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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 8  |  Issue : 3  |  Page : 237-243

Expression of mycolic acid in response to stress and association with differential clinical manifestations of tuberculosis


Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India

Date of Web Publication12-Sep-2019

Correspondence Address:
Dr Mandira Varma-Basil
Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi - 110 007
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_69_19

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  Abstract 


Background: Extrapulmonary tuberculosis (EPTB), accounting for 10%–20% of all cases of tuberculosis (TB), is known to be determined by host immunity. However, the contribution of bacterial factors to the development of EPTB has not been studied extensively. Mycolic acids are predominant lipids constituting the cell wall of Mycobacterium tuberculosis, and keto-mycolic acid is involved in the synthesis of foamy macrophages that facilitate persistence of mycobacteria. Hence, the present study was performed to gain an insight into variable expression of mycolic acids in clinical isolates of M. tuberculosis under stress. Methods: Pansusceptible clinical isolates of M. tuberculosis from patients with lymph node TB (LNTB) (n = 10) and pulmonary TB (PTB) (n = 10) were subjected to sodium dodecyl sulfate (SDS) stress, and the expression of mycolic acid and its biosynthetic genes was compared. Any bias arising due to the genotype of the clinical isolates was ruled out by performing single-nucleotide polymorphism cluster grouping (SCG), wherein no significant difference was observed between the SCG of LNTB or PTB isolates. Results: The expression of α-mycolic acid during the exposure to SDS was high in 7/10 (70%) LNTB and 6/10 (60%) PTB isolates. Methoxy mycolic acid showed an increased expression in 7/10 (70%) LNTB isolates and 4/10 (40%) PTB isolates. Increased expression of keto-mycolic acid on exposure with SDS was observed in 8/10 (80%) M. tuberculosis LNTB and 3/10 (30%) PTB isolates. Similarly, the mycolic acid synthesis gene, fas, was upregulated more in LNTB isolates than PTB isolates in vitro and ex vivo. SCG 3a was the most common SCG observed in 40% (8/20) of the isolates, followed by SCG 3b in 30% (6/20) of the isolates. There was no significant difference between the SCG of LNTB or PTB isolates. Conclusion: The higher expression of keto-mycolic acid in LNTB as against PTB isolates may indicate better survival in LNTB isolates in the presence of stress.

Keywords: Lymph node tuberculosis, mycolic acid, pulmonary tuberculosis, surface stress


How to cite this article:
Sharma NK, Rathor N, Sinha R, Gupta S, Tyagi G, Garima K, Pathak R, Singh P, Jain A, Bose M, Varma-Basil M. Expression of mycolic acid in response to stress and association with differential clinical manifestations of tuberculosis. Int J Mycobacteriol 2019;8:237-43

How to cite this URL:
Sharma NK, Rathor N, Sinha R, Gupta S, Tyagi G, Garima K, Pathak R, Singh P, Jain A, Bose M, Varma-Basil M. Expression of mycolic acid in response to stress and association with differential clinical manifestations of tuberculosis. Int J Mycobacteriol [serial online] 2019 [cited 2019 Sep 20];8:237-43. Available from: http://www.ijmyco.org/text.asp?2019/8/3/237/266495




  Introduction Top


Extrapulmonary tuberculosis (EPTB) accounts for 10%–20% of all TB cases in regions with a comprehensive diagnostic and reporting system.[1] The incidence is even higher in patients with HIV infection.[2] The factors that determine the development of EPTB in a patient are not clearly understood. Impaired host immunity has been assumed to be a causative factor. However, very few investigations have searched for bacterial factors determining the clinical presentation of EPTB. A study from India found no association between the phylogenetic lineage and EPTB.[3] Another study from Brazil concluded that risk factors associated with EPTB were related more to host factors than bacterial factors.[4]

However, Garcia de Viedma et al.[5] demonstrated that the same patient could be infected by two different strains of Mycobacterium tuberculosis at the same time, and the strains could be compartmentalized into different sites of the human body.[5] In this study, the isolates of M. tuberculosis from the extrapulmonary site were more efficient in invading macrophages or multiplying in them as compared to the pulmonary isolate.[5]

