Vanaja Kumar

Origins of Highly Mosaic Mycobacteriophage Genomes

Bacteriophages are the most abundant organisms in the biosphere and play major roles in the ecological balance of microbial life. The genomic sequences of ten newly isolated mycobacteriophages suggest that the bacteriophage population as a whole is amazingly diverse and may represent the largest unexplored reservoir of sequence information in the biosphere. Genomic comparison of these mycobacteriophages contributes to our understanding of the mechanisms of viral evolution and provides compelling evidence for the role of illegitimate recombination in horizontal genetic exchange. The promiscuity of these recombination events results in the inclusion of many unexpected genes including those implicated in mycobacterial latency, the cellular and immune responses to mycobacterial infections, and autoimmune diseases such as human lupus. While the role of phages as vehicles of toxin genes is well established, these observations suggest a much broader involvement of phages in bacterial virulence and the host response to bacterial infections.

Exploring the Mycobacteriophage Metaproteome: Phage Genomics as an Educational Platform

Bacteriophages are the most abundant forms of life in the biosphere and carry genomes characterized by high genetic diversity and mosaic architectures. The complete sequences of 30 mycobacteriophage genomes show them collectively to encode 101 tRNAs, three tmRNAs, and 3,357 proteins belonging to 1,536 “phamilies” of related sequences, and a statistical analysis predicts that these represent approximately 50% of the total number of phamilies in the mycobacteriophage population. These phamilies contain 2.19 proteins on average; more than half (774) of them contain just a single protein sequence. Only six phamilies have representatives in more than half of the 30 genomes, and only three—encoding tape-measure proteins, lysins, and minor tail proteins—are present in all 30 phages, although these phamilies are themselves highly modular, such that no single amino acid sequence element is present in all 30 mycobacteriophage genomes. Of the 1,536 phamilies, only 230 (15%) have amino acid sequence similarity to previously reported proteins, reflecting the enormous genetic diversity of the entire phage population. The abundance and diversity of phages, the simplicity of phage isolation, and the relatively small size of phage genomes support bacteriophage isolation and comparative genomic analysis as a highly suitable platform for discovery-based education.

Randomized Clinical Trial of Thrice-Weekly 4-Month Moxifloxacin or Gatifloxacin Containing Regimens in the Treatment of New Sputum Positive Pulmonary Tuberculosis Patients

Background Shortening tuberculosis (TB) treatment duration is a research priority. This paper presents data from a prematurely terminated randomized clinical trial, of 4-month moxifloxacin or gatifloxacin regimens, in South India. Methods Newly diagnosed, sputum-positive HIV-negative pulmonary TB patients were randomly allocated to receive gatifloxacin or moxifloxacin, along with isoniazid and rifampicin for 4 months with pyrazinamide for first 2 months (G or M) or isoniazid and rifampicin for 6 months with ethambutol and pyrazinamide for first 2 months (C). All regimens were administered thrice-weekly. Clinical and bacteriological assessments were done monthly during treatment and for 24 months post-treatment. The Data and Safety Monitoring Board recommended termination of the trial due to high TB recurrence rates in the G and M regimens. Results Of 416 patients in intent-to-treat analysis, 6 (5%) of 124, 2 (2%) of 110 and 2 (2%) of 137 patients with drug-susceptible TB in the G, M and C arms respectively had unfavorable response at the end of treatment; during the next 24 months, 17 (15%) of 115, 11 (11%) of 104 and 8 (6%) of 132 patients respectively, had TB recurrence. Of 38 drug-resistant patients 1 of 8 and 3 of 26 in the G and C arms respectively had unfavourable response at the end of treatment; and TB recurrence occurred in 2 of 7 and 2 of 23 patients, respectively. The differences in TB recurrence rates between the G and C arms was statistically significant (p = 0.02). Gastro-intestinal symptoms occurred in 23%, 22% and 9% of patients in the G, M and C arms respectively, but most reactions were mild and manageable with symptomatic measures; 1% required regimen modification. Conclusions 4-month thrice-weekly regimens of gatifloxacin or moxifloxacin with isoniazid, rifampicin and pyrazinamide, were inferior to standard 6-month treatment, in patients with newly diagnosed sputum positive pulmonary TB. Trial Registration Clinical Trials Registry of India CTRI/2012/10/003060

Novel 1,3,5-triphenyl-2-pyrazolines as anti-infective agents.

