Growth and Division Mechanisms by Which Genetic Resisters Emerge from the Rifampicin-Surviving Population of Differentially Antibiotic-Susceptible Mycobacterial Subpopulations
Authors: Jakkala Kishor , Paul Avraneel , Nair Rashmi Ravindran , Swaminath Sharmada , Pradhan Atul , Ajitkumar Parthasarathi 
Published: September 2022

Background:
We recently reported the de novo emergence of unusually high numbers of antibiotic resisters from the in vitro cultures of Mycobacterium tuberculosis and Mycobacterium smegmatis surviving in the presence of minimum bactericidal concentration (MBC) of antituberculosis antibiotics. The resisters emerged due to multiple asymmetric divisions of elongated mother cells containing multiple nucleoids and multiple septae. We had earlier found a minor subpopulation of short-sized cells (SCs) and a major subpopulation of normal-sized cells (NCs) (10% and 90%, respectively, of the whole population), with significant difference in antibiotic susceptibility and resister generation frequency, in the in vitro cultures of M. tuberculosis, M. smegmatis, and Mycobacterium xenopi, as well as in pulmonary tuberculosis patients' sputum. However, the mechanisms of growth and division promoting the emergence of antibiotic resisters from these subpopulations remained unknown. Therefore, here, we took up the first-time study to find out the mechanism of growth and division by which antibiotic resisters emerge from the antibiotic-surviving population of the two subpopulations of M. smegmatis.

Methods:

M. smegmatis SCs and NCs were fractionated from mid-log phase cultures using Percoll gradient centrifugation; their purity was checked and exposed to 10×, 2×, and 0.4× MBC of rifampicin for 120 h. The colony-forming units (CFUs) were determined on rifampicin-free plates for the total population and on rifampicin-containing plates for scoring rifampicin resisters. The phenotype and the morphology of the cells at various stages of the exposure were determined using transmission electron microscopy. The dynamic growth and division mechanisms of the cells to emerge as rifampicin resisters were monitored using live-cell time-lapse imaging. The rifampicin resisters were sequenced for mutations in the rifampicin resistance determining region of rpoB gene. Statistical significance was calculated using two-tailed paired t-test, with *P ≤ 0.05 and **P ≤ 0.01.

Results:
Multinucleated and multiseptated elongated cells emerged from their respective antibiotic-surviving populations. They produced a large number of sibling-daughter cells through multiple asymmetric divisions in short durations, showing abnormally high spurts in CFUs of antibiotic resisters. The CFUs were several-fold higher than that expected from the mass-doubling time of the subpopulations. Despite this commonality, the subpopulations showed specific differences in their response to different multiples of their respective MBC of rifampicin.

Conclusions:
Mycobacterial subpopulations come out of rifampicin stress by undergoing multiple nucleoid replications, multiple septation for nucleoid segregation, and acquisition of antibiotic target-specific mutations, followed by multiple asymmetric divisions to generate unusually a large number of rifampicin resisters. Because we had earlier shown that SCs and NCs are present in the pulmonary tuberculosis patients' sputum, the present findings have clinical relevance on the mechanism of emergence of antibiotic-resistant strains from mycobacterial subpopulations.