Mycobacteria face a number of stresses inside the human host, namely oxidative, nutritional, or acidic.[6] Although mycobacteria are intrinsically resistant to a variety of these stresses due to their thick waxy cell wall, they also respond differently to environmental stress by the induction of specific genes.[6] It is suggested that M. tuberculosis modifies its cell wall integrity, including its thickness, alterations in the cell wall lipids, and protein composition in response to the in vitro stresses.[7],[8] The M. tuberculosis strains causing pulmonary TB (PTB) may not be genetically different from the ones responsible for EPTB.[9] However, due to the difference in the biological environment faced by mycobacteria infecting pulmonary and extrapulmonary sites, M. tuberculosis might adapt to the local condition by differential expression of genes, especially those required for lipid metabolism and survival.

Since lymph node TB (LNTB) is one of the most common manifestations of EPTB,[10] the present study was an attempt to explore the biological basis of variable manifestations of M. tuberculosis infecting the lung and lymph nodes.


  Methods Top


Bacterial strains and growth conditions

The study included a total of 247 M. tuberculosis isolates obtained from the same number of patients with PTB, and 13 M. tuberculosis isolates obtained from patients with LNTB. These isolates were obtained from the repository of the Department of Microbiology, Vallabhbhai Patel Chest Institute. Patients suffering from tuberculosis (TB) were recruited in the Outpatient Facility of the Department of Respiratory Medicine at Vallabhbhai Patel Chest Institute, which serves as a referral center for patients with respiratory diseases in North India, and the DOTS center at Rajan Babu Institute of Pulmonary Medicine and TB in Delhi between January 2011 and December 2014. Only patients ≥15 years of age were enrolled in the study. Culture and identification were performed at the Vallabhbhai Patel Chest Institute. All isolates were confirmed to be M. tuberculosis by niacin, nitrate, catalase tests,[11] and polymerase chain reaction (PCR) restriction analysis.[12] Informed consent and detailed history of contact were obtained from each patient before the collection of samples, following clearance from the Institutional Ethical Committee.

H37Rv and the clinical isolates of M. tuberculosis were grown in Middlebrook 7H9 broth medium supplemented with 0.2% glycerol at 37°C with shaking at 96 rpm for further tests.[13]

Drug susceptibility profile of clinical isolates

Drug susceptibility testing to isoniazid, rifampicin (RIF), streptomycin (SM), and ethambutol was performed by the proportion method as described by the Revised National Tuberculosis Control Program of India.[14]

Single-nucleotide polymorphism cluster groups assignment

Each M. tuberculosis isolate was assigned to one of seven phylogenetically distinct single-nucleotide polymorphism (SNP) cluster groups (SCG) and three subgroups using nine SNP markers as described previously.[15],[16] The allele of each SNP marker was identified using hairpin primer PCR assays as described previously.[17]

Effect of stress on Mycobacterium tuberculosis clinical isolates obtained from lymph node tuberculosis and pulmonary tuberculosis patients

M. tuberculosis H37Rv and all PTB and LNTB clinical isolates were grown to an optical density of 0.4 at 600 nm (OD600) in Middlebrook 7H9 medium. The culture was divided into two aliquots of 50 ml each, and the aliquots were washed twice with phosphate-buffered saline to provide sodium dodecyl sulfate (SDS) stress. The washed fraction of cells was resuspended in Middlebrook 7H9 medium with or without 0.05% SDS at 37°C for 1.5 h for further assays.

Mycolic acid profiling under stress

After exposure to SDS, the M. tuberculosis LNTB and PTB clinical isolates were analyzed for mycolic acids. The SDS exposed and unexposed cultures of M. tuberculosis were autoclaved, and the bacterial cell pellets were collected and dried at 80°C for 16 h. The mycolic acids were extracted and methylated by acid methanolysis as described previously.[18]

Thin-layer chromatography analysis

Methyl mycolates were developed in one-dimensional-thin-layer chromatography (TLC) using hexane/diethyl ether (85:15, v/v) three times.[18] TLC plates were sprayed with 10% (w/v) sulfuric acid (concentrated) in absolute ethanol and heated at 120°C. Charred TLC plates were scanned using a Gel Doc™ imaging system (BIO-RAD). Developed mycolic acid spots were quantified (Densitometry) using Image Lab™ software of BIO-RAD Gel Doc system. Relative proportion of mycolic acids was calculated.

