Identification of Novel Gene S Responsible for Drug Resistance in Mycobacterium Fortuitum

Abstract

Mycobacterium fortuitum is rapidly growing, nontuberculous mycobacteria (NTM) which is ubiquitously present in nature. It is one of the most important pathogenic species, representing more than 80% of clinical isolates of rapidly growing mycobacteria. To add up insult to injury, an obstruction is imposed on the way to its treatment as these organisms are evolving into a drug resistant species. Prolonged antibiotic therapy is now a necessity for M. fortuitum infection. Considering these crisis, our study aims to identify the resistant gene(s) and the mechanism associated, and the subsequent use of these findings in establishing effective drug targets. As the genomic sequence of M. fortuitum is not known, random mutagenesis technology (using TnphoA) was employed. The plasmid (pRT291) containing transposon Tn5 was electroporated in M. fortuitum wild type strains which resulted in formation of 50 blue mutant colonies showing mutation. These mutants were further screened by β-galactosidase assay. 10 mutants (i.e. KM1 to KM10) having high β-galactosidase activity were shortlisted for drug profiling where Ciprofloxacin, Amikacin, Rifampicin, Isoniazid and Ethambutol were utilized as these are most commonly used for treatment of M. fortuitum related infections. The minimum inhibitory concentration (MIC) of mutant strain KM4 for Rifampicin was observed to increase by 4-fold whereas the strain KM5 was sensitive to it. All the mutants had same MIC as that of wild type when Isoniazid was used, but they were sensitive to Amikacin (4-fold decrease in MIC). Genomic analysis of these mutants was done by restriction digestion of their genomic DNA, cloning of the insert containing transposon into pUC19, followed by sequencing. Comparative genomic analysis of mutants revealed two hypothetical genes of M. fortuitum i.e. “Antranilate synthase” and “NusA ribosome maturation factor RimP” which may be used as potential drug targets.