Avraneel Paul

We had earlier reported the de novo emergence of genetic resisters of Mycobacterium tuberculosis and Mycobacterium smegmatis to rifampicin and moxifloxacin from the antibiotic-surviving population containing elevated levels of the non-DNA-specific mutagenic reactive oxygen species (ROS) hydroxyl radical. Since hydroxyl radical is generated by Fenton reaction between Fe(II) and H ₂ O ₂ , which is produced by superoxide dismutation, we here report significantly elevated levels of these three ROS and Fe(II) in the M. smegmatis rifampicin-surviving population.

Antibiotics exposed bacteria acquire genetic mutations and emerge as antibiotic resistant clones that thwart treatment of bacterial diseases. Genome-wide mutations are inflicted by the reactive oxygen species (ROS), hydroxyl radical, formed in most of the antibiotic exposed bacteria. Hydroxyl radical is generated through Fenton reaction of Fe (II) with H2O2, which is formed by the dismutation of superoxide. This implied that antibiotic exposed bacteria would contain these three ROS, promoting resister generation. In the present study, we examined Escherichia coli exposed independently to gentamicin and moxifloxacin for the presence of the three ROS and consequential emergence of genetic resisters to the antibiotics. Here we show that the three ROS are formed in E. coli exposed independently to bactericidal concentrations of gentamicin and moxifloxacin for prolonged duration. Resisters to these antibiotics were found to emerge from the respective antibiotic surviving population. The antibiotic unexposed cultures did not show these responses. The Gram-positive ESKAPE pathogen, Staphylococcus aureus, also showed a response similar to that of E. coli upon prolonged exposure to bactericidal concentrations of rifampicin and moxifloxacin. The similar responses of E. coli and S. aureus to antibiotics indicated a common mechanism of ROS generation in the emergence of resisters against antibiotics.

Bacterial antibiotic persister cells tolerate lethal concentrations of antibiotics but emerge as the antibiotic-sensitive population upon antibiotics withdrawal. However, the possibility of antibiotic-resistant genetic mutants emerging from the antibiotic persister population in the continued exposure to microbicidal concentrations of antibiotics needed investigation. We explored this possibility using the fast-growing Mycobacterium smegmatis as a model organism for Mycobacterium tuberculosis biology, as it is known to incur antibiotic-resistant mutations identical to and at identical target positions as found in the clinical isolates of M. tuberculosis. Here we report that the moxifloxacin (MXF) persister population generate significantly elevated levels of hydroxyl radical. Hydroxyl radical being a sequence-non-specific mutagen, resulted in the emergence of moxifloxacin-resistant genetic mutants at 8-log 10 higher frequency from the persister population. Luria-Delbruck experiment (in modified format) confirmed that MXF-resistant mutants emerged de novo from the persister population and were not pre-existent. The nature of the mutations in the quinolone resistance determining region indicated that they were generated due to oxidative stress. These mutations were identical to and at identical positions as found in the clinical isolates of MXF-resistant M. tuberculosis. Interestingly, from the MXF persister population, resisters to microbicidal concentrations of ethambutol and isoniazid could also be selected. These observations implied that the significantly high levels of hydroxyl radical might have generated genome-wide mutations, creating a pool of mutants in the MXF persister population, facilitating selection of resisters to other antibiotics also. These findings may be of clinical relevance to the emergence of drug-resistant strains during prolonged tuberculosis treatment regimen with high doses of multiple antibiotics.