Efflux Pump Inhibitory Activity Research Articles

Mycobactin analogue interacting with siderophore efflux-pump protein: insights from molecular dynamics simulations and whole-cell assays

IntroductionIn response to continued public health emergency of antimicrobial resistance (AMR), a significant key strategy is the discovery of novel mycobacterial efflux-pump inhibitors (EPIs) as potential adjuvants in combination drug therapy. Interest in identifying new chemotypes which could potentially synergize with the existing antibiotics and can be deployed as part of a combination therapy. This strategy could delay the emergence of resistance to existing antibiotics and increase their efficacy against resistant strains of mycobacterial species. In recent decades, notable approaches have been accounted for EPI development and have resulted in the discovery of several EPIs including SQ109 and AU1235. In context, to accelerate newer EPIs with novel mode of action here we have discussed mycobactin analogues and highlighted in silico binding orientation with siderophore efflux-pump proteins MmpL4/5.Methods3-(2-hydroxyphenyl)-5-(aryl)-pyrazoline series was investigated for whole-cell efflux-pump inhibitory activity against Mycobacterium smegmatis and Mycobacterium abscessus. Machine learning and molecular dynamics were performed to construct a MmpL4/5 complex embedded in a lipid bilayer to identify the putative binding site and to predict ligand-protein binding energetics. Furthermore, the identified HIT compound was investigated in synergistic assay with bedaquiline.ResultsCompound Il, 2-(5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenol, was identified as the most potent efflux pump inhibitor against M. smegmatis in whole-cell efflux-pump investigation. Followed HIT Il employed against M. abscessus for efflux-pump inhibition investigations and notable whole-cell efflux-pump inhibitory profile has been observed. The theoretical investigations predicted compound Il to be selective towards MmpL4, with significant hydrogen bonding and π-π stacking interactions effectively blocking a critical Asp-Tyr dyad interaction network necessary for proton translocation. Compound Il with bedaquiline highlighted an additive profile against the M. abscessus pathogen.ConclusionsMD simulations and whole-cell assays are indicating potential development of compound Il as an adjunct to the existing therapeutic regimen against mycobacterial infections.

Evaluation of phyto-gallic acid as a potential inhibitor of Staphylococcus aureus efflux pump mediated tetracycline resistance: an in-vitro and in-silico study

Plants contain many bioactive compounds with potent antibacterial and efflux pump inhibitory activity (EPI). In this study, gallic acid extracted from pomegranate molasses by analytical HPLC holds promise as an EPI drug for Staphylococcus aureus mediated tetracycline resistance, it lowered the bacterial resistance and reversed the mechanism via tet family efflux pump, using molecular technique and in-silico molecular docking analysis. Extracted gallic acid combined with tetracycline demonstrated a significant decrease in the minimal inhibitory concentration MIC compared to its single activity. Similarly, little growth and lower fluorescence of S. aureus were observed on ethidium bromide (2.5 mg/mL) agar plates, indicating a reversible efflux pump mechanism and a potent EPI activity. Molecular docking demonstrated a promising affinity binding energy between gallic acid and tet efflux genes, opening a new baseline in bacterial infection treatment. PCR for tetK and Qac A/B genes failed to show any relation between tet genes and gallic acid.

Discovery of novel dihydronaphthalene—imidazole ligands as potential inhibitors of Staphylococcus aureus multidrug resistant NorA efflux pump: A combination of experimental and in silico molecular docking studies

