INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, July 1981, p. 263-275 Vol. 31, No. 3 0020-7713/81/030263-13$02.00/0 Numerical Analysis of Rapidly Growing, Scotochromogenic Mycobacteria, Including Mycobacterium o buense sp. nov., norn. rev., Mycobacterium rhodesiae sp. nov., nom. rev., Mycobacterium aichiense sp. nov., norn. rev., Mycobacterium chubuense sp. nov., norn. rev., and Mycobacterium tokaiense sp. nov., nom. rev. MICHIO TSUKAMURA, SHOJI MIZUNO, AND SUM10 TSUKAMURA The National Chubu Hospital, Obu, Aichi, Japan 474 We performed numerical analyses of 155 strains of rapidly growing, scotochrom- ogenic mycobacteria by two different methods; in one method we used 104 characters, and in the other we used 84 characters. The following taxa appeared as distinct clusters: Myco bacterium thermoresistibile, Myco bacterium flavescens, Mycobacterium duvalii, Mycobacterium phlei, “Mycobacterium o buense,” My- co bacterium parafortuitum, Mycobacterium vaccae, Mycobacterium sphagni, “Mycobacterium aichiense,” “Mycobacterium rhodesiae,” Mycobacterium neoaurum, “Mycobacterium chubuense,” “Mycobacterium tokaiense,” and My- cobacterzum komossense (names in quotation marks are not on the Approved Lists of Bacterial Names). M. flavescens strains were divided into two subgroups, one consisting of strains isolated in Japan and the other consisting of strains isolated in Rhodesia and strains received from the American Type Culture Collection, including the type strain of M. flavescens (ATCC 14474). We found that there are many species of rapidly growing, scotochromogenic mycobacteria, and we believe that new species should be recognized and named on the basis of at least three strains. The following species appeared to be distinct from all presently named species: “Mycobacteriumgallinarum,” “Mycobacterium armen- tun,” “Mycobacterium pelpallidurn,” and “Mycobacterium taurus.” However, each of these species was proposed on the basis of only one or two strains. Because of the small number of strains studied, formal proposals for these species should not be made until more strains of each taxon are studied. Furthermore, we consider “Mycobacterium magistrae” and “Mycobacterium auratum” to be syn- onyms of M. flavescens, “Mycobacterium pallidum” to be a synonym of M. parafortuitum, “Mycobacterium liquefactum” to be a synonym of M. vaccae, and “Mycobacterium sufflavum” to be a synonym of M. neoaurum. “M. obuense,” “M.rhodesiae,” “M. aichiense,” “M. chubuense,” and “M.tokaiense” are not on the Approved Lists of Bacterial Names, but based on the results of this study, we consider these organisms distinct species. Therefore, these names are revived for the same organisms with which they were originally associated. The type strains of these species are strain 47001 (=ATCC 27023 =NCTC 10778), strain 02002 (=ATCC 27024 -NCTC 10779), strain 49005 (=ATCC 27280 =NCTC 10820), strain 48013 (=ATCC 27278 =NCTC 10819), and strain 47503 (=ATCC 27282 =NCTC 10821), respectively. Mycobacterium flavescens Bojalil et al. (1) were published before 1 January 1980, were not was proposed as a new species in 1962. Bojalil et included in the Approved Lists of Bacterial al. also proposed Mycobacterium acapulcensis Names [9], and have not been validly published as a new species, but later Tsukamura (18) since 1 January 1980). This organism resembles showed that this organism is identical to M. M. flavescens, and this name was considered by flavescens. At present, the name M. flavescens Tsukamura (unpublished data) to be a synonym is used for this organism. Tsukamura et al. (33) of M. flavescens. However, Lechevalier et al. (6) proposed “Mycobacterium gallinarum” as a differentiated M. flavescens and “M. gallina- new species in 1967 (names in quotation marks rum” on the basis of their lipid compositions. 263 264 TSUKAMURA, MIZUNO, AND TSUKAMURA INT. J. SYST.BACTERIOL, Tsukamura and Miznuo (26) reported that thin- gave the same result) for differentiating among the layer chromatography of ethyl ether-ethanol cel- test strains: strong acid fastness; weak or partial acid lular extracts after incubation with [35S]methi- fastness; glucose as C source (glutamate N); acetate as onine was useful in differentiating among my- C source (glutamate N); succinate as C source (gluta- mate N); pyruvate as C source (glutamate N); per- cobacterial species. When this technique was manent mycelium; fragmenting mycelium; cord for- used with these organisms, they were classified mation; production of niacin; and benzamide as simul- into two subgroups; subgroup one consisted of taneous N and C sources. The first six of these char- “M. gallinarum” and some strains labeled M. acters were positive in all test strains, and the remain- flavescens, and the other consisted of the re- ing five were negative in all test strains. maining strains labeled M. flavescens (28).Thus, We used two systems of numerical classification; in M. flavescens was regarded as consisting of two one, we used all 104 characters, and in the other we subgroups. used 84 characters. The 20 characters omitted in the The purpose of this study was to clarify the 84-character system were all of the ineffective char- taxonomy of M. flauescens by using numerical acters described above except niacin production and utilization of benzamide as simultaneous N and C analyses. Furthermore, we studied a number of sources, four characters for which almost all of the strains recently received as members of new strains showed positive results (growth on 1 pg of species, some of which are on the Approved Lists thiophene-2-carboxylic acid hydrazide per ml, growth of Bacterial Names (9) and some of which are on 0.5 mg of salicylate per ml, growth on 1%Tween not, together with strains of older named species. 