Kinetics of recA and recX induction in drug-susceptible and MDR clinical strains of Mycobacterium tuberculosis.

OBJECTIVES: To investigate and compare the expression of recA and recX, components of the SOS pathway, following rifampicin treatment in drug-susceptible and MDR clinical strains of Mycobacterium tuberculosis. METHODS: Strains (M. tuberculosis and Mycobacterium smegmatis) were subjected to rifampicin- and mitomycin-induced stress for 36 h followed by RNA extraction. recA and recX in the RNA extract were estimated using qRT-PCR. RESULTS: The MDR clinical strain induced faster (24 h) and higher (7-fold) levels of recA as compared with the drug-susceptible strain (36 h) in response to rifampicin. recX levels were found to rise with an increase in levels of recA; however, the levels were relatively higher than recA. CONCLUSIONS: Drug-susceptible and MDR strains have different kinetics of induction of DNA repair.

Evaluating the anti Mycobacterium tuberculosis activity of Alpinia galanga (L.) Willd. axenically under reducing oxygen conditions and in intracellular assays.

BACKGROUND: In tuberculosis (TB), the steadily increasing bacterial resistance to existing drugs and latent TB continue to be major concerns. A combination of conventional drugs and plant derived therapeutics can serve to expand the antimicrobial spectrum, prevent the emergence of drug resistant mutants and minimize toxicity. Alpinia galanga, used in various traditional medicines, possesses broad spectrum antibacterial properties. The study was undertaken to assess the antimycobacterial potential of A. galanga in axenic (under aerobic and anaerobic conditions) and intracellular assays. METHODS: Phytochemical analysis was done using HPTLC. The acetone, aqueous and ethanolic extracts (1, 10, 25, 50 and 100 µg/ml) of A. galanga were tested axenically using Microplate Alamar Blue Assay (MABA) against Mycobacterium tuberculosis (M.tb) H37Rv and three drug sensitive and three multi drug resistant clinical isolates. The activity of the extracts was also evaluated intracellularly in A549 cell line against these strains. The extracts active under intracellular conditions were further tested in an axenic setup under reducing oxygen concentrations using only H37Rv. RESULTS: 1´ acetoxychavicol acetate, the reference standard used, was present in all the three extracts. The acetone and ethanolic extracts were active in axenic (aerobic and anaerobic) and intracellular assays. The aqueous extract did not demonstrate activity under the defined assay parameters. CONCLUSION: A. galanga exhibits anti M.tb activity with multiple modes of action. Since the activity of the extracts was observed under reducing oxygen concentrations, it may be effective in treating the dormant and non-replicating bacteria of latent TB. Though the hypothesis needs further testing, A. galanga being a regular dietary component may be utilized in combination with the conventional TB therapy for enhanced efficacy.

Point-of-care detection of tuberculosis using magnetoresistive biosensing chip.

In this paper, a highly sensitive and specific technique based on the principle of giant magnetoresistance (GMR) has been proposed for the early stage Tuberculosis (TB) diagnostics. This GMR biosensing assay employs monoclonal antibodies against M. tuberculosis specific ESAT-6 antigen with the use of magnetic nanoparticles (MNPs) as labels. MNPs bind to the GMR sensor in presence of ESAT-6 and the binding is proportional to the ESAT-6 protein concentration leading to the change in overall resistance of GMR sensor. GMR biosensor simulation showed that ESAT-6 concentration can be detected in the range of pg/mL in comparison to the other transduction techniques available for ESAT-6 detection and further, the signal strength increased with the increase in the concentration. This work has shown that the GMR biosensing strategy is pertinent for the TB detection at the primitive phases when compared with other magnetic techniques used for TB diagnostics.

Elucidating the biosynthetic pathways of volatile organic compounds in Mycobacterium tuberculosis through a computational approach.

Microbial volatile organic compounds (VOCs) have gained prominence in the recent past for their potential use as disease markers. The discovery of microbial VOCs has benefited 'difficult to detect' diseases such as tuberculosis (TB). Few of the identified VOCs of Mycobacterium tuberculosis (Mtb) are currently being explored for their diagnostic potential. However, very little is known about the biosynthesis of these small lipophilic molecules. Here, we propose putative biosynthetic pathways in Mycobacterium tuberculosis for three VOCs, namely methyl nicotinate, methyl phenylacetate and methyl p-anisate, using computational approaches. In particular, we identify S-adenosyl methionine (SAM) transferases that play a crucial role in esterification of the acids to the final product. Our results provide important insights into the specificity of these pathways to Mtb species.

Activity of Medicinal Plant Extracts on Multiplication of Mycobacterium tuberculosis under Reduced Oxygen Conditions Using Intracellular and Axenic Assays.

Aim. Test the activity of selected medicinal plant extracts on multiplication of Mycobacterium tuberculosis under reduced oxygen concentration which represents nonreplicating conditions. Material and Methods. Acetone, ethanol and aqueous extracts of the plants Acorus calamus L. (rhizome), Ocimum sanctum L. (leaf), Piper nigrum L. (seed), and Pueraria tuberosa DC. (tuber) were tested on Mycobacterium tuberculosis H37Rv intracellularly using an epithelial cell (A549) infection model. The extracts found to be active intracellularly were further studied axenically under reducing oxygen concentrations. Results and Conclusions. Intracellular multiplication was inhibited ≥60% by five of the twelve extracts. Amongst these 5 extracts, in axenic culture, P. nigrum (acetone) was active under aerobic, microaerophilic, and anaerobic conditions indicating presence of multiple components acting at different levels and P. tuberosa (aqueous) showed bactericidal activity under microaerophilic and anaerobic conditions implying the influence of anaerobiosis on its efficacy. P. nigrum (aqueous) and A. calamus (aqueous and ethanol) extracts were not active under axenic conditions but only inhibited intracellular growth of Mycobacterium tuberculosis, suggesting activation of host defense mechanisms to mediate bacterial killing rather than direct bactericidal activity.