Overexpression of the efflux pump is a predominant mechanism by which bacteria show antimicrobial resistance (AMR) and leads to the global emergence of multidrug resistance (MDR). In this work, the inhibitory potential of library of dihydronapthyl scaffold-based imidazole derivatives having structural resemblances with some known efflux pump inhibitors (EPI) were designed, synthesized and evaluated against efflux pump inhibitor against overexpressing bacterial strains to study the synergistic effect of compounds and antibiotics. Out of 15 compounds, four compounds (Dz-1, Dz-3, Dz-7, and Dz-8) were found to be highly active. DZ-3 modulated the MIC of ciprofloxacin, erythromycin, and tetracycline by 128-fold each against 1199B, XU212 and RN4220 strains of S. aureus respectively. DZ-3 also potentiated tetracycline by 64-fold in E. coli AG100 strain. DZ-7 modulated the MIC of both tetracycline and erythromycin 128-fold each in S. aureus XU212 and S. aureus RN4220 strains. DZ-1 and DZ-8 showed the moderate reduction in MIC of tetracycline in E. coli AG100 only by 16-fold and 8-fold, respectively. DZ-3 was found to be the potential inhibitor of NorA as determined by ethidium bromide efflux inhibition and accumulation studies employing NorA overexpressing strain SA-1199B. DZ-3 displayed EPI activity at non-cytotoxic concentration to human cells and did not possess any antibacterial activity. Furthermore, molecular docking studies of DZ-3 was carried out in order to understand the possible binding sites of DZ-3 with the active site of the protein. These studies indicate that dihydronaphthalene scaffolds could serve as valuable cores for the development of promising EPIs.

Efflux Pump Inhibition-Based Screening and Anti-Infective Evaluation of Punica granatum Against Bacterial Pathogens.

Drug efflux pumps contribute to bacterial multidrug resistance (MDR), reducing antibiotic effectiveness and causing treatment failures. Besides their role in MDR, efflux pumps also assist in the transportation of quorum sensing (QS) signal molecules and increased the tolerance of biofilms. Recently, the search for efflux pump inhibitors from natural sources, including anti-infective plants, has gained attention as a potential therapy against drug-resistant bacteria. In this study, 19 traditional Indian medicinal plants were screened for their efflux pump inhibitory activity against Escherichia coli TGI. The promising extract, i.e., Punica granatum was subsequently fractioned in the solvents of increasing polarity. Among them, at sub-MIC active EPI fraction was PGEF (P. granatum ethyl acetate fraction), further investigated for anti-infective potential against Chromobacterium violaceum 12,472, Pseudomonas aeruginosa PAO1, and Serratia marcescens MTCC 97. PGEF was also evaluated for in vivo efficacy in Caenorhabditis elegans model. Major phytocompounds were analyzed by mass spectroscopic techniques. At respective Sub-MIC, PGEF reduced violacein production by 71.14% in C. violaceum 12,472. Moreover, PGEF inhibited pyocyanin (64.72%), pyoverdine (48.17%), protease (51.35%), and swarming motility (44.82%) of P. aeruginosa PAO1. Furthermore, PGEF reduced the production of prodigiosin and exoprotease by 64.73% and 61.80%, respectively. Similarly, at sub-MIC, PGEF inhibited (≥ 50%) biofilm development in all test pathogens. The key phytocompounds detected in active fraction include 5-hydroxymethylfurfural, trans-p-coumaric acid 4- glucoside, (-)-Epicatechin 3'-O-glucuronide, and ellagic acid. Interestingly, PGEF also demonstrated anti-infective efficacy against the PAO1-infected C. elegans test model and highlighting its therapeutic potential as an anti-infective agent to combat drug-resistant problems.

Rationally Designed Novel Phenyloxazoline Synthase Inhibitors: Chemical Synthesis and Biological Evaluation to Accelerate the Discovery of New Antimycobacterial Antibiotics.