Primer designing

Sequences of mycolic acid synthesis genes, namely fas (Rv2524), fabH (Rv0533c), kasA (Rv2245), inhA (Rv1484), fabG1 (Rv1483), and pks13 (Rv3800c) were retrieved from TubercuList a whole genome data base [19] and the primers were designed using Gene Runner Version 3.01 software (Developed by Frank Buquicchio and Michael Spruyt; http://www.generunner.net/) [Table 1]. Previously published primer sequences were used for the housekeeping gene sigA[20] [Table 1].
Table 1: Genes primer sequences, product length and melting temperature (Tm)

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Expression analysis of mycolic acid synthesis genes

The SDS exposed and unexposed M. tuberculosis clinical isolates were pelleted down and resuspended in RNA protection buffer (Qiagen GambH, Hilden, Germany) and incubated for 10 min before RNA isolation. RNA was extracted using RNeasy Mini Kit (Qiagen GambH, Hilden, Germany) according to manufacturer's instructions and subsequently treated with DNase I (Thermo Fischer Scientific Inc., Waltham, MA). The quantity and quality of the RNA extracted from each condition was assessed by virtual gel electrophoresis on the DNR Bio-Imaging Systems (MiniLumi) as well as by spectrophotometric measurement of the A260/A280 ratio. Only RNA extracts with no visible amplified product were used for subsequent experiments. The cDNA was synthesized using random hexamer primers provided with the first strand cDNA synthesis kit (Fermentas Life Sciences, Lithuania) as per the manufacturer's instructions.

Real-time polymerase chain reaction

Quantitative reverse transcription-PCR (qRT-PCR) was performed to quantify the expression of mycolic acid synthesis genes, namely fas, kasA, inhA, fabH, fabG1, and pks13 using QuantiTect™ SYBR Green Master Mix kit (Roche Applied Science, Indianapolis, USA) in a LightCycler ® 480 II Real-Time PCR System (Roche Applied Science, Indianapolis, USA). The housekeeping sigma factor gene sigA was used as internal control in qRT-PCR assays.[21],[22] Melt curve analysis was performed after each run to confirm the specificity of the primers. Each qRT-PCR experiment was carried out on duplicate biological samples that were further assayed in triplicates. The quantity of cDNA for the amplification of mycolic acid synthesis genes and the reference genes were equalized for each sample. Relative quantification in clinical isolates was done to determine the overexpression of genes in cultures exposed to SDS as compared to unexposed cultures, by 2-ΔΔCt method.[23] The data were analyzed using the built-in quantification software. A relative expression equal to one indicated that the expression level was identical to the control.

Infection of THP1 cells with Mycobacterium tuberculosis isolates

Human monocytic cell line THP-1 was maintained and infected as reported previously.[24] Briefly, THP-1 cell line was maintained and grown in Roswell Park Memorial Institute (RPMI) with 10% fetal bovine serum under the standard incubation conditions at 37°C and 5% CO2. Prior to infection with M. tuberculosis, 1 million cells of THP-1 cell line were calculated using Neubauer's chamber, plated in 25 cm culture flask in the presence of phorbol 12-myristate 13-acetate (20 ng/ml), and incubated overnight to make them adherent. The culture growth of M. tuberculosis was measured at OD at 600 nm with Infinite F200 Pro (Tecan, Mannedrof, Zurich, Switzerland) spectrophotometer. A multiplicity of infection of 1:10 was used to infect THP1 cells with H37Rv, two LNTB, and two PTB clinical isolates. Extracellular bacteria were removed by treatment with amikacin at a concentration of 25 μg/ml for 2 h. This time point was taken as 0 h of internalization, and RNA isolation was done at 0, 24, and 72 h after infection and qRT-PCR was performed as described above.

Data analysis

Fisher's exact test was used to assess the statistical significance of the comparison between experimental groups using GraphPad Software (GraphPad Software Inc., La Jolla, CA, USA).


