Antimicrobial Resistance (AMR) and Efflux Pump Inhibitors: A Potential Solution to Combat Drug-Resistant Infections

Antimicrobial resistance (AMR) is a global issue that continues to escalate, posing significant challenges to public health worldwide. One of the mechanisms leading to resistance is the removal of antibiotics from bacterial cells through efflux pump macromolecular machineries. These efflux pumps can confer resistance to various classes of antimicrobials, making them major contributors to AMR. In light of this, inhibiting efflux pumps holds great promise for reversing resistance and treating drug-resistant infections. While some efflux inhibitors have been identified, their clinical use is hindered by harmful side effects. Thus, the development of novel and safe inhibitors is crucial.

The Rising Challenge of Antimicrobial Resistance

Antimicrobial resistance has become a pressing public health challenge on a global scale. Certain infections, such as pneumonia, tuberculosis (TB), gonorrhoea, and salmonellosis, are becoming increasingly difficult to treat due to resistance. For instance, a concerning number of new TB cases exhibit resistance to rifampicin, the most effective first-line drug. Furthermore, there are growing fears that Neisseria gonorrhoeae has developed resistance to all currently recommended treatments. The urgent need for new antibiotics to combat these highly resistant infections is evident. Unfortunately, the process of drug discovery is slow and costly, leading to a limited number of new antibiotics being developed in recent years.

Additionally, the misuse of antibiotics contributes to the rapid emergence of resistance once new drugs are introduced clinically. Therefore, alongside antibiotic development, alternative strategies are vital to combat the continuous arms race of drug resistance. Antimicrobial resistance can occur through acquired or intrinsic mechanisms. Acquired resistance often results from horizontal gene transfer or spontaneous mutations, leading to alterations in drug targets or the production of enzymes that degrade antibiotics. In contrast, intrinsic resistance refers to non-specific mechanisms of antibiotic resistance that have evolved ancestrally, such as the presence of efflux pumps that remove drugs from bacterial cells.

Efflux Pumps and Their Role in Antibiotic Resistance

Efflux pumps play a significant role in bacterial resistance to antimicrobial agents. There are six families of bacterial efflux pumps identified, each with distinct mechanisms of action. The ATP-binding cassette (ABC) family directly hydrolyzes ATP to drive drug efflux, while the other five families utilize transmembrane ion gradients. While the RND family effluxes antibiotics across both membranes, the other families only transport antibiotics across the inner membrane.

Efflux pumps are often non-specific, providing resistance to a wide range of antimicrobials. They have been implicated in contributing to multi-drug resistant phenotypes in various bacterial species, including Mycobacterium tuberculosis, Pseudomonas aeruginosa, Neisseria gonorrhoeae, and Streptococcus pneumoniae. Inhibition of drug efflux presents an exciting prospect for treating drug-resistant bacteria and potentially revitalizing the use of old antibiotics in clinical settings.

The Potential of Efflux Pump Inhibitors

Efflux pump inhibitors have been explored as adjuvants to aid in the treatment of resistant infections. Despite the identification of potent efflux inhibitors, none have been successfully implemented clinically, primarily due to their toxicity at effective inhibitory concentrations. Therefore, there is an urgent need to develop novel efflux inhibitors that are safe for clinical use.

To achieve this goal, validation assays are essential to confirm the inhibitory activity of candidate compounds. Standard antibiotic susceptibility testing, such as the resazurin-based microplate assay, can be utilized to determine if a putative inhibitor, at sub-MIC concentrations, can lower the MIC of a known antibiotic. While this method is relatively easy and high-throughput, it lacks sensitivity and may not directly attribute changes in MIC to efflux inhibition.

Accumulation Assays and Efflux Assays: Techniques for Identifying Efflux Inhibitors

Two common laboratory techniques used to identify and characterize candidate efflux inhibitors are accumulation assays and efflux assays. These techniques provide valuable insights into the inhibitory potential of novel compounds.

1. Accumulation Assays

Accumulation assays follow the movement of an efflux pump substrate, often a fluorescent molecule, into and out of bacterial cells. These assays can use different molecules, such as ethidium bromide and Nile red, which fluoresce differently in extra- and intracellular environments. This fluorescence difference allows for a sensitive indication of the rate of efflux from the cell, aiding in the elimination of background fluorescence.

The assay starts with no dye added to the bacteria. The dye is then introduced, and its accumulation within the cells is monitored over time. Eventually, an equilibrium is achieved between influx and efflux of the dye, resulting in a steady-state fluorescence. The addition of efflux inhibitors during the assay can lead to increased dye accumulation within the cells, validating the inhibitory activity of candidate efflux inhibitors.

While accumulation assays are widely used for validation, they have limitations. They do not directly measure efflux, and the results may be influenced by the permeability of bacterial membranes, which can vary between different strains.

2. Efflux Assays

Efflux assays provide a more direct measure of efflux. These assays involve preloading cells with a dye or an efflux pump substrate, followed by the subsequent efflux of the dye after removal of any extracellular inhibitor. The movement of the dye out of the cells is then recorded, allowing for the determination of efflux rates.

Efflux assays offer a quantitative measure of efflux rates and are more applicable for a broader range of bacteria. However, they may not be suitable for non-fermenter bacteria, like Pseudomonas and Acinetobacter, as these cannot be easily reenergized. In such cases, accumulation assays are preferred.

Towards High-Throughput Screening of Efflux Inhibitors

One of the challenges in developing novel efflux inhibitors is the lack of high-throughput assays to validate candidate compounds. Both accumulation and efflux assays, while useful, are limited in throughput. To address this limitation, recent efforts have been made to develop more high-throughput screening assays, such as the BacPK assay, which uses a 96-well plate format combined with mass spectrometry. Such advancements are crucial for the discovery of effective and safe efflux inhibitors on a larger scale.

The Hope for Clinical Efflux Inhibitors

The development of clinical efflux inhibitors used as antibiotic adjuvants holds the potential to shift the balance in the battle against antibiotic resistance. By inhibiting efflux pumps, these inhibitors can render bacterial pathogens susceptible to existing antibiotics once again, prolonging the usefulness of these drugs in the fight against infections.

In conclusion, antimicrobial resistance remains a significant threat to global health, necessitating innovative solutions. Efflux pumps have emerged as critical players in antibiotic resistance, making efflux inhibitors promising candidates for combating drug-resistant infections. Accumulation assays and efflux assays serve as valuable tools in the identification and validation of potential inhibitors. The ongoing efforts to optimize high-throughput screening methods will undoubtedly drive the development of safe and effective efflux inhibitors, paving the way for improved strategies in the battle against antimicrobial resistance.

References

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