Efflux Pumps in Bacteria: What They Do and How We Can Stop Them

A special issue of Antibiotics (ISSN 2079-6382). This special issue belongs to the section "Antibiofilm Strategies".

Deadline for manuscript submissions: closed (15 May 2025) | Viewed by 35461

Special Issue Editor


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Guest Editor
Department of Pharmaceutical Sciences, University of Perugia, 06125 Perugia, Italy
Interests: medicinal chemistry; organic chemistry; chemical biology; antibiotics; antiviral agents; anticancer agents; efflux pump inhibitors; polymerase inhibitors
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Special Issue Information

Dear Colleagues,

Antimicrobial resistance (AMR) represents a growing risk for our global health, jeopardizing our lifestyle and the world economy. Among different mechanisms by which AMR can develop, efflux pumps play a central role. Indeed, microbial efflux pumps can contribute to AMR by extruding noxious agents, such as antibacterials, from the cytoplasm, thus producing a reduction of the antibacterial effect. Accordingly, this decrease of antimicrobials inside the bacterial cells can promote the development of more specific mechanisms of resistance, such as target modification or antibiotic-modifying or -degrading enzymes. In addition, microbial efflux pumps have been identified as important players in the formation of biofilm as well as in the complex signaling mechanism called quorum sensing. Therefore, the identification of new non-antibiotic molecules able to block microbial efflux pumps represents a promising approach to fight AMR. Indeed, efflux pump inhibitors (EPIs) have the potential to synergize with common antibiotics that are efflux pump substrates, thus restoring their antibacterial effect. In addition, EPIs could reduce the development of resistance by i) preventing the first nonspecific extrusion of antibiotics, ii) reducing biofilm formation and iii) modulating quorum sensing.

This Special Issue aims to collect high-quality reviews and original papers aiming at enriching our knowledge about microbial efflux pumps, their role in the development of AMR, and strategies to block them by EPI molecules.

Dr. Tommaso Felicetti
Guest Editor

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Keywords

  • new antibiotics
  • bioactive medicinal agents
  • classes of antibiotics
  • antibiotic resistance and misuse
  • natural antibiotics
  • microbial efflux pumps
  • efflux pump inhibitors

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Published Papers (8 papers)

