With the advent of antibiotics, bacterial infections were supposed to be a thing of past. However, this instead led to the selection and evolution of bacteria with mechanisms to counter the action of antibiotics. Antibiotic efflux is one of the major mechanisms, whereby bacteria pump out the antibiotics from their cellular interior to the external environment using special transporter proteins called efflux pumps. Inhibiting these pumps seems to be an attractive strategy at a time when novel antibiotic supplies are dwindling. Molecules capable of inhibiting these pumps, known as efflux pump inhibitors (EPIs), have been viewed as potential therapeutic agents that can rejuvenate the activity of antibiotics that are no longer effective against bacterial pathogens. EPIs follow some general mechanisms of efflux inhibition and are derived from various natural as well as synthetic sources. This review focuses on EPIs and identifies the challenges that have kept these futuristic therapeutics away from the commercial realm so far.

Efflux pump inhibitors for bacterial pathogens: From bench to bedside.

During active tuberculosis a spectrum of physiologically different Mycobacterium tuberculosis bacilli reside in human tissues. Subpopulations of the pathogen survive antibiotic treatment for a prolonged time in a dormant state of phenotypic drug resistance, a phenomenon independent of genetic mutations. Here, we used an established culture model of nutrient deprivation to shift down M. tuberculosis from growth to nonreplicating survival, which is characterized by a drastic loss of drug susceptibility. Liquid chromatography coupled with mass spectrometry techniques were employed to quantify drug penetration in replicating and nutrient-starved nonreplicating bacilli. We found that intracellular concentrations of fluoroquinolones, rifamycins, and linezolid were lower in nonreplicating M. tuberculosis. Studies with pump inhibitors suggest that the observed differences were independent of efflux processes. We conclude that decreased drug permeability contributes to phenotypic drug resistance of dormant M. tuberculosis.

Reduced drug uptake in phenotypically resistant nutrient-starved nonreplicating Mycobacterium tuberculosis

Drug tolerance likely represents an important barrier to tuberculosis treatment shortening. We previously implicated the Mycobacterium tuberculosis efflux pump Rv1258c as mediating macrophage-induced tolerance to rifampicin and intracellular growth. In this study, we infected the human macrophage-like cell line THP-1 with drug-sensitive and drug-resistant M. tuberculosis strains and found that tolerance developed to most antituberculosis drugs, including the newer agents moxifloxacin, PA-824, linezolid, and bedaquiline. Multiple efflux pump inhibitors in clinical use for other indications reversed tolerance to isoniazid and rifampicin and slowed intracellular growth. Moreover, verapamil reduced tolerance to bedaquiline and moxifloxacin. Verapamil's R isomer and its metabolite norverapamil have substantially less calcium channel blocking activity yet were similarly active as verapamil at inhibiting macrophage-induced drug tolerance. Our finding that verapamil inhibits intracellular M. tuberculosis growth and tolerance suggests its potential for treatment shortening. Norverapamil, R-verapamil, and potentially other derivatives present attractive alternatives that may have improved tolerability.

Verapamil, and Its Metabolite Norverapamil, Inhibit Macrophage-induced, Bacterial Efflux Pump-mediated Tolerance to Multiple Anti-tubercular Drugs

The emergence of drug resistance continues to plague TB control, with a global increase in the prevalence of MDR-TB. This acts as a gateway to XDR-TB and thus emphasizes the urgency for drug development and optimal treatment options. Bedaquiline is the first new anti-TB drug approved by the FDA in 40 years and has been shown to be an effective treatment option for MDR Mycobacterium tuberculosis infection. Bedaquiline has also recently been included in clinical trials for new regimens with the aim of improving and shortening treatment periods. Alarmingly, efflux-mediated bedaquiline resistance, as well as efflux-mediated cross-resistance to clofazimine, has been identified in treatment failures. This mechanism of resistance results in efflux of a variety of anti-TB drugs from the bacterial cell, thereby decreasing the intracellular drug concentration. In doing so, the bacillus is able to render the antibiotic treatment ineffective. Recent studies have explored strategies to reverse the resistance phenotype conferred by efflux pump activation. It was observed that the addition of efflux pump inhibitors partially restored drug susceptibility in vitro and in vivo. This has significant clinical implications, especially in MDR-TB management where treatment options are extremely limited. This review aims to highlight the current efflux pump inhibitors effective against M. tuberculosis, the effect of efflux pump inhibitors on mycobacterial growth and the clinical promise of treatment with efflux pump inhibitors and standard anti-TB therapy.