80, and growth on 0.5 mg of p-nitrobenzoic acid per ml), three related characters (24) (growth on 1%picric MATERIALS AND METHODS acid, growth on 0.2% NaN02, and nitrate reduction at Bacterial strains. We used 155 strains of rapidly 6 h), and four tests whose results were regarded as not growing, scotochromogenicmycobacteria in this study easily reproducible because of difficulties in reading the results (cross-barring, acetamde as N source, urea (Table 1). Phenotypic characters. For each strain, we ex- as N source, and pyrazinamide as N source). The two amined 104 characters (Table 2). We tested several ineffective characters which were retained were useful strains in duplicate to confirm the reliability of the in differentiating the strains of rapidly growing, non- tests; however, only one set of data was used for each photochromogenic mycobacteria which were included of these strains (Table 3). The methods which we used in a subsequent study. have been described previously (15, 17,21). Media. We used Ogawa egg medium and a modified Sauton agar medium as basal media. The composition RESULTS of the Ogawa egg medium was as follows: basal solu- Almost identical results were obtained with tion (1%KH2P04 and 1%sodium glutamate), 100 ml; the two numerical classification systems used. whole eggs, 200 ml; glycerol, 6 ml; and a 2% aqueous Therefore, only the data from the 84-character solution of malachite green, 6 ml. This medium was method are shown in Fig. 1 to 3. Nine strains poured in 7-ml quantities into tubes (165 by 16.5 mm), were tested in duplicate. Of these, seven had 95 and after sterilization at 90°C for 60 min, the medium was made into slants. The composition of the modified to 99% matching coefficients, thus showing the Sauton agar was as follows: glycerol, 30 ml, KHzP04, reproducibility of the test results. Two strain 0.5 g; MgS04.7H20, 0.5 g; citric acid, 2.0 g; ferric pairs (strains 57 and 58 and strains 95 and 96) ammonium citrate, 0.05 g; sodium glutamate, 4.0 g; showed 92 or 93%similarity in both systems. purified agar, 20.0 g; and distilled water, 970 ml. Before Numerical classification with 104 char- sterilization, the pH of this medium was adjusted to acters. At a similarity level of 91%, we differ- 7.0 by adding 10% KOH; 7 ml of medium was poured entiated 14 clusters, containing the following into each tube, which was slanted after sterilization of species: (i) Myco bacterium thermoresistibile; the medium at 120°C for 20 min. Unless noted other- wise, the tubes were observed for growth after incu- (ii) Mycobacterium duvalii, M. flavescens, and bation at 37°C for 14 days. Acid formation from car- two strains received as “Mycobacterium magis- bohydrates was determined after incubation at 28°C true” and “Mycobacterium auratum;” (iii) My- for 14 days. cobacterium phlei; (iv) “Mycobacterium ob- Numerical classification. Matching coefficients uense” (except one strain); (v) Mycobacterium were calculated as described previously (30). Cluster- parafortuitum;(vi) Myco bacterium vaccae; (vii) ing was done by the single-linkage method (10). The Mycobacterium sphagni (4) (received as Sph computer used was an IBM 53-370 (model 135), which group); (viii) “Mycobacterium aichiense;” (ix) is located in the Nagoya Factory of the Sumitomo “Mycobacterium rhodesiae;” (x) Myco bacte- Machinery Co., Obu, Aichi, Japan. The data for the (xi) original “strain x characters” matrix have been depos- rium neoaurum; Mycobacterium aurum ited with the World Data Center for Microorganisms, (only one strain tested); (xii) “Mycobacterium Brisbane, Australia. chu buense;” (xiii) “Mycobacterium tokaiense;” Of the 104 phenotypic characters examined, 93 were and (xiv) Mycobacterium komossense (5) (re- used to differentiate among the test strains. The fol- ceived as MA group). lowing 11 characters were ineffective (i.e., all strains M. flauenscens and M. duvalii could be sep- VOL. 31,1981 RAPIDLY GROWING SCOTOCHROMOGENIC MYCOBACTERIA 265 TABLE1. Strains used in this study Strain Labora- Origin Received as: Sourceb no.a tory no. Habitat Country 1 01002 M. thermosresistibile ATCC 19528 ATCC soil 2 01039 M. thermoresistibile ATCC 19527 (type) ATCC soil 3 01022 M. thermoresistibile MT soil 4 01023 M. thermoresistibile MT soil 5 01024 M. thermoresistibile MT soil 6 01025 M. thermoresistibile MT soil 7 01027 M. thermoresistibile MT soil 8 01028 M.