Transport across two membrane bilayers in E. coli by efflux pumps of different dimensions

Abstract Background: Bacterial infections involving the multidrug resistant (MDR) strains are among the top leading causes of death throughout the world. Healthcare system across the globe has been suffering from an extra-ordinary burden in terms of looking for the new and more potent antimicrobial compounds. The aim of the present study was to determine the antibacterial activity of some Cameroonian edible plants (Garcinia lucida bark, Phoenix dactylifera pericarps, Theobroma cacao pod, Solanum macrocarpon leaves and Termitomyces titanicus whole plant) and their antibiotics-potentiation effects against some MDR Gram-negative bacteria phenotypes expressing efflux pumps (Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa and Providencia stuartii strains). Methods: The antibacterial activities of plant extract alone and in combination with usual antibiotics were carried out using the micro-dilution method. The effects of the most active plant extract (Garcinia lucida bark) on H+-ATPase-mediated proton pumps and on bacterial growth kinetic were performed using experimental protocols, while qualitative reference methods were used to highligh the major groups of secondary metabolites present in the extracts. Results: Qualitative phytochemical screening of plant extracts indicated that all analysed secondary metabolites were present in Theobroma cacao and Termitomyces titanicus while one (saponins) of them was absent in Garcinia lucida and Solanum macrocarpon. Only three of them (polyphenols, flavonoids and saponins) were detected in Phoenix dactylifera. Antibacterial essays showed that G. lucida was the most active plant as it inhibited the growth of all studied bacteria with strong activity (MIC<100 µg/mL) against E. coli ATCC8739, significant activity (100≤MIC≤512 µg/mL) against 80% of bacteria and moderate activity (512<MIC≤2048 µg/mL) against E. coli AG100A and E. aerogenes (EA289 and CM64). It was followed by T. cacao and S. macrocarpon extracts which exhibited an antibacterial potential against 95% and 80% of bacterial strains, respectively. These three extracts exhibited a bactericidal effect on a few bacteria. Extracts from T. titanicus and P. dactylifera were less active as they moderately (512<MIC≤2048 µg/mL) inhibited the growth of 35% and 10% of bacteria. All extracts selectively potentiated the activities of all antibiotics with improvement activity factors (IAF) ranging from 2 to 256. G. lucida, T. cacao and S. macrocarpon potentiated the activities of 100%, 89% and 67% of antibiotics respectively against more than 70%, suggesting that they contain bioactive compounds which could be considered as efflux pumps inhibitors. Whereas T. titanicus and P. dactylifera improved the activities of almost 40% and 20% of antibiotics, respectively. This increase of activities also characterizes synergistic effects between antibiotics and these bioactive compounds. G. lucida extract at all tested concentrations, strongly inhibited the growth of bacterial strain E. coli ATCC8739 and exhibited an inhibitory effect on this bacterial H+-ATPase-mediated proton pumps increasing the pH of the medium. Conclusion: The overall results indicated that food plants among which G. lucida, T. cacao and S. macrocarpon could have a benefit interest in combatting resistant types of bacteria. Keywords: Food plants; infectious diseases; MDR bacteria; efflux pumps; antibiotics; secondary metabolites.

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The Role of AcrAB–TolC Efflux Pumps on Quinolone Resistance of E. coli ST131

Current Microbiology ◽

10.1007/s00284-018-1577-y ◽

2018 ◽

Vol 75 (12) ◽

pp. 1661-1666 ◽

Cited By ~ 7

Author(s):

N. Atac ◽

O. Kurt-Azap ◽

I. Dolapci ◽

A. Yesilkaya ◽

O. Ergonul ◽

...

Keyword(s):

Efflux Pumps ◽

Quinolone Resistance ◽

E Coli

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The Evolutionary Conservation of Escherichia coli Drug Efflux Pumps Supports Physiological Functions

Journal of Bacteriology ◽

10.1128/jb.00367-20 ◽

2020 ◽

Vol 202 (22) ◽

Cited By ~ 2

Author(s):

Tanisha Teelucksingh ◽

Laura K. Thompson ◽

Georgina Cox

Keyword(s):

