[Subspecies identification for Mycobacterium abscessus group].

OBJECTIVE: To identify the subspecies of Mycobacterium abscessus (M.abscessus) group.

METHODS: The corresponding genes (hsp65 and rpoB) in 19 clinical isolates (CIs) of M.abscessus group susceptible to clarithromycin were amplified by PCR. Phylogenetic analyses and subspecies identification of the hsp65 gene and rpoB gene were conducted separately and jointly by using the Mega program and PhyML program, and the conflicting species were further identified by the internally transcribed spacer (ITS) sequencing. The rrl and erm(41) of M.abscessus group detection were performed by PCR sequencing, and MICs of inducible resistance to clarithromycin were determined by the broth microdilution method, and then the phenotypic patterns of 19 isolates were analyzed.

RESULTS: Comparisons between hsp65 and rpoB sequences of the 19 clinical isolates led to the identification of 10 CIs as Mycobacterium (M). massiliense, 4 CIs as Mycobacterium (M). abscessus, and 1 CI as Mycobacterium (M). bolletii, while the other 4 isolates were identified as M. massiliense by hsp65 gene sequencing and as M.abscessus by rpoB gene sequencing. Ultimately the 4 conflicting isolates were identified as M. massiliense by ITS sequencing. No mutations in the rrl gene with clarithromycin resistance were found. The -35 sequence of the erm (41) promoter of M.abscessus was different from that of M. bolletii and M. massiliense, and the nucleotide at position 28 was polymorphic (T28 or C28); -35 sequence and the nucleotide at position 28 were the same in erm (41) for M. bolletii and M. massiliense. Fourteen M. massiliense strains shared 100% (14/14) homology for erm (41) with 276 bp deletions and 2 bp deletions, and no deletions were found in 4 CIs as M.abscessus and 1 CIs as M. bolletii. The clarithromycin inducible resistance test showed that 3 M.abscessus with T28 (CI02, CI04, CI12) and 1 M. bolletii (CI18) strains were highly resistant, and the other 14 M. massiliense and 1 M.abscessus with polymorphic C28 (CI17) strains remained susceptible. No correlations between -35 sequence of erm (41) promoter and clarithromycin inducible resistance were found.

CONCLUSIONS: Hsp65 is applicable to the identification of the subspecies of M.abscessus group, and due to the fact that subspecies of M.abscessus group shows distinct genotytpcally feature in erm (41) sequencing and phenotypic feature in clarithromycin susceptibility, hsp65 and erm (41) can be applied to the identification of subspecies of M.abscessus group, and the resulting data may be useful for clinical diagnosis and treatments.