Reversing drug resistance made possible

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Drug-resistant E. coli become sensitive to antibiotics when H2S synthesis is inhibited

Indian researchers have unravelled the mechanism by which hydrogen sulphide (H2S) gas produced by bacteria protects them from antibiotics and plays a key role in helping bacteria develop drug resistance. And by blocking/disabling the enzyme that triggers the biosynthesis of hydrogen sulphide in bacteria, the researchers from Bengaluru’s Indian Institute of Science (IISc) and Indian Institute of Science Education and Research (IISER) Pune, have been able to reverse antibiotic resistance in E. coli bacteria; E. coli bacteria were isolated from patients suffering from urinary tract infection. The results were published in the journal Chemical Science.

Antibiotics kill by increasing the levels of reactive oxygen species (oxidative stress) inside bacterial cells. So any mechanism that detoxifies or counters reactive oxygen species generated by antibiotics will reduce the efficacy of antibiotics. “Hydrogen sulphide does this to nullify the effect of antibiotics,” says Dr. Amit Singh from the Department of Microbiology and Cell Biology at IISc and one of the corresponding authors of the paper. “When bacteria face reactive oxygen species a protective mechanism in the bacteria kicks in and more hydrogen sulphide is produced.” Hydrogen sulphide successfully counters reactive oxygen species and reduces the efficacy of antibiotics.

The researchers carried out simple experiments to establish this. They first ascertained that regardless of the mode of action of antibiotics, the drugs uniformly induce reactive oxygen species formation inside E. coli bacteria. Then to test if increased levels of hydrogen sulphide gas inside bacteria counter reactive oxygen species produced upon treatment with antibiotics, a small molecule that produces hydrogen sulphide in a controlled manner inside the bacteria was used. “Hydrogen sulphide released by the molecule was able to counter reactive oxygen species and reduce the ability of antibiotics to kill bacteria,” says Dr. Singh.

The small molecule was synthesised by a team led by Prof. Harinath Chakrapani from the Department of Chemistry, IISER, Pune; he is one of the corresponding authors of the paper. “We designed the small molecule keeping in mind that synthesis should be easy, efficiency in producing hydrogen sulphide should be high and the molecule should release hydrogen sulphide only inside bacteria and not mammalian cells,” says Vinayak S. Khodade from the Department of Chemistry, IISER, Pune and one of the authors of the paper who contributed equally like the first author. The researchers were able to selectively increase hydrogen sulphide levels inside a wide variety of bacteria.

To reconfirm hydrogen sulphide’s role in countering reactive oxygen species, the team took multidrug-resistant, pathogenic strains of E. coli from patients suffering from urinary tract infection and measured the hydrogen sulphide levels in these strains. “We found the drug-resistant strains were naturally producing more hydrogen sulphide compared with drug-sensitive E. coli,” says Prashant Shukla from the Department of Microbiology and Cell Biology at IISc and the first author of the paper. So the team used a chemical compound that inhibits an enzyme responsible for hydrogen sulphide production. “There was nearly 50% reduction in drug-resistance when hydrogen sulphide production was blocked,” Dr. Singh says.

“Bacteria that are genetically resistant to antibiotics actually become sensitive to antibiotics when hydrogen sulphide synthesis is inhibited,” says Prof. Chakrapani. The multidrug-resistant E. coli regained its ability to survive antibiotics when hydrogen sulphide was once again supplied by introducing the small molecule synthesised by Prof. Chakrapani.

“As a result of our study, we have a found new mechanism to develop a new class of drug candidates that specifically target multidrug-resistant bacteria,” says Prof. Chakrapani. The researchers already have a few inhibitors that seem capable of blocking hydrogen sulphide production. But efforts are on to develop a library of inhibitors to increase the chances of success.

How H2S acts

The researchers identified that E. coli has two modes of respiration involving two different enzymes. The hydrogen sulphide gas produced shuts down E. coli’s aerobic respiration by targeting the main enzyme (cytochrome bo oxidase (CyoA)) responsible for it. E. coli then switches over to an alternative mode of respiration by relying on a different enzyme — cytochrome bd oxidase (Cydb). Besides enabling respiration, the Cydb enzyme detoxifies the reactive oxygen species produced by antibiotics and blunts the action of antibiotics.

“So we found that hydrogen sulphide activates the Cydb enzyme, which, in turn, is responsible for increasing resistance towards antibiotics,” says Dr. Singh. “If we have a drug-like molecule(s) that blocks hydrogen sulphide production and inhibits Cydb enzyme activity then the combination will be highly lethal against multidrug-resistant bacteria.”

This combination can also be used along with antibiotics to effectively treat difficult-to-cure bacterial infections.

The link between hydrogen sulphide and Cydb enzyme in the emergence of drug resistance is another key finding of the study.

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