Sixteen 1,3,5-triphenyl-2-pyrazolines were synthesized and their anti-infective activities (against Mycobacterium tuberculosis H(37)Rv, six bacterial and four fungal strains) were tested. Only compound with SO(2)CH(3) in the para position of the A-ring was active against the tubercular strain at 100 microg/ml concentration. All compounds showed good anti infective activity against Escherichia coli and poor activity against Staphylococcus aureus. Compounds 4, 12, 13 and 14 exhibited reasonable activity against all the organisms tested (<0.309 microM except against S. aureus. The activity of these compounds correlated with their lipophilic/hydrophilic nature. Compounds 4, 10 and 16 showed very good activity (>88% reduction) against four fungi studied at 2mg/ml. All these compounds possess halogen substitutions. Compound 11 showed very high activity (>90%) against three fungi. Majority of the compounds showed more than 90% inhibition against one or two fungi. Since pyrazolines are reported to inhibit the activity of p-glycoprotein, they may prevent drug resistance developed by microorganism.

Construction and evaluation of luciferase reporter phages for the detection of active and non-replicating tubercle bacilli.

The luciferase reporter phages (LRP) show great promise for diagnostic mycobacteriology. Though conventional constructs developed from lytic phages such as D29 and TM4 are highly specific, they lack sensitivity. We have isolated and characterized Che12, the first true temperate phage infecting M. tuberculosis. Since the tuberculosis (TB) cases among HIV infected population result from the reactivation of latent bacilli, it would be useful to develop LRP that can detect dormant bacteria. During dormancy, pathogenic mycobacteria switch their metabolism involving divergent genes than during normal, active growth phase. Since the promoters of these genes can potentially function during dormancy, they were exploited for the construction of novel mycobacterial luciferase reporter phages. The promoters of hsp60, isocitrate lyase (icl), and alpha crystallin (acr) genes from M. tuberculosis were used for expressing firefly luciferase gene (FFlux) in both Che12 and TM4 phages and their efficiency was evaluated in detecting dormant bacteria from clinical isolates of M. tuberculosis. These LRP constructs exhibited detectable luciferase activity in dormant as well as in actively growing M. tuberculosis. The TM4 ts mutant based constructs showed about one log increase in light output in three of the five tested clinical isolates and in M. tuberculosis H37Rv compared to conventional lytic reporter phage, phAE129. By refining the LRP assay format further, an ideal rapid assay can be designed not only to diagnose active and dormant TB but also to differentiate the species and to find their drug susceptibility pattern.

Synonymous Codon Usage Analysis of Thirty Two Mycobacteriophage Genomes

Synonymous codon usage of protein coding genes of thirty two completely sequenced mycobacteriophage genomes was studied using multivariate statistical analysis. One of the major factors influencing codon usage is identified to be compositional bias. Codons ending with either C or G are preferred in highly expressed genes among which C ending codons are highly preferred over G ending codons. A strong negative correlation between effective number of codons (Nc) and GC3s content was also observed, showing that the codon usage was effected by gene nucleotide composition. Translational selection is also identified to play a role in shaping the codon usage operative at the level of translational accuracy. High level of heterogeneity is seen among and between the genomes. Length of genes is also identified to influence the codon usage in 11 out of 32 phage genomes. Mycobacteriophage Cooper is identified to be the highly biased genome with better translation efficiency comparing well with the host specific tRNA genes.

Characterization of temperate phage Che12 and construction of a new tool for diagnosis of tuberculosis

A temperate phage, Che12, able to infect Mycobacterium tuberculosis, was isolated from soil samples taken from tuberculosis sanatorium area in Chennai, India. The plaque morphology of this phage showed varying grades of turbidity on lawns of M. tuberculosis. The temperate nature of Che12 was established by super infection immunity. Phage integration into the host genomic DNA was confirmed by Southern hybridization using Che12 DNA as a probe. PCR amplification and sequencing of a part of the integrated phage genome in a M. tuberculosis lysogen also confirmed the temperate nature of Che12. The morphology of the phage particles was observed by electron microscopy, revealing similarities to other mycobacteriophages like L5, D29 and TM4. A luciferase reporter phage, phAETRC16, was constructed by cloning firefly luciferase gene into Che12. Infection of viable M. tuberculosis cells by phAETRC16 resulted in expression of luciferase leading to sustained light output. Che12, a true temperate phage infecting M. tuberculosis, is thus ideally suited for developing a diagnostic tool facilitating rapid diagnosis of M. tuberculosis.

