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The emergence of drug-resistant tuberculosis is a significant global health issue. The presence of heteroresistant

Background: Rapidly growing mycobacteria (RGM) are increasingly being recognized as potential pathogens. RGM, particularly Mycobacterium abscessus, Mycobacterium fortuitum, and Mycobacterium chelonae, have been observed in both pulmonary and extrapulmonary infections including cutaneous, soft-tissue, and wound infections. However, there are limited reports of these potential pathogens from skin and soft-tissue infections. Moreover, the drug susceptibility profile of RGM is largely unknown in several regions of the world. Methods: We analyzed reports on RGM isolated from skin and soft-tissue infections globally for details of RGM species and drug susceptibility profile. We also analyzed the drug susceptibility profile of four RGM isolates, obtained from skin and soft-tissue infections in our laboratory, by broth microdilution method. Results: In the reports reviewed, the most common RGM isolated from skin and soft-tissue infections were M. abscessus (184/475, 38.7%), M. fortuitum (150/475, 31.5%), M. chelonae (72/475, 15%), and M. chelonae–M. abscessus complex (46/475, 9.6%). However, drug susceptibility was tested only in 26/39 (66.6%) reports. In our own laboratory, we obtained three isolates of M. abscessus and one isolate of M. fortuitum from one case of breast abscess and three cases of postsurgical wound infections. Maximum susceptibility of M. abscessus was observed to clarithromycin, amikacin, and linezolid. The M. fortuitum isolate was susceptible to clarithromycin, amikacin, clofazimine, and linezolid. Conclusion: Paucity of information available on RGM isolated from skin and soft-tissue infections highlights the need to be aware of the pathogenic potential and the drug susceptibility profile of these organisms.

Aim: Timely and reliable diagnostic test for tuberculosis (TB) is immediately required. Attempts were made to improve the technology and diagnostic potential of real-time immuno-PCR (RT-I-PCR). Methods: We designed gold nanoparticle (GNP)-based RT-I-PCR (GNP-RT-I-PCR) assay for the detection of Mycobacterium tuberculosis CFP-10 (Rv3874) protein in clinical samples of TB patients. Results: A wide quantitative detection range of CFP-10 was found to be 0.5–5 × 104 pg/ml in bodily fluids of TB patients, which can evaluate the progression of disease. Moreover, sensitivities of 83.7 and 76.2% were observed in pulmonary (n = 49) and extrapulmonary TB (n = 42) patients, respectively, with specificities of 93.5–93.8% (n = 63). Conclusion: Conjugation of detection antibodies and oligonucleotides to functionalized GNPs of GNP-RT-I-PCR is relatively easier, compared with streptavidin-biotin/succinimidyl-4-( N-maleimidomethyl) cyclohexane-1-carboxylate system employed in RT-I-PCR. Our assay also showed better diagnostic performance than RT-I-PCR, which may provide a viable platform for the development of an efficient TB diagnostic test.

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.

We report the draft genome sequence of Mycobacterium simiae, a slowly growing nontuberculous mycobacterium (NTM) isolated from a mouthwash sample of a healthy person. This genome of 6,603,693 bp exhibited a 66.13% GC content and 6,391 genes with 6,257 coding sequences, 3 rRNAs, and 78 tRNAs.

Background Genome sequencing and genetic polymorphism analysis of clinical isolates of M. tuberculosis is carried out to gain further insight into molecular pathogenesis and host-pathogen interaction. Therefore the functional evaluation of the effect of single nucleotide variation (SNV) is essential. At the same time, the identification of invariant sequences unique to M. tuberculosis contributes to infection detection by sensitive methods. In the present study, genome analysis is accompanied by evaluation of the functional implication of the SNVs in a MDR clinical isolate VPCI591. Result By sequencing and comparative analysis of VPCI591 genome with 1553 global clinical isolates of M. tuberculosis (GMTV and tbVar databases), we identified 141 unique strain specific SNVs. A novel intergenic variation in VPCI591 in the putative promoter/regulatory region mapping between embC (Rv3793) and embA (Rv3794) genes was found to enhance the expression of embAB, which correlates with the high resistance of the VPCI591 to ethambutol. Similarly, the unique combination of three genic SNVs in RNA polymerase β gene (rpoB) in VPCI591 was evaluated for its effect on rifampicin resistance through molecular docking analysis. The comparative genomics also showed that along with variations, there are genes that remain invariant. 173 such genes were identified in our analysis. Conclusion The genetic variation in M. tuberculosis clinical isolate VPCI591 is found in almost all functional classes of genes. We have shown that SNV in rpoB gene mapping outside the drug binding site along with two SNVs in the binding site can contribute to quantitative change in MIC for rifampicin. Our results show the collective effect of SNVs on the structure of the protein, impacting the interaction between the target protein and the drug molecule in rpoB as an example. The study shows that intergenic variations bring about quantitative changes in transcription in embAB and in turn can lead to drug resistance.