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Research

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22 pages, 4097 KiB  
Article
In Vitro and In Vivo Investigations into the Potential of Quinazoline and Quinoline Derivatives as NorA Efflux Pump Inhibitors Against Resistant Staphylococcus aureus Strains
by Nishtha Chandal, Nidhi Sharma, Giada Cernicchi, Tommaso Felicetti, Tommaso Rondini, Mattia Acito, Hemraj Nandanwar and Stefano Sabatini
Antibiotics 2025, 14(4), 339; https://doi.org/10.3390/antibiotics14040339 - 26 Mar 2025
Viewed by 564
Abstract
Background: Staphylococcus aureus is a highly lethal Gram-positive bacterium that is responsible for over one million deaths annually. As a member of the ESKAPE pathogens, its methicillin-resistant strains (MRSA) are prevalent worldwide and exhibit significant antimicrobial resistance (AMR). Bacterial efflux pumps play a [...] Read more.
Background: Staphylococcus aureus is a highly lethal Gram-positive bacterium that is responsible for over one million deaths annually. As a member of the ESKAPE pathogens, its methicillin-resistant strains (MRSA) are prevalent worldwide and exhibit significant antimicrobial resistance (AMR). Bacterial efflux pumps play a pivotal role in the development of AMR by facilitating the expulsion of a range of antimicrobial agents. Methods: The S. aureus strain SA-1199B, which overexpresses NorA and carries a GrlA mutation, was utilized to comprehensively profile the mechanism of the compounds PQQ16P and PQK4F. To assess the toxicity and genotoxicity of these compounds, RAW macrophages, HEK 293T, and HepG2 cell lines were utilized. Female BALB/c mice were utilized to assess the in vivo synergism of EPIs with CPX, Results: NorA efflux pump inhibitors (EPIs), PQQ16P and PQK4F, enhanced the efficacy of the antibacterial ciprofloxacin (CPX) against resistant S. aureus strains. The mechanism of EPIs involved the inhibition of NorA efflux pump, without compromising bacterial membrane permeability, ATP levels, or mammalian calcium channels. Moreover, the EPIs significantly augmented the bactericidal and post-antibiotic effects of CPX, elevating its mutation prevention concentration without manifesting substantial toxicity to human cells. Furthermore, the EPIs reduced S. aureus invasiveness in macrophages, indicating a role for NorA in bacterial virulence. Notably, the in vivo synergism of these EPIs with CPX was observed in a mouse infection model. Conclusions: This study provides substantial evidence for the potential of employing EPIs in a combination with CPX to counteract AMR, both in vitro and in vivo. Full article
(This article belongs to the Special Issue Efflux Pumps in Bacteria: What They Do and How We Can Stop Them)
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27 pages, 9267 KiB  
Article
Selective Serotonin Reuptake Inhibitors: Antimicrobial Activity Against ESKAPEE Bacteria and Mechanisms of Action
by Thiago Hideo Endo, Mariana Homem de Mello Santos, Sara Scandorieiro, Bruna Carolina Gonçalves, Eliana Carolina Vespero, Márcia Regina Eches Perugini, Wander Rogério Pavanelli, Gerson Nakazato and Renata Katsuko Takayama Kobayashi
Antibiotics 2025, 14(1), 51; https://doi.org/10.3390/antibiotics14010051 - 8 Jan 2025
Cited by 1 | Viewed by 1278
Abstract
Background: Multidrug-resistant bacteria cause over 700,000 deaths annually, a figure projected to reach 10 million by 2050. Among these bacteria, the ESKAPEE group is notable for its multiple resistance mechanisms. Given the high costs of developing new antimicrobials and the rapid emergence of [...] Read more.
Background: Multidrug-resistant bacteria cause over 700,000 deaths annually, a figure projected to reach 10 million by 2050. Among these bacteria, the ESKAPEE group is notable for its multiple resistance mechanisms. Given the high costs of developing new antimicrobials and the rapid emergence of resistance, drug repositioning offers a promising alternative. Results: This study evaluates the antibacterial activity of sertraline and paroxetine. When tested against clinical and reference strains from the ESKAPEE group, sertraline exhibited minimum inhibitory concentration (MIC) values between 15 and 126 μg/mL, while the MIC values for paroxetine ranged from 60 to 250 μg/mL. Both drugs effectively eradicated bacterial populations within 2 to 24 h and caused morphological changes, such