Previous investigations demonstrated three taxonomic groups among 22 clinical isolates of Mycobacterium smegmatis. These studies were expanded to 71 clinical isolates, of which 35 (49%) (group 1) were identical to five ATCC reference strains including the type strain ATCC 19420'. Twenty-eight isolates (39%) were group 2, and eight isolates (11 %) were group 3. Isolates of groups 2 and 3 were most often associated with post-traumatic or post-surgical wound infections including osteomyelitis, were susceptible to sulfamethoxazole, amikacin, imipenem and the tetracyclines, variably resistant to clarithromycin, and susceptible (group I ) , intermediately resistant (group 2) or resistant (group 3) to tobramycin. The three groups were similar by routine biochemical and growth characteristics, but had different mycolic acid dimethoxy-4-coumarinylmethyl ester elution patterns by HPLC and different PCR-restriction enzyme patterns of a 439 bp fragment of the hsp-65 gene. Group 3 isolates differed from group 1 by 18 bp by 165 rRNA sequencing and exhibited < 25% homology by DNA-DNA hybridization, being most closely related to Mycobacterium mageritense.

Previous studies have indicated that the conventional tests used for the identification of mycobacteria may (i) frequently result in erroneous identification and (ii) underestimate the diversity within the genus Mycobacterium. To address this issue in a more systematic fashion, a study comparing phenotypic and molecular methods for the identification of mycobacteria was initiated. Focus was given to isolates which were difficult to identify to species level and which yielded inconclusive results by conventional tests performed under day-today routine laboratory conditions. Traditional methods included growth rate, colonial morphology, pigmentation, biochemical profiles, and gas-liquid chromatography of short-chain fatty acids. Molecular identification was done by PCR-mediated partial sequence analysis of the gene encoding the 16S rRNA. A total of 34 isolates was included in this study; 13 of the isolates corresponded to established species, and 21 isolates corresponded to previously uncharacterized taxa. For five isolates, phenotypic and molecular analyses gave identical results. For five isolates, minor discrepancies were present; four isolates remained unidentified after biochemical testing. For 20 isolates, major discrepancies between traditional and molecular typing methods were observed. Retrospective analysis of the data revealed that the discrepant results were without exception due to erroneous biochemical test results or interpretations. In particular, phenotypic identification schemes were compromised with regard to the recognition of previously undescribed taxa. We conclude that molecular typing by 16S rRNA sequence determination is not only more rapid (12 to 36 h versus 4 to 8 weeks) but also more accurate than traditional typing.

A number of mycobacterial strains with similar growth characteristics, metabolic properties, and lipid compositions, which were previously placed in the Helsinki group (E. Brander, E. Jantzen, R. Huttunen, A.Juntunen, and M.-L. Katila, J. Clin. Microbiol. 301972-1975Microbiol. 301972- , 1992, were characterized by performing 16s rRNA gene sequencing. Of the 14 strains studied, 9 had a unique, previously undescribed sequence in the variable region of 16s rRNA. These nine strains, all of which were isolated from respiratory tract specimens, were nonpigmented and grew at 25 C to 45"C, reaching full colony size after 2 to 3 weeks. They produced arylsulfatase, nicotinamidase, and pyrazinamidase and were negative for lbeen 80 hydrolysis, catalase, urease, and nitrate reductase activities, and niacin. Their glycolipid patterns were identical. A mycolic acid analysis performed by using thin-layer chromatography showed that these organisms contained alpha-mycolates, ketomycolates, and carboxy mycolates. Gas-liquid chromatography revealed that 2-eicosanol was the major alcohol and hexacosanoic acid was the major mycolic acid cleavage product. On the basis of their growth, biochemical, and lipid characteristics and their unique 16s rRNA sequence, we propose that these organisms should be assigned to a new species, Mycobacerium branderi. Comparative 16s rRNA sequencing revealed that this new species is closely related to Mycobacterium celahcm, Mycobacterium cmkii, and Mycobacterium xenopi.Strains 52157T (T = type strain) and 43548 have been deposited in the American Type Culture Collection as strains ATCC 51789 and ATCC 51788, respectively.The taxonomic position of a collection of 14 strains of slowly growing mycobacteria that were identified previously on the basis of biochemical activity and lipid composition as members of a potential new species (the "Helsinki group") (3) was determined by analyzing of their 16s rFWA gene sequences. These strains were isolated from clinical samples in Finland between 1972 and 1990 and were stored at -70°C because their taxonomic identities were ambiguous. These organisms resembled Mycobacteriurn xenopi, members of the Mycobacterium avium complex, and Mycobacten'um shirnoidei in growth characteristics (3,20) but could be distinguished from them by their fatty acid and alcohol compositions and the results of glycolipid analyses. By using gene sequencing, the group was found to comprise two genetic entities, one identical to the recently described species Mycobacterium celaturn (4) and the other an undescribed taxon. In this paper we describe the + -The data for all taxa except M. branderi and M. celatum are from references 7, 11, 17, 20, and 21. 14-day test. Semiquantitative catalase test (<45 mm was a negative reaction). dResults of analyses of nine strains whose sequences were identical. The catalase test was negative (<30 mm) for all strains. In the nicotinamidase and pyrazinamidase tests, the strains exhibited trace to 2+ reactions. Symbols: +, more than 80% of the strains were posit...