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Efflux pump inhibitors: targeting mycobacterial efflux systems to enhance TB therapy.

Background
The detection of mutations in the gyrA and gyrB genes in the Mycobacterium tuberculosis genome that have been demonstrated to confer phenotypic resistance to fluoroquinolones is the most promising technology for rapid diagnosis of fluoroquinolone resistance.

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Frequency and Geographic Distribution of gyrA and gyrB Mutations Associated with Fluoroquinolone Resistance in Clinical Mycobacterium Tuberculosis Isolates: A Systematic Review

Mycobacterium tuberculosis, the aetiological agent of tuberculosis (TB), has re-emerged as a killer pathogen in western countries after the increase of HIV-AIDS cases and development of resistance to various anti-tubercular drugs. This increase in the number of multi-drug resistant M. tuberculosis isolates has drawn the attention towards the identification of alternate drugs like fluoroquinolones (FQs) for the treatment of TB. It is known that M. tuberculosis commonly acquires drug resistant phenotype by accumulation of mutations in the structural genes encoding the drug target or the enzymes involved in drug activation. Other known cause of drug resistance in mycobacteria is efflux of drug molecules1. The principal cellular target of the FQs is the DNA gyrase encoded by gyrA and gyrB genes. Mutation in the quinolone resistance determining region (QRDR) of gyrA was the most common cause of FQ resistance in various organisms2,3. However, studies carried out in India have reported that only 11.74 and 45 per cent5 of ofloxacin resistant M. tuberculosis isolates harbour mutations in their gyrA gene and no mutation was found in gyrB gene. As mutations in DNA gyrase alone do not account for the mechanism(s) of resistance in a significant proportion of FQs resistant M. tuberculosis isolates, it suggests the need to investigate the role of alternate mechanisms, like efflux pumps. The upregulation of efflux systems can significantly decrease the intracellular concentration of many antibiotics, reducing their clinical efficacy. For this reason attention has been focused on identifying inhibitors of the efflux systems of Gram-negative and Gram-positive bacteria that could potentially be used in combination with antibiotics to improve efficacy and abolish resistance1. Banerjee and co-workers6 observed that carbonyl cyanide m chlorophenyl hydrazone (CCCP), verapamil and 2,4-dinitro phenol (DNP) increased the accumulation of drug possibly due to inhibition of active efflux. Several mycobacterial efflux pumps associated with FQs resistance have been described. These efflux pumps include the pumps of Major Facilitator Superfamily (MFS) family (lfrA, Rv1634 and Rv1258c) and ATP Binding Cassette (ABC) transporters (DrrAB, PstB and Rv2686c-2687c-2688c)1. For better understanding of drug resistance and to find out the newer drugs and/or identify suitable drug targets for better treatment of TB, there is a need to understand the exact mechanism(s) of resistance to FQs in M. tuberculosis. In the present study we have studied the effect of certain efflux inhibitors on in vitro susceptibility levels in ofloxacin (OFL)-resistant clinical M. tuberculosis isolates.

Ramdas

Papers

Effect of efflux pump inhibitors on drug susceptibility of ofloxacin resistant Mycobacterium tuberculosis isolates.

Quebrachitol from Putranjiva roxburghii Wall. (Putranjivaceae) a potent antimalarial: Pre-clinical efficacy and its interaction with PfLDH.