scholarly journals The 2019 Garrod Lecture: MDR efflux in Gram-negative bacteria—how understanding resistance led to a new tool for drug discovery

2019 ◽  
Vol 74 (11) ◽  
pp. 3128-3134 ◽  
Author(s):  
Laura J V Piddock
Keyword(s):  
Efflux Pump ◽  
Single Amino Acid ◽  
Lon Protease ◽  
Gram Negative Bacteria ◽  
Efflux System ◽  
Chemical Library ◽  
Gene Encoding ◽  
Gram Negative ◽  
Fluorescent Reporters ◽  
Pump Component

Abstract The AcrAB-TolC MDR efflux system confers intrinsic MDR and overproduction confers clinically relevant resistance to some antibiotics active against Gram-negative bacteria. The system is made up of three components, namely AcrA, AcrB and TolC, otherwise known as the AcrAB-TolC tripartite system. Inactivation or deletion of a gene encoding one of the constituent proteins, or substitution of a single amino acid in the efflux pump component AcrB that results in loss of efflux function, confers increased antibiotic susceptibility. Clinically relevant resistance can be mediated by a mutation in acrB that changes the way AcrB substrates are transported. However, it is more common that resistant clinical and veterinary isolates overproduce the AcrAB-TolC MDR efflux system. This is due to mutations in genes such as marR and ramR that encode repressors of transcription factors (MarA and RamA, respectively) that when produced activate expression of the acrAB and tolC genes thereby increasing efflux. The Lon protease degrades MarA and RamA to return the level of efflux to that of the WT. Furthermore, the levels of AcrAB-TolC are regulated by CsrA. Studies with fluorescent reporters that report levels of acrAB and regulatory factors allowed the development of a new tool for discovering efflux inhibitors. Screens of the Prestwick Chemical Library and a large library from a collaborating pharmaceutical company have generated a number of candidate compounds for further research.

scholarly journals A Simple Method for Assessment of MDR Bacteria for Over-Expressed Efflux Pumps

2013 ◽  
Vol 7 (1) ◽  
pp. 72-82 ◽  
Author(s):  
Marta Martins ◽  
Matthew P McCusker ◽  
Miguel Viveiros ◽  
Isabel Couto ◽  
Séamus Fanning ◽  
...  
Keyword(s):  
Ethidium Bromide ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Outer Cell ◽  
Gram Negative Bacteria ◽  
Drug Resistant ◽  
Topoisomerase Iv ◽  
Simple Method ◽  
Gram Negative ◽  
Over Expression

It is known that bacteria showing a multi-drug resistance phenotype use several mechanisms to overcome the action of antibiotics. As a result, this phenotype can be a result of several mechanisms or a combination of thereof. The main mechanisms of antibiotic resistance are: mutations in target genes (such as DNA gyrase and topoisomerase IV); over-expression of efflux pumps; changes in the cell envelope; down regulation of membrane porins, and modified lipopolysaccharide component of the outer cell membrane (in the case of Gram-negative bacteria). In addition, adaptation to the environment, such as quorum sensing and biofilm formation can also contribute to bacterial persistence. Due to the rapid emergence and spread of bacterial isolates showing resistance to several classes of antibiotics, methods that can rapidly and efficiently identify isolates whose resistance is due to active efflux have been developed. However, there is still a need for faster and more accurate methodologies. Conventional methods that evaluate bacterial efflux pump activity in liquid systems are available. However, these methods usually use common efflux pump substrates, such as ethidium bromide or radioactive antibiotics and therefore, require specialized instrumentation, which is not available in all laboratories. In this review, we will report the results obtained with the Ethidium Bromide-agar Cartwheel method. This is an easy, instrument-free, agar based method that has been modified to afford the simultaneous evaluation of as many as twelve bacterial strains. Due to its simplicity it can be applied to large collections of bacteria to rapidly screen for multi-drug resistant isolates that show an over-expression of their efflux systems. The principle of the method is simple and relies on the ability of the bacteria to expel a fluorescent molecule that is substrate for most efflux pumps, ethidium bromide. In this approach, the higher the concentration of ethidium bromide required to produce fluorescence of the bacterial mass, the greater the efflux capacity of the bacterial cells. We have tested and applied this method to a large number of Gram-positive and Gram-negative bacteria to detect efflux activity among these multi-drug resistant isolates. The presumptive efflux activity detected by the Ethidium Bromide-agar Cartwheel method was subsequently confirmed by the determination of the minimum inhibitory concentration for several antibiotics in the presence and absence of known efflux pump inhibitors.

scholarly journals MexAB-OprM Efflux Pump Interaction with the Peptidoglycan of Escherichia coli and Pseudomonas aeruginosa

