AUTHOR=Vijay Srinivasan , Nair Rashmi Ravindran , Sharan Deepti , Jakkala Kishor , Mukkayyan Nagaraja , Swaminath Sharmada , Pradhan Atul , Joshi Niranjan V. , Ajitkumar Parthasarathi 

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

JOURNAL=Frontiers in Microbiology

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2017.00463

DOI=10.3389/fmicb.2017.00463

ISSN=1664-302X

ABSTRACT=<p>The present study shows the existence of two specific sub-populations of <italic>Mycobacterium smegmatis</italic> and <italic>Mycobacterium tuberculosis</italic> cells differing in size and density, in the <underline><bold>m</bold></underline>id-<underline><bold>l</bold></underline>og <underline><bold>p</bold></underline>hase (MLP) cultures, with significant differential susceptibility to antibiotic, oxidative, and nitrite stress. One of these sub-populations (~10% of the total population), contained <underline><bold>s</bold></underline>hort-sized <underline><bold>c</bold></underline>ells (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 <underline><bold>n</bold></underline>ormal/long-sized <underline><bold>c</bold></underline>ells (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 <underline><bold>SC</bold></underline>s-enriched <underline><bold>f</bold></underline>raction (SCF) at lower density (probably indicating lipid-richness) and the <underline><bold>NC</bold></underline>s-enriched <underline><bold>f</bold></underline>raction (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), H<sub>2</sub>O<sub>2</sub> (oxidative stress),and acidified NaNO<sub>2</sub> (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 H<sub>2</sub>O<sub>2</sub>, 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 <italic>M. tuberculosis</italic> 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.</p>