The study was conducted between January 2006 and June 2010 at the TB Laboratory, Division of Clinical Microbiology and Molecular Medicine, Department of Laboratory Medicine, All India Institute of Medical Sciences (AIIMS), New Delhi, India. This study was approved by the Institutional Ethics Committee of AIIMS and written informed consent was obtained from the patient. The patient was being treated at the designated microscopy and DOTS (directly observed treatment-short course) centres of Shahpurjat, New Delhi. This patient (22 yr old male) was diagnosed as having pulmonary TB on the basis of clinical and radiological findings and sputum smear microscopy. He was prescribed with anti-TB treatment (ATT) under the DOTS programme. The thrice a week treatment regimen comprised isoniazid, rifampicin, pyrazinamide (PZA) and ethambutol (EMB) (category I treatment) in intensive phase for two months followed by four month treatment with two (isoniazid and rifampicin) drugs regimen. Pre-treatment sputum specimen was used for isolation of Mycobacterium sp. by BACTEC MGIT-960 (Becton Dickinson, Sparks, MD, USA), which was positive. The isolate was identified as Mtb by conventional phenotypic and in-house PCR method. This culture was labelled as isolate A, and was subjected to 16sRNA gene sequencing. The patient though took full six months course of treatment but became irregular in taking drugs after initial improvement in his clinical symptoms. After three months of cessation of treatment (6+3= 9 month, his condition again deteriorated and his sputum culture was again positive for Mtb. We labelled this second culture as isolate B. He was re-treated with isoniazid, rifampicin, pyrazinamide, ethambutol and streptomycin (SM) (category II regimen). Within two months his clinical condition improved but he again defaulted. After an asymptomatic period of about four months his symptoms reappeared. His sputum was again culture positive and this culture was labelled as isolate C. The patient was again prescribed with the same treatment for 12 months after counselling but he stopped treatment after six months. His condition further deteriorated and he died of multisystem failure. The fourth sample was received just before his death and the isolate from this sputum sample was labelled as isolate D.
All the four clinical isolates (A, B, C & D) were identified as Mtb using standard protocols. The anti-mycobacterial drug susceptibility testing was performed on all the isolates by both BACTEC  MGIT-960 (Becton Dickinson, Sparks, MD, USA) and proportional method using Middlebrook 7H10 (Difco, USA) agar plates containing first-line anti-TB drugs (SM 2.0 microg/ml, INH 0.2 microg/ml, RIF 1.0 microg/ml, EMB 6.0 microg/ml). All four isolates were also genotyped by spoligotyping and identified using SITVIT-WEB database. The rpoB, inhA and katG gene targets were sequenced using the primers as described elsewhere.
Preparation of mycobacterial whole cell lysate: All Mtb isolates were grown without shaking in Middlebrook 7H9 medium supplemented with 0.2 per cent (v/v) glycerol, 10 per cent oleic acid, albumin-dextrose and catalase (OADC, Difco, USA) at 37 C for two weeks. Whole cell lysate was prepared according to protocol of Sharma et al. Cells were washed three times with normal saline and then suspended in sonication buffer [50 mM tris-HCl containing 10 mM MgCl2, 0.1% sodium azide, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1mM ethylene glycol tetra acetic acid (EGTA); PH 7.4] at a concentration of 1g wet cell mass per 5ml, and then broken by intermittent sonication for 15 min at 4 C using sonicator (Sonics & Materials Inc, USA). The homogenate was centrifuged at 12,000xg for 20 min at 4 C. The pellets were discarded and supernatant was stored at -70 C until further use.
Protein precipitation with sodium dodecyl sulphate (SDS)-trichloroacetic acid (TCA)-acetone: The cell lysates were treated with 1 per cent SDS and then subjected to TCA-acetone precipitation procedure. The protein pellet was suspended in appropriate volume of two-dimensional rehydration buffer (Bio-Rad, USA), and the protein concentration was estimated using the Bradford method.
Two-dimensional gel electrophoresis: Isoelectric focusing (IEF) was done using the in-gel rehydration method (Bio-Rad, USA).
2D gels were analysed using PDQuest Advanced software (version 8.0) (Bio-Rad, USA). After acquisition, the images were analyzed using step-wise spot detection and spot matching followed by differential expression analysis. The quantity of each spot was normalized by total valid spot intensity. The expression differences for all four mycobacterial isolates were compared using the same software. Images for sensitive and resistant isolates were manually checked for artifactual spots, merged spots and missed spots, and spots with more isolate-specific variability were omitted in the downstream processing. Equal amount of protein was loaded in all gels and experiments were repeated three times with three independent biological replicates.
In-gel digestion of protein spots with trypsin: Protein spots of interest were excised from the coomassie brilliant blue R250 stained 2D gels using spot picker Investigator ProPic (Genomic Solutions Ltd., Huntingdon, UK). Digestion of proteins and spotting of peptides on matrix assisted LASER desorption/ionization-time of flight (MALDI-TOF) target plate was carried out using protein digester investigator ProPrep (Genomic Solutions, Huntingdon, UK).
