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 5 1 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 interjecturn, a pathogen that resembles the ilonpathogen Mycobacterium gurdonae 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.Approximately 17 years ago the International Working Group on Mycobacterial Taxonomy (IWGMT) began a cooperative open-ended study in which phenotypically unusual strains of s l o~l y growing mycobacteria were collected on a continuing basis. These strains were distributed to participants in the open-ended study for characterization by a broad range of predominantly phenotypic tests, and at intervals the data obtained were subjected to numerical taxonomic (NT) analyses (29-32). The purpose of studying an expanding set of cultures was to characterize slowly growing mycobacterial strains that either represented uncommonly encountered species that had not been represented in previous cooperative studies (10, 25, 28) or belonged to clusters of previously unrecognized taxa. These analyses yielded expanded phenotypic characterizations of members of some clusters that had not been thoroughly characterized before, such as the clusters that included the type strains of Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium asiaticum, and Mycobacterium malmoense (3 1). However, some individual strains and phenotypic clusters emerged that exhibited no unequivocal aff...

The open-ended study of the International Working Group on Mycobacterial Taxonomy is an ongoing project to characterize slowly growing strains of mycobacteria that do not belong to well-established or thoroughly characterized species. In this fourth report we describe two numerical taxonomic clusters that represent subspecies or biovars of Mycobacterium simiae, one cluster that encompasses the erstwhile type strain of the presently invalid species "Mycobacterium parafinicum," one cluster that is phenotypically very similar to Mycobacterium avium and Mycobacterium intraceltulare but may be a separate genospecies, one cluster that appears to be phenotypically distinct from M . aviurn but reacts with a nucleic acid probe specific for M . avium, and three tentatively defined clusters in proximity to a cluster that encompasses the type strain of Mycobacterium malmoense. Of special practical interest is the fact that one of the latter three clusters is composed of clinically significant scotochromogenic bacteria that can be misidentified as the nonpathogenic organism Mycobacterium gordonae if insufficient biochemical tests are performed.In their first cooperative studies, the members of the International Working Group on Mycobacterial Taxonomy (IWGMT) applied numerical taxonomic (NT) analysis methods to discrete and limited sets of mycobacteria to provide broad-based characterizations of clusters that corresponded to previously established species (5,10,12,16,26,31). Subsequently, a cooperative open-ended study was initiated in which new strains of slowly growing mycobacteria were introduced on a continuing basis, and the data were analyzed at intervals (32-34). The purpose of this study of an expanding set of cultures was to characterize slowly growing mycobacterial strains that represented uncommonly encountered species that had not been represented in the first six studies, as well as to recognize clusters of previously unknown taxa.A total of 38 new strains have been added to the openended study since the most recent report (341, and most of these fell into groups that exhibited phenotypic affinities to clusters that encompass the type strains of Mycohacterium simiae, M . intracellulare, M . avium, M . scrofulaceum, M . malmoense, and the presently invalid species " M . parafinicum." As noted in the previous report (34), these clusters are phenotypically less clearly resolved from one another than clusters that represent other species of slowly growing mycobacteria, and in this study we analyzed the expanded data base for these organisms. MATERIALS AND METHODS Selection of strains.The criteria for introducing new cultures into this study have been described previously (32,34 Editing and analysis of data. The criteria and editing programs for suppression of irrelevant, redundant, and incomplete data that were used in generating the NT matrices have been described previously (25,(32)(33)(34). Features were coded according to the RKC scheme of Rogosa et al. (15).NT analyses were based on simple matching coefficients,...