. typhimurium cells using tetraphenylphosphonium (TPP(+) ) and ethidium cations as the efflux pump substrates. Results of our study indicated that efficiency of the inhibitors depends on the cell outer membrane permeability and method of the assay used. Temperature of the incubation medium and a solvent of the inhibitor used have only minor effect on results of the assay.

Mammalian Multidrug Resistance Lipopentasaccharide Inhibitors from Ipomoea alba Seeds

As part of an ongoing project to identify inhibitors of multidrug efflux pumps, three new resin glycosides, albinosides I-III (1-3), were isolated from a CHCl(3)-soluble extract from the seeds of moon vine (Ipomoea alba). Their structures were established through NMR spectroscopy and mass spectrometry as partially acylated branched pentasaccharides derived from three new glycosidic acids, named albinosinic acids A-C (4-6). The same oligosaccharide core formed by two D-quinovose, one D-glucose, and two L-rhamnose units was linked to either convolvulinolic or jalapinolic acid for 1 and 3, respectively. They were partially esterified with (2R,3R)-3-hydroxy-2-methylbutanoic, acetic, or 2-methyl-2-butenoic acid. Compound 2 has two D-quinovose and three L-rhamnose units, linked to convolvulinolic acid, and its esterifying residues were characterized as two units of 2-methyl-2-butenoic acid. The aglycone lactonization site was located at C-2 of the terminal rhamnose unit (Rha) for 1, at C-3 of the terminal rhamnose unit (Rha') for 2, and at C-3 of the second saccharide unit (Glc) for 3. Reversal of multidrug resistance by this class of plant metabolites was also evaluated in vinblastine-resistant human breast carcinoma cells (MCF-7/Vin). The noncytotoxic compound 3 exerted the strongest potentiation effect of vinblastine susceptibility to over 2140-fold, while a moderate activity was observed for 1 (3.1-fold) and 2 (2.6-fold) at a concentration of 25 μg/mL.

A Microfluidic Device for Simple and Rapid Evaluation of Multidrug Efflux Pump Inhibitors

Recently, multidrug-resistant pathogens have disseminated widely owing essentially to their increased multidrug efflux pump activity. Presently, there is a scarcity of new antibacterial agents, and hence, inhibitors of multidrug efflux pumps belonging to the resistance–nodulation–cell division (RND) family appear useful in the treatment of infections by multidrug-resistant pathogens. Moreover, recent progress in microfabrication technologies has expanded the application of nano/micro-devices to the field of human healthcare, such as the detection of infections and diagnosis of diseases. We developed a microfluidic channel device for a simple and rapid evaluation of bacterial drug efflux activity. By combining the microfluidic device with a fluorogenic compound, fluorescein-di-β-D-galactopyranoside, which is hydrolyzed to a fluorescent dye in the cytoplasm of Escherichia coli, we successfully evaluated the effects of inhibitors on the RND-type multidrug efflux pumps MexAB–OprM and MexXY–OprM from Pseudomonas aeruginosa in E. coli. Our new method successfully detected the MexB-specific inhibitory effect of D13-9001 and revealed an unexpected membrane-permeabilizing effect of Phe-Arg-β-naphthylamide, which has long been used as an efflux pump inhibitor.

Microbial Efflux Pump Inhibition: Tactics and Strategies

Traditional antimicrobials are increasingly suffering from the emergence of multidrug resistance among pathogenic microorganisms. To overcome these deficiencies, a range of novel approaches to control microbial infections are under investigation as potential alternative treatments. Multidrug efflux is a key target of these efforts. Efflux mechanisms are broadly recognized as major components of resistance to many classes of chemotherapeutic agents as well as antimicrobials. Efflux occurs due to the activity of membrane transporter proteins widely known as Multidrug Efflux Systems (MES). They are implicated in a variety of physiological roles other than efflux and identifying natural substrates and inhibitors is an active and expanding research discipline. One plausible alternative is the combination of conventional antimicrobial agents/antibiotics with small molecules that block MES known as multidrug efflux pump inhibitors (EPIs). An array of approaches in academic and industrial research settings, varying from high-throughput screening (HTS) ventures to bioassay guided purification and determination, have yielded a number of promising EPIs in a series of pathogenic systems. This synergistic discovery platform has been exploited in translational directions beyond the potentiation of conventional antimicrobial treatments. This venture attempts to highlight different tactical elements of this platform, identifying the need for highly informative and comprehensive EPI-discovery strategies. Advances in assay development genomics, proteomics as well as the accumulation of bioactivity and structural information regarding MES facilitates the basis for a new discovery era. This platform is expanding drastically. A combination of chemogenomics and chemoinformatics approaches will integrate data mining with virtual and physical HTS ventures and populate the chemical-biological interface with a plethora of novel chemotypes. This comprehensive step will expedite the preclinical development of lead EPIs.

