Differentiation of Mycobacterium ulcerans, M. marinum, and M. haemophilum: mapping of their relationships to M. tuberculosis by fatty acid profile analysis, DNA-DNA hybridization, and 16S rRNA gene sequence analysis

Abstract

Although Mycobacterium ulcerans, M. marinum, and M. haemophilum are closely related, their exact taxonomic placements have not been determined. We performed gas chromatography of fatty acids and alcohols, as well as DNA-DNA hybridization and 16S rRNA gene sequence analysis, to clarify their relationships to each other and to M. tuberculosis. M. ulcerans and M. marinum were most closely related to one another, and each displayed very strong genetic affinities to M. tuberculosis; they are actually the two mycobacterial species outside the M. tuberculosis complex most closely related to M. tuberculosis. M. haemophilum was more distinct from M. ulcerans and M. marinum, and it appeared to be as related to these two species as to M. tuberculosis. These results are important with regard to the development of diagnostic and epidemiological tools such as species-specific DNA probes and PCR assays for M. ulcerans, M. marinum, and M. haemophilum. In addition, the finding that M. ulcerans and M. marinum are more closely related to M. tuberculosis than are other pathogenic mycobacterial species suggests that they may be evaluated as useful models for studying the pathogenesis of M. tuberculosis. M. marinum may be particularly useful in this regard since strains of this species grow much more rapidly than M. tuberculosis and yet can cause systemic disease in immunocompromised hosts.

FIG. 1

Principal-component plots based on the fatty acid profiles of the individual mycobacterial strains. (A) Component 1 versus component 2. (B) Component 1 versus component 3. (C) Component 2 versus component 3. Each point represents one strain. M. tub., M. tuberculosis; M. ulc., M. ulcerans, M. haem., M. haemophilum.

FIG. 2

Graphic presentation of the mean RBRs for M. ulcerans (M. ulc.), M. marinum (M. mar.), M. tuberculosis (M. tb.), and M. haemophilum (M. haem.) with total genomic DNAs as radioactively labelled probes. For M. marinum the results obtained by using the genomic DNA probe (left bars) and the cosmid clone pMm5 (right bars) are shown.

FIG. 3

Phylogenetic tree based on the alignment of partial 16S rRNA gene sequences illustrating the positions of strains of M. ulcerans, M. marinum, M. tuberculosis, and M. haemophilum and other slowly growing mycobacterial species. The tree was rooted by use of N. asteroides as an outgroup. The bar indicates a 10-nucleotide (10-nt) difference.