Reduced Permeability to Rifampicin by Capsular Thickening as a Mechanism of Antibiotic Persistence inMycobacterium tuberculosis

Abstract

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 ...

A Minor Subpopulation of Mycobacteria Inherently Produces High Levels of Reactive Oxygen Species That Generate Antibiotic Resisters at High Frequency From Itself and Enhance Resister Generation From Its Major Kin Subpopulation

“…For confirming the high expression levels of NADH oxidase (MSMEG_6603), we generated an Msm /pAKMN2-P MSMEG_ 6603 -u gfp m 2+ strain carrying transcriptional fusion of the specific nadh oxidase promoter to mycobacterial codon-optimized unstable gfp m 2+ ( ugfp m 2+ ) (as a single copy integrated at the L5 mycobacteriophage att site in the genome) (Roy et al, 2012; Sebastian, 2016). To determine the uGFP m 2+ fluorescence in the pAKMN2-P MSMEG_ 6603 -u gfp m 2+ integrants of the SCs and the NCs using fluorescence microscopy, all the individual cells, which were of length ≤ 2.6 μm, were taken as the SCs while the cells of length > 2.6 μm were taken as the NCs.…”

“…For this purpose, we scored for hygromycin-resistant colonies from two different NLP mixtures reconstituted with SCF:NCF at 1:9 wherein the SCF and the NCF cells were differently tagged. For making the two differently tagged NLP mixtures, we first prepared SCF1, SCF2, and NCF cells from Msm wild type culture (WT) and from the culture of Msm /pAKMN2- ugfp m 2+ - hyg r integrant cells, carrying the stable single copy of the genome-integrated pAKMN2- ugfp m 2+ - hyg r plasmid, cultured in the absence of hygromycin (Roy et al, 2004, 2012; Sebastian, 2016). We mixed the SCF1-WT and the SCF2-WT cells with the NCF/pAKMN2- ugfp m 2+ - hyg r cells to obtain NLP Mixture 1.…”

“…The amplified sequence was first cloned in pBS(KS) and sequence verified. The promoter segment was then subcloned in pAKMN2- ugfp m 2+ vector as 5′ transcriptional fusion to the reporter gene ugfp m 2+ (Roy et al, 2012; Sebastian, 2016). Msm cells were electroporated with the recombinant pAKMN2-P MSMEG_ 6603 - ugfp m 2+ vector.…”

PerSort facilitates characterization and elimination of persister subpopulation in mycobacteria

“…Whereas after nutrient starvation, the proportion of the drug tolerant subpopulations increased to ∼75% for INH and ∼50% for RIF. The increase in delay of RIF killing under nutrient starved conditions, might reflect cell wall alterations that are required for adaptation to nutrient deprivation, but also restrict entry of RIF (18, 19).…”

Hypoxic Non-replicating Persistent Mycobacterium tuberculosis Develops Thickened Outer Layer That Helps in Restricting Rifampicin Entry

“…The conjugation of 5-carboxy fluorescein (5-FAM) to rifampicin to get 5-FAM-rifampicin (5-FAM-RIF) was custom made, as described (Sebastian, 2016). The 5-FAM-RIF molecule has maximum excitation at 488 nm and maximum emission at 519 nm.…”