MOLECULAR DEFINITION OF UNIQUE SPECIES STATUS OF MYCOBACTERIUM-W - A CANDIDATE LEPROSY VACCINE STRAIN

Mycobacterium w, a candidate leprosy vaccine strain, is an atypical cu ltivable mycobacterium. Based on its growth and metabolic properties, M. w was listed in Runyon Group IV, along with other rapid growers suc h as M. fortuitum, M. smegmatis, M. chelonae and M. vaccae. However, M . w was not fully identical to any one of these. In the present study, a molecular biology approach was used to define the species identity of M. w in a manner that allows reliable comparison to be made with ov er 30 known mycobacterial species. A 383-bp region, present at the ami no terminus of the conserved mycobacterial 65-kDa gene, has been polym erase chain reaction (PCR) amplified in M. w and the DNA sequence was determined. A comparison of the M. w DNA sequence with those of M. tub erculosis, M. avium, M. paratuberculosis and M. fortuitum revealed a s pecies-specific polymorphism, i.e., the presence of nucleotide substit utions unique to M. w. In an alternate approach, a 441-bp region, also a part of the 65-kDa gene, has been PCR amplified in M. w and a Hae I II restriction pattern was generated. The 142/127/59-bp Hae III patter n of M. w was found to be unique when compared with M. tuberculosis H3 7Rv, M. bovis, M. avium, M. intracellulare, M. scrofulaceum, M. kansas ii, M. gastri, M. gordonae, M. shimoidei, M. malmoense, M. haemophilum , M. terrae, M. nonchromogenicum, M. triviale, M. marinum, M. flavesce ns, M. simiae, M. szulgai, M. xenopi, M. asiaticum, M. aurum, M. smegm atis, M. vaccae, M. fortuitum subsp. fortuitum, M. fortuitum subsp. pe regrinum, M. chelonae subsp. chelonae, M. chelonae subsp. abscessus an d M. genavense; mycobacteria for which the 441-bp Hae III patterns hav e been documented in the literature. These results established the spe cies identity of M. w at the nucleotide level.