  Results Top


Drug susceptibility profile of Mycobacterium tuberculosis isolates

Of the 247 isolates obtained from PTB patients, 128 were pan-susceptible, 52 were monodrug resistant, 32 were polydrug resistant, and 35 were multidrug resistant. Of the 13 isolates obtained from LNTB patients, ten were susceptible to all the antimicrobial agents studied. One isolate was resistant to SM, while two were resistant to SM and RIF. For further experiments, ten pan-susceptible isolates from PTB patients and ten from LNTB patients were considered to avoid any bias due to drug resistance.

Single-nucleotide polymorphism cluster groups

Phylogenetic analysis using SNP markers has been established as a useful method to establish the lineages of M. tuberculosis isolates. An analysis of nine SNP markers enabled us to assign ten LNTB and ten PTB clinical isolates to one of the known SCG. The most prevalent SCG was 3a observed in 4/10 (40%) LNTB and 4/10 (40%) PTB isolates, followed by SCG 3b in 3/10 (30%) LNTB and 3/10 (30%) PTB isolates. SCG 3c was observed only in 2/10 (20%) LNTB isolates, SCG 6b in 1/10 (10%) LNTB and 1/10 (10%) PTB isolates, and SCG 1 and 6a in 1/10 (10%) PTB isolate each.

Mycolic acid expression during stress

The surface stress was created in vitro by the exposure of M. tuberculosis clinical isolates of LNTB and PTB to SDS. The expression of α-mycolic acid during the exposure to SDS, as observed in TLC [Figure 1], was high in 7/10 (70%) LNTB and 6/10 (60%) PTB isolates [Figure 2].
Figure 1: Representative thin-layer chromatography from lymph node tuberculosis and pulmonary tuberculosis isolates. Lane 1: Control, Lane 2: Surface stress (sodium dodecyl sulfate 0.05%). Equal quantities (5 μl) of extracted mycolic acids from each clinical isolate were spotted by capillary on silica gel-coated plates. Thin-layer chromatography was developed three times in n-Hexane and diethyl ether (85:15, v/v). Thin-layer chromatography (a) Mycolic acid of the standard strain Mycobacterium tuberculosis H37Rv; thin-layer chromatography (b and c) Mycolic acids extracted from lymph node tuberculosis; thin-layer chromatography (d and e) Mycolic acids extracted from pulmonary tuberculosis

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Figure 2: Densitometry of mycolic acids. Intensity of mycolic acid spot for untreated sample (control) was taken as 1. Each treated isolate was quantified relative to the control sample for surface stress

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Methoxy mycolic acid showed an increased expression in 7/10 (70%) of the M. tuberculosis LNTB isolates, while only 4/10 (40%) M. tuberculosis isolates from PTB patients showed an increased expression of methoxy mycolic acid content. Interestingly, 8/10 (80%) M. tuberculosis LNTB as against 3/10 (30%) PTB isolates were found to show increased expression of keto-mycolic acid on exposure to SDS, although the difference was not statistically significant (P = 0.0698; Fisher's exact test) [Figure 2]. The reference strain M. tuberculosis H37Rv showed an increased expression of α-mycolic acid after SDS stress, while the other two components of mycolic acid were reduced as compared to the untreated control.

Expression profile of mycolic acid synthesis genes

After the exposure to SDS, the gene fas was upregulated in 3/10 (30%) PTB isolates and 6/10 (60%) LNTB M tuberculosis clinical isolates [Figure 3]. inhA and pks13 were upregulated in 2/10 (20%) PTB isolates and 3/10 (30%) PTB isolates, respectively. All the other genes, namely kasA, fabH, and fabG1, were downregulated [Figure 3]. Only 1/10 (10%) LNTB isolate showed upregulation for kasA, while inhA, fabH, fabG1, and pks13 were downregulated.
Figure 3: Relative expression of mycolic acid synthesis genes in Mycobacterium tuberculosis H37Rv, lymph node tuberculosis, and pulmonary tuberculosis isolates treated with 0.05% sodium dodecyl sulfate as compared to untreated samples (control) grown in vitro, calculated by quantitative reverse transcription-polymerase chain reaction. sigA was used as an internal control. X-axis denotes the isolates used in the study. Y-axis denotes the fold change in expression. Fold expression equal to 1 corresponds to no alterations in expression as compared with unexposed control. Fold change ≥2.5 in gene expression, relative to the nonexposed control denotes significant overexpression