The uncontrolled spread of drug-resistant tuberculosis (DR-TB) clinical cases necessitates the urgent discovery of newer chemotypes with novel mechanisms of action. Here, we report the chemical synthesis of rationally designed novel transition-state analogues (TSAs) by targeting the cyclization (Cy) domain of phenyloxazoline synthase (MbtB), a key enzyme of the conditionally essential siderophore biosynthesis pathway. Following bio-assay-guided evaluation of TSA analogues preferentially in iron-deprived and iron-rich media to understand target preferentiality against a panel of pathogenic and non-pathogenic mycobacteria strains, we identified a hit, i.e., TSA-5. Molecular docking, dynamics, and MMPBSA calculations enabled us to comprehend TSA-5's stable binding at the active site pocket of MbtB_Cy and the results imply that the MbtB_Cy binding pocket has a strong affinity for electron-withdrawing functional groups and contributes to stable polar interactions between enzyme and ligand. Furthermore, enhanced intracellular killing efficacy (8 μg/mL) of TSA-5 against Mycobacterium aurum in infected macrophages is noted in comparison to moderate in vitro antimycobacterial efficacy (64 μg/mL) against M. aurum. TSA-5 also demonstrates whole-cell efflux pump inhibitory activity against Mycobacterium smegmatis. Identification of TSA-5 by focusing on the modular MbtB_Cy domain paves the way for accelerating novel anti-TB antibiotic discoveries.

Computational assessment and in vitro test of phytochemicals of Usnea aciculifera as potential inhibitors of Escherichia coli efflux pump AcrB

Lichens produce secondary metabolites that have many pharmaceutical activities such as antimicrobial, antioxidant, antiviral, anticancer, antigenotoxic, anti-inflammatory, analgesic and antipyretic activities. However, there is limited research on their efflux pump inhibitory activities. Twelve phytochemicals were isolated from Usnea aciculifera, and their activity of AcrAB-TolC efflux pump inhibition was evaluated. Four potential compounds, which are diffractaic acid (2), 8′ -O- methylstictic acid (5), 3-hydroxy-4-(methoxycarbonyl)-2,5-dimethylphenyl 2,4-dimethoxy-3,6-dimethylbenzoate (8) and 3-hydroxy-4-(methoxycarbonyl)-2,5-dimethylphenyl 2-hydroxy-4-methoxy-3,6-dimethylbenzoate (9), were found by virtual screening using pharmacophore and 2D-QSAR model. Compound 8 exhibited AcrB inhibition activity in vitro with an accumulation H33342 percentage compared with untreated control of 202% at a concentration of 50 µM and increased the antibacterial activity of levofloxacin by four-fold at a concentration of 200 µM. By molecular docking and molecular dynamics (MD) simulation, the binding affinity of depside and depsidone derivatives to AcrB was also clarified. Despite the poor docking score to the AcrB binding site, compound 8 was the most stable among the four complexes at 20 ns of MD simulation. The analysis of long MD at 100 ns indicated that compound 8 interacts strongly with the residues in the distal pocket, creating a stable complex with ΔGbind of −31.51 kcal.mol-1. According to the ADMETlab 2.0 web server’s predictions of pharmacokinetics and toxicities, compound 8 has the potential for drug development. Communicated by Ramaswamy H. Sarma

Antimycobacterial, antibiofilm and efflux pump inhibitory activity of extracts from selected Combretum species used in traditional medicine in Zimbabwe

Antimycobacterial, antibiofilm and efflux pump inhibitory activity of extracts from selected Combretum species used in traditional medicine in Zimbabwe

Combination Therapy with Ciprofloxacin and Pentamidine against Multidrug-Resistant Pseudomonas aeruginosa: Assessment of In Vitro and In Vivo Efficacy and the Role of Resistance-Nodulation-Division (RND) Efflux Pumps.