Escherichia Coli ◽

Efflux Pump ◽

Efflux Pumps ◽

Evolutionary Conservation ◽

Drug Efflux ◽

Physiological Functions ◽

Content Type ◽

Bacterial Physiology ◽

E Coli ◽

Drug Efflux Pumps

ABSTRACT Bacteria harness an impressive repertoire of resistance mechanisms to evade the inhibitory action of antibiotics. One such mechanism involves efflux pump-mediated extrusion of drugs from the bacterial cell, which significantly contributes to multidrug resistance. Intriguingly, most drug efflux pumps are chromosomally encoded components of the intrinsic antibiotic resistome. In addition, in terms of xenobiotic detoxification, bacterial efflux systems often exhibit significant levels of functional redundancy. Efflux pumps are also considered to be highly conserved; however, the extent of conservation in many bacterial species has not been reported and the majority of genes that encode efflux pumps appear to be dispensable for growth. These observations, in combination with an increasing body of experimental evidence, imply alternative roles in bacterial physiology. Indeed, the ability of efflux pumps to facilitate antibiotic resistance could be a fortuitous by-product of ancient physiological functions. Using Escherichia coli as a model organism, we here evaluated the evolutionary conservation of drug efflux pumps and we provide phylogenetic analysis of the major efflux families. We show the E. coli drug efflux system has remained relatively stable and the majority (∼80%) of pumps are encoded in the core genome. This analysis further supports the importance of drug efflux pumps in E. coli physiology. In this review, we also provide an update on the roles of drug efflux pumps in the detoxification of endogenously synthesized substrates and pH homeostasis. Overall, gaining insight into drug efflux pump conservation, common evolutionary ancestors, and physiological functions could enable strategies to combat these intrinsic and ancient elements.

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Evaluation of Physiological Effects Induced by Manuka Honey Upon Staphylococcus aureus and Escherichia coli

Microorganisms ◽

10.3390/microorganisms7080258 ◽

2019 ◽

Vol 7 (8) ◽

pp. 258 ◽

Cited By ~ 5

Author(s):

Patricia Combarros-Fuertes ◽

Leticia M. Estevinho ◽

Rita Teixeira-Santos ◽

Acácio G. Rodrigues ◽

Cidália Pina-Vaz ◽

...

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Membrane Potential ◽

Membrane Integrity ◽

Efflux Pumps ◽

Coli Strain ◽

Antibacterial Action ◽

Manuka Honey ◽

E Coli ◽

Action Mechanisms

Several studies have explored the antimicrobial properties of manuka honey (MkH). However, the data available regarding antibacterial action mechanisms are scarcer. The aim of this study was to scrutinize and characterize primary effects of manuka honey (MkH) upon the physiological status of Staphylococcus aureus and Escherichia coli (as Gram-positive and Gram-negative bacteria models, respectively), using flow cytometry (FC) to reveal its antibacterial action mechanisms. Effects of MkH on membrane potential, membrane integrity and metabolic activity were assessed using different fluorochromes in a 180 min time course assay. Time-kill experiments were carried out under the same conditions. Additionally, MkH effect on efflux pumps was also studied in an E. coli strain with an over-expression of several efflux pumps. Exposure of bacteria to MkH resulted in physiological changes related to membrane potential and membrane integrity; these effects displayed slight differences among bacteria. MkH induced a remarkable metabolic disruption as primary physiological effect upon S. aureus and was able to block efflux pump activity in a dose-dependent fashion in the E. coli strain.

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Expression of Pseudomonas aeruginosa Multidrug Efflux Pumps MexA-MexB-OprM and MexC-MexD-OprJ in a Multidrug-Sensitive Escherichia coli Strain

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.1.65 ◽

1998 ◽

Vol 42 (1) ◽

pp. 65-71 ◽

Cited By ~ 119

Author(s):

