Analysis of Biofilm Formation And Identification of Gene(S) Involved in Biofilm Formation In Mycobacterium Fortuitum

Abstract

Mycobacterium fortuitum is a non tuberculous, rapidly growing mycobacterium which is present ubiquitously in nature. M. fortuitum is an emergent pathogen attaining relevance in human health as it is one of the leading causes of opportunistic infections such as local cutaneous diseases, osteomyelitis, joint infections, ocular disease, post surgical infections etc. An obstruction is imposed on the way of its treatment as it is evolving resistance to the currently prescribed drugs. Growing incidence of nosocomial infections caused by M. fortuitum may be correlated with its ability to assemble biofilm on implantable devices and other surgical instruments. Biofilm formation plays an important in the pathogenesis of M. fortuitum, thus, the present study aims to identify genes responsible for biofilm formation in M. fortuitum by random mutagenesis. As the genomic sequence of M. fortuitum is not known, random mutagenesis of wild type M. fortuitum (ATCC 6841) was done by electroporation of vector pRT291 containing transposon Tn5 with kanamycin resistance. M. fortuitum mutants were screened on NAT plates containing Kanamycin, Cycloheximide, X-gal, IPTG and 5% glucose. Borrowed library containing 120 mutants was screened for biofilm formation using crystal violet assay, out of which 50 mutants showing minimum optical density were selected. These mutants were further examined for biofilm formation by carbol fuschin staining method to shortlist 5 mutants with attenuation in biofilm formation for further experiments. The mutants were individually subjected to the standardised biofilm forming protocol, to check for any deviation in the amount of biofilm formed. Genomic analysis followed by homology study of mutant was done, which revealed a hypothetical gene i.e. “Anthranilate phosphoribosyl transferase”, which may be involved in biofilm formation in M. fortuitum. Gene identified by this study can serve as potential drug targets for development of novel drugs or other intervention strategies.