For protein digestion, method of Shevchenko et al was followed with slight modifications. In brief, the gel plugs were de-stained and dehydrated by washing three times (10 min) with 25 mM NH4HCO3-50 per cent acetonitrile (ACN) (1:1v/v) solution and treated with freshly prepared 10 mM dithiothreitol (DTT) in 50 mM NH4HCO3 for 45 min at 56 C. After incubation, DTT was replaced with freshly prepared 55 mM iodoacetamide for 30 min and then dehydrated with 100 per cent ACN. The dried gel pieces were incubated for 12 h at 37  C with 25 mM NH4HCO3 containing 0.02 mug/mul of mass spectrometry grade trypsin (Promega, USA). The resulting peptides were extracted twice from the gel pieces, using peptide extraction buffer [1:1v/v mixture of 70% ACN and 0.1% trifluoroacetic acid (TFA)].
Mass spectrometric analysis: Mass spectrometry (MS) was carried out as described earlier. The digested samples were desalted and concentrated on C-18 ZipTips (Millipore, USA) using the manufacturer's protocol before mass spectrometric analysis. ZipTips were eluted on MTP 384 target plate with 2 mul of alpha-cyano-4-hydroxycinnamic acid (HCCA) (Sigma-Aldrich, USA) saturated solution dissolved in 50 per cent ACN and 0.2 per cent TFA. Mass spectra of digested proteins were acquired using Autoflex II TOF/TOF 50 (Bruker GmbH, Leipzig, Germany) in positive reflectron mode. AnchorChip target plate was placed in sample inlet of the instrument, controlled by flexControl 2.4 software (Bruker, Germany). The instrument was equipped with a 337 nm nitrogen LASER, delayed extraction electronics, and a 50Hz digitizer and percentage of LASER energy was maintained at 30-40 per cent. The pulse energy was 105 microJ and pulse duration was 1.3 nano sec. Final mass spectra were produced by averaging 1500-2500 LASER shots taken at different positions within each spot. The spectra were acquired in positive reflection mode in the mass range of 500-3000 m/z. Calibration was performed using peptide calibration standard II (Bruker, Germany). The proteolytic masses obtained, were processed through FlexAnalysis v.2.4 programme (Bruker, Germany) for peak detection. Initially, the spectra acquired were processed for baseline subtraction with 80 per cent baseline flatness followed by smoothening with threshold of signal-to-noise ratio (S/N) >5. The contaminant m/z peaks originating from human keratin, trypsin auto-digestion and matrix were removed from the spectra to generate the peptide mass list for the database search. Finally, the proteolytic masses obtained were evaluated using MASCOT, a peptide mass fingerprinting (PMF) tool (Matrix Sciences, UK). Peak detection in MALDI spectra and peak lists were submitted to the UniProtKB/Swiss-Prot database using the MASCOT search engine (http://www.matrixscience.com ) to identify the proteins from the annotated Mtb chromosome (strain H37Rv, EMBL/GenBank/DDBJ entry AL123456). Peptide mass tolerance was set in range of 50-100 ppm, with carbamidomethyl-cystein set as fixed modification, oxidation of methionine as variable modification and only 0 or 1 missed cleavage site was allowed. Further, matched precursor ions of identified proteins were selected for subsequent fragmentation using post source decay (PSD) for MS/MS. Lift_ATT method was performed in flex control software; parent peak mass spectrum was acquired by hitting LASER for 400-550 shots followed by acquisition of fragments of selected precursor ions for the same number of shots. Both parent and fragment spectra were pooled to generate MS/MS spectrum of a particular peptide. MS/MS spectrum was submitted to database using MASCOT wizard (Matrix Sciences, UK). The same parameters were used for MS/MS search in addition to the fragment mass tolerance from 0.5 to 2.0 Da.
Glycogen estimation: Logarithmic and stationary phase growth of Mtb sensitive (isolate A) and MDR (isolates B,C,D) isolates were collected by centrifugation (3 min at 5000 x g and at 4 C), and the pellet (15-20 mg wet weight) was re-suspended in 0.25M Na2CO3 and incubated at 95 C for 4 h. The glycogen content was estimated by following the procedure of Schulze et al.
Isolation of Mtb total RNA and real-time quantitative PCR (qRT-PCR): Mtb H37Rv was grown in Middlebrook 7H9 broth containing 10 per cent OADC, and was treated with INH (0.1microg/ml), RIF (1.0microg/ml), EMB (5microg/ml) and INH (0.1microg/ml) +RIF (1.0microg/ml). Total RNA was isolated using a TRI reagent (Sigma, USA) following manufacturer's instructions. To analyze mRNA expression, cDNA was synthesized from 1 microg of total RNA by using Superscript III (Invitrogen Life Technologies, USA) and random primers (Invitrogen, Life Technologies, USA), followed by amplification of the gene(s) by gene-specific primers, using master mix SYBR green (Applied Biological Materials Inc., Canada). The expression is represented in fold increase. 16sRNA was used as internal control for mRNA expression analysis of ahpC, Rv1827, pknA, pknB, pknG and wag31.
Each reaction was repeated thrice with three independent RNA samples in a smart cycler Cepheid machine (Cepheid, USA). RT-PCR conditions were as follows: an initial denaturation step of 10 min, followed by 40 amplification cycles of 30 sec at 95 C, 30 sec at 60 C and 30 sec at 72 C. Melting curve analysis was carried out to confirm the specificity of the amplified product. After baseline corrections and determination of threshold settings, calculation and statistical analyses were carried out using the 2-  CT Method. The results are shown as fold increase in expression profile.