Ramakrishnan Srikumar ◽

Tatiana Kon ◽

Naomasa Gotoh ◽

Keith Poole

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Crystal Violet ◽

Efflux Pump ◽

Efflux Pumps ◽

Multidrug Efflux ◽

Efflux System ◽

E Coli ◽

Multidrug Efflux Pumps

ABSTRACT The mexCD-oprJ and mexAB-oprM operons encode components of two distinct multidrug efflux pumps inPseudomonas aeruginosa. To assess the contribution of individual components to antibiotic resistance and substrate specificity, these operons and their component genes were cloned and expressed in Escherichia coli. Western immunoblotting confirmed expression of the P. aeruginosa efflux pump components in E. coli strains expressing and deficient in the endogenous multidrug efflux system (AcrAB), although only the ΔacrAB strain, KZM120, demonstrated increased resistance to antibiotics in the presence of the P. aeruginosa efflux genes. E. coli KZM120 expressing MexAB-OprM showed increased resistance to quinolones, chloramphenicol, erythromycin, azithromycin, sodium dodecyl sulfate (SDS), crystal violet, novobiocin, and, significantly, several β-lactams, which is reminiscent of the operation of this pump in P. aeruginosa. This confirmed previous suggestions that MexAB-OprM provides a direct contribution to β-lactam resistance via the efflux of this group of antibiotics. An increase in antibiotic resistance, however, was not observed when MexAB or OprM alone was expressed in KZM120. Thus, despite the fact that β-lactams act within the periplasm, OprM alone is insufficient to provide resistance to these agents. E. coli KZM120 expressing MexCD-OprJ also showed increased resistance to quinolones, chloramphenicol, macrolides, SDS, and crystal violet, though not to most β-lactams or novobiocin, again somewhat reminiscent of the antibiotic resistance profile of MexCD-OprJ-expressing strains ofP. aeruginosa. Surprisingly, E. coli KZM120 expressing MexCD alone also showed an increase in resistance to these agents, while an OprJ-expressing KZM120 failed to demonstrate any increase in antibiotic resistance. MexCD-mediated resistance, however, was absent in a tolC mutant of KZM120, indicating that MexCD functions in KZM120 in conjunction with TolC, the previously identified outer membrane component of the AcrAB-TolC efflux system. These data confirm that a tripartite efflux pump is necessary for the efflux of all substrate antibiotics and that the P. aeruginosa multidrug efflux pumps are functional and retain their substrate specificity in E. coli.

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The Rho-Dependent Transcription Termination Is Involved in Broad-Spectrum Antibiotic Susceptibility in Escherichia coli

Frontiers in Microbiology ◽

10.3389/fmicb.2020.605305 ◽

2020 ◽

Vol 11 ◽

Author(s):

Md. Hafeezunnisa ◽

Ranjan Sen

Keyword(s):

Broad Spectrum ◽

Antibiotic Susceptibility ◽

Transcription Termination ◽

Efflux Pumps ◽

Dependent Manner ◽

Broad Spectrum Antibiotic ◽

E Coli ◽

Distinct Cell ◽

High Level ◽

Termination Function

One of the major ways of acquiring multidrug resistance in bacteria is via drug influx and efflux pathways. Here, we show that E. coli with compromised Rho-dependent transcription termination function has enhanced broad-spectrum antibiotic susceptibility, which arises from the inefficient TolC-efflux process and increased permeability of the membrane. The Rho mutants have altered morphology, distinct cell surface, and increased levels of lipopolysaccharide in their outer membrane, which might have rendered the TolC efflux pumps inefficient. These alterations are due to the upregulations of poly-N-acetyl-glucosamine and lipopolysaccharide synthesis operons because of inefficient Rho functions. The Rho mutants are capable of growing on various dipeptides and carbohydrate sources, unlike their WT counterpart. Dipeptides uptake arises from the upregulations of the di-peptide permease operon in these mutants. The metabolomics of the Rho mutants revealed the presence of a high level of novel metabolites. Accumulation of these metabolites in these Rho mutants might titrate out the TolC-efflux pumps, which could further reduce their efficiency. We conclude that the transcription termination factor, Rho, regulates the broad-spectrum antibiotic susceptibility of E. coli through multipartite pathways in a TolC-dependent manner. The involvement of Rho-dependent termination in multiple pathways and its association with antibiotic susceptibility should make Rho-inhibitors useful in the anti-bacterial treatment regimen.

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Resistance and Virulence Mechanisms of Escherichia coli Selected by Enrofloxacin in Chicken

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01824-18 ◽

2019 ◽

Vol 63 (5) ◽

Cited By ~ 3

Author(s):

Jun Li ◽

Haihong Hao ◽

Menghong Dai ◽

Heying Zhang ◽

Jianan Ning ◽

...