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Expression of mycolic acid synthesis genes in Mycobacterium tuberculosis isolates infecting THP-1 cell line

Two isolates each from LNTB and PTB patients were inoculated into THP-1 cell line. On examining the mycolic acid genes through qRT-PCR, it was observed that fas and inhA were upregulated in both the LNTB isolates. LNTB-1 also upregulated kasA and fabG1 either at 24 h or 72 h or both. PTB-1 showed upregulation of fas, kasA, and inhA either at 24 h or 72 h or both. However, PTB-2 did not upregulate any of the mycolic acid synthesis genes ex vivo. M. tuberculosis H37Rv, used as a reference strain, also showed fas, kasA, and fabH upregulation [Figure 4].
Figure 4: Relative expression of mycolic acid synthesis genes in Mycobacterium tuberculosis H37Rv, lymph node tuberculosis, and pulmonary tuberculosis isolates at different time points grown ex vivo (THP-1 cells). sigA was used as an internal control. X-axis denotes the different time point and isolates used in the study. Y-axis denotes the fold change in expression. Fold expression equal to 1 corresponds to no alterations in expression as compared with unexposed control. Fold change ≥2.5 in gene expression, relative to the nonexposed control denotes significant overexpression

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  Discussion Top


EPTB is a challenge to physicians. Diagnostic assays available currently are limited in their accuracy in diagnosing EPTB, and the treatment is at times longer than that for PTB. Moreover, though EPTB contributes to the burden of the disease, it has only recently started getting attention in control strategies. Hence, though certain regions of the world report a reduction in cases of PTB, the incidence of EPTB has not decreased.[25]

LNTB is one of the most common manifestations of EPTB. Of these, cervical lymph nodes are the most common sites of involvement reported in 60%–90% patients with or without involvement of other lymphoid tissue.[10] Understanding the pathogenesis of EPTB in addition to PTB is important for proper control of TB. EPTB has been attributed to impaired host immunity,[5] however, the importance of bacterial factors in determining the clinical outcome of TB is still not clearly understood. Investigations into the molecular type of M. tuberculosis did not reveal any significant differences between the lineage of organism causing EPTB and that causing PTB.[9] In the present study, the isolates were subjected to SCG. Several studies have used SNPs for phylogenetic studies globally.[26],[27],[28],[29] Alland et al.[16] described nine SNP markers that enabled the placement of M. tuberculosis isolates into seven SCGs and five subgroups.[16] The most prevalent SCG in our study was 3a observed in 4/10 (40%) LNTB and PTB isolates each, followed by SCG 3b observed in 3/10 (30%) LNTB and PTB isolate each. SCG 3c was observed only in 2/10 (20%) LNTB isolates, while SCG 1 was present only in 1/10 (10%) PTB isolates. Thus, similar to earlier studies,[9] we did not find any major difference in the lineages of the M. tuberculosis isolates obtained from LNTB and PTB patients.

We continued our search for bacterial factors that could possibly be responsible for different manifestations of TB. Since of the total genome of M. tuberculosis, 30% genes encode for lipid synthesis or metabolism, many of which are activated during infection,[30],[31] we focused on the lipid profile of the LNTB and PTB isolates.

Lipids are the main components of the cell wall, which is the first line of defense of mycobacteria against most host-derived stresses and antimicrobial drugs. Mycolic acid is the major constituent of the cell wall lipids which make 40%–60% of dry weight of the cell [32] and is present in three forms (α, methoxy, and keto).[33] Mycolic acids are not only impermeability factors to host stresses and antimicrobial agents but also act as transactive molecules, which activate lipid sensing receptor TR4 and increase foamy macrophage formation.[34] Acting as a transactivation molecule, the different types of mycolic acids may differ in their ability to attack neutrophils, induce foamy macrophages, or adopt an antigenic structure for antibody recognition, depending on the chemical functions modulated by the attachment of the meromycolic chain, cis/trans-cyclopropanes, and oxygenated groups. Modification of mycolic acids may also contribute to resistance against oxidative stress,[35] suggesting that variations in mycolic acid composition between strains may result in altered levels of protection from the hostile environment encountered within macrophages and in vitro. In our study, we observed differential expression of mycolic acid in different strains of M. tuberculosis from patients with LNTB and PTB.

