Mycobacterium smegmatis moxifloxacin persister cells produce high levels of hydroxyl radical, generating genetic resisters selectable not only with moxifloxacin, but also with ethambutol and isoniazid

Swaminath, Sharmada; Paul, Avraneel; Pradhan, Atul; Sebastian, Jees; Nair, Rashmi Ravindran; Ajitkumar, Parthasarathi

doi:10.1099/mic.0.000874

Cited by 18 publications

(31 citation statements)

References 71 publications

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“…The rifampicin resister generation frequency of ϳ10 Ϫ3 was ϳ5-log 10 -fold higher than the natural resister generation frequency of 10 Ϫ8 of M. smegmatis against rifampicin (11,18). The high resister generation frequency alluded to elevated levels of the DNA-nonspecific mutagen, hydroxyl radical (20)(21)(22), as found by us in the rifampicin/moxifloxacin-exposed M. tuberculosis and M. smegmatis antibioticsurviving cells (9)(10)(11) and in the rifampin exposed M. tuberculosis cells (23) and by others in other bacterial systems exposed to antibiotics (12)(13)(14).…”

Section: Figmentioning

confidence: 56%

“…Based on the CFU on antibiotic-free plates at specific intervals of the exposure period, the killing phase with exponential reduction in the CFU, the antibiotic-surviving phase with no appreciable change in the CFU, and the regrowth phase with a steady rise in the CFU were temporally demarcated, as described earlier (10,11). The cells from the antibiotic-surviving and the regrowing populations were analyzed for hydroxyl radical production using specific fluorescence dye, genetic mutations inflicted by hydroxyl radical by DNA sequencing, regrowth of the mutants using CFU determination on antibiotic plates, fluorescence and transmission electron microscopy and livecell time-lapse imaging of cell division process, and determination of the expression levels of the genes involved in cell division, DNA replication and repair, SOS regulon, and so on.…”

Section: Resultsmentioning

confidence: 99%

“…In parallel, biological triplicates of M. smegmatis cultures were exposed to 10ϫ, 3.75ϫ, and 1ϫ MBC of moxifloxacin (an antitubercular drug against gyrase) for 120 h, for the purpose of comparison of the physiological mechanisms. Here, 10 8 cells/ml were used to preempt the natural resisters against moxifloxacin that arise at 10 Ϫ9 frequency (10). However, plating of the moxifloxacin-exposed cells was performed only on moxifloxacin-free plates and not on moxifloxacin-containing plates.…”

Section: Resultsmentioning

confidence: 99%

“…Also, the cells in the colonies were not sequenced for genetic mutations on the moxifloxacin-specific targets, gyrA/gyrB. This was because the gyrA mutations in the moxifloxacin resisters formed from the moxifloxacin-surviving cells were determined in our recent study on M. smegmatis cells surviving in the presence of 3.75ϫ MBC of moxifloxacin (10).…”

Section: Resultsmentioning

confidence: 99%

“…We recently reported the de novo emergence of rifampicin/moxifloxacin-resistant genetic mutants of Mycobacterium tuberculosis and Mycobacterium smegmatis from the respective antibiotic-surviving populations in the continued presence of bactericidal concentrations of antibiotics for prolonged duration in vitro (9)(10)(11). Besides genomewide mutations, these antibiotic-selected genetic resisters carried hydroxyl radicalinflicted mutations at the rifampicin resistance-determining region (RRDR) in rpoB and the quinolone resistance-determining region (QRDR) in gyrA, respectively.…”

mentioning

confidence: 99%

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Unique Mode of Cell Division by the Mycobacterial Genetic Resister Clones Emerging De Novo from the Antibiotic-Surviving Population

Jakkala

Paul

Pradhan

et al. 2020

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The emergence of antibiotic genetic resisters of pathogenic bacteria poses a major public health challenge. The mechanism by which bacterial antibiotic genetic resister clones formed de novo multiply and establish a resister population remained unknown. Here, we delineated the unique mode of cell division of the antibiotic genetic resisters of Mycobacterium smegmatis and Mycobacterium tuberculosis formed de novo from the population surviving in the presence of bactericidal concentrations of rifampicin or moxifloxacin. The cells in the rifampicin/moxifloxacin-surviving population generated elevated levels of hydroxyl radical-inflicting mutations. The genetic mutants selected against rifampicin/moxifloxacin became multinucleated and multiseptated and developed multiple constrictions. These cells stochastically divided multiple times, producing sister-daughter cells phenomenally higher in number than what could be expected from their generation time. This caused an abrupt, unexpectedly high increase in the rifampicin/moxifloxacin resister colonies. This unique cell division behavior was not shown by the rifampicin resisters formed naturally in the actively growing cultures. We could detect such abrupt increases in the antibiotic resisters in others’ and our earlier data on the antibiotic-exposed laboratory/clinical M. tuberculosis strains, M. smegmatis and other bacteria in in vitro cultures, infected macrophages/animals, and tuberculosis patients. However, it went unnoticed/unreported in all those studies. This phenomenon occurring in diverse bacteria surviving against different antibiotics revealed the broad significance of the present study. We speculate that the antibiotic-resistant bacillary clones, which emerge in patients with diverse bacterial infections, might be using the same mechanism to establish an antibiotic resister population quickly in the continued presence of antibiotics. IMPORTANCE The bacterial pathogens that are tolerant to antibiotics and survive in the continued presence of antibiotics have the chance to acquire genetically resistant mutations against the antibiotics and emerge de novo as antibiotic resisters. Once the antibiotic resister clone has emerged, often with compromise on growth characteristics, for the protection of the species, it is important to establish an antibiotic-resistant population quickly in the continued presence of the antibiotic. In this regard, the present study has unraveled multinucleation and multiseptation followed by multiple constrictions as the cellular processes used by the bacteria for quick multiplication to establish antibiotic-resistant populations. The study also points out the same phenomenon occurring in other bacterial systems investigated in our laboratory and others’ laboratories. Identification of these specific cellular events involved in quick multiplication offers additional cellular processes that can be targeted in combination with the existing antibiotics’ targets to preempt the emergence of antibiotic-resistant bacterial strains.

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