Screening for Inhibitors of Bacterial Multidrug Efflux Pumps
Microbial resistance to drug therapy is an area of increasing concern in clinical microbiology and infectious disease treatment. The emergence of antimicrobial resistance (AMR) in microorganisms can occur via several mechanisms [1], with one prominent mechanism being drug efflux mediated by membrane transporter proteins—commonly known as Multidrug Efflux Systems (MES). These efflux systems actively pump a wide range of drug-like molecules out of bacterial cells, including many clinically relevant antimicrobial agents.
Multidrug Efflux Systems (MES)
Figure 1. Representations of known families of MES [2].
A) The MFS, SMR, and MATE families are powered by chemiosmotic energy, pumping drugs out of the cell while pumping H⁺ or Na⁺ into the cell. B) The ABC family of pumps is powered by ATP. C) The RND family is a multisubunit complex spanning the inner and outer membranes in Gram-negative bacteria.
Efflux systems serve many essential biological roles, but they are also a major contributor to antibiotic resistance. Overall, bacterial MES fall into five superfamilies: major facilitator (MFS), small multidrug resistance (SMR), multi-antimicrobial extrusion protein (MATE), ATP-binding cassette (ABC), and resistance-nodulation cell division (RND) (Figure 1). The natural substrates, structural preferences, and biological functions of each efflux pump family vary widely. Some commonly studied systems and their substrates are summarized in Table 1.
| Superfamily | Pump(s) | Organism | Antibiotic substrates |
| SMR | Smr/QacC | S. aureus | QA |
| SMR | EmrE | E. coli | AC |
| MF | TetA | E. coli | TC |
| MF | NorA | S. aureus | AC, CA, FQ, PM, QA |
| MATE | YdhE | E. coli | CF, KM, NF, SM |
| ABC | MacB | E. coli | AZ, CL, EM, OL |
| RND | AcrB/AcrA/TolC | E. coli | AC, BL, CA, EM, FA, FQ, MC, NA, NV, TC |
| RND | MexB/MexA/OprM | P. aeruginosa | AZ, CF, RP |
Table 1. Substrate abbreviations: AC, acriflavin; AZ, azithromycin; BL, β-lactams; CA, chloramphenicol; CF, ciprofloxacin; CL, clarithromycin; EM, erythromycin; FQ, fluoroquinolones; FA, fusidic acid; KM, kanamycin; MC, mitomycin; NA, nalidixic acid; NF, norfloxacin; NV, novobiocin; OL, oleandomycin; PM, puromycin; QA, quaternary ammonium compounds (including benzalkonium chloride and cetyltrimethylammonium bromide); RP, rifampicin; SM, streptomycin; TC, tetracycline. [3]
Research on MES has led to the development of efflux pump inhibitors (EPIs) that can block the function of specific efflux systems. Combining EPIs with standard antimicrobials holds promise for creating new treatment options against drug-resistant bacteria and multidrug-resistant (MDR) pathogens.
Emery Pharma offers several efflux pump inhibitor screening assays based on current research into MES and EPI technologies. Our approach utilizes a panel of microbial strains specifically designed to study efflux pump activity. MES-knockout mutants can be used to determine whether a test compound is a substrate of a particular efflux system. By comparing the Minimum Inhibitory Concentration (MIC) of a compound in a wild-type strain versus a MES-knockout mutant, we can infer the degree to which efflux impacts drug efficacy.
In the example shown in Table 2, compounds 1–3 all exhibit an MIC of 32 µg/mL against wild-type Pseudomonas aeruginosa. However, compound 1 is a strong substrate of the MexB/MexA/OprM efflux system but displays strong intrinsic activity. Compound 2 is a weak substrate with intermediate activity. Compound 3 is not a substrate and exhibits poor activity, showing how efflux profiling can elucidate antimicrobial performance.
| Compound | MIC, wild-type P. aeruginosa (ug/ml) | MIC, ΔMexB, ΔMexA, ΔOprM P. aeruginosa (ug/ml) |
| 1 | 32 | 0.5 |
| 2 | 32 | 16 |
| 3 | 32 | 32 |
Table 2. Comparison of MICs against wild-type and efflux-knockout mutants yields information about MES specificity (posted with permission).
Emery Pharma also offers specialized support in developing and optimizing screening assays for efflux pump inhibitors, tailored to unique research and regulatory needs. MES have been linked to the formation and persistence of bacterial biofilms, which further complicate treatment of chronic infections. Our team provides a range of biofilm quantification and testing services, including MBEC assays, drip-flow reactors, and custom in-house biofilm reactor models—all of which are adaptable to your compound screening and validation requirements. Contact us today to learn how we can help you advance your research!
About the Author
Originally authored by Dr. Dmitri Debabov. This article was reviewed and updated on June 2, 2025 by Dr. Janet Liu, current Director of Biology.
References
- Debabov, D. (2013). Antibiotic Resistance: Origins, Mechanisms, Approaches to Counter. Applied Biochemistry and Microbiology, 49, 1–7.
- Tegos, G. P., Haynes, M., Strouse, J. J., Khan, M. T., Bologa, C. G., Oprea, T. I., & Sklar, L. A. (2013). Curr Pharm Des, 17, 1291–1302.
- Borges-Walmsley, M. I., McKeegan, K. S., & Walmsley, A. R. (2003). Structure and function of efflux pumps that confer resistance to drugs. The Biochemical Journal, 376, 313–338.
