Chronic pneumonia due to Klebsiella oxytoca mimicking pulmonary tuberculosis

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

Single nucleotide polymorphism in the genes of mce1 and mce4 operons of Mycobacterium tuberculosis: analysis of clinical isolates and standard reference strains

<p>Abstract</p> <p>Background</p> <p>The presence of four mammalian cell entry (<it>mce</it>) operons in <it>Mycobacterium tuberculosis </it>suggests the essentiality of the functions of the genes in these operons. The differential expression of the four <it>mce </it>operons in different phases of <it>in vitro </it>growth and in infected animals reported earlier from our laboratory further justifies the apparent redundancy for these genes in the genome.</p> <p>Here we investigate the extent of polymorphism in eight genes in the <it>mce1 </it>and <it>mce4 </it>operons of <it>M. tuberculosis </it>from four standard reference strains (H37Rv, H37Ra, LVS (Low Virulent Strain) and BCG) and 112 clinical isolates varying in their drug susceptibility profile, analysed by direct sequencing and Sequenom MassARRAY platform.</p> <p>Results</p> <p>We discovered 20 single nucleotide polymorphisms (SNPs) in the two operons. The comparative analysis of the genes of <it>mce1 </it>and <it>mce4 </it>operons revealed that <it>yrbE1A </it>[<it>Rv0167</it>] was most polymorphic in <it>mce1 </it>operon while <it>yrbE4A </it>[<it>Rv3501c</it>] and <it>lprN </it>[<it>Rv3495c</it>] had the highest number of SNPs in the <it>mce4 </it>operon. Of 20 SNPs, 12 were found to be nonsynonymous and were further analysed for their pathological relevance to <it>M. tuberculosis </it>using web servers PolyPhen and PMut, which predicted five deleterious nonsynonymous SNPs. A mutation from proline to serine at position 359 of the native Mce1A protein was most deleterious as predicted by both PolyPhen and PMut servers. Energy minimization of the structure of native Mce1A protein and mutated protein was performed using InsightII. The mutated Mce1A protein showed structural changes that could account for the effects of this mutation.</p> <p>Conclusions</p> <p>Our results show that SNPs in the coding sequences of <it>mce1 </it>and <it>mce4 </it>operons in clinical isolates can be significantly high. Moreover, <it>mce4 </it>operon is significantly more polymorphic than <it>mce1 </it>operon (p < 0.001). However, the frequency of nonsynonymous substitutions is higher in <it>mce1 </it>operon and synonymous substitutions are more in <it>mce4 </it>operon. <it>In silico </it>modeling predict that nonsynonymous SNP at <it>mce1A </it>[<it>Rv0169</it>], a virulence gene could play a pivotal role in causing functional changes in <it>M. tuberculosis </it>that may reflect upon the biology of the bacteria.</p

Comparative Genetic Association Analysis of Human Genetic Susceptibility to Pulmonary and Lymph Node Tuberculosis

Background: Tuberculosis (TB) manifests itself primarily in the lungs as pulmonary disease (PTB) and sometimes disseminates to other organs to cause extra-pulmonary TB, such as lymph node TB (LNTB). This study aimed to investigate the role of host genetic polymorphism in immunity related genes to find a genetic basis for such differences. Methods: Sixty-three, Single nucleotide polymorphisms (SNPs) in twenty-three, TB-immunity related genes including eleven innate immunity (SLCA11, VDR, TLR2, TLR4, TLR8, IRGM, P2RX7, LTA4H, SP110, DCSIGN and NOS2A) and twelve cytokine (TNFA, IFNG, IL2, Il12, IL18, IL1B, IL10, IL6, IL4, rs1794068, IL8 and TNFB) genes were investigated to find genetic associations in both PTB and LNTB as compared to healthy community controls. The serum cytokine levels were correlated for association with the genotypes. Results: PTB and LNTB showed differential genetic associations. The genetic variants in the cytokine genes (IFNG, IL12, IL4, TNFB and IL1RA and TLR2, 4 associated with PTB susceptibility and cytokine levels but not LNTB (p LTA4H, P2RX7, DCSIGN and SP110 showed susceptibility to LNTB and not PTB. Pathway analysis showed abundance of cytokine related variants for PTB and apoptosis related variants for LNTB. Conclusions: PTB and LNTB outcomes of TB infection have a genetic component and should be considered for any future functional studies or studies on susceptibility to pulmonary and extra-pulmonary TB