Additional file 4. Intergenic Variations identified in VPCI591 (Type II).

Additional file 5. Invariant genes in global clinical isolates (including VPCI591).

Additional file 6. Unique genic variations in VPCI591.

Additional file 7. Inter-genic variations unique to VPCI591.

Additional file 3. Intergenic Variations in VPCI591 (Type I).

Lipid metabolism forms the heart and soul of Mycobacterium tuberculosis life cycle. Starting from macrophage invasion at cholesterol rich micro-domains to a sustainable survival for infection by utilizing cholesterol, Mycobacterium displays the nexus of metabolic pathways around host derived lipids. mce4 operon acts as cholesterol import system in M. tuberculosis and here we demonstrate role of mce4A gene of this operon in cholesterol catabolism. Here M. tuberculosis H37Rv overexpressing Rv3499c (mce4A) recombinant was used as a model to decipher the metabolic flux during intake and utilization of host lipids by mycobacteria. We analysed the impact of mce4A expression on carbon shift initiated during cholesterol utilization necessary for long term survival of mycobacterium. Through transcriptional analysis, upregulation in methylcitrate cycle (MCC) and methylmalonyl pathway (MMP) genes was observed in Rv3499c overexpressing recombinants of M. tuberculosis H37Rv. Up-regulation of methylmalonyl pathway associated enzyme encoding genes increased accumulation of virulence associated mycobacterial lipids phthiocerol dimycocerates (PDIM) and sulfolipid (SL1). We demonstrate that MCC and MMP associated enzyme encoding genes are upregulated upon mce4A overexpression and lead to enhanced accumulation of PDIM and SL1 which are responsible for pathogenicity of M. tuberculosis.

Atypical pathogens including Mycoplasma pneumoniae and Legionella pneumophila are increasingly recognized as important causes of community-acquired pneumonia (CAP). Mycoplasma pneumoniae accounts for 20-40% of all CAP and L. pneumophila is responsible for 3-15% of cases. The paucity of data from India in this regard prompted us to conduct this prospective multicentric analysis to detect the prevalence of M. pneumoniae and L. pneumophila in our geographical region. A total of 453 patients with symptoms of pneumonia and 90 controls with no history of lower respiratory tract infections were included in the study. A duplex polymerase chain reaction (PCR) targeting 543 bp region of P1 adhesin gene of M. pneumoniae and 375 bp region of macrophage infectivity potentiator (mip) gene of L. pneumophila was standardized for simultaneous detection of these atypical pathogens. Respiratory secretions, blood, and urine samples were collected from each patient and control and were subjected to duplex PCR, culture and serology for M. pneumoniae and L. pneumophila. Urine samples were subjected for detecting L. pneumophila antigen. Among the 453 patients investigated for M. pneumoniae, 52 (11.4%) were positive for IgM antibodies, 17 were positive by culture, and seven tested positive by PCR (P1 gene). Similarly for L. pneumophila, 50 cases (11%) were serologically positive for IgM antibodies, one was positive by PCR (mip gene) and urine antigen detection. A total of eight samples were positive by duplex PCR for M. pneumoniae P1 gene (N = 7) and L. pneumophila mip gene (N = 1). Of the 90 controls, two samples (2.2%) showed IgM positivity, and 15 (16.7%) showed IgG positivity for M. pneumoniae. For L. pneumophila, three samples (3.3%) tested positive for IgM, and 12 (13.3%) tested positive for IgG antibodies. The study findings indicate the presence of M. pneumoniae and L. pneumophila in our geographical region, and a combination of laboratory approaches including PCR, culture, and serology is required for effective detection of these agents.