2021 ◽  
Vol 22 (10) ◽  
pp. 5328
Author(s):  
Miao Ma ◽  
Margaux Lustig ◽  
Michèle Salem ◽  
Dominique Mengin-Lecreulx ◽  
Gilles Phan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Cell Division ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Efflux Pump ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Active Efflux

One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e., the active efflux of drugs. In Gram-negative bacteria, these proteins are inserted in the inner membrane and form a tripartite assembly with an outer membrane factor and a periplasmic linker in order to cross the two membranes to expulse molecules outside of the cell. A lot of information has been collected to understand the functional mechanism of these pumps, especially with AcrAB-TolC from Escherichia coli, but one missing piece from all the suggested models is the role of peptidoglycan in the assembly. Here, by pull-down experiments with purified peptidoglycans, we precise the MexAB-OprM interaction with the peptidoglycan from Escherichia coli and Pseudomonas aeruginosa, highlighting a role of the peptidoglycan in stabilizing the MexA-OprM complex and also differences between the two Gram-negative bacteria peptidoglycans.

scholarly journals Effect of Bile on the Cell Surface Permeability Barrier and Efflux System of Vibrio cholerae

2004 ◽  
Vol 186 (20) ◽  
pp. 6809-6814 ◽  
Author(s):  
Arpita Chatterjee ◽  
Sohini Chaudhuri ◽  
Gargi Saha ◽  
Satadeepa Gupta ◽  
Rukhsana Chowdhury
Keyword(s):  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Efflux Pump ◽  
Permeability Barrier ◽  
Synergistic Activity ◽  
Efflux System ◽  
Gram Negative ◽  
Outer Leaflet ◽  
Hydrophobic Compounds ◽  
Energy Dependent

ABSTRACT Gram-negative bacteria are inherently impermeable to hydrophobic compounds, due to the synergistic activity of the permeability barrier imposed by the outer membrane and energy dependent efflux systems. The gram-negative, enteric pathogen Vibrio cholerae appears to be deficient in both these activities; the outer membrane is not an effective barrier to hydrophobic permeants, presumably due to the presence of exposed phospholipids on the outer leaflet of the outer membrane, and efflux systems are at best only partially active. When V. cholerae was grown in the presence of bile, entry of hydrophobic compounds into the cells was significantly reduced. No difference was detected in the extent of exposed phospholipids on the outer leaflet of the outer membrane between cells grown in the presence or absence of bile. However, in the presence of energy uncouplers, uptake of hydrophobic probes was comparable between cells grown in the presence or absence of bile, indicating that energy-dependent efflux processes may be involved in restricting the entry of hydrophobic permeants into bile grown cells. Indeed, an efflux system(s) is essential for survival of V. cholerae in the presence of bile. Expression of acrAB, encoding an RND family efflux pump, was significantly increased in V. cholerae cells grown in vitro in the presence of bile and also in cells grown in rabbit intestine.

scholarly journals The carbon source influences the efflux pump-mediated antimicrobial resistance in clinically important Gram-negative bacteria

2012 ◽  
Vol 67 (4) ◽  
pp. 921-927 ◽  
Author(s):  
N. A. Villagra ◽  
J. A. Fuentes ◽  
M. R. Jofre ◽  
A. A. Hidalgo ◽  
P. Garcia ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Carbon Source ◽  
Efflux Pump ◽  
Gram Negative Bacteria ◽  
Gram Negative

A Tobramycin Vector Enhances Synergy and Efficacy of Efflux Pump Inhibitors against Multidrug-Resistant Gram-Negative Bacteria

2017 ◽  
Vol 60 (9) ◽  
pp. 3913-3932 ◽  
Author(s):  
Xuan Yang ◽  
Sudeep Goswami ◽  
Bala Kishan Gorityala ◽  
Ronald Domalaon ◽  
Yinfeng Lyu ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Multidrug Resistant ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Efflux Pump Inhibitors

scholarly journals Antibacterial and Antibiotic Modifying Potential of Crude Extracts, Fractions, and Compounds from Acacia polyacantha Willd. against MDR Gram-Negative Bacteria

2019 ◽  
Vol 2019 ◽  
pp. 1-13 ◽  
Author(s):  
Flora T. Mambe ◽  
Jean Na-Iya ◽  
Ghislain W. Fotso ◽  
Fred Ashu ◽  
Bathélémy Ngameni ◽  
...  
Keyword(s):  
Oleanolic Acid ◽  
Bacterial Infections ◽  
Efflux Pump ◽  
Synergistic Effects ◽  
Bacterial Species ◽  
Gram Negative Bacteria ◽  
Efflux Pump Inhibitor ◽  
Gram Negative ◽  
Acacia Polyacantha

