Screening a small-molecule library of about 11,000 compounds, researchers at the Indian Institute of Technology (IIT) Roorkee identified a potent molecule that exhibits broadspectrum bactericidal activity against multidrug-resistant bacteria — Escherichia coli,Acinetobacter baumannii, Klebsiella pneumoniae and Mycobacterium tuberculosis. The molecule also shows antibacterial activity against Staphylococcus aureus and diarrhoea causing Clostridium difficile.
In mice infected with sepsis-causing bacteria A. baumannii, the molecule was found to significantly reduce the bacterial load in the spleen, lungs, kidney and liver at half the dose of a well known drug nitrofurantoin. The results were published in Journal of Antimicrobial Chemotherapy.
The molecule belongs to the nitrofuran class of antibiotics — nitrofurantoin and furazolidone — which are routinely used for treating urinary tract infections and intestinal ailments, respectively.
The team led by Ranjana Pathania from the Department of Biotechnology at IIT Roorkee found that the molecule kills the bacteria by damaging their DNA as well as by inhibiting cell division. When half the concentration required to kill the bacteria was used, the researchers found the daughter cells were unable to separate on cell division, leading to the bacteria forming into long filaments. “Since the molecule targets two pathways to kill the bacteria, microbes are less prone to resistance generation or would take a longer time to develop resistance,” says Prof. Pathania.
“Even at 16-fold less concentration, the molecule was more effective in killing E. coli compared with nitrofurantoin,” she says. The molecule was found to be effective against both gram-negative and Gram-positive bacteria as well as against anaerobic bacteria such as C. dificile. Compared with nitrofurantoin and furazolidone drugs, the molecule was able to kill anaerobic bacteria at many times lower concentration.
The team generated mutants to the molecule and ascertained that the molecule was a pro-drug like the rest of the nitrofuran class of antibiotics. Bacteria are less likely to develop resistance against a pro-drug as it becomes active only after getting inside the bacteria. The active components formed from the pro-drug are potent and short-lived, thus not giving the bacteria sufficient time to develop resistance.
Besides killing actively dividing bacteria, the molecule was effective against persister bacteria that remain in a dormant state. Persister bacteria can survive high doses of antibiotic treatment and are responsible for causing recurring bacterial infections. The researchers generated E. coli persisters and tested the ability of the molecule to kill them using two, four and eight times the minimum dosage required to kill the bacteria. “By the end of 12 hours, there’s a significant reduction in the persisters at four and eight times the MIC,” says Timsy Bhando from IIT Roorkee and the first author. “Pro-drug does not differentiate between dormant and metabolically active bacteria and so they get into even the dormant bacteria. The molecule then kills the dormant bacteria by damaging the DNA.”
Not only was the molecule able to inhibit biofilm formation (which helps bacteria to protect themselves from the action of antibiotics), but was also effective in disrupting the already formed biofilms. Compared with the molecule, both nitrofurantoin and furazolidone drugs displayed “poor ability” to eradicate already formed biofilms.
Bacteria generally can tolerate antibiotics by flushing out the drugs using the efflux pumps. “Even when we shut down the efflux pumps using an inhibitor, the amount of molecule required to kill the bacteria did not reduce much. So the efflux pumps are ineffective in pushing out the molecule from the inside the bacteria,” says Dr. Bhando.
Generally, reactive oxygen species (ROS) generated by a drug helps kill the bacteria. But in the case of the molecule identified by the team, generation of the reactive oxygen species followed bacterial killing. To ascertain if ROS generation killed the bacteria or was produced after the death of the bacteria, the researchers pre-treated E. coli with vitamin C, an antioxidant that removes the ROS. “Even after pre-treatment with vitamin C, bacteria were dying. So ROS is a consequence of DNA damage and was not the cause of bacteria death,” Pathania says. The study was done in collaboration with Government Medical College and Hospital, Chandigarh, AIIMS, Bhopal and AIIMS, Delhi.