The aim of this work was to (i) evaluate the efficacy of a combination treatment of pentamidine with ciprofloxacin against Galleria mellonella larvae infected with an MDR strain of P. aeruginosa and (ii) determine if pentamidine acts as an efflux-pump inhibitor. Resistant clinical isolates, mutant strains overexpressing one of three RND efflux pumps (MexAB-OprM, MexCD-OprJ, and MexEF-OprN), and a strain with the same three pumps deleted were used. MIC assays confirmed that the clinical isolates and the mutants overexpressing efflux pumps were resistant to ciprofloxacin and pentamidine. The deletion of the three efflux pumps induced sensitivity to both compounds. Exposure to pentamidine and ciprofloxacin in combination resulted in the synergistic inhibition of all resistant strains in vitro, but no synergy was observed versus the efflux-pump deletion strain. The treatment of infected G. mellonella larvae with the combination of pentamidine and ciprofloxacin resulted in enhanced efficacy compared with the monotherapies and significantly reduced the number of proliferating bacteria. Our measurement of efflux activity from cells revealed that pentamidine had a specific inhibitory effect on the MexCD-OprJ and MexEF-OprN efflux pumps. However, the efflux activity and membrane permeability assays revealed that pentamidine also disrupted the membrane of all cells. In conclusion, pentamidine does possess some efflux-pump inhibitory activity, in addition to a more general disruptive effect on membrane integrity that accounts for its ability to potentiate ciprofloxacin activity. Notably, the enhanced efficacy of combination therapy with pentamidine and ciprofloxacin versus MDR P. aeruginosa strains in vivo merits further investigation into its potential to treat infections via this pathogen in patients.

Plant Secondary Metabolites on Efflux-Mediated Antibiotic Resistant Stenotrophomonas Maltophilia: Potential of Herbal-Derived Efflux Pump Inhibitors.

During the process of adapting to metal contamination, plants produce secondary metabolites that have the potential to modulate multidrug-resistant (MDR) phenotypes; this is achieved by inhibiting the activity of efflux pumps to reduce the minimum inhibitory concentrations (MICs) of antimicrobial substrates. Our study evaluated the effect of secondary metabolites of belowground parts of Pteris vittata L. and Fallopia japonica, two metal-tolerant plants from northern Vietnam, on six antibiotic-resistant Stenotrophomonas maltophilia strains possessing efflux pump resistance mechanisms that were isolated from soil and clinical samples. The chemical composition of aqueous and dichloromethane (DCM) fractions extracted from P. vittata and F. japonica was determined using UHPLC-DAD-ESI/QTOF analysis. The antibacterial and efflux pump inhibitory activities of the four fractions were evaluated for the six strains (K279a, 0366, BurA1, BurE1, PierC1, and 502) using a microdilution assay at fraction concentrations of 62.5, 125, and 250 μg/mL. The DCM fraction of F. japonica exhibited remarkable antibacterial activity against strain 0366, with a MIC of 31.25 μg/mL. Furthermore, this fraction also significantly decreased gentamicin MIC: four-fold and eight-fold reductions for BurA1 and BurE1 strains, respectively (when tested at 250 μg/mL), and two-fold and eight-fold reductions for K279a and BurE1 strains, respectively (when tested at 125 μg/mL). Pure emodin, the main component identified in the DCM fraction of F. japonica, and sennidine A&B only reduced by half the MIC of gentamicin (when tested at 30 μg/mL). Our results suggest that the DCM fraction components of F. japonica underground parts may be potential candidates for new bacterial efflux pump inhibitors (EPIs).

Discovery of AcrAB-TolC pump inhibitors: Virtual screening and molecular dynamics simulation approach

AcrAB-TolC tripartite efflux pump, which belongs to the RND superfamily, is a main multi-drug efflux system of Escherichia coli (E. coli) because of the broad resistance on various antibiotics. With the discovering of efflux pump inhibitors (EPIs), a combination between these and antibiotics is one of the most promising therapies. Therefore, building a virtual screening model with prediction capacities for the efflux pump inhibitory activities of candidates from DrugBank and ZINC15 dataset, is one of the key goals of this project. Based on the database of 170 diverse chemical structures collected from 28 research journals, two 2D-QSAR models and a 3D-pharmacophore model have been performed. On the AcrB protein (PDB 4DX7), two binding sites have been discovered that match to the hydrophobic trap in the distal pocket and the switch loop in the proximal pocket. After virtual screening processes, twenty candidate AcrAB-TolC inhibitors have been subjected to molecular dynamics simulations, binding free energy calculations and ADMET predictions. The results indicate that three compounds namely DB09233, DB02581, and DB15224 are potential inhibitors with ΔGbind of −42.30 ± 4.58, −40.76 ± 7.30 and −31.06 ± 7.63 kcal.mol−1, respectively. Communicated by Ramaswamy H. Sarma

Evaluation of the antibacterial activity of hecogenin acetate and its inhibitory potential of NorA and MepA efflux pumps from Staphylococcus aureus.

