Clinical Impact of the Over-Expression of Efflux Pump in Nonfermentative Gram-Negative Bacilli, Development of Efflux Pump Inhibitors

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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An alkylaminoquinazoline restores antibiotic activity in Gram-negative resistant isolates

Microbiology ◽

10.1099/mic.0.045716-0 ◽

2011 ◽

Vol 157 (2) ◽

pp. 566-571 ◽

Cited By ~ 17

Author(s):

Abdallah Mahamoud ◽

Jacqueline Chevalier ◽

Milad Baitiche ◽

Elissavet Adam ◽

Jean-Marie Pagès

Keyword(s):

Efflux Pump ◽

Bacterial Species ◽

Efflux Pumps ◽

Multidrug Resistant ◽

Side Chain ◽

Structural Class ◽

Gram Negative ◽

Activity Spectrum ◽

Efflux Pump Inhibitors ◽

Drug Efflux Pump

To date, various bacterial drug efflux pump inhibitors (EPIs) have been described. They exhibit variability in their activity spectrum with respect to antibiotic structural class and bacterial species. Among the various 4-alkylaminoquinazoline derivatives synthesized and studied in this work, one molecule, 1167, increased the susceptibility of important human-pathogenic, resistant, Gram-negative bacteria towards different antibiotic classes. This 4-(3-morpholinopropylamino)-quinazoline induced an increase in the activity of chloramphenicol, nalidixic acid, norfloxacin and sparfloxacin, which are substrates of the AcrAB-TolC and MexAB-OprM efflux pumps that act in these multidrug-resistant isolates. In addition, 1167 increased the intracellular concentration of chloramphenicol in efflux pump-overproducing strains. The rate of restoration depended on the structure of the antibiotic, suggesting that different sites in the efflux pumps may be involved. A molecule exhibiting a morpholine functional group and a propyl extension of the side chain was more active.

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

Journal of Medicinal Chemistry ◽

10.1021/acs.jmedchem.7b00156 ◽

2017 ◽

Vol 60 (9) ◽

pp. 3913-3932 ◽

Cited By ~ 28

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

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ToxR mediates the antivirulence activity of phenyl-arginine-β-naphthylamide to attenuate Vibrio cholerae virulence.

Infection and Immunity ◽

10.1128/iai.00147-21 ◽

2021 ◽

Author(s):

Yuding Weng ◽

Thomas F. Bina ◽

X. Renee Bina ◽

James E. Bina

Keyword(s):

Antimicrobial Resistance ◽

Vibrio Cholerae ◽

Virulence Factor ◽

Efflux Pump ◽

Feedback Mechanism ◽

Gram Negative ◽

Factor Production ◽

Regulatory Circuit ◽

Efflux Pump Inhibitors ◽

Virulence Factor Production

Multidrug efflux systems belonging to the resistance-nodulation-cell division (RND) family are ubiquitous in Gram negative bacteria and critical for antimicrobial resistance. This realization has led to efforts to develop efflux pump inhibitors (EPI) for use as adjuvants for antibiotic treatment of resistant organisms. However, the functions of RND transporters extend beyond antimicrobial resistance to include physiological functions that are critical for pathogenesis, suggesting that EPIs could also be used as antivirulence therapeutics. This was documented in the enteric pathogen Vibrio cholerae where EPIs were shown to attenuate the production of the critical virulence factors cholera toxin (CT) and the toxin coregulated pilus (TCP). In this study we investigated the antivirulence mechanism of action of the EPI phenyl-arginine-β-naphthylamide (PAβN) on V. cholerae. Using bioassays, we documented that PAβN inhibited virulence factor production in three epidemic V. cholerae isolates. Transcriptional reporter studies and mutant analysis indicated that PAβN initiated a ToxR-dependent regulatory circuit to activate leuO expression and that LeuO repressed the expression of the critical virulence activator aphA to attenuate CT and TCP production. The antivirulence activity of PAβN was found to be dependent on the ToxR periplasmic sensing domain suggesting that a feedback mechanism was involved in its activity. Collectively the data indicated that PAβN inhibited V. cholerae virulence factor production by activating a ToxR-dependent metabolic feedback mechanism to repress the expression of the ToxR virulence regulon. This suggests that efflux pump inhibitors could be used as antivirulence therapeutics for the treatment of cholera and perhaps other gram negative pathogens.