as protrusions and cellular fragmentation, as shown by electron scanning microscopy. Regarding their mechanisms of action as antibacterials, for the first time, increased membrane permeability was detected, as evidenced by heightened dye absorption, along with the increased presence of total proteins and dsDNA in the extracellular medium of Escherichia coli ATCC2 25922 and Staphylococcus aureus ATCC 25923, and oxidative stress was also detected in bacteria treated with sertraline and paroxetine, with reduced efficiency observed in the presence of antioxidants and higher levels of oxygen-reactive species evidenced by their reaction with 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate. The drugs also inhibited bacterial efflux pumps, increasing ethidium bromide accumulation and enhancing tetracycline activity in resistant strains. Conclusions: These findings indicate that sertraline and paroxetine could serve as alternative treatments against multidrug-resistant bacteria, as well as efflux pump inhibitors (EPIs), and they support further development of antimicrobial agents based on these compounds. Full article
(This article belongs to the Special Issue Efflux Pumps in Bacteria: What They Do and How We Can Stop Them)
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12 pages, 595 KiB  
Article
Inhibition of Staphylococcus pseudintermedius Efflux Pumps by Using Staphylococcus aureus NorA Efflux Pump Inhibitors
by Elisa Rampacci, Tommaso Felicetti, Giada Cernicchi, Valentina Stefanetti, Stefano Sabatini and Fabrizio Passamonti
Antibiotics 2023, 12(5), 806; https://doi.org/10.3390/antibiotics12050806 - 24 Apr 2023
Cited by 3 | Viewed by 1996
Abstract
One promising approach in treating antibiotic-resistant bacteria is to “break” resistances connected with antibacterial efflux by co-administering efflux pump inhibitors (EPIs) with antibiotics. Here, ten compounds, previously optimized to restore the susceptibility to ciprofloxacin (CIP) of norA-overexpressing Staphylococcus aureus, were evaluated [...] Read more.
One promising approach in treating antibiotic-resistant bacteria is to “break” resistances connected with antibacterial efflux by co-administering efflux pump inhibitors (EPIs) with antibiotics. Here, ten compounds, previously optimized to restore the susceptibility to ciprofloxacin (CIP) of norA-overexpressing Staphylococcus aureus, were evaluated for their ability to inhibit norA-mediated efflux in Staphylococcus pseudintermedius and synergize with CIP, ethidium bromide (EtBr), gentamycin (GEN), and chlorhexidine digluconate (CHX). We focused efforts on S. pseudintermedius as a pathogenic bacterium of concern within veterinary and human medicine. By combining data from checkerboard assays and EtBr efflux inhibition experiments, the hits 2-arylquinoline 1, dihydropyridine 6, and 2-phenyl-4-carboxy-quinoline 8 were considered the best EPIs for S. pseudintermedius. Overall, most of the compounds, except for 2-arylquinoline compound 2, were able to fully restore the susceptibility of S. pseudintermedius to CIP and synergize with GEN as well, while the synergistic effect with CHX was less significant and often did not show a dose-dependent effect. These are valuable data for medicinal chemistry optimization of EPIs for S. pseudintermedius and lay the foundation for further studies on successful EPIs to treat staphylococcal infections. Full article
(This article belongs to the Special Issue Efflux Pumps in Bacteria: What They Do and How We Can Stop Them)
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15 pages, 2268 KiB  
Article
Plant Secondary Metabolites on Efflux-Mediated Antibiotic Resistant Stenotrophomonas Maltophilia: Potential of Herbal-Derived Efflux Pump Inhibitors
by Thi Huyen Thu Nguyen, Ngoc Anh Thơ Nguyen, Hai Dang Nguyen, Thi Thu Hien Nguyen, Mai Huong Le, Minh Quan Pham, Huu Nghi Do, Kim Chi Hoang, Serge Michalet, Marie-Geneviève Dijoux-Franca and Hoang Nam Pham
Antibiotics 2023, 12(2), 421; https://doi.org/10.3390/antibiotics12020421 - 20 Feb 2023
Cited by 5 | Viewed by 3110
Abstract
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 [...] Read more.
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). Full article
(This article belongs to the Special Issue Efflux Pumps in Bacteria: What They Do and How We Can Stop Them)
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Review

