Using a small molecule screened from a synthetic library of 8,000 molecules, researchers at the Indian Institute of Technology (IIT) Roorkee have been able to reverse drug resistance and restore the efficacy of fluoroquinolone-group of antibiotics by inhibiting the proton gradient which drives the efflux pump. Antibiotic-resistant bacteria use the efflux pumps to expel antibiotics from the intracellular environment thus preventing antibiotics from reaching the target thus helping the bacteria to survive.
By inhibiting the proton gradient using the small molecule, the team led by Prof. Ranjana Pathania from the Department of Biotechnology at IIT Roorkee was able to inactivate the efflux, leading to an effective build-up of antibiotic inside the bacteria and subsequent bacterial death. The results were published in the International Journal of Antimicrobial Agents.
The team studied the efficiency of the small molecule in multidrug-resistant bacteria Acinetobacter baumannii. While the small molecule did not inhibit the growth of the bacteria per se, it was able to enhance the activity of a few antibiotics such as ciprofloxacin and norfloxacin in fluoroquinolone-resistant clinical isolates of A. baumannii. A. baumannii causes pneumonia, meningitis and urinary tract infections and is one of the most prevalent hospital-acquired infections across the world.
ESKAPE pathogen
“The reason for using small molecule to target A. baumannii is because it is among the six ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) that cause the most hospital-acquired infections,” says Atin Sharma from the Department of Biotechnology at IIT Roorkee and one first author of the paper.
“Since discovering new antibiotics takes a long time, boosting the activity of existing antibiotics by inhibiting the mechanism that prevents the drugs from acting will be a viable alternative,” Sharma says.
“Since the molecule inhibits the proton gradient, it and can potentially inhibit a wide variety of proton-driven efflux pumps in many multidrug-resistant pathogens,” says Prof. Pathania.
They found that lower dosage of antibiotics were sufficient to kill the bacteria when used along with the small molecule. In the case of clinical isolates of A. baumannii, when 25 micromolar of the inhibitor was used along with the antibiotic, there was a 64-fold reduction in the minimum inhibitory concentration (MIC) (the lowest concentration of the compound required to inhibit the visible growth of a pathogen) of both ciprofloxacin and norfloxacin.
“The use of small molecule inhibitor not only restores the efficacy of antibiotics but also decreases the frequency of resistant bacteria,” she says. Ciprofloxacin in combination with 50 micromolar of the inhibitor exhibited “significantly lower” mutation selection frequency compared with ciprofloxacin used alone at the same concentration.
The molecule appears safe to mammalian cells at minimum effective concentration of 16 micromolar and 32 micromolar for ciprofloxacin and norfloxacin respectively. The IC50 (a measure of toxicity) of the small molecule for human embryonic kidney cells is about 133 micromolar, which is about ten times more than the effective concentration.
“Most of the PMF [proton-motive force] inhibitors are associated with high toxicity. But the small molecule is not an inhibitor of PMF as it targets only the proton gradient and hence is not toxic to mammalian cells,” says Prof. Pathania.
The molecule has also been tested in mice models for safety and efficacy. “We could revive the activity of ciprofloxacin and norfloxacin on mice model of A. baumannii,” she says.
