Bacterial persisters are a subpopulation of cells that can tolerate lethal concentrations of antibiotics. However, the possibility of the emergence of genetically resistant mutants from antibiotic persister cell populations, upon continued exposure to lethal concentrations of antibiotics, remained unexplored. In the present study, we found that Mycobacterium tuberculosis cells exposed continuously to lethal concentrations of rifampin (RIF) or moxifloxacin (MXF) for prolonged durations showed killing, RIF/MXF persistence, and regrowth phases. RIF-resistant or MXFresistant mutants carrying clinically relevant mutations in the rpoB or gyrA gene, respectively, were found to emerge at high frequency from the RIF persistence phase population. A Luria-Delbruck fluctuation experiment using RIF-exposed M. tuberculosis cells showed that the rpoB mutants were not preexistent in the population but were formed de novo from the RIF persistence phase population. The RIF persistence phase M. tuberculosis cells carried elevated levels of hydroxyl radical that inflicted extensive genome-wide mutations, generating RIF-resistant mutants. Consistent with the elevated levels of hydroxyl radical-mediated genome-wide random mutagenesis, MXF-resistant M. tuberculosis gyrA de novo mutants could be selected from the RIF persistence phase cells. Thus, unlike previous studies, which showed emergence of genetically resistant mutants upon exposure of bacteria for short durations to sublethal concentrations of antibiotics, our study demonstrates that continuous prolonged exposure of M. tuberculosis cells to lethal concentrations of an antibiotic generates antibiotic persistence phase cells that form a reservoir for the generation of genetically resistant mutants to the same antibiotic or another antibiotic. These findings may have clinical significance in the emergence of drug-resistant tubercle bacilli.

Recently, several reports showed that about 80 % of mid-log phase Mycobacterium smegmatis, Mycobacterium marinum, and Mycobacterium bovis BCG cells divide symmetrically with 5-10 % deviation in the septum position from the median. However, the mode of cell division of the pathogenic mycobacterial species, Mycobacterium tuberculosis, remained unclear. Therefore, in the present study, using electron microscopy, fluorescence microscopy of septum- and nucleoid-stained live and fixed cells, and live cell time-lapse imaging, we show the occurrence of asymmetric cell division with unusually deviated septum/constriction in 20 % of the 15 % septating M. tuberculosis cells in the mid-log phase population. The remaining 80 % of the 15 % septating cells divided symmetrically but with 2-5 % deviation in the septum/constriction position, as reported for M. smegmatis, M. marinum, and M. bovis BCG cells. Both the long and the short portions of the asymmetrically dividing M. tuberculosis cells with unusually deviated septum contained nucleoids, thereby generating viable short and long cells from each asymmetric division. M. tuberculosis short cells were acid fast positive and, like the long cells, further readily underwent growth and division to generate micro-colony, thereby showing that they were neither mini cells, spores nor dormant forms of mycobacteria. The freshly diagnosed pulmonary tuberculosis patients' sputum samples, which are known for the prevalence of oxidative stress conditions, also contained short cells at the same proportion as that in the mid-log phase population. The probable physiological significance of the generation of the short cells through unusually deviated asymmetric cell division is discussed.

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17Persisters constitute a subpopulation of bacteria that can tolerate lethal concentrations 18 of antibiotics. Multiple mechanisms have been suggested for bacterial persistence 19 against antibiotics. With mycobacteria being no exception to this behaviour, we had 20 reported the de novo emergence of genetically antibiotic-resistant Mycobacterium 21 tuberculosis from persister cells upon prolonged exposure to microbicidal 22 concentrations of the anti-tuberculosis drugs, rifampicin and moxifloxacin. Here, we 23 present evidence for reduced permeability to rifampicin as a mechanism for 24 persistence of Mycobacterium tuberculosis in vitro. We observed that rifampicin 25 persistent M. tuberculosis cells developed a thick outer layer (TOL) capsule. The TOL 26 restricted the entry of fluorochrome-conjugated rifampicin, 5-carboxyfluorescein-27 rifampicin (5-FAM-rifampicin), which retained only 2.5% of its original bactericidal 28 activity, but high levels of permeability, on actively growing mid-log phase cells. Gentle 29 mechanical removal of TOL significantly enhanced 5-FAM-rifampicin entry into the 30 persister cells. The level of 5-FAM-rifampicin in the persister cells was not affected by 31 the pre-incubation of the cells with verapamil, a drug efflux pump inhibitor, ruling out 32 the involvement of efflux pumps in the reduced intracellular concentration of 5-FAM-33 rifampicin. GC-MS analysis of TOL showed the presence of ~7-fold, ~5-fold and ~2-34 fold higher levels of α-D-glucopyranoside, 1,2,5-linked-mannitol, and 3,4-linked 35 mannose, respectively, among ~2-fold higher levels of derivatives of several other 36 types of sugars such as arabinose and galactose. Taken together, the present study 37 reveals that rifampicin-persistent M. tuberculosis cells develop TOL that enables the 38 bacilli to restrict entry of rifampicin and thereby remain tolerant to the antibiotic in vitro.39 40 65 4unknown strategy to ensure sub-lethal concentration of antibiotics inside persister cells 66 even though they remain exposed to lethal concentrations of the antibiotics. In order 67 to verify this possibility, we examined whether the permeability of rifampicin into M. 68 tuberculosis persister cells was affected by any ultrastructural changes. The present 69 study reports the ultrastructural changes of rifampicin persistent M. tuberculosis cells 70 and their contribution to the restricted permeability of rifampicin, in comparison with 71 the entry of rifampicin into actively growing mid-log phase cells. 72 73 MATERIALS AND METHODS 74 75 Rifampicin treatment of Mycobacterium tuberculosis 76 Mycobacterium tuberculosis H37Ra (National JALMA Institute of Leprosy and Other 77 Mycobacterial Diseases, Agra, India) was used in all the experiments. Bacteria were 78 grown in Middlebrook 7H9 broth (BD) supplemented with 0.2% glycerol, 0.05% 79 Tween-80 and 10% ADS (albumin, dextrose, sodium chloride) until mid-log phase 80 (MLP, 0.6 at OD600 nm) at 37ºC under shaking at 170 rpm. Cultures were then exposed 81 to 1 µg/ml (10x MBC; 12) of ...