Madhu Goyal

Overexpression of heat-shock proteins reduces survival of Mycobacterium tuberculosis in the chronic phase of infection

Elevated expression of heat-shock proteins (HSPs) can benefit a microbial pathogen struggling to penetrate host defenses during infection, but at the same time might provide a crucial signal alerting the host immune system to its presence. To determine which of these effects predominate, we constructed a mutant strain of Mycobacterium tuberculosis that constitutively overexpresses Hsp70 proteins. Although the mutant was fully virulent in the initial stage of infection, it was significantly impaired in its ability to persist during the subsequent chronic phase. Induction of microbial genes encoding HSPs might provide a novel strategy to boost the immune response of individuals with latent tuberculosis infection.

Cytokine mRNA expression in cattle infected with different dosages of Mycobacterium bovis

Cytokine mRNA expression of pro-inflammatory cytokines, i.e., interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and anti-inflammatory cytokines, i.e., interleukin-4 (IL-4), interleukin-10 (IL-10) was quantified using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in cattle infected with different doses (1-1000 colony-forming units (cfu)) of Mycobacterium bovis. RNA was extracted from the Hepes glutamic acid buffer mediated organic solvent protection effect (HOPE) fixed lymph node tissues using Trizol method. The expression levels of all the four cytokines gradually increased in cattle infected with 1 cfu-1000 cfu. Statistical significance (P<0.05) was observed for the cytokines IFN-gamma, IL-4 and IL-10 between the cattle infected with 1 cfu and 1000 cfu. Though there was an increase in the expression levels of TNF-alpha from cattle infected with 1 cfu-1000 cfu, this difference in expression was not statistically significant (P>0.05). The increase in the levels of IFN-gamma indicates that the host may be responding to control the infection and the increased level of IL-4 and IL-10 which are anti-inflammatory cytokines, suggests that these cytokines are trying to protect the host by reducing the inflammation and also by controlling the levels of TNF-alpha (the cytokine that may cause tissue damage).

Synthesis and Antimicrobial Activity of 1,2-Benzothiazine Derivatives

A number of 1,2-benzothiazines have been synthesized in a three-step process. Nine chalcones 1–9 bearing methyl, fluoro, chloro and bromo substituents were chlorosulfonated with chlorosulfonic acid to generate the chalcone sulfonyl chlorides 10–18. These were converted to the dibromo compounds 19–27 through reaction with bromine in glacial acetic acid. Compounds 19–27 were reacted with ammonia, methylamine, ethylamine, aniline and benzylamine to generate a library of 45 1,2-benzothiazines 28–72. Compounds 28–72 were evaluated for their antimicrobial activity using broth microdilution techniques against two Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) and two Gram-negative bacteria (Proteus vulgaris and Salmonella typhimurium). The results demonstrated that none of the compounds showed any activity against Gram-negative bacteria P. vulgaris and S. typhimurium; however, compounds 31, 33, 38, 43, 45, 50, 53, 55, 58, 60, 63 and 68 showed activity against Gram-positive bacteria Bacillus subtilis and Staphylococcous aureus. The range of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) was 25–600 µg/mL, though some of the MIC and MBC concentrations were high, indicating weak activity. Structure activity relationship studies revealed that the compounds with a hydrogen atom or an ethyl group on the nitrogen of the thiazine ring exerted antibacterial activity against Gram-positive bacteria. The results also showed that the compounds where the benzene ring of the benzoyl moiety contained a methyl group or a chlorine or bromine atom in the para position showed higher antimicrobial activity. Similar influences were identified where either a bromine or chlorine atom was in the meta position.

