Molecular definition of unique species status of Mycobacterium w; A candidate leprosy vaccine strain

Mycobacterium w, a candidate leprosy vaccine strain, is an atypical cultivable mycobacterium. Based on its growth and metabolic properties, M. w was listed in Runyon Group IV, along with other rapid growers such 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 over 30 known mycobacterial species. A 383-bp region, present at the amino terminus of the conserved mycobacterial 65-kDa gene, has been polymerase 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. tuberculosis, M. avium, M. paratuberculosis and M. fortuitum revealed a species-specific polymorphism, i.e., the presence of nucleotide substitutions 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 III restriction pattern was generated. The 142/127/59-bp Hae III pattern of M. w was found to be unique when compared with M. tuberculosis H37Rv, M. bovis, M. avium, M. intracellulare, M. scrofulaceum, M. kansasii, M. gastri, M. gordonae, M. shimoidei, M. malmoense, M. haemophilum, M. terrae, M. nonchromogenicum, M. triviale, M. marinum, M. flavescens, M. simiae, M. szulgai, M. xenopi, M. asiaticum, M. aurum, M. smegmatis, M. vaccae, M. fortuitum subsp. fortuitum, M. fortuitum subsp. peregrinum, M. chelonae subsp. chelonae, M. chelonae subsp. abscessus and M. genavense; mycobacteria for which the 441-bp Hae III patterns have been documented in the literature. These results established the species identity of M. w at the nucleotide level.