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

Abstract

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.

Deletion of rifampicin-inactivating mono-ADP-ribosyl transferase gene of Mycobacterium smegmatis globally altered gene expression profile that favoured increase in ROS levels and thereby antibiotic resister generation

“…The CFU scores, from the plates with and without rifampicin, overlapped consistently from the 39 hr and 48 hr of exposure of arr -KO and WT cultures, respectively (n = 3 biological replicates in each case). The overlapping showed that the regrowing cells would all have originated from rifampicin-resistant clones, divided using unusual cell division mechanisms and grown, as reported by us recently ( Jakkala et al., 2020 ). Despite both the cultures behaving in a similar manner, the mass doubling time of arr- KO from 39 hr onwards was 4.10 hrs and that of WT from 48 hr onwards was 5.25 hrs (Fig.…”

“…Fourthly, there was no possibility of hypoxia setting in in the well aerated cultures with 170 rpm shaking. Further, when the cells in the rifampicin surviving population gained rpoB mutation, they regrew to establish a rifampicin-resistant population, as reported by us ( Jakkala et al., 2020 ). Thus, taken together, the data suggested very low probability of adaptation of the arr -KO cultures to hypoxia under the culture conditions used in the study.…”

The evolving biology of Mycobacterium tuberculosis drug resistance

“…Environmental stresses such as hypoxia, nutrient deprivation, and host immune effectors can generate genome-wide mutations in subsets of cells, leading to heritable genetic resistance ( Sebastian et al., 2017 ; Ragheb et al., 2019 ; Hicks et al., 2020 ; Jakkala et al., 2020 ; Swaminath et al., 2020 ). The activity and efficiency of different DNA repair pathways influences the rate at which these cells are a reservoir from which resistant isolates emerge.…”

Clinically encountered growth phenotypes of tuberculosis-causing bacilli and their in vitro study: A review

“…SCs were found to be more susceptible than NCs to antibiotics (rifampin and isoniazid), H 2 O 2 , and acidified NaNO 2 . Additionally, drug resistant bacteria display a distinct mode of cell division and cell length heterogeneity when compared with drug sensitive bacteria ( Vijay et al., 2017b ; Jakkala et al., 2020 ). Notably, mycobacteria exhibit a unique kind of heterogeneity under stress where it asymmetrically distributes irreversibly oxidized proteins within bacteria and between their progeny ( Vaubourgeix et al., 2015 ).…”