Keyword(s):

Escherichia Coli ◽

Efflux Pumps ◽

Genetic Alterations ◽

Site Directed Mutagenesis ◽

Single Mutation ◽

Genetic Characteristics ◽

Content Type ◽

E Coli ◽

Low Level ◽

High Level

ABSTRACT This study aimed to investigate the genetic characteristics, antibiotic resistance patterns, and novel mechanisms involved in fluoroquinolone (FQ) resistance in commensal Escherichia coli isolates. The E. coli isolates were recovered from a previous clinical study and subjected to antimicrobial susceptibility testing and molecular typing. Known mechanisms of FQ resistance (target site mutations, plasmid-mediated quinolone resistance [PMQR] genes, relative expression levels of efflux pumps and porins) were detected using DNA sequencing of PCR products and real-time quantitative PCR. Whole-genome shotgun sequencing was performed on 11 representative strains to screen for single nucleotide polymorphisms (SNPs). The function of a key SNP (A1541G) was investigated by site-directed mutagenesis and allelic exchange. The results showed that long-term enrofloxacin treatment selected multidrug-resistant (MDR) E. coli isolates in the chicken gut and that these E. coli isolates had diverse genetic backgrounds. Multiple genetic alterations, including double mutations on GyrA (S83L and D87N), a single mutation on ParC (S80I) and ParE (S458E), activation of efflux pumps, and the presence of the QnrS1 protein, contributed to the high-level FQ resistance (enrofloxacin MIC [MICENR] ≥ 128 μg/ml), while the relatively low-level FQ resistance (MICENR = 8 or 16 μg/ml) was commonly mediated by decreased expression of the porin OmpF, besides enhancement of the efflux pumps. No significant relationship was observed between resistance mechanisms and virulence genes. Introduction of the A1541G mutation on aegA was able to increase FQ susceptibility by 2-fold. This study contributes to a better understanding of the development of MDR and the differences underlying the mechanisms of high-level and low-level FQ resistance in E. coli.

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Characterization of a Novel Pyranopyridine Inhibitor of the AcrAB Efflux Pump of Escherichia coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01866-13 ◽

2013 ◽

Vol 58 (2) ◽

pp. 722-733 ◽

Cited By ~ 91

Author(s):

Timothy J. Opperman ◽

Steven M. Kwasny ◽

Hong-Suk Kim ◽

Son T. Nguyen ◽

Chad Houseweart ◽

...

Keyword(s):

Escherichia Coli ◽

Efflux Pump ◽

Efflux Pumps ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Resistance Nodulation Division ◽

Rnd Efflux Pumps ◽

Acrab Efflux Pump

ABSTRACTMembers of the resistance-nodulation-division (RND) family of efflux pumps, such as AcrAB-TolC ofEscherichia coli, play major roles in multidrug resistance (MDR) in Gram-negative bacteria. A strategy for combating MDR is to develop efflux pump inhibitors (EPIs) for use in combination with an antibacterial agent. Here, we describe MBX2319, a novel pyranopyridine EPI with potent activity against RND efflux pumps of theEnterobacteriaceae. MBX2319 decreased the MICs of ciprofloxacin (CIP), levofloxacin, and piperacillin versusE. coliAB1157 by 2-, 4-, and 8-fold, respectively, but did not exhibit antibacterial activity alone and was not active against AcrAB-TolC-deficient strains. MBX2319 (3.13 μM) in combination with 0.016 μg/ml CIP (minimally bactericidal) decreased the viability (CFU/ml) ofE. coliAB1157 by 10,000-fold after 4 h of exposure, in comparison with 0.016 μg/ml CIP alone. In contrast, phenyl-arginine-β-naphthylamide (PAβN), a known EPI, did not increase the bactericidal activity of 0.016 μg/ml CIP at concentrations as high as 100 μM. MBX2319 increased intracellular accumulation of the fluorescent dye Hoechst 33342 in wild-type but not AcrAB-TolC-deficient strains and did not perturb the transmembrane proton gradient. MBX2319 was broadly active againstEnterobacteriaceaespecies andPseudomonas aeruginosa. MBX2319 is a potent EPI with possible utility as an adjunctive therapeutic agent for the treatment of infections caused by Gram-negative pathogens.

