MOLECULAR CHARACTERIZATION OF MYCOBACTERIUM TUBERCULOSIS STRAINS ISOLATED IN PAKISTAN

DNA fingerprints were generated and analyzed to determine the genetic diversity of local Mycobacterium. tuberculosis (M. tuberculosis) clinical isolates. IS6110 based RFLP analysis and VNTR typing were applied for characterization of M. tuberculosis specific molecular markers.\ud \ud RFLP analysis of 237 clinical isolates revealed the highly variable nature of IS6110 copy numbers, ranging from 1-21. Fifteen percent of the isolates carried a single copy, while 85 % isolates carried multiple copies of the insertion sequence. Of the multiple-copy isolates, 94.1 % carried 6-21 copies while 5.9% carried 2-5 copies. A bimodal distribution of IS6110 copy numbers was seen, with peaks at one and 14 copies per genome. All strains with multiple copies showed unique RFLP patterns. IS6110 negative strains were not found in this study. Four isolates showed RFLP patterns similar to those of "Beijing family" genotype.\ud \ud Cluster analysis was performed on isolates with more than five IS6110 copies, and clusters assigned on the basis of greater than 60% similarity in RFLP patterns. 5imilarity values distributed 74% of the isolates into 27 clusters, while 26% remained unclustered. Clustering was more common in isolates with more then nine IS6110 copies with 76% showing clustering, while 67% of isolates with less then nine copies remained unclustered.\ud \ud M.tuberculosis isolates carrying less than five IS6110 copies were sub-typed by the VNTR method. VNTR typing identified 38 isolates as unique resulting in 38 unique VNTR profiles, while 5 profiles were shared by two strains each. Thus typing of VNTR loci can very effectively differentiate low IS6110 copy number isolates. 5ix new VNTR profiles are reported in this study.\ud \ud Results of this study show a high degree of genetic relatedness in M. tuberculosis isolates prevalent in local population. Very similar DNA fingerprints obtained by both RFLP patterns and VNTR profiles exhibit this relatedness. Though the majority of RFLP profiles showed a high degree of similarity, no identical patterns were seen in this study.\ud \ud The highly related but non-identical patterns most likely reflect the evolution of few clones circulating in this area. The majority of strains analyzed shared at least two-thirds of the IS6110 containing PvuII fragments. A significant number of these differed by the presence or absence of a single IS6110 copy indicating the highly transposable nature of this insertion sequence during the transmission of infection