Characterization of mycobacteria from a major Brazilian outbreak suggests that revision of the taxonomic status of members of the Mycobacterium chelonae-M. abscessus group is needed

Abstract

An outbreak of postsurgical infections caused by rapidly growing mycobacteria has been ongoing in Brazil since 2004. The degrees of similarity of the rpoB and hsp65 sequences from the clinical isolates and the corresponding sequences from both the Mycobacterium massiliense and the M. bolletii type strains were above the accepted limit for interspecies variability, leading to conflicting identification results. Therefore, an extensive characterization of members of the M. chelonae-M. abscessus group was carried out. The M. abscessus, M. chelonae, M. immunogenum, M. massiliense, and M. bolletii type strains and a subset of clinical isolates were analyzed by biochemical tests, high-performance liquid chromatography, drug susceptibility testing, PCR-restriction enzyme analysis of hsp65 (PRA-hsp65), rpoB, and hsp65 gene sequencing and analysis of phylogenetic trees, DNA-DNA hybridization (DDH), and restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene (RFLP-16S rRNA). The clinical isolates and the M. abscessus, M. massiliense, and M. bolletii type strains could not be separated by phenotypic tests and were grouped in the phylogenetic trees obtained. The results of DDH also confirmed the >70% relatedness of the clinical isolates and the M. abscessus, M. massiliense, and M. bolletii type strains; and indistinguishable RFLP-16S rRNA patterns were obtained. On the contrary, the separation of clinical isolates and the M. abscessus, M. massiliense, and M. bolletii type strains from M. chelonae and M. immunogenum was supported by the results of PRA-hsp65, DDH, and RFLP-16S rRNA and by the rpoB and hsp65 phylogenetic trees. Taken together, these results led to the proposition that M. abscessus, M. massiliense, and M. bolletii represent a single species, that of M. abscessus. Two subspecies are also proposed, M. abscessus subsp. abscessus and M. abscessus subsp. massiliense, and these two subspecies can be distinguished by two different PRA-hsp65 patterns, which differ by a single HaeIII band, and by differences in their rpoB (3.4%) and hsp65 (1.3%) sequences.

FIG. 1.

Comparison of representative HPLC mycolic acid patterns of members of the M. chelonae-M. abscessus group. HMWIS, high-molecular-weight internal standard.

FIG. 2.

Phylogenetic tree based on rpoB (a) and hsp65 (b) gene sequences, showing the relationships between Brazilian clinical isolates (isolates B5, B31, B60, B61, B66, and B67) and other type strains of selected rapidly growing mycobacteria. GenBank accession numbers are shown in parentheses. M. tuberculosis ATCC 27294^T was included as the outgroup.

FIG. 3.

RFLP-16S rRNA patterns of mycobacterial DNAs digested with the restriction enzymes BamHI (a) and PstI (b). Lanes 1 to 11, isolates 1 to 11, respectively, as described in footnote a of Table 1; lane 12, M. fortuitum ATCC 6841.