Isolation and Characterization of Native Plasmids from Clinical Isolates of Mycobacterium avium intracellulare

Mycobacterium avium intracellulare (MAI), a pathogen of birds, have also been found in human clinical isolates. These pathogens prevalent in the Asia, Europe and the US, cause a pulmonary nontuberculous mycobacterial disease in humans and frequently seen as disseminated infection among immunocompromised patients. Plasmids have been found in the clinical isolates from MAI-infected patients. Investigators have indicated a possible relation of antibiotic resistance, virulence, and pathogenesis to the presence of plasmids in the clinical isolates. This study was undertaken to investigate the presence of plasmids in clinical isolates from patients attending a chest diseases hospital at Delhi. Out of the 200 clinical isolates that were positive for NTM Runyon group III, 50 were biochemically identified as MAI. Thirty of the 50 isolates were screened for the presence of plasmids. Various methods were attempted to isolate plasmids with inconsistent and poor recovery of plasmid DNA. Since high molecular weight plasmids have been reported in MAI, pulsed-field gel electrophoresis was performed for the isolation. Of the thirty human isolates of MAI screened for plasmids using a protocol developed in our laboratory, five isolates indicated the presence of plasmids. One of the plasmids gave multiple bands using this protocol. The plasmids from the isolates screened had sizes between 16 and 20 kb. One of the prominent plasmids was purified and partially characterized was found to have a size of ~ 20 kb. A 650 bp amplicon was obtained with primers from M. fortuitum plasmid pMF1 and a 600 bp amplicon with primers of a linear plasmid pCLP from M. celatum. Cloning and partial sequencing revealed homology to a RAPD marker of Salmonella dublin. Plasmids have potential as an epidemiological tool and can be used as probes for detection of MAI in clinical specimens.