Purpose. We explored the efficiency of Rv1458c, the gene encoding a putative ABC drug transporter specific for the Mycobacterium tuberculosis complex (MTBC), as a diagnostic marker. Methodology. A 190 bp region of Rv1458c and a 300 bp region of hsp65 were targeted in a novel duplex PCR assay and the results were compared with those for PCR restriction analysis(PRA) using the restriction enzymes NruI and BamHI. Species identification of a subset of the isolates (n=50) was confirmed by sequencing. Clinical isolates of M. tuberculosis (n=426) obtained from clinically suspected patients of pulmonary tuberculosis and mycobacterial (n=13) and non-mycobacterial (n=8) reference strains were included in the study. Results. The duplex PCR assay correctly identified 320/426 isolates as MTBC and 106/426 isolates as non-tuberculous mycobacteria(NTM). The test was 100 % specific and sensitive when compared with NruI/BamHI PCR restriction analysis and highlighted the use of Rv1458c as a diagnostic marker for MTBC. Conclusion. The duplex PCR assay could be developed for use as a screening test to identify MTBC in clinical specimens in peripheral laboratories with limited resources.

A complicated epidemiological situation characterized by significantly high tuberculosis (TB) morbidity is observed in West Siberia. This study was aimed to investigate the genetic characteristics of Mycobacterium tuberculosis circulating in the southern part of West Siberia (in the Omsk region).From March 2013 to January 2015, 100 isolates of M. tuberculosis were obtained from patients with pulmonary TB living in the Omsk region. Drug susceptibility testing was performed on Lowenstein-Jensen medium (absolute concentration method). Genetic typing of isolates was carried out by variable number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR) typing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The genetic types and characteristics of cluster strains were determined using 15 MIRU-VNTR loci.Thirty six VNTR types were found. Twenty six (26.0%) isolates had a unique profile, and the remaining 74 were grouped in 10 clusters containing from 2 to 23 isolates. The Beijing genotype was found in 72 isolates, 61 (85.0%) of which were part of five clusters that included two large clusters containing 23 isolates. Other genetic families, such as Latin-American Mediterranean (LAM, 11.0%), S family (2.0%) and Haarlem (4.0%), were also detected. The genetic family of 11 isolates could not be determined. Six different VNTR profiles were found in these non-classified isolates. Only 16 per cent of isolates were sensitive to anti-TB drugs. The katG315 (94.8%) and rpoB531 (92.2%) mutations were identified in 77 multidrug-resistant M. tuberculosis isolates.This study showed that the M. tuberculosis population in the Omsk region was heterogeneous. The Beijing genotype predominated and was actively spreading. The findings obtained point to the need for the implementation of more effective preventive measures to stop the spread of drug-resistant M. tuberculosis strains.

Worldwide, tuberculosis (TB) is still a serious and significant health concern, more so with the emergence of multidrug-resistant-TB. The inability of mankind to control this infection stems from the fact that the vaccines and drugs that were once effective against TB are no longer efficacious. This has led to a search for new antituberculous agents and adjuvant therapy. Vitamins are being revisited for their role in pathogenicity as well as for their antimycobacterial properties. Vitamins such as biotin and thiamin are essential for Mycobacterium tuberculosis and are required for establishment of infection. On the other hand, vitamins such as Vitamin C and Vitamin D have been shown to possess antimycobacterial properties. To combat M. tuberculosis, innovative strategies need to be devised, keeping in mind the efficacy of the agent to be used. Vitamins can prove to be useful agents capable of modifying the life cycle and biology of M. tuberculosis. We present here a brief overview of the available knowledge on thiamin, biotin, Vitamin C, and Vitamin D, keeping TB treatment and control in perspective.