Role of Vitamins B, C, and D in the fight against tuberculosis

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

Contribution of putative efflux pump genes to isoniazid resistance in clinical isolates of Mycobacterium tuberculosis

Background: Isoniazid (INH) resistance in Mycobacterium tuberculosis has been mainly attributed to mutations in katG (64%) and inhA (19%). However, 20%–30% resistance to INH cannot be explained by mutations alone. Hence, other mechanisms besides mutations may play a significant role in providing drug resistance. Here, we explored the role of 24 putative efflux pump genes conferring INH-resistance in M. tuberculosis. Materials and Methods: Real-time expression profiling of the efflux pump genes was performed in five INH-susceptible and six high-level INH-resistant clinical isolates of M. tuberculosis exposed to the drug. Isolates were also analyzed for mutations in katG and inhA. Results: Four high-level INH-resistant isolates (minimum inhibitory concentration [MIC] ≥2.5 mg/L) with mutations at codon 315 (AGC-ACC) of katG showed upregulation of one of the efflux genes Rv1634, Rv0849, efpA, or p55. Another high-level INH-resistant isolate (MIC 1.5 mg/L), with no mutations at katG or inhA overexpressed 8/24 efflux genes, namely, Rv1273c, Rv0194, Rv1634, Rv1250, Rv3823c, Rv0507, jefA, and p55. Five of these, namely, Rv0194, Rv1634, Rv1250, Rv0507, and p55 were induced only in resistant isolates. Conclusion: The high number of efflux genes overexpressed in an INH-resistant isolate with no known INH resistance associated mutations, suggests a role for efflux pumps in resistance to this antituberculous agent, with the role of Rv0194 and Rv0507 in INH resistance being reported for the first time

Expression profile of mce4 operon of Mycobacterium tuberculosis following environmental stress

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

Potential impact of efflux pump genes in mediating rifampicin resistance in clinical isolates of Mycobacterium tuberculosis from India.

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

Ultrasensitive Detection of Multidrug-Resistant Mycobacterium tuberculosis Using SuperSelective Primer-Based Real-Time PCR Assays

The emergence of drug-resistant tuberculosis is a significant global health issue. The presence of heteroresistant Mycobacterium tuberculosis is critical to developing fully drug-resistant tuberculosis cases. The currently available molecular techniques may detect one copy of mutant bacterial genomic DNA in the presence of about 1&ndash;1000 copies of wild-type M. tuberculosis DNA. To improve the limit of heteroresistance detection, we developed SuperSelective primer-based real-time PCR assays, which, by their unique assay design, enable selective and exponential amplification of selected point mutations in the presence of abundant wild-type DNA. We designed SuperSelective primers to detect genetic mutations associated with M. tuberculosis resistance to the anti-tuberculosis drugs isoniazid and rifampin. We evaluated the efficiency of our assay in detecting heteroresistant M. tuberculosis strains using genomic DNA isolated from laboratory strains and clinical isolates from the sputum of tuberculosis patients. Results show that our assays detected heteroresistant mutations with a specificity of 100% in a background of up to 104 copies of wild-type M. tuberculosis genomic DNA, corresponding to a detection limit of 0.01%. Therefore, the SuperSelective primer-based RT-PCR assay is an ultrasensitive tool that can efficiently diagnose heteroresistant tuberculosis in clinical specimens and contributes to understanding the drug resistance mechanisms. This approach can improve the management of antimicrobial resistance in tuberculosis and other infectious diseases

Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India

The aim of the present study was to compare polymerase chain reaction (PCR)-based methods - spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing - with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligotype International Types (SITs)26 [Central-Asian (CAS)1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI) 3-Indian lineage]. A large number of isolates (75%) belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains) resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study