The Impact of Efflux Pump Inhibitors on the Activity of Selected Non-Antibiotic Medicinal Products against Gram-Negative Bacteria

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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Effects of Efflux Pump Inhibitors on Colistin Resistance in Multidrug-Resistant Gram-Negative Bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00248-16 ◽

2016 ◽

Vol 60 (5) ◽

pp. 3215-3218 ◽

Cited By ~ 42

Author(s):

Wentao Ni ◽

Yanjun Li ◽

Jie Guan ◽

Jin Zhao ◽

Junchang Cui ◽

...

Keyword(s):

Stenotrophomonas Maltophilia ◽

Efflux Pump ◽

Multidrug Resistant ◽

Gram Negative Bacteria ◽

Colistin Resistance ◽

Gram Negative ◽

Content Type ◽

Resistant Strains ◽

Efflux Pump Inhibitors ◽

Time Kill

ABSTRACTWe tested the effects of various putative efflux pump inhibitors on colistin resistance in multidrug-resistant Gram-negative bacteria. Addition of 10 mg/liter cyanide 3-chlorophenylhydrazone (CCCP) to the test medium could significantly decrease the MICs of colistin-resistant strains. Time-kill assays showed CCCP could reverse colistin resistance and inhibit the regrowth of the resistant subpopulation, especially inAcinetobacter baumanniiandStenotrophomonas maltophilia. These results suggest colistin resistance in Gram-negative bacteria can be suppressed and reversed by CCCP.

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Conformational study of (Z)-5-(4-chlorobenzylidene)-2-[4-(2-hydroxyethyl)piperazin-1-yl]-3H-imidazol-4(5H)-one in different environments: insight into the structural properties of bacterial efflux pump inhibitors

Acta Crystallographica Section C Structural Chemistry ◽

10.1107/s2053229617016461 ◽

2017 ◽

Vol 73 (12) ◽

pp. 1151-1157 ◽

Cited By ~ 4

Author(s):

Ewa Żesławska ◽

Wojciech Nitek ◽

Jadwiga Handzlik

Keyword(s):

Multidrug Resistance ◽

Hydrogen Bonds ◽

Crystal Structures ◽

Efflux Pump ◽

Resistant Bacteria ◽

Gram Negative Bacteria ◽

Carboxylate Group ◽

Piperazine Ring ◽

Gram Negative ◽

Efflux Pump Inhibitors

The 2-amine derivatives of 5-arylidene-3H-imidazol-4(5H)-one are a new class of bacterial efflux pump inhibitors, the chemical compounds that are able to restore antibiotic efficacy against multidrug resistant bacteria. 5-Arylidene-3H-imidazol-4(5H)-ones with a piperazine ring at position 2 reverse the mechanisms of multidrug resistance (MDR) of the particularly dangerous Gram-negative bacteria E. coli by inhibition of the efflux pump AcrA/AcrB/TolC (a main multidrug resistance mechanism in Gram-negative bacteria, consisting of a membrane fusion protein, AcrA, a Resistant-Nodulation-Division protein, AcrB, and an outer membrane factor, TolC). In order to study the influence of the environment on the conformation of (Z)-5-(4-chlorobenzylidene)-2-[4-(2-hydroxyethyl)piperazin-1-yl]-3H-imidazol-4(5H)-one, (3), two different salts were prepared, namely with picolinic acid {systematic name: 4-[(Z)-4-(4-chlorobenzylidene)-5-oxo-3,4-dihydro-1H-imidazol-2-yl]-1-(2-hydroxyethyl)piperazin-1-ium pyridine-2-carboxylate, C16H20ClN4O2 +·C6H4NO2 −, (3 a )} and 4-nitrophenylacetic acid {systematic name: 4-[(Z)-4-(4-chlorobenzylidene)-5-oxo-3,4-dihydro-1H-imidazol-2-yl]-1-(2-hydroxyethyl)piperazin-1-ium 2-(4-nitrophenyl)acetate, C16H20ClN4O2 +·C8H6NO4 −, (3 b )}. The crystal structures of the new salts were determined by X-ray diffraction. In both crystal structures, the molecule of (3) is protonated at an N atom of the piperazine ring by proton transfer from the corresponding acid. The carboxylate group of picolinate engages in hydrogen bonds with three molecules of the cation of (3), whereas the carboxylate group of 4-nitrophenylacetate engages in hydrogen bonds with only two molecules of (3). As a consequence of these interactions, different orientations of the hydroxyethyl group of (3) are observed. The crystal structures are additionally stabilized by both C—H...N [in (3 a )] and C—H...O [in (3 a ) and (3 b )] intermolecular interactions. The geometry of the imidazolone fragment was compared with other crystal structures possessing this moiety. The tautomer observed in the crystal structures presented here, namely 3H-imidazol-4(5H)-one [systematic name: 1H-imidazol-5(4H)-one], is also that most frequently observed in other structures containing this heterocycle.

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A Review on Efflux Pump Inhibitors of Gram-Positive and Gram-Negative Bacteria from Plant Sources

International Journal of Current Microbiology and Applied Sciences ◽

10.20546/ijcmas.2016.506.092 ◽

2016 ◽

Vol 5 (6) ◽

pp. 837-855 ◽

Cited By ~ 6

Author(s):

Rakesh Kumar ◽

Savita Jandaik Pooja Patial

Keyword(s):

Efflux Pump ◽

Gram Negative Bacteria ◽

Gram Positive ◽

Gram Negative ◽

Efflux Pump Inhibitors ◽

Plant Sources

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

The Open Microbiology Journal ◽

10.2174/1874285801307010072 ◽

2013 ◽

Vol 7 (1) ◽

pp. 72-82 ◽

Cited By ~ 39

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.