Timely diagnosis of paucibacillary tuberculosis (TB) which includes smear-negative pulmonary TB (PTB) and extra-pulmonary TB (EPTB) remains a challenge. This study was performed to assess the diagnostic utility of stool as a specimen of choice for detection of mycobacterial DNA in paucibacillary TB patients in a TB-endemic setting. Stool samples were collected from 246 subjects including 129 TB patients (62 PTB and 67 EPTB) recruited at TB hospital in Delhi, India. Diagnostic efficacy of stool IS6110 PCR (n = 228) was measured, using microbiologically/clinically confirmed TB as the reference standard. The clinical sensitivity of stool PCR was 97.22% (95% confidence interval (CI), 85.47-99.93) for detection of Mycobacterium tuberculosis in stool samples of smear-positive PTB patients and 76.92% (CI, 56.35–91.03) in samples from smear-negative PTB patients. Overall sensitivity of PCR for EPTB was 68.66% (CI, 56.16–79.44), with the highest sensitivity for stool samples from patients with lymph node TB (73.5%), followed by abdominal TB (66.7%) and pleural effusion (56.3%). Stool PCR presented a specificity of 95.12%. The receiver operating characteristic curve also indicated the diagnostic utility of stool PCR in TB detection (AUC: 0.882). The performance characteristic of the molecular assay suggests that stool DNA testing has clinical value in detection of TB.

Aim: There is an urgent need to design a reliable diagnostic test for tuberculosis (TB). Methods: Real-time immuno-PCR (RT-I-PCR) assay was devised for the quantitative detection of a cocktail of mycobacterial MPT64 (Rv1980c) and PstS1 (Rv0934) in TB patients. Results: A broad dynamic range of 0.95 pg/ml–95 ng/ml of MPT64+PstS1 was detected in TB patients. In smear-positive (n = 59) and smear-negative (n = 42) pulmonary TB cases, sensitivities of 93.2 and 83.3% were observed, respectively with 92.8% specificity, whereas a sensitivity of 77.9% and a specificity of 91.3% were observed in extrapulmonary TB cases (n = 86). Furthermore, significantly reduced MPT64+PstS1 concentrations (p

Aims and objectives: Multidrug resistant TB (MDR-TB) and extensively drug resistant (XDR) are responsible for significant obstruction to global control of tuberculosis (TB). Drug resistance in Mycobacterium tuberculosis mainly occurs through chromosomal mutations in genes encoding for drug target or drug activating enzymes. However, alternative mechanisms, such as efflux pumps may also be responsible for drug resistance. Methods: Insight into the mechanisms of drug resistance would help in limiting the disease through the use and development of better diagnostic and therapeutic tools. The importance of rapid and effective methods of identification of drug resistance in M. tuberculosis cannot be over emphasized. Traditional laboratory techniques are time consuming and cumbersome and not sufficiently sensitive or specific to fight this menace. Results: Though rapid molecular methods such as GeneXpert and line probe assays are vital tools in the fight against drug resistant TB, major advances in next generation sequencing (NGS) technology are allowing increasingly rapid and accurate sequencing of entire bacterial genomes, providing unprecedented depth of information. Conclusion: NGS has the ability to cause a revolutionary paradigm shift in the diagnosis of drug resistant tuberculosis and in understanding the evolution of drug resistance. However, certain challenges need to be overcome to enable the use of this promising technology in routine diagnosis and research, especially in resource limited regions of the world.

Lipoprotein signal peptidase, lspA (Rv1539), is the only known gene in mycobacterial genome for cleaving the signal sequence from prolipoprotein to form mature lipoprotein. It has been implicated in maintaining the virulence of Mycobacterium tuberculosis . The regulation of lspA had not been studied so far. Here, we identify a novel operon lspA –Rv1540 in M. tuberculosis . We detected co-transcription of the open reading frames of lspA –Rv1540 in in-vitro as well as in ex-vivo conditions. Analysis of the sequence upstream to lspA revealed a strong promoter activity that was shown to be induced significantly by surface stress and acidic environment.