Antimicrobial drugs are of great importance in the control of bacterial infections. Its indiscriminate use contributes to the consolidation of bacterial resistance. Its applicability is due to its secondary metabolites, such as saponins, which are compounds with relevant antibacterial action. Hecogenin acetate is a saponin present in plants of the agave genus with analgesic, antioxidant, antinociceptive, cardioactive, anticancer, antifungal and antimicrobial activity. The present work aimed to identify the antibacterial activity of hecogenin acetate against strains of E. coli, P. aeruginosa and S. aureus and to investigate the NorA and MepA efflux pump inhibitory activity of S. aureus strains. The Minimum Inhibitory Concentration was evaluated by broth microdilution. The Antibiotic Activity Modifier effect and the assessment of efflux pump inhibition were evaluated by microdilution with sub-inhibitory concentrations. Hecogenin acetate showed minimal inhibitory concentration without significant relevance. In the evaluation of the potentiating activity of the antibiotic action, a greater antagonistic behavior is noticed. In the analyzes performed with the efflux pump, it was noticed that the hecogenin acetate does not interfere in the efflux pump mechanism of the analyzed bacteria.

Evaluation of efflux pump inhibitory activity of some plant extracts and using them as adjuvants to potentiate the inhibitory activity of some antibiotics against Staphylococcus aureus

Antibiotic-resistant pathogens became a real global threat to human and animal health. This needs to concentrate the efforts to minimize and control these organisms. Efflux pumps are considered one of the important strategies used by bacteria to exclude harmful materials from the cell. Inhibition of these pumps can be an active strategy against multidrug resistance pathogens. There are two sources of efflux pump inhibitors that can be used, chemical and natural inhibitors. The chemical origin efflux pump inhibitors have many toxic side effects while the natural origin is characterized by a wide margin of safety for the host cell. In this study, the ability of some plant extracts like (propolis show rosemary, clove, capsaicin, and cumin) to potentiate the inhibitory activity of some antibiotics such as (ciprofloxacin, erythromycin, gentamycin, tetracycline, and ampicillin) against Staphylococcus aureus pathogen were tested. Efflux pump inhibitory activity of the selected plant extracts was tested using an ethidium bromide (EtBr) accumulation assay. The results have shown that Propolis has a significant synergistic effect in combination with ciprofloxacin, erythromycin, and gentamycin. While it has no effect with tetracycline or ampicillin. Also, no synergic effect was noticed in a combination of the minimum inhibitory concentration for the selected plant extracts (rosemary, clove, capsaicin, and cumin) with any of the tested antibiotics. Interestingly, according to the results of the EtBr accumulation assay, Propolis has potent inhibitory activity against the S. aureus (MRS usa300) pump system. This study suggests that Propolis might act as a resistance breaker that is able to restore the activity of ciprofloxacin, erythromycin, and gentamycin against S. aureus strains, in case of the efflux-mediated antimicrobial resistance mechanisms.

Plants Derived Efflux Pump Inhibitors: An approach against Multidrug-Resistant Gram-negative bacteria Klebsiella pneumoniae

The AcrAB/TolC efflux pump-mediated drug resistance in gram-negative bacteria might be controlled by efflux pump inhibitors (EPIs). This study was planned to discover a compound from plants that can be used as an EPI. Forty isolates of K. pneumoniae and 33 different plants were collected. The methanolic extracts of parts of plants were prepared. Chloramphenicol, tetracycline, and Ciprofloxacin were used for the sensitivity assay. Plant extracts were tested for EPI activity using Berberine and Et

Evaluation of the antibacterial and inhibitory activity of NorA and MepA efflux pumps from Staphylococcus aureus by diosgenin