Antimycobacterial Efficacy of Andrographis paniculata Leaf Extracts Under Intracellular and Hypoxic Conditions.

The inhibition of the growth of Mycobacterium tuberculosis by the extracts of Andrographis paniculata has been studied using intracellular and axenic hypoxic conditions. The inhibition (confirmed using the gold standard colony forming unit assay) was found to increase with "double stimuli" or higher concentration of the extract. Organic solvent extracts were found to inhibit bacterial growth more than the aqueous extracts under microaerophilic conditions mimicked through axenic and intracellular assays. This could be further explored to evaluate the potential of the plant to be used against nonreplicating/dormant bacilli.

Fitness of acquired drug resistant Mycobacterium tuberculosis isolates from DOTS compliant patients.

The increasing levels of multi drug resistant tuberculosis (MDRTB) in endemic regions are a cause of concern. When MDR strains exhibit higher fitness they threaten global tuberculosis control. Evaluation of factors other than the particular non-synonymous substitutions that contribute to the fitness of strains may have an important bearing on understanding the spread of the disease. The aim of the study was to determine the fitness of MDRTB strains from TB endemic region, acquiring drug resistance during the course of treatment. Paired isolates of different lineages (one that was drug resistant and one that was drug susceptible) from 5 patients, which acquired resistance during antibiotic treatment and bearing drug resistance mutations in the rpoß and katG/inhA regions were mixed axenically. Fitness of Mycobacterium tuberculosis (M.tb) strains was evaluated using the difference in generation times of drug resistant and susceptible population obtained by enumeration of CFU. The drug resistant strains bore no significant fitness cost. The selection of specific allelic combinations in multiple drug resistance governing loci may attribute the enhanced/retained fitness of the drug resistant strains.

Global transcriptional profiling of longitudinal clinical isolates of Mycobacterium tuberculosis exhibiting rapid accumulation of drug resistance.

The identification of multidrug resistant (MDR), extensively and totally drug resistant Mycobacterium tuberculosis (Mtb), in vulnerable sites such as Mumbai, is a grave threat to the control of tuberculosis. The current study aimed at explaining the rapid expression of MDR in Directly Observed Treatment Short Course (DOTS) compliant patients, represents the first study comparing global transcriptional profiles of 3 pairs of clinical Mtb isolates, collected longitudinally at initiation and completion of DOTS. While the isolates were drug susceptible (DS) at onset and MDR at completion of DOTS, they exhibited identical DNA fingerprints at both points of collection. The whole genome transcriptional analysis was performed using total RNA from H37Rv and 3 locally predominant spoligotypes viz. MANU1, CAS and Beijing, hybridized on MTBv3 (BuG@S) microarray, and yielded 36, 98 and 45 differentially expressed genes respectively. Genes encoding transcription factors (sig, rpoB), cell wall biosynthesis (emb genes), protein synthesis (rpl) and additional central metabolic pathways (ppdK, pknH, pfkB) were found to be down regulated in the MDR isolates as compared to the DS isolate of the same genotype. Up regulation of drug efflux pumps, ABC transporters, trans-membrane proteins and stress response transcriptional factors (whiB) in the MDR isolates was observed. The data indicated that Mtb, without specific mutations in drug target genes may persist in the host due to additional mechanisms like drug efflux pumps and lowered rate of metabolism. Furthermore this population of Mtb, which also showed reduced DNA repair activity, would result in selection and stabilization of spontaneous mutations in drug target genes, causing selection of a MDR strain in the presence of drug pressures. Efflux pump such as drrA may play a significant role in increasing fitness of low level drug resistant cells and assist in survival of Mtb till acquisition of drug resistant mutations with least fitness cost.

Estimating fitness by competition assays between drug susceptible and resistant Mycobacterium tuberculosis of predominant lineages in Mumbai, India.

BACKGROUND: Multi Drug Resistant Tuberculosis (MDR TB) is a threat to global tuberculosis control. A significant fitness cost has been associated with DR strains from specific lineages. Evaluation of the influence of the competing drug susceptible strains on fitness of drug resistant strains may have an important bearing on understanding the spread of MDR TB. The aim of this study was to evaluate the fitness of MDR TB strains, from a TB endemic region of western India: Mumbai, belonging to 3 predominant lineages namely CAS, Beijing and MANU in the presence of drug susceptible strains from the same lineages. METHODOLOGY: Drug susceptible strains from a single lineage were mixed with drug resistant strain, bearing particular non synonymous mutation (rpoB D516V; inhA, A16G; katG, S315T1/T2) from the same or different lineages. Fitness of M.tuberculosis (M.tb) strains was evaluated using the difference in growth rates obtained by using the CFU assay system. CONCLUSION/SIGNIFICANCE: While MANU were most fit amongst the drug susceptible strains of the 3 lineages, only Beijing MDR strains were found to grow in the presence of any of the competing drug susceptible strains. A disproportionate increase in Beijing MDR could be an alarm for an impending epidemic in this locale. In addition to particular non synonymous substitutions, the competing strains in an environment may impact the fitness of circulating drug resistant strains.