Atypical pathogens including Mycoplasma pneumoniae and Legionella pneumophila are increasingly recognized as important causes of community-acquired pneumonia (CAP). Mycoplasma pneumoniae accounts for 20–40% of all CAP and L. pneumophila is responsible for 3–15% of cases. The paucity of data from India in this regard prompted us to conduct this prospective multicentric analysis to detect the prevalence of M. pneumoniae and L. pneumophila in our geographical region. A total of 453 patients with symptoms of pneumonia and 90 controls with no history of lower respiratory tract infections were included in the study. A duplex polymerase chain reaction (PCR) targeting 543 bp region of P1 adhesin gene of M. pneumoniae and 375 bp region of macrophage infectivity potentiator (mip) gene of L. pneumophila was standardized for simultaneous detection of these atypical pathogens. Respiratory secretions, blood, and urine samples were collected from each patient and control and were subjected to duplex PCR, culture and serology for M. pneumoniae and L. pneumophila. Urine samples were subjected for detecting L. pneumophila antigen. Among the 453 patients investigated for M. pneumoniae, 52 (11.4%) were positive for IgM antibodies, 17 were positive by culture, and seven tested positive by PCR (P1 gene). Similarly for L. pneumophila, 50 cases (11%) were serologically positive for IgM antibodies, one was positive by PCR (mip gene) and urine antigen detection. A total of eight samples were positive by duplex PCR for M. pneumoniae P1 gene (N = 7) and L. pneumophila mip gene (N = 1). Of the 90 controls, two samples (2.2%) showed IgM positivity, and 15 (16.7%) showed IgG positivity for M. pneumoniae. For L. pneumophila, three samples (3.3%) tested positive for IgM, and 12 (13.3%) tested positive for IgG antibodies. The study findings indicate the presence of M. pneumoniae and L. pneumophila in our geographical region, and a combination of laboratory approaches including PCR, culture, and serology is required for effective detection of these agents.

Determination of lipid content of any biological sample is essential for various kinds of studies related to pathogenicity and drug development. Thus, reliable methods for the quantitative extraction of lipids are of critical importance. The mycobacterial cell wall is largely composed of lipids. Commonly used methods to extract lipids, such as the Bligh and Dyer method or the Folch method, yield a low amount of lipids when applied to mycobacterial cells. This study presents an efficient modification of Chandramauli’s method, a less known method developed at this institute earlier that is able to yield a considerably higher concentration of mycobacterial lipids.

Global burden of latent TB infection comprises one-third of the world population. Identifying potential Mycobacterium tuberculosis (Mtb) latency associated antigens that can generate protective immunity against the pathogen is crucial for designing an effective TB vaccine. Usually the immune system responds to a small number of amino acids as MHC Class I or Class II peptides. The precision to trigger epitope specific protective T-cell immune response could therefore be achieved with synthetic peptide-based subunit vaccine. In the present study we have considered an immunoinformatic approach using available softwares (ProPred, IEDB, NETMHC, BIMAS, Vaxijen2.0) and docking and visualizing softwares (CABSDOCK, HEX, Pymol, Discovery Studio) to select 10 peptides as latency antigens from 4 proteins (Rv2626, Rv2627, Rv2628, and Rv2032) of DosR regulon of Mtb. As Intracellular IFN-γ secreted by T cells is the most essential cytokine in Th1 mediated protective immunity, these peptides were verified as potential immunogenic epitopes in Peripheral Blood Mononuclear Cells (PBMCs) of 10 healthy contacts of TB patients (HTB) and 10 Category I Pulmonary TB patients (PTB).The antigen-specific CD4 and CD8 T cells expressing intracellular IFN-γ were analyzed using monoclonal antibodies in all subjects by multi-parameter flow cytometry. Both, PTB and HTB individuals responded to DosR peptides by showing increased frequency of IFN-γ+CD4 and IFN-γ+CD8 T cells. The T-cell responses were significantly higher in PTB patients in comparision to the HTB individuals. Additionally, our synthetic peptides and pools showed higher frequencies of IFN-γ+CD4 and IFN-γ+CD8 T cells than the peptides of Ag85B. This pilot study can be taken up further in larger sample size which may support the untapped opportunity of designing Mtb DosR inclusive peptide based post-exposure subunit vaccine.