The increase in bacterial resistance to available antibiotics has driven several researchers to search for new agents with therapeutic properties. Diosgenin is a naturally occurring steroidal saponin that has demonstrated several pharmacological properties. In the present study, we report the antimicrobial activity of diosgenin against the standard and multidrug-resistant bacteria of Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, in addition to the efflux pump inhibitory activity against Staphylococcus aureus strains carrying NorA and MepA pumps. For this purpose, the broth microdilution method was used, from which the value of the Minimum Inhibitory Concentration (MIC) was obtained, and this was associated with subinhibitory concentration (MIC/8) with antibiotic of clinical use and ethidium bromide for strains carrier by efflux pump. Diosgenin showed antimicrobial activity for standard S. aureus bacteria and potentiating activity in association with gentamicin and ampicillin for P. aeruginosa multidrug-resistant bacteria, it also showed potentiation in association with norfloxacin against the E. coli strain and gentamicin against the S. aureus strain. Antimicrobial activity against efflux pump-bearing strains revealed that saponin did not interfere with the efflux pump mechanism or intervened antagonistically. Thus, saponin has shown to be very promising against bacterial resistance in association with aminoglycoside, fluoroquinolones and beta-lactam, however additional studies should be carried out to better elucidate the mechanism of action of diosgenin.

Promising FDA-approved drugs with efflux pump inhibitory activities against clinical isolates of Staphylococcus aureus.

Background and objectivesStaphylococcus aureus is an opportunistic pathogen that causes wide range of nosocomial and community-acquired infections which have spread worldwide leading to an urgent need for developing effective anti-staphylococcal agents. Efflux is an important resistance mechanism that bacteria used to fight the antimicrobial action. This study aimed to investigate the efflux mechanism in S. aureus and assess diclofenac, domperidone, glyceryl trinitrate and metformin as potential efflux pump inhibitors that can be used in combination with antibiotics for treating topical infections caused by S. aureus.Materials and methodsEfflux was detected qualitatively by the ethidium bromide Cart-Wheel method followed by investigating the presence of efflux genes by polymerase chain reaction. Twenty-six isolates were selected for further investigation of efflux by Cart-Wheel method in absence and presence of tested compounds followed by quantitative efflux assay. Furthermore, antibiotics minimum inhibitory concentrations in absence and presence of tested compounds were determined. The effects of tested drugs on expression levels of efflux genes norA, fexA and tetK were determined by quantitative real time-polymerase chain reaction.ResultsEfflux was found in 65.3% of isolates, the prevalence of norA, tetK, fexA and msrA genes were 91.7%, 77.8%, 27.8% and 6.9%. Efflux assay revealed that tested drugs had potential efflux inhibitory activities, reduced the antibiotic’s MICs and significantly decreased the relative expression of efflux genes.ConclusionDiclofenac sodium, domperidone and glyceryl trinitrate showed higher efflux inhibitory activities than verapamil and metformin. To our knowledge, this is the first report that shows that diclofenac sodium, glyceryl trinitrate and domperidone have efflux pump inhibitory activities against S. aureus.

Efflux Pump Inhibitory Potential of Vitexin 2"-O-xyloside Against Gram Positive Bacteria Staphylococcus aureus

Expression of bacterial efflux pump is one of the major causes for developing antibiotic resistance in a variety of pathogens. Efflux pump inhibitors (EPIs) are potential molecules that can antagonize those pumps and help in alleviating the resistance problem. The study was aimed to explore the EPI activity of vitexin 2"-O-xyloside, an apigenin flavone glycoside. A total 15 clinical isolates of Staphylococcus aureus were collected and their antibiotic resistance profiles were detected by Kirby-Bauer disc diffusion assay and MIC values were determined through broth microdilution technique. Prevalence of efflux pump activity were examined through ethidium bromide agar-cartwheel method. Fractional inhibitory assay was carried out in combination with tetracycline and ciprofloxacin against clinical isolates of S. aureus with efflux pump activity. Finally, molecular docking approach was carried out in the active binding sites of NorA efflux pump protein by Autodock Vina. All of the clinical isolates showed resistance to cefixime, ciprofloxacin and tetracycline antibiotics whereas all of them were sensitive to chloramphenicol. Efflux pump was found active among 20% of the clinical isolates. The tested compound showed additive effective (ƩFIC value 0.625 – 0.75) when co-treated with tetracycline in the efflux pump active isolates which was similar to reserpine. Molecular docking studies showed that vitexin 2"-O-xyloside may bind to the different binding sites, opening the door for it to be considered as a potential EPI. Dhaka Univ. J. Pharm. Sci. 20(3): 307-315, 2022 (June) Centennial Special Issue