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

International Journal of Molecular Sciences ◽

10.3390/ijms22105328 ◽

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.

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Impact of a Single Plain Finger Ring on the Bacterial Load on the Hands of Healthcare Workers

Infection Control and Hospital Epidemiology ◽

10.1086/520739 ◽

2007 ◽

Vol 28 (10) ◽

pp. 1191-1195 ◽

Cited By ~ 14

Author(s):

Mette Fagernes ◽

Egil Lingaas ◽

Per Bjark

Keyword(s):

Healthcare Workers ◽

Paired Comparison ◽

Bacterial Load ◽

Physical Contact ◽

Gram Negative Bacteria ◽

Quantitative Culture ◽

Control Groups ◽

Gram Negative ◽

The Impact ◽

Paired Analysis

Objective.To investigate the impact of a single plain finger ring on the number and types of bacteria on the hands of healthcare workers (HCWs).Design.Nonequivalent control groups, posttest only (preexperimental).Methods.A total of 121 HCWs wearing 1 plain ring and 113 HCWs wearing no rings had both hands sampled by the “glove juice” technique. Quantitative culture of the samples was performed and microorganisms were identified.Setting.Two Norwegian acute care hospitals.Participants.A total of 234 HCWs who had physical contact with patients.Results.Total bacterial counts did not differ when hands with rings and hands without rings were compared, both according to nonpaired analysis (which compared the ring-bearing hands of ring-wearing HCWs to the hands of HCWs who did not wear rings [P= .661]) and according to paired analysis (which compared the ring-bearing and ring-free hands of ring-wearing HCWs [P= .071]).Staphylococcus aureuswas recovered from 18.6% of the hands sampled, belonging to 26.9% of the HCWs, but neither paired nor nonpaired analysis showed any association with ring wearing. Gram-negative bacteria were recovered from 20.3% of the hands sampled, belonging to 28.6% of the HCWs. Ring-wearing HCWs were significantly more likely to be carriers of Enterobacteriaceae (P= .006), but paired comparison of the ring-bearing and ring-free hands of these HCWs did not show significant differences (P= .180). Carriage of nonfermentative gram-negative rods did not differ between the 2 groups, by either paired or nonpaired analysis.Conclusions.Wearing a single plain finger ring did not increase the total bacterial load on the hands, nor was it associated with an increased rate of carriage ofS. aureusor nonfermentative gram-negative rods. However, plain rings were associated with an increased rate of Enterobacteriaceae carriage.

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The Impact of Risk Factors on Treatment Outcomes of Nosocomial Pneumonia Due to Gram-Negative Bacteria in the Intensive Care Unit

Pulmonary Therapy ◽

10.1007/s41030-021-00175-4 ◽

2021 ◽

Author(s):

Thu Vo-Pham-Minh ◽

Van Duong-Thi-Thanh ◽

Thang Nguyen ◽

Quyen Phan-Tran-Xuan ◽

Hoang Phan-Thi ◽

...

Keyword(s):

Risk Factors ◽

Intensive Care Unit ◽

Intensive Care ◽

Treatment Outcomes ◽

Nosocomial Pneumonia ◽

Gram Negative Bacteria ◽

Gram Negative ◽

The Impact

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Towards an Understanding of Ligand Induced Functional Conformational Changes of MexB Efflux Transporter

10.26434/chemrxiv.14175383 ◽

2021 ◽

Author(s):

Khondaker Miraz Rahman ◽

Shirin Jamshidi ◽

J Mark Sutton ◽

Sara Jambarsang

Keyword(s):

Conformational Changes ◽

Efflux Pump ◽

Vital Role ◽

Gram Negative Bacteria ◽

Efflux Transporter ◽

Structural Rearrangements ◽

Protonation State ◽

New Information ◽

Cytotoxic Molecules ◽

Efflux Pump Inhibitors

<p>MexB, an RND-superfamily efflux pump, plays a vital role in conferring resistance to cytotoxic molecules, including antibiotics, upon Gram-negative bacteria. Although the principal mechanistic elements of switching between the access, binding and extrusion conformers of the protomers of tripartite efflux transporters have been described previously, details surrounding the further mechanism that ends in either substrate extrusion or pump inhibition are limited to observations based on the type of ligand bound to the transporter. A central but missing link in the structure/mechanism relationship is a description of how ligand-induced conformational changes in the presence of a membrane and changing transporter protonation state lead to either substrate extrusion or inhibition of the pump. Here, we report that differences in conformational changes are governed by ligand binding to the transporter. The current study describes important new information about ligand-induced structural rearrangements and conformational changes of MexB in relation to the protonation state of critical acidic residues. We used tetracycline (TET) as a model substrate of MexB and phenylalanine-arginine beta-naphthylamide (PAβN) as a model inhibitor of MexB to study the aforementioned conformational changes. This new information will contribute to the design of new, effective and selective efflux pump inhibitors that could play key roles in reversing antimicrobial resistance.</p>

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