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The development of efflux pump inhibitors to treat Gram-negative infections

Expert Opinion on Drug Discovery ◽

10.1080/17460441.2018.1514386 ◽

2018 ◽

Vol 13 (10) ◽

pp. 919-931 ◽

Cited By ~ 5

Author(s):

Paula Blanco ◽

Fernando Sanz-García ◽

Sara Hernando-Amado ◽

José Luis Martínez ◽

Manuel Alcalde-Rico

Keyword(s):

Efflux Pump ◽

Gram Negative ◽

Efflux Pump Inhibitors

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

PeerJ ◽

10.7717/peerj.3168 ◽

2017 ◽

Vol 5 ◽

pp. e3168 ◽

Cited By ~ 14

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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The Influence of Efflux Pump Inhibitors on the Activity of Non-Antibiotic NSAIDS against Gram-Negative Rods

PLoS ONE ◽

10.1371/journal.pone.0147131 ◽

2016 ◽

Vol 11 (1) ◽

pp. e0147131 ◽

Cited By ~ 24

Author(s):

Agnieszka E. Laudy ◽

Agnieszka Mrowka ◽

Joanna Krajewska ◽

Stefan Tyski

Keyword(s):

Efflux Pump ◽

Gram Negative ◽

Efflux Pump Inhibitors

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Effectiveness of Efflux Pump Inhibitors as Biofilm Disruptors and Resistance Breakers in Gram-negative (ESKAPEE) Bacteria

10.20944/preprints201910.0255.v1 ◽

2019 ◽

Author(s):

Akif Reza ◽

J. Mark Sutton ◽

Khondaker Miraz Rahman

Keyword(s):

Antibiotic Resistance ◽

Antimicrobial Resistance ◽

Efflux Pump ◽

Protein Structures ◽

Underlying Mechanism ◽

Resistance Mechanisms ◽

Healthcare Provision ◽

Gram Negative ◽

Environmental Threats ◽

Efflux Pump Inhibitors

Antibiotic resistance represents a significant threat to the modern healthcare provision. The ESKAPEE pathogens, in particular, have proven to be especially challenging to treat, due to their intrinsic and acquired ability to rapidly develop resistance mechanisms in response to environmental threats. The development of biofilm has been characterised as an essential contributing factor towards antimicrobial-resistance and tolerance. Several studies have implicated the involvement of efflux pumps in antibiotic resistance, both directly, via drug extrusion and indirectly, through the formation of biofilm. As a result, the underlying mechanism of these pumps has attracted considerable interest due to the potential of targeting these protein structures and developing novel adjunct therapies. Subsequent investigations have revealed the ability of efflux pump-inhibitors (EPIs) to block drug-extrusion and disrupt biofilm formation, thereby, potentiating antibiotics and reversing resistance of pathogen towards them. This review will discuss the potential of EPIs as a possible solution to antimicrobial resistance, examining different challenges to the design of these compounds, with an emphasis on Gram-negative ESKAPEE pathogens.

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Effectiveness of Efflux Pump Inhibitors as Biofilm Disruptors and Resistance Breakers in Gram-Negative (ESKAPEE) Bacteria

Antibiotics ◽

10.3390/antibiotics8040229 ◽

2019 ◽

Vol 8 (4) ◽

pp. 229 ◽

Cited By ~ 8

Author(s):

Akif Reza ◽

J. Mark Sutton ◽

Khondaker Miraz Rahman

Keyword(s):

Antibiotic Resistance ◽

Antimicrobial Resistance ◽

Efflux Pump ◽

Protein Structures ◽

Underlying Mechanism ◽

Resistance Mechanisms ◽

Healthcare Provision ◽

Gram Negative ◽

Environmental Threats ◽

Efflux Pump Inhibitors

Antibiotic resistance represents a significant threat to the modern healthcare provision. The ESKAPEE pathogens (Enterococcus faecium., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. and Escherichia coli), in particular, have proven to be especially challenging to treat, due to their intrinsic and acquired ability to rapidly develop resistance mechanisms in response to environmental threats. The development of biofilm has been characterised as an essential contributing factor towards antimicrobial-resistance and tolerance. Several studies have implicated the involvement of efflux pumps in antibiotic resistance, both directly, via drug extrusion and indirectly, through the formation of biofilm. As a result, the underlying mechanism of these pumps has attracted considerable interest due to the potential of targeting these protein structures and developing novel adjunct therapies. Subsequent investigations have revealed the ability of efflux pump-inhibitors (EPIs) to block drug-extrusion and disrupt biofilm formation, thereby, potentiating antibiotics and reversing resistance of pathogen towards them. This review will discuss the potential of EPIs as a possible solution to antimicrobial resistance, examining different challenges to the design of these compounds, with an emphasis on Gram-negative ESKAPEE pathogens.

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