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Characterisation of Hybrid Polymersome Vesicles Containing the Efflux Pumps NaAtm1 or P-Glycoprotein

Polymers ◽

10.3390/polym12051049 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1049

Author(s):

Sarah Rottet ◽

Shagufta Iqbal ◽

Paul A. Beales ◽

Anran Lin ◽

Jiwon Lee ◽

...

Keyword(s):

Protein Function ◽

Gradient Centrifugation ◽

Efflux Pumps ◽

Passive Permeability ◽

E Coli ◽

Quantitative Measurements ◽

Hydrophobic Compounds ◽

P Glycoprotein ◽

Poly Ethylene Oxide ◽

Poly Ethylene

Investigative systems for purified membrane transporters are almost exclusively reliant on the use of phospholipid vesicles or liposomes. Liposomes provide an environment to support protein function; however, they also have numerous drawbacks and should not be considered as a “one-size fits all” system. The use of artificial vesicles comprising block co-polymers (polymersomes) offers considerable advantages in terms of structural stability; provision of sufficient lateral pressure; and low passive permeability, which is a particular issue for transport assays using hydrophobic compounds. The present investigation demonstrates strategies to reconstitute ATP binding cassette (ABC) transporters into hybrid vesicles combining phospholipids and the block co-polymer poly (butadiene)-poly (ethylene oxide). Two efflux pumps were chosen; namely the Novosphingobium aromaticivorans Atm1 protein and human P-glycoprotein (Pgp). Polymersomes were generated with one of two lipid partners, either purified palmitoyl-oleoyl-phosphatidylcholine, or a mixture of crude E. coli lipid extract and cholesterol. Hybrid polymersomes were characterised for size, structural homogeneity, stability to detergents, and permeability. Two transporters, NaAtm1 and P-gp, were successfully reconstituted into pre-formed and surfactant-destabilised hybrid polymersomes using a detergent adsorption strategy. Reconstitution of both proteins was confirmed by density gradient centrifugation and the hybrid polymersomes supported substrate dependent ATPase activity of both transporters. The hybrid polymersomes also displayed low passive permeability to a fluorescent probe (calcein acetomethoxyl-ester (C-AM)) and offer the potential for quantitative measurements of transport activity for hydrophobic compounds.

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Effects of Chlorophyll-Derived Efflux Pump Inhibitor Pheophorbide a and Pyropheophorbide a on Growth and Macrolide Antibiotic Resistance of Indicator and Anaerobic Swine Manure Bacteria

International Journal of Antibiotics ◽

10.1155/2014/185068 ◽

2014 ◽

Vol 2014 ◽

pp. 1-14 ◽

Cited By ~ 3

Author(s):

Mareike Kraatz ◽

Terence R. Whitehead ◽

Michael A. Cotta ◽

Mark A. Berhow ◽

Mark A. Rasmussen

Keyword(s):

Bacterial Resistance ◽

Efflux Pump ◽

Growth Parameters ◽

Swine Manure ◽

Efflux Pumps ◽

Resistant Bacteria ◽

Pheophorbide A ◽

Efflux Pump Inhibitor ◽

E Coli ◽

Pyropheophorbide A

Natural plant compounds, such as the chlorophyll a catabolites pheophorbide a (php) and pyropheophorbide a (pyp), are potentially active in the gastrointestinal tracts and manure of livestock as antimicrobial resistance-modifying agents through inhibition of bacterial efflux pumps. To investigate whether php, a known efflux pump inhibitor, and pyp influence bacterial resistance, we determined their long-term effects on the MICs of erythromycin for reference strains of clinically relevant indicator bacteria with macrolide or multidrug resistance efflux pumps. Pyp reduced the final MIC endpoint for Staphylococcus (S.) aureus and Escherichia (E.) coli by up to 1536 and 1024 μg erythromycin mL−1 or 1.4- and 1.2-fold, respectively. Estimation of growth parameters of S. aureus revealed that pyp exerted an intrinsic inhibitory effect under anaerobic conditions and was synergistically active, thereby potentiating the effect of erythromycin and partially reversing high-level erythromycin resistance. Anaerobe colony counts of total and erythromycin-resistant bacteria from stored swine manure samples tended to be lower in the presence of pyp. Tylosin, php, and pyp were not detectable by HPLC in the manure or medium. This is the first study showing that pyp affects growth and the level of sensitivity to erythromycin of S. aureus, E. coli, and anaerobic manure bacteria.

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