Despite the consideration of chromosomal mutations as the major cause of rifampicin (RIF) resistance in M. tuberculosis, the role of other mechanisms such as efflux pumps cannot be ruled out. We evaluated the role of four efflux pumps viz., MmpL2 (Rv0507), MmpL5 (Rv0676c), Rv0194 and Rv1250 in providing RIF resistance in M. tuberculosis. The real time expression of the efflux pumps was analyzed in 16 RIF resistant and 11 RIF susceptible clinical isolates of M. tuberculosis after exposure to RIF. Expression of efflux pumps in these isolates was also correlated with mutations in the rpoB gene and MICs of RIF in the presence and absence of efflux pump inhibitors. Under RIF stress, Rv0194 was induced in 8/16 (50%) RIF resistant and 2/11 (18%) RIF susceptible isolates; mmpL5 in 7/16 (44%) RIF resistant and 1/11 (9%) RIF susceptible isolates; Rv1250 in 4/16 (25%) RIF resistant and 2/11 (18%) RIF susceptible isolates; and mmpL2 was upregulated in 2/16 (12.5%) RIF resistant and 1/11 (9%) RIF susceptible isolates. This preliminary study did not find any association between Rv0194, MmpL2, MmpL5 and Rv1250 and RIF resistance. However, the overexpression of Rv0194 and mmpL5 in greater number of RIF resistant isolates as compared to RIF susceptible isolates and expression of Rv0194 in wild type (WT) resistant isolates suggests a need for further investigations.

The limitations of conventional methods of identification of Mycobacterium tuberculosis have led to the development of several nucleic acid amplification techniques which have the advantage of being rapid, sensitive, and specific. However, their expense or the need for technical expertise makes it difficult to use them in regions in which tuberculosis is endemic. A novel PCR restriction analysis (PRA) of the hsp65 gene was therefore developed for rapid screening of clinical isolates to identify Mycobacterium spp. The restriction enzymes NruI and BamHI were selected to obtain a limited number of restriction patterns to further differentiate between Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM). Three hundred ten isolates from clinical specimens and 24 reference strains were tested. The assay correctly identified 295 of the 310 culture isolates as MTBC, while the remaining 15 isolates were identified as NTM. Of the isolates tested, 135 MTBC strains and all 15 NTM were also confirmed by PRA using Sau96I and CfoI. Thirty-eight randomly selected MTBC strains and all 15 NTM were further confirmed by sequencing. The NruI/BamHI PRA was simple, as it did not require any elaborate analyses. It was cost-effective, rapid, highly sensitive, and specific and did not require technical expertise. The assay can, therefore, be used as a simple screening test not only to detect Mycobacterium spp. but also to differentiate MTBC from NTM in peripheral laboratories with minimal availability of funds.

Background The mce4 operon is one of the four mce operons with eight genes ( yrbE4A , yrbE4B , mce4A , mce4B , mce4C , mce4D , mce4E and mce4F ) of Mycobacterium tuberculosis . It expresses in the later phase of infection and imports cholesterol for long term survival of the bacilli. To cause latent infection, M. tuberculosis undergoes metabolic reprogramming of its genes to survive in the hostile environment like low availability of oxygen and nutrition depletion inside the host. Objective To analyze real time expression profile of mce4 operon under various stress conditions. Methods M. tuberculosis H37Rv was exposed to surface stress (0.1% SDS for 30 min and 90 min in late log and stationary phase of culture), hypoxia (5, 10, 15 and 20 days) and grown in the presence of either glycerol or cholesterol as sole source of carbon. The expression profile of genes of mce4 operon was analyzed by real time PCR. Results Surface stress induced expression of mce4C and yrbE4B in late log phase on 30 min and 90 min exposure respectively. The SDS exposure for 30 min induced mce4C , mce4D and mce4F in stationary phase. All eight genes were induced significantly on 10th and 15th days of hypoxia and in the presence of cholesterol. Conclusion Hypoxia and cholesterol are potent factors for the expression of mce4 operon of M. tuberculosis .

