Sequencing of hsp65 distinguishes among subsets of the Mycobacterium avium complex

Abstract

The Mycobacterium avium complex consists of epidemiologically distinct subsets. The classification of these subsets is complicated by a number of factors, including the ambiguous results obtained with phenotypic and genetic assays and the recent appreciation that human and avian strains appear to be distinct. In previous work, sequencing based on a 441-bp portion of the hsp65 gene has proven to efficiently classify isolates within the Mycobacterium genus but provides low resolution for distinguishing among members of the M. avium complex. Therefore, in this study, we have targeted the more variable 3' region of the hsp65 gene to determine whether it can effectively discriminate M. avium complex isolates at the levels of species and subspecies. Primers designed for this target consistently generated amplicons for all organisms classified as M. avium complex. Sequences obtained indicate that M. intracellulare is genetically divergent from M. avium organisms, and distinct sequevars were obtained for M. avium subsets, including M. avium subsp. avium (bird type), M. avium subsp. hominissuis, and M. avium subsp. paratuberculosis. In addition, sequence differences served to distinguish bovine from ovine strains of M. avium subsp. paratuberculosis. A unique profile for M. avium subsp. silvaticum was not obtained. These results indicate that sequencing the 3' region of the hsp65 gene can simply and unambiguously distinguish species and subspecies of the M. avium complex.

FIG. 1.

(A) Phylogenetic analyses of hsp65 sequence data spanning 360 bp of the 441-bp fragment with previously described sequevars (GenBank accession numbers in parentheses) (31), with M. kansasii ATCC 12478^T used as the outgroup. This region corresponds to hsp65 positions 190 to 549. (B) Phylogenetic analysis of the 3′ region of hsp65, from positions 612 to 1588, for the M. avium complex subsets shown in panel A. For the 3′ region, M. intracellulare ATCC 13950^T (code 10) is provided as the outgroup to emphasize the distinctions among codes 1 through 9. The bar scale represents the number of base pair variations.