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

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

doi:10.3389/fmicb.2017.00463

Srinivasan Vijay, Atul Pradhan + Show 7 more

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https://doi.org/10.3389/fmicb.2017.00463

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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.

Highlights

Bacteria maintain population heterogeneity by generating subpopulations of phenotypically different but genetically identical members with “division of labour” for the survival under diverse stress conditions (Hallez et al, 2004; Aertsen and Michiels, 2005; Zgur-Bertok, 2007; Davidson and Surette, 2008; Diard et al, 2013)
In parallel, using live cell imaging, the susceptibility/tolerance of the individual members of the following three different sister daughter pairs of cells was determined in terms of the time taken by the individual sister daughter cells for the growth and division post-exposure to the stress: (i). the sized cells (SCs)-normal/long-sized cells (NCs) pairs of sister daughter cells, formed from the highly-deviated asymmetric cell division (ACD) of normal/long-sized mother cells; (ii). the SC-SC pairs of sister daughter cells formed from the symmetric cell division (SCD) of short-sized mother cells; and (iii). the NCNC pairs of sister daughter cells formed from the SCD of normal/long-sized mother cells
Our studies show that M. smegmatis mc2155 (Msm) and M. tuberculosis H37Ra (Mtb) mid-log phase (MLP) populations naturally harbor a very high level of heterogeneity, with the two subpopulations of SCs and NCs, standing out in terms of their difference in size, density, and uniform differential susceptibility to three prominent stress conditions, the antibiotic, oxidative and nitrite stress that are faced by mycobacteria in their natural habitats

Summary

Introduction

Bacteria maintain population heterogeneity by generating subpopulations of phenotypically different but genetically identical members with “division of labour” for the survival under diverse stress conditions (Hallez et al, 2004; Aertsen and Michiels, 2005; Zgur-Bertok, 2007; Davidson and Surette, 2008; Diard et al, 2013). Heterogeneity in cell size, culturability, morphology, and growth rate has been the hallmark of Mycobacterium tuberculosis, Mycobacterium bovis BCG, Mycobacterium smegmatis, and Mycobacterium xenopi cells irrespective of their habitat in in vitro cultures, infected macrophages, animal models, or in TB patients, under the diverse stress conditions existent in these environments (McCarthy, 1974; Nyka, 1974; Khomenko, 1987; Smeulders et al, 1999; Thanky et al, 2007; Davidson and Surette, 2008; Anuchin et al, 2009; Deb et al, 2009; Ghosh et al, 2009; Farnia et al, 2010; Ryan et al, 2010; Aldridge et al, 2012; Markova et al, 2012; Vijay et al, 2014a,b; Wu et al, 2016). A recent live cell imaging study showed the presence of rifampicin-susceptible M. smegmatis cells, one of which was highly-susceptible and the other divided once but stopped further growth or division (Richardson et al, 2016)

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