A large number of potentially pathogenic non-tuberculous mycobacteria (NTM) encountered in the clinical laboratory makes it necessary to identify their species to ensure appropriate treatment. However, labor-intensive conventional methods of speciation are not used in every laboratory, and hence NTM infections are often ignored. Polymerase chain reaction (PCR) restriction analysis (PRA) was applied in this study for early identification and speciation of mycobacterial species on 306 cultures of acid-fast bacilli isolated from patients suspected of suffering from tuberculosis. Mycobacterium tuberculosis was identified in 85.6% of the isolates. The NTM isolated most commonly was Mycobacterium kansasii/gastri group (3.5%), followed by Mycobacterium fortuitum (3.2%). Four of the M. fortuitum were grown from cultures obtained on the same day, but from samples from different patients and were probably laboratory contaminants. Mycobacterium intracellulare and Mycobacterium avium were identified in 2.94% and 2.28% of the isolates, respectively. Three isolates of M. avium and two isolates of M. intracellulare were obtained in repeated cultures from sputum samples of the same patients and were thus pathogenic. A single isolate of Mycobacterium abscessus was obtained from a breast abscess. A rare pathogen Mycobacterium phocaicum was isolated from one patient with epididymitis. However, whether it was the causative agent of epididymitis in this patient remains doubtful. The results of this study highlight the importance of speciation of mycobacteria for appropriate diagnosis and the importance of including molecular assays to augment conventional methods of diagnosis of mycobacterial diseases for rapid identification of NTM so that these potential pathogens are not overlooked in routine diagnostic procedures.

A rapid and sensitive loop-mediated isothermal amplification assay for the sdaA gene of Mycobacterium tuberculosis was developed using a dUTP-uracil- N -glycosylase (dUTP-UNG) strategy to prevent carryover contamination. Evaluation of the assay using clinical specimens ( n = 648) showed high specificity (97.2%) and sensitivity (100%), demonstrating its potential as a diagnostic test for tuberculosis, especially in resource-limited settings.

Eighty per cent of the cases of acute exacerbation of chronic obstructive pulmonary disease (AECOPD) have an infective aetiology, atypical bacteria including Mycoplasma pneumoniae accounting for 5–10 % of these. However, the importance of association of M. pneumoniae with episodes of AECOPD still remains doubtful. The present study was therefore undertaken to delineate the extent of involvement of M. pneumoniae in patients with AECOPD at a referral hospital in Delhi, India. Sputum samples and throat swabs from a total of 100 AECOPD patients attending the Clinical Research Center of Vallabhbhai Patel Chest Institute, Delhi, were collected during a 2-year period (January 2004–June 2006). The samples were investigated for the presence of aerobic bacterial pathogens and M. pneumoniae. Diagnosis of infection with M. pneumoniae was based on culture, serology, direct detection of M. pneumoniae specific antigen and PCR. Bacterial aetiology could be established in 16 of the 100 samples studied. Pseudomonas spp. were recovered from eight cases, Streptococcus pneumoniae from four and Klebsiella spp. from two cases. Acinetobacter sp. and Moraxella catarrhalis were isolated from one case each. Serological evidence of M. pneumoniae infection and/or detection of M. pneumoniae specific antigen were seen in 16 % of the cases. One case with definite evidence of M. pneumoniae infection also had coinfection with Pseudomonas spp. However, no direct evidence of M. pneumoniae infection was found in our study population as defined by culture isolation or PCR. In conclusion, although the serological prevalence of M. pneumoniae infection in our study population was significantly higher than in the control group, there was no direct evidence of it playing a role in AECOPD.

Klebsiella species infrequently cause acute community acquired pneumonia (CAP). The chronic form of the disease caused by K. pneumoniae (Friedlander’s bacillus) was occasionally seen in the pre-antibiotic era. K. oxytoca is a singularly uncommon cause of CAP. The chronic form of the disease caused by K. oxytoca has been documented only once before. A 50-year-old immunocompetent male smoker presented with haemoptysis for 12 months. Imaging demonstrated a cavitary lesion in the right upper lobe with emphysematous changes. Sputum stains and cultures for Mycobacterium tuberculosis were negative. However, three sputum samples for aerobic culture as well as bronchial aspirate cultured pure growth of K. oxytoca. A diagnosis of chronic pneumonia due to K. oxytoca was established and with appropriate therapy, the patient was largely asymptomatic. The remarkable clinical and radiological similarity to pulmonary tuberculosis can result in patients with chronic Klebsiella pneumonia erroneously receiving anti-tuberculous therapy.

