Origin and Dynamics of Mycobacterium tuberculosis Subpopulations That Predictably Generate Drug Tolerance and Resistance.

Initial responses to tuberculosis treatment are poor predictors of final therapeutic outcomes in drug-susceptible disease, suggesting that treatment success depends on features that are hidden within a small minority of the overall infecting Mycobacterium tuberculosis population. We developed a multitranswell robotic system to perform numerous parallel cultures of genetically barcoded M. tuberculosis exposed to steady-state concentrations of rifampicin to uncover these difficult-to-eliminate minority populations. We found that tolerance emerged repeatedly from at least two subpopulations of barcoded cells, namely, one that could not grow on solid agar media and a second that could form colonies, but whose kill curves diverged from the general bacterial population within 4 and 16 days of drug exposure, respectively. These tolerant subpopulations reproducibly passed through a phase characterized by multiple unfixed resistance mutations followed by emergent drug resistance in some cultures. Barcodes associated with drug resistance identified an especially privileged subpopulation that was rarely eliminated despite 20 days of drug treatment even in cultures that did not contain any drug-resistant mutants. The association of this evolutionary scenario with a defined subset of barcodes across multiple independent cultures suggested a transiently heritable phenotype, and indeed, glpK phase variation mutants were associated with up to 16% of the resistant cultures. Drug tolerance and resistance were eliminated in a ΔruvA mutant, consistent with the importance of bacterial stress responses. This work provides a window into the origin and dynamics of bacterial drug-tolerant subpopulations whose elimination may be critical for developing rapid and resistance-free cures. IMPORTANCE Tuberculosis is unusual among bacterial diseases in that treatments which can rapidly resolve symptoms do not predictably lead to a durable cure unless treatment is continued for months after all clinical and microbiological signs of disease have been eradicated. Using a novel steady-state antibiotic exposure system combined with chromosomal barcoding, we identified small hidden Mycobacterium tuberculosis subpopulations that repeatedly enter a state of drug tolerance with a predisposition to develop fixed drug resistance after first developing a cloud of unfixed resistance mutations. The existence of these difficult-to-eradicate subpopulations may explain the need for extended treatment regimen for tuberculosis. Their identification provides opportunities to test genetic and therapeutic approaches that may result in shorter and more effective TB treatments.

A Study on the Impact of the Novel Biochemical Parameter-Calcium Score in Preventing the Progression of the Cardiovascular Diseases to Invasive Interventions.

Cardiovascular diseases is increasing its pace day by day. Though the traditional biomarkers are made available the novel biomarkers are being incorporated to predict the risk of cardiovascular diseases for earlier detection. The present study aimed to investigate the impact of calcium score level the novel biochemical parameter in preventing the progression of the cardiovascular patients to PTCA (percutaneous transluminal coronary angioplasty) and CABG (coronary artery bypass grafting). Four hundred cardiovascular patients irrespective of sex were randomly selected from Visakhapatnam district Andhrapradesh. Information of subjects was collected using an interview schedule. Data collected were consolidated and tabulated. From this group a sub sample of 50 patients was selected and grouped as primordial, secondary and post PTCA. The subjects were then analyzed for their biochemical parameters before and after intervention. Statistical analysis was done and interpreted. An extensive evidence of calcium score was shown among 61.2% of the cardiovascular patients, a minimum evidence of 22.2% and moderate evidence of about 16.5%. The paired sample t-test is employed to observe any statistical significant difference between the before and after treatment effects. The analysis for the calcium score level was found to be significantly lower (mean difference=424.0134; t=13.297; df=49; p=0.01) in post intervention (mean=88.3766±88.40) than pre-intervention (512.39±260.79812). The present study identified calcium score the novel biochemical parameter as a key preventive measure among the usual biochemical management conducted by the clinicians to diagnosis and confirm the progression of the disease.