A method for the enrichment, isolation and validation of Mycobacterium smegmatis population surviving in the presence of bactericidal concentrations of rifampicin and moxifloxacin.
The bacterial populations surviving in the presence of antibiotics contain cells that have gained genetic resistance, phenotypic resistance and tolerance to antibiotics. Isolation of live bacterial population, surviving against antibiotics, from the milieu of high proportions of dead/damaged cells will facilitate the study of the cellular/molecular processes used by them for survival. Here we present a Percoll gradient centrifugation based method for the isolation of enriched population of Mycobacterium smegmatis surviving in the presence of bactericidal concentrations of rifampicin and moxifloxacin. From the time of harvest, throughout the enrichment and isolation processes, and up to the lysis of the cells for total RNA preparation, we maintained the cells in the presence of the antibiotic to avoid changes in their metabolic status. The total RNA extracted from the enriched population of live antibiotic-surviving population showed structural integrity and purity. We analysed the transcriptome profile of the antibiotic-surviving population and compared it with the orthologue genes of Mycobacterium tuberculosis that conferred antibiotic tolerance on tubercle bacilli isolated from the tuberculosis patients under treatment with four antitubercular antibiotics. Statistically significant comparability between the gene expression profiles of the antibiotic tolerance associated genes of M. smegmatis and M. tuberculosis validated the reliability/utility of the method.
['Pradhan|Atul|A|', 'Swaminath|Sharmada|S|', 'Jakkala|Kishor|K|', 'Ajitkumar|Parthasarathi|P|0000-0001-9743-0186']
D000995:Antitubercular Agents D001431:Bacteriological Techniques D004361:Drug Tolerance D020869:Gene Expression Profiling D006801:Humans D008826:Microbial Sensitivity Tests D050296:Microbial Viability D000077266:Moxifloxacin D009165:Mycobacterium Infections, Nontuberculous D020102:Mycobacterium smegmatis D015203:Reproducibility of Results D012293:Rifampin
rifampicin moxifloxacin antibiotic-surviving population gene expression profile Mycobacteria microarray