Topoisomerase mutations and efflux are associated with fluoroquinolone resistance in Enterococcus faecalis

To understand better the mechanisms of fluoroquinolone resistance in Enterococcus faecalis, fluoroquinolone-resistant mutants isolated from Ent. faecalis ATCC 29212 by stepwise selection with sparfloxacin (SPX) and norfloxacin (NOR) were analysed. The results showed the following. (i) In general, fluoroquinolone-resistance mechanisms in Ent. faecalis are similar to those in other Gram-positive bacteria, such as Staphylococcus aureus and Streptococcus pneumoniae, namely, mutants with amino acid changes in both GyrA and ParC exhibited high fluoroquinolone resistance, and single GyrA mutants and a single ParC mutant were more resistant to SPX and NOR, respectively, than the parent strain, indicating that the primary targets of SPX and NOR in Ent. faecalis are DNA gyrase and topoisomerase IV, respectively. (ii) Alterations in GyrB (DeltaKGA, residues 395-397) and ParE (Glu-459 to Lys) were associated with fluoroquinolone resistance in some mutants. Moreover, the facts that the NOR MIC, but not the SPX MIC, decreased in the presence of multidrug efflux pump inhibitors, that NOR accumulation decreased in the cells, and that the EmeA mRNA expression level did not change, strongly suggested that a NorA-like efflux pump, rather than EmeA, was involved in resistance to NOR.

Susceptibility of Campylobacter hyointestinalis subsp. hyointestinalis to antimicrobial agents and characterization of quinolone-resistant strains

To study the susceptibility of Campylobacter hyointestinalis subsp. hyointestinalis to several antimicrobial agents and to investigate the mechanisms of nalidixic acid and ciprofloxacin resistance. The disc diffusion method was employed to study the susceptibility of 49 C. hyointestinalis subsp. hyointestinalis strains of reindeer and bovine origin to 12 different antimicrobial agents. In addition, the MICs of nalidixic acid and ciprofloxacin were determined. The nucleotide sequence of a 270 bp fragment of the gyrA gene was determined in ciprofloxacin-susceptible and -resistant strains. The effect of a multidrug efflux pump inhibitor Phe-Arg-beta-naphthylamide (PA beta N) on the MICs of ciprofloxacin and nalidixic acid was also studied. The only decreased susceptibility for antimicrobial agents of this study was observed for sulphonamide compound and streptomycin (24% and 32% of the strains, respectively), and this phenomenon was observed exclusively in the bovine strains. In sequence studies, a Thr-86-->Ile change was found in strains with MICs of ciprofloxacin of > or = 64 mg/L, but this mutation was absent in strains with lower resistance levels. The use of PA beta N did not affect the MIC of ciprofloxacin but decreased the MIC of nalidixic acid 2-4-fold. The Finnish C. hyointestinalis subsp. hyointestinalis strains are susceptible to a majority of the antimicrobials of veterinary importance. The mechanism of ciprofloxacin resistance at lower levels (< or = 32 mg/L) is not associated with a specific mutation in the quinolone resistance-determining region of the gyrA gene. Finally, there are distinct differences in the mechanisms of ciprofloxacin resistance compared with nalidixic acid resistance within the studied species.

A Multidrug Efflux Pump Inhibitor Reduces Fluoroquinolone Resistance in Pseudomonas aeruginosa Isolates

In general, resistance to fluoroquinolones (FQs) in gram-negative bacteria is acquired either by mutations in DNA gyrase and topoisomerase IV or by active export of the agents via antibiotic efflux pumps. Reduced porin expression is also proposed to be another mechanism leading to resistance. In this study, interaction between levofloxacin, ofloxacin, and ciprofloxacin with MC-207,110 (multidrug efflux pump inhibitor) was investigated by a checkerboard assay using Pseudomonas aeruginosa. Levofloxacin, ofloxacin, and ciprofloxacin were tested at different concentrations (0.06–64 µg/ml) and MC-207,110 was tested at a concentration range of 4–128 µg/ml. In the presence of MC-207,110 (at 128, 64, 32, 16 µg/ml) resistance to FQs was inhibited significantly and MIC values were decreased, except at 8 and 4 µg/ml of MC-207,110. When MC-207,110 was used, resistance of P. aeruginosa to FQs in vitro was inhibited significantly, suggesting that MC-207,110 may be useful for use in clinical treatment protocols to overcome FQs resistance.