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Mycobacterium Austroafricanum Sp

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Mycobacterium Austroafricanum Sp INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, July 1983, p. 460-469 Vol. 33, No. 3 0020-7713/83/030460-10$02.00/0 Copyright 0 1983, International Union of Microbiological Societies Numerical Taxonomy of Rapidly Growing, Scotochromogenic Mycobacteria of the Mycobacterium parafortuitum Complex: Mycobacterium austroafricanum sp. nov. and Mycobacterium diernhoferi sp. nov., nom. rev. MICHIO TSUKAMURA,’* HERMINA J. VAN DER MEULEN,2 AND WILHELM 0. K. GRABOW3 National Chubu Hospital, Obu, Aichi, Japan 474l: South African Institute for Medical Research, Johannesburg 2001, South Africa2; and National Institute for Water Research, Pretoria 0002, South Africa3 A numerical analysis of phenetic data collected from rapidly growing, scotoch- romogenic mycobacterial strains isolated from water in South Africa and from strains of the taxa Mycobacterium parafortuitum, Mycobacterium aurum, Myco- bacterium neoaurum, and “Mycobacterium diernhoferi” indicated that all of these organisms belong to the Mycobacterium parqfortuitum complex. Our results also indicated that each of the taxa mentioned above is worthy of species status within the complex. The name Mycobacterium austroafricanum sp. nov. is proposed for the South African isolates, and the characteristics of these isolates are described; the type strain is E9789-SA12441 (= ATCC 33464). The name Mycobacterium diernhoferi is revived for the organism described originally by Bonicke and Juhasz in 1965; the type strain of this species is strain 41001 (= ATCC 19340). Three species of rapidly growing, scotochro- at -20°C and were subcultured at 6-month intervals. mogenic mycobacteria, Mycobacterium para- Only the strains deposited in the American Type fortuitum (i6), Mycobacte&m aurum (7), and Culture Collection, Rockville, Md., are shown in Mycobacteriurn neoaurum (9), have not been Table 1. The type strains of all named species of clearly differentiated from each other in previ- rapidly growing, scotochromogenic mycobacteria were also included id this study (Table 1). ous numerical classification schemes (5, 14) and A total of 23 strains of rapidly growing, scotochro- have been considered members of a Mycobacte- mogenic mycobacteria were isolated from water in rium parafortuitum complex. Saito et al. (5) South Africa. These strains were isolated as follows. showed that strains of “Mycobacterium diern- Samples of water were pretreated by adding 1.25% hoferi” formed one cluster together with strains oxalic kid, which was neutralized after 10 min with of M. parafortuitum. Based on this finding, “M. 2% sodium hydroxide. After filtration, the filters were diernhoferi” was regarded as a synonym of M. incubated at 37°C for 9 days on enriched Middlebrook parafortuitum. Recently, Kusunose et al. (M. 7H9 medium contdining c ycloheximide (2). This medi- Kusunose, E. Kusunose, I. Yano, S. Toriyama, um contained 4.7 g of Middlebrook 7H9 broth base (BBL Microbiology Systems), 1 mg of malachite H. Saito, and M. Tsukamura, Kekkaku 54:219- green, 1 g of sodium propionate, 15 g of agar, 100 ml of 220,1979) found that M.parafortuitum contains Middlebrook OADC enrichment (Difco Laboratories), 54- to 60-carbon mycolic acids, whereas ‘M. 25 mg of nalidixic acid, 25,000 U of penicillin, 50,000 U diernhoferi’ ’ contains 68- to 76-carbon my colic of mycostatin, 100 mg of cycloheximide, and 1,000 ml acids. Furthermore, a number of strains of rap- of distilled water. idly growing, scotochromogenic mycobacteria A total of 107 biochemical or physiological charac- have been isolated from water in South Africa. ters were studied for each strain. Of these, 104 were These strains seem to represent a new taxon. used In asprevious study (15), with methods described The purpose of the present study was to previously (10, 12). The three additional characters used were growth at 42”C, resistance to 5-fluorouracil examine the taxonomic relationships among the (20 pg/ml) in Ogawa egg medium, and resistance to three species of the M. parafortuitum complex, mitomycin C (5 kg/ml) in Ogawa egg medium. Growth five strains of “M. diernhoferi,” and a cluster of at 42°C was assayed by incubating test strains at 42 k 23 South African strains isolated from water. 03°C for 7 days. Numerical analysis. Numerical classification was MATERIALS AND METHODS carried out by using a method described previously Bacterial strains and cultivation techniques. All (15). Clustering was made by the single-linkage meth- strains of M. parafortuitum, M. aurum, and M. od (6). According to Orchard and Goodfellow (4), neoaururn were isolated in the laboratory of the Na- single- and average-linkage methods give the same tional Chubu Hospital. These strains were maintained clusters. 