Iodine-Mediated C═C Double Bond Cleavage toward Pyrido[2,1-b]quinazolinones.

A transition-metal-free C═C double bond cleavage reaction employing molecular iodine is described. In the presence of K2CO3 as the base, I2-mediated C═C bond cleavage followed by intramolecular annulation of N-(2-vinylaryl)pyridin-2-amine substrates produces pyrido[2,1-b]quinazolinones and related heterocyclic compounds. This reaction can be completed on a gram scale and has been successfully applied to the synthesis of compounds with important biological properties, including efflux pump inhibitory and antiallergic activities.

Phenolic Compound Ethyl 3,4-Dihydroxybenzoate Retards Drug Efflux and Potentiates Antibiotic Activity.

The World Health Organization indicated that antibiotic resistance is one of the greatest threats to health, food security, and development in the world. Drug resistance efflux pumps are essential for antibiotic resistance in bacteria. Here, we evaluated the plant phenolic compound ethyl 3,4-dihydroxybenzoate (EDHB) for its efflux pump inhibitory (EPI) activity against drug-resistant Escherichia coli. The half-maximal inhibitory concentration, modulation assays, and time-kill studies indicated that EDHB has limited antibacterial activity but can potentiate the activity of antibiotics for drug-resistant E. coli. Dye accumulation/efflux and MALDI-TOF studies showed that EDHB not only significantly increases dye accumulation and reduces dye efflux but also increases the extracellular amount of antibiotics in the drug-resistant E. coli, indicating its interference with substrate translocation via a bacterial efflux pump. Molecular docking analysis using AutoDock Vina indicated that EDHB putatively posed within the distal binding pocket of AcrB and in close interaction with the residues by H-bonds and hydrophobic contacts. Additionally, EDHB showed an elevated postantibiotic effect on drug-resistant E. coli. Our toxicity assays showed that EDHB did not change the bacterial membrane permeability and exhibited mild human cell toxicity. In summary, these findings indicate that EDHB could serve as a potential EPI for drug-resistant E. coli.

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.

Identified Seaweed Compound Diphenylmethane Serves as an Efflux Pump Inhibitor in Drug-Resistant Escherichia coli.

Drug efflux pumps are one of the major elements used by antibiotic-resistant bacteria. Efflux pump inhibitors (EPIs) are potential therapeutic agents for adjunctive therapy, which can restore the activity of antibiotics that are no longer effective against pathogens. This study evaluated the seaweed compound diphenylmethane (DPM) for its EPI activity. The IC50 and modulation results showed that DPM has no antibacterial activity but can potentiate the activity of antibiotics against drug-resistant E. coli. Time-kill studies reported that a combination of DPM and erythromycin exhibited greater inhibitory activity against drug-resistant Escherichia coli. Dye accumulation and dye efflux studies using Hoechst 33342 and ethidium bromide showed that the addition of DPM significantly increased dye accumulation and reduced dye efflux in drug-resistant E. coli, suggesting its interference with dye translocation by an efflux pump. Using MALDI-TOF, it was observed that the addition of DPM could continuously reduce antibiotic efflux in drug-resistant E. coli. Additionally, DPM did not seem to damage the E. coli membranes, and the cell toxicity test showed that it features mild human-cell toxicity. In conclusion, these findings showed that DPM could serve as a potential EPI for drug-resistant E. coli.