The present study aimed to assess the in vitro antibacterial and antibiotic modifying activities of methanol extracts prepared from the leaf (APL) and bark (APB) of Acacia polyacantha, fractions (APLa-d) and compounds isolated from APL against a panel of multidrug resistant (MDR) Gram-negative bacteria. Leaf extract was subjected to column chromatography for compounds isolation; antibacterial assays were performed on samples alone and with an efflux pump inhibitor (EPI), respectively, and several antibiotics on the tested bacteria. The phytochemical investigation of APL led to the isolation of stigmasterol (1), β-amyrin (2), 3-O-β-D-glucopyranosylstigmasterol (3), 3-O-methyl-D-chiro-inositol (4), epicatechin (5), quercetin-3-O-glucoside (6), 3-O-[β-D-xylopyranosyl-(1→4)-β-D-galactopyranosyl]-oleanolic acid (7), and 3-O-[β-galactopyranosyl-(1→4)-β-D-galactopyranosyl]-oleanolic acid (8). APL and APB had minimal inhibitory concentration (MIC) values ≤ 1024 μg/mL on 73.3% and 46.7% of the tested bacteria, respectively. APLb and APLd were effective against 88.9% of tested bacterial species with compound 8 showing the highest activity inhibiting 88.9% of tested bacteria. The EPI, phenylalanine-arginine-β-naphthylamide (PAßN), strongly improved the activity of APL, APLb, APLd, and compound 8 on all tested bacteria. Synergistic effects were obtained when APL and compounds 7 and 8 were combined with erythromycin (ERY), gentamycin (GEN), ciprofloxacin (CIP), and norfloxacin (NOR). The present study demonstrates the antibacterial potential of Acacia polyacantha and its constituents to combat bacterial infections alone or in combination with EPI.

scholarly journals Sensor histidine kinase is a β-lactam receptor and induces resistance to β-lactam antibiotics

2016 ◽  
Vol 113 (6) ◽  
pp. 1648-1653 ◽  
Author(s):  
Lu Li ◽  
Qiyao Wang ◽  
Hui Zhang ◽  
Minjun Yang ◽  
Mazhar I. Khan ◽  
...  
Keyword(s):  
Cell Wall ◽  
Histidine Kinase ◽  
Vibrio Parahaemolyticus ◽  
Response Regulator ◽  
Strong Support ◽  
Binding Pocket ◽  
Single Amino Acid ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Direct Recognition

β-Lactams disrupt bacterial cell wall synthesis, and these agents are the most widely used antibiotics. One of the principle mechanisms by which bacteria resist the action of β-lactams is by producing β-lactamases, enzymes that degrade β-lactams. In Gram-negative bacteria, production of β-lactamases is often induced in response to the antibiotic-associated damage to the cell wall. Here, we have identified a previously unidentified mechanism that governs β-lactamase production. In the Gram-negative enteric pathogenVibrio parahaemolyticus, we found a histidine kinase/response regulator pair (VbrK/VbrR) that controls expression of a β-lactamase. Mutants lacking either VbrK or VbrR do not produce the β-lactamase and are no longer resistant to β-lactam antibiotics. Notably, VbrK autophosphorylation is activated by β-lactam antibiotics, but not by other lactams. However, single amino acid substitutions in the putative periplasmic binding pocket of VbrK leads its phosphorylation in response to both β-lactam and other lactams, suggesting that this kinase is a β-lactam receptor that can directly detect β-lactam antibiotics instead of detecting the damage to cell wall resulting from β-lactams. In strong support of this idea, we found that purified periplasmic sensor domain of VbrK binds penicillin, and that such binding is critical for VbrK autophosphorylation and β-lactamase production. Direct recognition of β-lactam antibiotics by a histidine kinase receptor may represent an evolutionarily favorable mechanism to defend against β-lactam antibiotics.

scholarly journals Quantum dot probes for the quantitative study of drug transport by the MacAB TolC efflux pump in lipid scaffolds

2020 ◽  
Author(s):  
Hager Souabni ◽  
William Batista dos Santos ◽  
Quentin Cece ◽  
Dhenesh Puvanendran ◽  
Martin Picard
Keyword(s):  
Energy Consumption ◽  
Membrane Protein ◽  
Virulence Factors ◽  
Quantitative Study ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Efflux Pump ◽  
Coupling Efficiency ◽  
Gram Negative Bacteria ◽  
Gram Negative

AbstractABC tripartite efflux pumps are macromolecular membrane protein machineries that expel a large variety of drugs and export virulence factors from Gram negative bacteria. Using a lipid scaffold mimicking the two-membrane environment of the transporter and designing spectroscopic conditions allowing the monitoring of both ATP hydrolysis and substrate transport in real time, we show that MacAB-TolC accommodates transport and energy consumption with high coupling efficiency.

scholarly journals Bacillus phage endolysin, lys46, bactericidal properties against Gram-negative bacteria