Mycolic acid is also a surface active lipid [36] which leads to an investigation of its role during various stresses imposed by the host macrophage, dendritic cells, or during in vitro culture of M. tuberculosis isolated from LNTB and PTB patients.

During infection, macrophages thrust antibacterial peptides, proteins, lysozyme, pH stress, and oxidative damage on the mycobacterial cell wall which may challenge the integrity of the cell wall of M. tuberculosis and affect its survival. We analyzed the three forms of mycolic acid, namely α-, methoxy-, and keto-mycolic acids in LNTB and PTB isolates. When LNTB and PTB M. tuberculosis isolates were exposed to surface stress, LNTB M. tuberculosis isolates showed increased expression of mycolic acid for all three forms as compared to PTB isolates. Interestingly, an increased expression of keto-mycolic acid was found in 8/10 (80%) of LNTB M. tuberculosis isolates when exposed to surface stress as compared to only 3/10 (30%) PTB isolates [Figure 2]. In correlation with this, the mycolic acid synthesis gene, fas, was upregulated more in LNTB isolates than PTB isolates in vitro [Figure 3]. Upregulation of fas under stress and ex vivo has also been observed earlier in M. tuberculosis H37Rv.[37],[38] The remaining mycolic acid synthesis genes, namely kasA, inhA, and pks13 were upregulated in 1–2 isolates of either LNTB or PTB [Figure 3], whereas fabH and fabG 1 were not upregulated in vitro [Figure 3]. To mimic the infectious processes taking place in the biological host, we also cultured M. tuberculosis clinical isolates on THP1 cells and analyzed the response of mycolic acid synthesis genes ex vivo. Mycolic acid synthesis genes were upregulated in both LNTB isolates but in only one PTB isolate.

Thus, although not significant statistically, a noticeable difference was observed in the content of mycolic acids and upregulation of mycolic acid biosynthetic genes between LNTB and PTB isolates when exposed to SDS. SDS is a surface active agent that may affect the formation of cell wall lipids. Since mycolic acids are major cell wall lipids, strains of M. tuberculosis may increase the expression of mycolic acids for better survival under surface stress faced in vivo. Previously, Garcia de Viedma et al.[5] reported that extrapulmonary M. tuberculosis isolates in their study were more virulent as well as infective to macrophages than pulmonary isolates.

Moreover, earlier studies of Peyron et al.[39] and D'Avila et al.[40] reported the induction of foamy macrophages by M. tuberculosis and Bacillus Calmette–Guérin Trehalose dimycolate, a mycolic acid containing compound known to induce foamy macrophage and granuloma formation.[39] The bacteria containing only oxygenated mycolic acids (methoxy and keto) induced the formation of foamy macrophages. Oxygenated mycolic acid triggers the differentiation of human monocyte-derived macrophages into foamy macrophages that could constitute a shelter for persisting bacilli.[39] The relevance of keto-mycolic acid in foamy macrophage formation was emphasized by the fact that Mycobacterium smegmatis which lacks keto-mycolic acid, induces only ~5% of the foamy macrophage formation in comparison to M. tuberculosis.[34]

In the present study, increased expression of α-, keto-, and methoxy-mycolic acids was observed in higher number of LNTB isolates than PTB isolates, with the maximum difference observed for keto-mycolic acid on stress. Our findings suggest that the ability to survive in vivo may be better in LNTB isolates than PTB isolates, since they expressed more mycolic acid under stress.


  Conclusions Top


To conclude, the present study highlights the altered expression of keto-mycolic acid during stress in LNTB and PTB M. tuberculosis isolates which may protect the mycobacteria during stress. This may be more evident in the lymph nodes where the harsher environment persists in comparison to the lungs. Further investigations into the bacterial factors responsible for the clinical manifestations of M. tuberculosis may pave the way for the development of biomarkers for diagnosis of EPTB.

Acknowledgments

N.K.S. is thankful to the Indian Council of Medical Research, India, for Senior Research Fellowship (80/907/2014-ECD-I).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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