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35 pages, 2104 KiB  
Review
Antimicrobial Resistance: Two-Component Regulatory Systems and Multidrug Efflux Pumps
by Giuseppe Valerio De Gaetano, Germana Lentini, Agata Famà, Francesco Coppolino and Concetta Beninati
Antibiotics 2023, 12(6), 965; https://doi.org/10.3390/antibiotics12060965 - 26 May 2023
Cited by 42 | Viewed by 9648
Abstract
The number of multidrug-resistant bacteria is rapidly spreading worldwide. Among the various mechanisms determining resistance to antimicrobial agents, multidrug efflux pumps play a noteworthy role because they export extraneous and noxious substrates from the inside to the outside environment of the bacterial cell [...] Read more.
The number of multidrug-resistant bacteria is rapidly spreading worldwide. Among the various mechanisms determining resistance to antimicrobial agents, multidrug efflux pumps play a noteworthy role because they export extraneous and noxious substrates from the inside to the outside environment of the bacterial cell contributing to multidrug resistance (MDR) and, consequently, to the failure of anti-infective therapies. The expression of multidrug efflux pumps can be under the control of transcriptional regulators and two-component systems (TCS). TCS are a major mechanism by which microorganisms sense and reply to external and/or intramembrane stimuli by coordinating the expression of genes involved not only in pathogenic pathways but also in antibiotic resistance. In this review, we describe the influence of TCS on multidrug efflux pump expression and activity in some Gram-negative and Gram-positive bacteria. Taking into account the strict correlation between TCS and multidrug efflux pumps, the development of drugs targeting TCS, alone or together with already discovered efflux pump inhibitors, may represent a beneficial strategy to contribute to the fight against growing antibiotic resistance. Full article
(This article belongs to the Special Issue Efflux Pumps in Bacteria: What They Do and How We Can Stop Them)
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19 pages, 1019 KiB  
Review
Efflux, Signaling and Warfare in a Polymicrobial World
by Ay’sha Moore-Machacek, Antje Gloe, Niall O’Leary and F. Jerry Reen
Antibiotics 2023, 12(4), 731; https://doi.org/10.3390/antibiotics12040731 - 8 Apr 2023
Cited by 6 | Viewed by 2795
Abstract
The discovery void of antimicrobial development has occurred at a time when the world has seen a rapid emergence and spread of antimicrobial resistance, the ‘perfect storm’ as it has often been described. While the discovery and development of new antibiotics has continued [...] Read more.
The discovery void of antimicrobial development has occurred at a time when the world has seen a rapid emergence and spread of antimicrobial resistance, the ‘perfect storm’ as it has often been described. While the discovery and development of new antibiotics has continued in the research sphere, the pipeline to clinic has largely been fed by derivatives of existing classes of antibiotics, each prone to pre-existing resistance mechanisms. A novel approach to infection management has come from the ecological perspective whereby microbial networks and evolved communities already possess small molecular capabilities for pathogen control. The spatiotemporal nature of microbial interactions is such that mutualism and parasitism are often two ends of the same stick. Small molecule efflux inhibitors can directly target antibiotic efflux, a primary resistance mechanism adopted by many species of bacteria and fungi. However, a much broader anti-infective capability resides within the action of these inhibitors, borne from the role of efflux in key physiological and virulence processes, including biofilm formation, toxin efflux, and stress management. Understanding how these behaviors manifest within complex polymicrobial communities is key to unlocking the full potential of the advanced repertoires of efflux inhibitors. Full article
(This article belongs to the Special Issue Efflux Pumps in Bacteria: What They Do and How We Can Stop Them)
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33 pages, 17163 KiB  
Review
Update on the Discovery of Efflux Pump Inhibitors against Critical Priority Gram-Negative Bacteria
by Nina Compagne, Anais Vieira Da Cruz, Reinke T. Müller, Ruben C. Hartkoorn, Marion Flipo and Klaas M. Pos
Antibiotics 2023, 12(1), 180; https://doi.org/10.3390/antibiotics12010180 - 15 Jan 2023
Cited by 60 | Viewed by 8616
Abstract
Antimicrobial resistance (AMR) has become a major problem in public health leading to an estimated 4.95 million deaths in 2019. The selective pressure caused by the massive and repeated use of antibiotics has led to bacterial strains that are partially or even entirely [...] Read more.
Antimicrobial resistance (AMR) has become a major problem in public health leading to an estimated 4.95 million deaths in 2019. The selective pressure caused by the massive and repeated use of antibiotics has led to bacterial strains that are partially or even entirely resistant to known antibiotics. AMR is caused by several mechanisms, among which the (over)expression of multidrug efflux pumps plays a central role. Multidrug efflux pumps are transmembrane transporters, naturally expressed by Gram-negative bacteria, able to extrude and confer resistance to several classes of antibiotics. Targeting them would be an effective way to revive various options for treatment. Many efflux pump inhibitors (EPIs) have been described in the literature; however, none of them have entered clinical trials to date. This review presents eight families of EPIs active against Escherichia coli or Pseudomonas aeruginosa. Structure–activity relationships, chemical synthesis, in vitro and in vivo activities, and pharmacological properties are reported. Their binding sites and their mechanisms of action are also analyzed comparatively. Full article
(This article belongs to the Special Issue Efflux Pumps in Bacteria: What They Do and How We Can Stop Them)
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26 pages, 1346 KiB  
Review
What Approaches to Thwart Bacterial Efflux Pumps-Mediated Resistance?
by Armel Jackson Seukep, Helene Gueaba Mbuntcha, Victor Kuete, Yindi Chu, Enguo Fan and Ming-Quan Guo
Antibiotics 2022, 11(10), 1287; https://doi.org/10.3390/antibiotics11101287 - 21 Sep 2022
Cited by 21 | Viewed by 5034
Abstract
An effective response that combines prevention and treatment is still the most anticipated solution to the increasing incidence of antimicrobial resistance (AMR). As the phenomenon continues to evolve, AMR is driving an escalation of hard-to-treat infections and mortality rates. Over the years, bacteria [...] Read more.
An effective response that combines prevention and treatment is still the most anticipated solution to the increasing incidence of antimicrobial resistance (AMR). As the phenomenon continues to evolve, AMR is driving an escalation of hard-to-treat infections and mortality rates. Over the years, bacteria have devised a variety of survival tactics to outwit the antibiotic’s effects, yet given their great adaptability, unexpected mechanisms are still to be discovered. Over-expression of efflux pumps (EPs) constitutes the leading strategy of bacterial resistance, and it is also a primary driver in the establishment of multidrug resistance (MDR). Extensive efforts are being made to develop antibiotic resistance breakers (ARBs) with the ultimate goal of re-sensitizing bacteria to medications to which they have become unresponsive. EP inhibitors (EPIs) appear to be the principal group of ARBs used to impair the efflux system machinery. Due to the high toxicity of synthetic EPIs, there is a growing interest in natural, safe, and innocuous ones, whereby plant extracts emerge to be excellent candidates. Besides EPIs, further alternatives are being explored including the development of nanoparticle carriers, biologics, and phage therapy, among others. What roles do EPs play in the occurrence of MDR? What weapons do we have to thwart EP-mediated resistance? What are the obstacles to their development? These are some of the core questions addressed in the present review. Full article
(This article belongs to the Special Issue Efflux Pumps in Bacteria: What They Do and How We Can Stop Them)
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