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During previous cooperative numerical taxonomic studies of slowly growing mycobacteria, the International Working Group on Mycobacterial Taxonomy described a number of strains whose taxonomic status was ambiguous. A new study of DNA, RNA, and proteins from 66 of these organisms was performed to correlate their properties with phenotypic clustering behavior; the results of this study permitted 51 of the strains studied to be assigned to known species. The methods used to characterize the semantides included nucleotide sequencing and assessment of levels of semantide relatedness by affinity binding techniques, including whole DNA-DNA hybridization, probe hybridization, and antibody binding. There was good overall agreement between the phenotypic and chemotaxonomic clusters and the groups of organisms identified by semantide analyses. Our results supported the conclusion that we should continue to rely on polyphasic taxonomy to provide satisfactory systematic resolution of members of the genus Mycobacterium. We identified no single 16S rRNA interstrain nucleotide sequence difference value that unequivocally defined species boundaries. DNA-DNA hybridization remains the gold standard, but common resources are needed to permit DNA-DNA hybridization analyses to be made available to laboratories that are not prepared to use this technology. One of the large novel clusters which we studied corresponds to the recently described species Mycobacterium interjectum, a pathogen that resembles the nonpathogen Mycobacterium gordonae phenotypically. We also identified strains that appear to represent ribovars of Mycobacterium intracellulare which do not react with the commercial diagnostic probes that are currently used for identification of this species. Other branches or clusters consisted of too few strains to permit a decision about their taxonomic status to be made.

Strains of a new type of slowly growing scotochromogenic, rose-pink-pigmented mycobacterium were isolated repeatedly from sphagnum vegetation, true moss, and soil in Ireland. These strains grew at 22, 31, and 37 degrees C but not at 45 degrees C and possessed acid phosphatase and arylsulfatase activities. They reduced nitrate, tolerated 0.1% NaNO2, did not split amides, and were resistant to most of the antituberculous drugs tested, except ethambutol. They did not form acid from glucose and mannose. Their internal phenetic similarity was 97.08% +/- 2.07%. The whole mycolate pattern confirmed the homogeneity of the taxa sharing similar mycolate types with several other mycobacterial species. However, on the basis of the nature of the major pyrolysis esters, the taxon appeared unique. The phylogenetic analysis based on evolutionary distance values revealed that the strains belong to a new species of slowly growing mycobacteria. The DNA-DNA hybridization values confirmed that these strains differ significantly from Mycobacterium nonchromogenicum, M. terrae, M. triviale, and M. thermoresistibile. The strains produced a unique rose-pink pigment and were nonpathogenic for mice, guinea pigs, and rabbits, but they provoked a nonspecific hypersensitivity reaction to bovine tuberculin in guinea pigs and cattle. Hence, they are considered a member of a new species of nonpathogenic slowly growing mycobacteria, for which the name Mycobacterium hiberniae is proposed. Strain Hi 11 is the type strain, a culture of which has been deposited in the American Type Culture Collection as strain ATCC 49874.