Microwave-assisted synthesis, molecular docking and antitubercular activity of 1,2,3,4-tetrahydropyrimidine-5-carbonitrile derivatives.

Based on bioisosteric similarities with isoniazid, a series of 1,2,3,4-tetrahydropyrimidine-5-carbonitrile derivatives has been designed. The target compounds have been synthesized by multicomponent reaction which involves one-pot organic reactions using ethylcyanoacetate, urea/thiourea and arylaldehydes in presence of ethanolic K(2)CO(3). Two methodologies, conventional and microwave-assisted, have been adopted for the synthesis. The later strategy gave high yields in less than 10 min as compared to long hours using the former approach. Molecular docking of the target compounds into the enzyme Mycobacterium tuberculosis enoyl reductase (InhA) revealed important structural information on the plausible binding interactions. Major binding interactions were found to be of dispersion type (residues Tyr158, Ile215, Met103 and Met199) and a hydrogen bond with Tyr158. Binding poses of the all the compounds were energetically favorable and showed good interactions with the active site residues. Few selected compounds were also evaluated for antitubercular activity in vitro against drug-sensitive M. tuberculosis H37Rv strain and clinically isolated S, H, R and E resistant M. tuberculosis by luciferase reporter phage (LRP) assay method. Some compounds displayed promising antimycobacterial activity comparable or less than the standard drugs isoniazid and rifampicin.

Sputum Culture Conversion With Moxifloxacin-Containing Regimens in the Treatment of Patients With Newly Diagnosed Sputum-Positive Pulmonary Tuberculosis in South India

BACKGROUND Rapid sputum culture conversion at 2 months indicates the sterilizing capacity and potential of regimens to shorten duration of tuberculosis treatment. We compared results of sputum culture conversion by moxifloxacin and control regimens and identified factors affecting sputum culture positivity after 2 months of treatment. METHODS Human immunodeficiency virus-uninfected adults with newly diagnosed smear-positive pulmonary tuberculosis were randomized to receive a 3- or 4-month moxifloxacin regimen (moxifloxacin [M], isoniazid [H], rifampicin [R], pyrazinamide [Z], ethambutol [E]) or the control regimen (RHZE thrice weekly). Bacteriological assessments were done at 15, 30, 45, and 60 days of treatment. Because all patients in the moxifloxacin groups received 2 months of daily RHZEM, they were grouped together for analysis. Statistical methods included χ(2) test and logistic regression analysis. RESULTS Sputum culture conversion was analyzed in 780 (616 in the moxifloxacin group and 164 in the control group) of 801 enrolled patients. Ninety-five percent of 590 patients in the moxifloxacin group and 81% of 151 patients in the control group had negative sputum cultures at month 2 (P < .001). The control regimen, age (≥35 years), initial sputum culture grade (2+ or 3+), and male sex were significantly associated with higher odds of positive sputum cultures at 2 months. CONCLUSIONS A 5-drug daily regimen with moxifloxacin results in significantly higher sputum culture conversion in the first 2 months compared with a thrice-weekly, 4-drug regimen in patients with newly diagnosed sputum-positive pulmonary tuberculosis.

Phage lysin as a substitute for antibiotics to detect Mycobacterium tuberculosis from sputum samples with the BACTEC MGIT 960 system

Phage lysin was evaluated as a substitute for antibiotics in sputum samples processed by a modified Petroffs method for the detection of Mycobacterium tuberculosis with the MGIT 960 system. One hundred and fifty sputum samples were processed, inoculated onto two slopes of Lowenstein-Jensen medium, and divided in to two aliquots of 0.5 mL each. One aliquot was added to 7 mL of MGIT medium containing polymyxin B, amphotericin B, nalidixic acid, trimethoprim and azlocillin (PANTA) (MGIT-PANTA) and the other was added to 7 mL of MGIT medium containing 0.8 mL of lysin (MGIT-Lysin). The samples were randomized and incubated at 37°C in the MGIT 960 system. The sensitivity and specificity of MGIT-Lysin were 97% and 88%, respectively, as compared with MGIT-PANTA. The average times to detection with MGIT-Lysin and MGIT-PANTA were 9.3 and 8.6 days, respectively. The rate of contamination with MGIT-PANTA and MGIT-Lysin were 16% and 7.3%, respectively. Phage lysin can be substituted for antibiotics in processed sputum samples for the detection of M. tuberculosis.