Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress

Abstract

The present study shows the existence of two specific sub-populations of Mycobacterium smegmatis and Mycobacterium tuberculosis cells differing in size and density, in the mid-log phase (MLP) cultures, with significant differential susceptibility to antibiotic, oxidative, and nitrite stress. One of these sub-populations (~10% of the total population), contained short-sized cells (SCs) generated through highly-deviated asymmetric cell division (ACD) of normal/long-sized mother cells and symmetric cell divisions (SCD) of short-sized mother cells. The other sub-population (~90% of the total population) contained normal/long-sized cells (NCs). The SCs were acid-fast stainable and heat-susceptible, and contained high density of membrane vesicles (MVs, known to be lipid-rich) on their surface, while the NCs possessed negligible density of MVs on the surface, as revealed by scanning and transmission electron microscopy. Percoll density gradient fractionation of MLP cultures showed the SCs-enriched fraction (SCF) at lower density (probably indicating lipid-richness) and the NCs-enriched fraction (NCF) at higher density of percoll fractions. While live cell imaging showed that the SCs and the NCs could grow and divide to form colony on agarose pads, the SCF, and NCF cells could independently regenerate MLP populations in liquid and solid media, indicating their full genomic content and population regeneration potential. CFU based assays showed the SCF cells to be significantly more susceptible than NCF cells to a range of concentrations of rifampicin and isoniazid (antibiotic stress), H2O2 (oxidative stress),and acidified NaNO2 (nitrite stress). Live cell imaging showed significantly higher susceptibility of the SCs of SC-NC sister daughter cell pairs, formed from highly-deviated ACD of normal/long-sized mother cells, to rifampicin and H2O2, as compared to the sister daughter NCs, irrespective of their comparable growth rates. The SC-SC sister daughter cell pairs, formed from the SCDs of short-sized mother cells and having comparable growth rates, always showed comparable stress-susceptibility. These observations and the presence of M. tuberculosis SCs and NCs in pulmonary tuberculosis patients' sputum earlier reported by us imply a physiological role for the SCs and the NCs under the stress conditions. The plausible reasons for the higher stress susceptibility of SCs and lower stress susceptibility of NCs are discussed.

Influence of Stress and Antibiotic Resistance on Cell-Length Distribution in Mycobacterium tuberculosis Clinical Isolates

“…In some studies mycobacterial cell length and elongation rates are associated with differential susceptibility to host- and antibiotic-induced stress ( Aldridge et al, 2012 ; Richardson et al, 2016 ; Vijay et al, 2017 ). The exact mechanisms contributing to such stress tolerance is not clear.…”

“…In M. smegmatis sub-populations of cells were observed to grow, divide, and die during the persistence phase of isoniazid killing, and this was independent of single-cell growth rates ( Wakamoto et al, 2013 ). It is also observed that cell size- and density-specific subpopulations exist in mycobacteria, and long cell size is associated with tolerance to host and antibiotic stress conditions ( Vijay et al, 2017 ). Antibiotic tolerant cells can also give rise to antibiotic-resistant cells in M. tuberculosis during antibiotic treatment, possibly due to growth under antibiotic selection pressure ( Wakamoto et al, 2013 ; Sebastian et al, 2017 ).…”

“…Ultrastructural studies have revealed enhanced asymmetric cell division, and other cellular adaptations associated with multidrug resistance ( Farnia et al, 2010 ). Hence, cell-length heterogeneity in mycobacterial population is established during cell elongation and division, generating long and short cells, contributing to antibiotic tolerance ( Vijay et al, 2014a , b , 2017 ).…”

Tolerance and Persistence to Drugs: A Main Challenge in the Fight Against Mycobacterium tuberculosis

“…The molecular mechanism underneath this phenomenon has been described, with LamA (formerly Mmps3), a member of the divisome, inhibiting growth at nascent new poles ( Rego et al, 2017 ). Several evidences indicated that Mycobacterium smegmatis cells of different size, generated by asymmetric division, had different sensitivity to drugs: accelerator cells were shown to be more susceptible than alternators to drugs targeting the cell wall biosynthesis such as cycloserin, meropenem, and INH, however, they were less susceptible to rifampicin ( Aldridge et al, 2012 ; Richardson et al, 2016 ; Vijay et al, 2017a ), suggesting that asymmetric cell division can contribute to the formation bacterial subpopulations more tolerant to drugs. Interestingly, deletion of lamA in M. tuberculosis leads to the loss of single-cell heterogeneity and faster killing by vancomycin and rifampicin ( Rego et al, 2017 ).…”

Watching antibiotics in action: Exploiting time-lapse microfluidic microscopy as a tool for target-drug interaction studies inMycobacterium

“…One serious challenge in the treatment of TB caused by both susceptible and resistant Mtb strains is the high population heterogeneity of mycobacterial cells (30)(31)(32)(33)(34)(35)(36)(37)(38). Asymmetric growth (mycobacteria elongate preferentially from the old pole) and asymmetric septum placement give rise to daughter cells of unequal sizes and growth rates and, as indicated by some reports, different susceptibilities to antibiotics (30,39,40). Moreover, exposure to stress further diversifies the population (41,42), suggesting that mycobacteria utilize heterogeneity as key survival strategy under stressful conditions.…”