Genetic diversity of Mycobacterium tuberculosis complex strains isolated from patients with pulmonary tuberculosis in Anambra State, Nigeria

In this study, we analyzed Mycobacterium tuberculosis complex (MTC) genetic diversity in Anambra State, Nigeria based on spoligotyping followed by 5-loci exact tandem repeats (ETRs). Spoligotyping of 180 MTC strains isolated in 2009–2011 from pulmonary tuberculosis (TB) patients led to a total of 31 distinct patterns. A comparison with the SITVIT2 international database showed that all the 31 patterns could be classified as Shared-types (SITs) in this database; briefly, 26/31 SITs (n =174 isolates) matched a preexisting shared-type in the database, whereas 5/31 SITs (n =6 isolates) were newly created due to 2 or more strains belonging to an identical new pattern within this study (SIT3396) or after a match with an orphan in the database (SIT3397, SIT3398, SIT3399 and SIT3400). A total of 18/31 SITs containing 167 or 92.8% isolates were clustered within this study (2–89 isolates per cluster) while 13/31 SITs contained unique strains. Using VNTR typing, a total of 36 distinct patterns were identified; 27 patterns (n =157 isolates) matched a pattern already reported in the SITVIT2 database. Combination of both the methods generated 47 combined patterns for the 180 strains: 17 belonged to clustered isolates (n =127 isolates or 70.5%) while 30 corresponded to as many unique strains (note 23 strains could not be typed using 5-loci ETRs). No correlation was found between the spoligotyping pattern and the HIV status of the patient or drug sensitivity of the strain. This study showed that the LAM10-CAM prototype SIT61 accounted for highest number of isolates (n =89) in Anambra State, showing its relative contribution to the TB burden in the study.

Molecular Approaches in Mycobacterium tuberculosis and Other Infections Caused by Mycobacterium Species

The genus Mycobacterium consists of a diverse group of organisms that are ubiquitous and are believed to be some of the oldest bacteria on earth. They may exist as free-living commensals inhabiting soil and water, but they are also potentially pathogenic to man and other animals, being transmitted by airborne or droplet spread. At least 25 species of mycobacteria have been associated with human disease. Robert Koch in 1882, identified the acid-fast bacterium (AFB), Mycobacterium tuberculosis as the causative agent of tuberculosis (TB). TB is an ancient disease that remains a significant global health problem With improved living standards and the introduction of chemotherapy in 195Os, the incidence of TB in most industrialized countries showed a progressive decline, with very little mortality by the mid 1980s. This pattern has changed over the last decade.

Synthesis and Spectral Characterization of Benzo-[6,7][1,5]diazocino[2,1-a]isoindol-12-(14H)-one Derivatives

A simple synthetic route affording 27%–85% yields of benzo[6,7][1,5]diazocino[2,1-a]isoindol-12(14H)-one ring systems from readily available 3-(2-oxo-2-phenylethyl) isobenzofuran-1(3H)-ones and 2-(aminomethyl)aniline starting materials in toluene and catalysed by p-toluene-sulfonic acid is developed. The 1H- and 13C-NMR spectra of the final products were assigned using a variety of one and two-dimensional NMR experiments. The distinction between the two potential isomers of the final products was made on the basis of heteronuclear multiple bond connectivity (HMBC) NMR spectra.

Synthesis and antibacterial activity of benzo[4,5]isothiazolo[2,3-a]pyrazine-6,6-dioxide derivatives

Using a routine procedure, a number of derivatives of the benzo[4,5]isothiazolo[2,3-a]pyrazine-6,6-dioxide ring system have been synthesized from readily available starting materials. A series of chalcones were synthesized, which were subsequently reacted with chlorosulfonic acid to generate chalcone sulfonyl chlorides. The chalcone sulfonyl chlorides were then treated with bromine to generate dibromo chalcone sulfonyl chlorides. These were subsequently reacted with 1,2-diaminopropane and 2-methyl-1,2-diaminopropane in boiling ethanol resulting in compounds 2–10 and 11–19 respectively, in 12–80% yields. The products were characterized by spectral analysis and the definitive structure of compound 11 was determined by X-ray crystallography. The synthesized compounds were screened for potential antibacterial properties against Bacillus subtilis, Escherichia coli, Proteus vulgaris and Staphylococcus aureus.