Purpose Diagnosis of extrapulmonary tuberculosis (EPTB) poses serious challenges. A careful selection of appropriate gene targets is essential for designing a multiplex-polymerase chain reaction (M-PCR) assay. Materials and methods We compared several gene targets of Mycobacterium tuberculosis, including IS6110, devR, and genes encoding MPB-64 (mpb64), 38kDa (pstS1), 65kDa (hsp65), 30kDa (fbpB), ESAT-6 (esat6), and CFP-10 (cfp10) proteins, using PCR assays on 105 EPTB specimens. From these data, we chose the two best gene targets to design an M-PCR. Results Among all gene targets tested, mpb64 showed the highest sensitivity (84% in confirmed cases and 77.5% in clinically suspected cases), followed by IS6110, hsp65, 38kDa, 30kDa, esat6, cfp10, and devR. We used mpb64+IS6110 for designing an M-PCR assay. Our M-PCR assay demonstrated a high sensitivity of 96% in confirmed EPTB cases and 88.75% in clinically suspected EPTB cases with a high specificity of 100%, taking clinical diagnosis as the gold standard. Conclusion These M-PCR results along with the clinical findings may facilitate an early diagnosis of EPTB patients and clinical management of disease.

Mutations at position 306 of embB ( embB 306) have been proposed as a marker for ethambutol resistance in Mycobacterium tuberculosis ; however, recent reports of embB 306 mutations in ethambutol-susceptible isolates caused us to question the biological role of this mutation. We tested 1,020 clinical M. tuberculosis isolates with different drug susceptibility patterns and of different geographical origins for associations between embB 306 mutations, drug resistance patterns, and major genetic group. One hundred isolates (10%) contained a mutation in embB 306; however, only 55 of these mutants were ethambutol resistant. Mutations in embB 306 could not be uniquely associated with any particular type of drug resistance and were found in all three major genetic groups. A striking association was observed between these mutations and resistance to any drug ( P < 0.001), and the association between embB 306 mutations and resistance to increasing numbers of drugs was highly significant ( P < 0.001 for trend). We examined the association between embB 306 mutations and IS 6110 clustering (as a proxy for transmission) among all drug-resistant isolates. Mutations in embB 306 were significantly associated with clustering by univariate analysis (odds ratio, 2.44; P = 0.004). In a multivariate model that also included mutations in katG 315, katG 463, gyrA 95, and kasA 269, only mutations in embB 306 (odds ratio, 2.14; P = 0.008) and katG 315 (odds ratio, 1.99; P = 0.015) were found to be independently associated with clustering. In conclusion, embB 306 mutations do not cause classical ethambutol resistance but may predispose M. tuberculosis isolates to the development of resistance to increasing numbers of antibiotics and may increase the ability of drug-resistant isolates to be transmitted between subjects.

Little is known about the intracellular events that occur following the initial inhibition of Mycobacterium tuberculosis by the first-line antituberculosis drugs isoniazid (INH) and ethambutol (EMB). Understanding these pathways should provide significant insights into the adaptive strategies M. tuberculosis undertakes to survive antibiotics. We have discovered that the M. tuberculosis iniA gene (Rv 0342) participates in the development of tolerance to both INH and EMB. This gene is strongly induced along with iniB and iniC (Rv 0341 and Rv 0343) by treatment of Mycobacterium bovis BCG or M. tuberculosis with INH or EMB. BCG strains overexpressing M. tuberculosis iniA grew and survived longer than control strains upon exposure to inhibitory concentrations of either INH or EMB. An M. tuberculosis strain containing an iniA deletion showed increased susceptibility to INH. Additional studies showed that overexpression of M. tuberculosis iniA in BCG conferred resistance to ethidium bromide, and the deletion of iniA in M. tuberculosis resulted in increased accumulation of intracellular ethidium bromide. The pump inhibitor reserpine reversed both tolerance to INH and resistance to ethidium bromide in BCG. These results suggest that iniA functions through an MDR-pump like mechanism, although IniA does not appear to directly transport INH from the cell. Analysis of two-dimensional crystals of the IniA protein revealed that this predicted transmembrane protein forms multimeric structures containing a central pore, providing further evidence that iniA is a pump component. Our studies elucidate a potentially unique adaptive pathway in mycobacteria. Drugs designed to inhibit the iniA gene product may shorten the time required to treat tuberculosis and may help prevent the clinical emergence of drug resistance.