2020 ◽  
Author(s):  
Mohammad Reza Sarjoughian ◽  
Fereshte Rahmani ◽  
Shamsozoha Abolmaali ◽  
Shakiba Darvish Alipour Astaneh
Keyword(s):  
Bacillus Subtilis ◽  
Screening Program ◽  
Inhibitory Effect ◽  
Indicator Bacteria ◽  
Subtilis Strain ◽  
Gram Negative Bacteria ◽  
Gene Encoding ◽  
Gram Negative ◽  
Antibacterial Screening ◽  
Bacillus Phage

Background and Objectives: The great potential of bacteriophage for removing pathogen bacteria via targeting the cell wall is highly concerned. With a priority for overcoming drug-resistance, we screened against endolysins targeting Gram-negative bacteria to introduce a new antibacterial agent. This study was aimed to identify endolysins from the lysogenic phage of the Siphoviridea family in Bacillus subtilis. Materials and Methods: The Bacillus subtilis strain DDBCC46 was isolated from a preliminary antibacterial screening program. The endolysin (s) was extracted, concentrated with ammonium sulfate saturation, and their activity evaluated against the indicator bacteria. The phage particles were extracted from the bacteria using the minimum inhibition concentration of mitomycin C, followed by testing the phage inhibitory effect on the growth of indicator bacteria. The NCBI, Virus-Host DB, and EXPASY databases were used to obtain and confirm the sequences of the genes encoding PG hydrolases in Siphoviridea phages hosted in B. subtilis. Results: An 816 bp gene encoding an endolysin enzyme, was approved in the B. subtilis DDBCC 46, with specific primers of Bacillus phage SPP1. The purified-endolysin indicated antibacterial activity against Klebsiella pneumoniae, Salmonella typhimurium, Proteus (sp), and Escherichia coli. SDS-PAGE profiling followed by silica gel purification, led to introduceLys4630 as a therapeutic product and food preservative. Conclusion: lys4630 showed antibacterial effects on the common Gram-negative pathogens in clinics and food industries; E. coli, P. aeruginosa and Salmonella (sp).

scholarly journals Mode of action of the 2-phenylquinoline efflux inhibitor PQQ4R againstEscherichia coli

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3168 ◽  
Author(s):  
Diana Machado ◽  
Laura Fernandes ◽  
Sofia S. Costa ◽  
Rolando Cannalire ◽  
Giuseppe Manfroni ◽  
...  
Keyword(s):  
Mode Of Action ◽  
Bacterial Infections ◽  
Efflux Pump ◽  
Resistant Bacteria ◽  
Gram Negative Bacteria ◽  
Synergistic Activity ◽  
Dependent Manner ◽  
Gram Negative ◽  
E Coli ◽  
Efflux Pump Inhibitors

Efflux pump inhibitors are of great interest since their use as adjuvants of bacterial chemotherapy can increase the intracellular concentrations of the antibiotics and assist in the battle against the rising of antibiotic-resistant bacteria. In this work, we have described the mode of action of the 2-phenylquinoline efflux inhibitor (4-(2-(piperazin-1-yl)ethoxy)-2-(4-propoxyphenyl) quinolone – PQQ4R), againstEscherichia coli,by studding its efflux inhibitory ability, its synergistic activity in combination with antibiotics, and compared its effects with the inhibitors phenyl-arginine-β-naphthylamide (PAβN) and chlorpromazine (CPZ). The results showed that PQQ4R acts synergistically, in a concentration dependent manner, with antibiotics known to be subject to efflux inE. colireducing their MIC in correlation with the inhibition of their efflux. Real-time fluorometry assays demonstrated that PQQ4R at sub-inhibitory concentrations promote the intracellular accumulation of ethidium bromide inhibiting its efflux similarly to PAβN or CPZ, well-known and described efflux pump inhibitors for Gram-negative bacteria and whose clinical usage is limited by their levels of toxicity at clinical and bacteriological effective concentrations. The time-kill studies showed that PQQ4R, at bactericidal concentrations, has a rapid antimicrobial activity associated with a fast decrease of the intracellular ATP levels. The results also indicated that the mode of action of PQQ4R involves the destabilization of theE. coliinner membrane potential and ATP production impairment, ultimately leading to efflux pump inhibition by interference with the energy required by the efflux systems. At bactericidal concentrations, membrane permeabilization increases and finally ATP is totally depleted leading to cell death. Since drug resistance mediated by the activity of efflux pumps depends largely on the proton motive force (PMF), dissipaters of PMF such as PQQ4R, can be regarded as future adjuvants of conventional therapy againstE. coliand other Gram-negative bacteria, especially their multidrug resistant forms. Their major limitation is the high toxicity for human cells at the concentrations needed to be effective against bacteria. Their future molecular optimization to improve the efflux inhibitory properties and reduce relative toxicity will optimize their potential for clinical usage against multi-drug resistant bacterial infections due to efflux.

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