460 In addition, a numerical analysis was also carried Mdtchiny Coefficient 18) out to define the ranges of species (taxon), as follows. 75 21 0 85 90 95 Lpo Ranges were measured by calculating M t 2 SD or M 2 1.7 where M is the mean matching coefficient (mean SD,M value) for individual strains of each species relative to a hypothetical median organism pattern (HMO) (3) within that species or another species and a SD is the standard deviation. The range M +- 2 SD 9 contains ca. 95% of the individual strains within a 10 * 11 species (11). For example, if two species (taxa), spe- 12 13 cies A and B, are distinct, the range for species A 14 relative to the HMO of species A should be higher than 15 16 the range for species B relative to the HMO of species 17 18 A. There should be no overlap, or the overlap should 19 occur in less than 5% of the strains within the pro- 20 21 posed species. When we calculate M - 2 SD for 22 23 species A relative to its own HMO, a range higher than I24 the lower limit of the range should contain ca. 97.5% of 25 26 the individual strains in species A. On the other hand, 27 when we calculate M for species B relative to 28 + 2 SD 29 the HMO of species A, a range lower than the upper 30 31 limit for the species should contain 97.5% of the 11 33 strains in species B. Thus, if the lower limit of species 34 A is higher than the higher limit of species B, the two 35 36 species are regarded as distinctly separate and have 37 38 either no overlap or an overlap of less than 2.5% of the 39 individual strains within the species. If the lower limit 40 41 of species A is measured as M - 1.7 SD, the higher 42 limit of species B is measured as M + 1.7 SD, and the 41 former limit is higher than the latter limit, the two 44 4546 1 species are regarded as separate. Again, there should 4) be no overlap, or an overlap of less than 5% of the 48 49 individual strains within each species (8). 50 51 52 RESULTS AND DISCUSSION 53 54 5s The results of our numerical analysis are 56 5L 57 0” shown in Fig. 1 as a dendrogram. Five clusters 58 a were observed at a matching coefficient (M 59 : 60 . value) level of 91%, although a few intermediate 61 67 strains were present between the clusters. The 63 - first cluster was composed of the strains isolated from water from South Africa, and the remain- ing four clusters contained the type strains of M. aururn, M. neoaurum, “M. diernhoferi,” and M. parafortuitum. Therefore, the latter four clusters were considered to be a complex of four distinct species, and the five groups could be combined 7 b I, into one large cluster at a level of 90%. Only one R type strain of another species, Mycobucterium 79 RO rhodesiae, was included in this large cluster. 1L a1 01 The strains and species in the five clusters are e I84 shown in Table 1, whereas the biochemical and L: I05 86 physiological properties of the groups are sum- 81 marized in Table 2. The M. parafortuiturn clus- XH 84 ter was composed of two subgroups; one con- 1 I1 91 tained the type strain of the species, and the 9’ II’ 9, other contained three strains named by Saito et J1 [J ‘j al. (5) as Kanazawa strains. 9 f, qi The M. aurum and M. neoaururn clusters each w, contained a number of strains labeled as other 0’1 1 Ull species, and these two species have been fre- 1 IJI 1111 quently confused with each other in identifica- 10 1 tion techniques. This situation was clarified by 1 u4 10, our study. The major distinguishing features of 1,717 10: these two species are summarized in Table 3. 1 ii:1 111’1 ll/l 11; I ,’ FIG. 1. Dendrogram showing relationships among 1 11 I strains studied. See Table 1 for strain serial numbers. 1!1 461 462 TSUKAMURA, VAN DER MEULEN, AND GRABOW INT.J. SYST.BACTERIOL. TABLE 1. Mycobacterial strains used and cluster arrangement Serial Laboratory Received as: Sourceb Habitat‘ Cluster no. no. ‘’ M. austroafri- 1 E9789B M. austroafricanurn SA12441T 1 Water, South Africa canurn (= ATCC 33464T) 2 E9790B M. austroafricanurn SA12442 1 Water, South Africa (= ATCC 33465) 3 E9789A M. austroafricanurn SA12441T 1 Water. South Africa (= ATCC 33464T) 4 E9790A M. austroafricanurn SA12442 1 Water, South Africa (= ATCC 33465) M. aururn 8 10005 M. neoaururn ATCC 25799 2 Soil, Japan 9 10006 M. neoaururn ATCC 25800d 2 Soil, Japan 19 10004 M. neoaururn ATCC 25798 2 Soil, Japan 23 10001 M. neoaururn ATCC 25791 2 Soil, Japan 31 15012 M. aururn Lausanne 708 3 Soil, Japan 32 15061 M. aururn ATCC 25792 2 Soil, Japan 34 15035 M. aururn ATCC 25803 2 Soil, Japan 37 15013 M. aururn ATCC 25793 2 Soil, Japan 40 15006 M. aururn ATCC 23366T 2 Soil, Japan (= NCTC 10437T) 41 15067 M.
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