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RAPIDLY GROWING, ACID FAST BACTERIA' Original 21 of This Species RAPIDLY GROWING, ACID FAST BACTERIA' II. SPEcIES' DESCRPTION OF Mycobacteriumfortuitum CRUZ RUTH E. GORDON AND MILDRED M. SMITH Institute of Microbiology, Rutgers University, the State University of New Jersey, New Brunswick, New Jersey Received for publication October 13, 1954 The taxonomic study of the acid fast bacteria the following medium, a modification of Koser's capable of comparatively rapid growth on citrate agar (1924): NaCl, 1 g; MgSO4, 0.2 g; ordinary media, first reported in 1953 by Gordon (NH4)2HP04, 1 g; KH2PO4, 0.5 g; Na benzoate, and Smith, has been continued. Additional 2 g; agar, 15 g; distilled water, 1,000 ml. The strains have been examined and other tests ap- pH of the agar was adjusted to 7.0, and 20 ml plied to all the strains. A few supplementary of a 0.04 per cent solution of phenol red were characteristics of the two previously delineated added. An alkaline reaction of the medium in- species, Mycobacterium phlei Lehmann and dicated use of the benzoate. Neumanm and Mycobacterium smgmatis (Trevi- Acid from carbohydrats. Maltose and trehalose san) Lehmann and Neumann, are presented, and were used in conjunction with the carbohydrates the strains newly assigned to these species are previously listed. listed. As the work progresed, a third group of strains DESCRIPONS OF SPECIES emerged. The strains of this taxon seemed The collection2 of mycobacteria forming the closely related to each other and sufficiently basis of this taxonomic study increased from distinct from the other strains of the collection 124 of first to 195. The to warrant their separation into a species. A the strains the report description of this third species, Mycobacterium named strains, as received, represented 23 fortuitum Cruz, is given and compared with different species; the unnamed ones were isolated those of M. and M. from a variety of sources. Of the 195 strains, 68 phlei smegmatis. per cent were assigned to the following three MATERIALS AND METHODS taxa: In addition to the observations and tests used Mycobacterium phle Lehmann and Neumann by Gordon and Smith (1953), the following ones were employed: The six strains (three received as M. phlei Decomposition of tyrosine. The cultures were and three unnamed isolations) added to the streaked on plates of tyrosine agar and examined original 21 of this species (Gordon and Smith, after 14 and 21 days' incubation at 28 C for 1953, p. 43) are listed in table 1. General agree- the disappearance of the tyrosine crystals un- ment on the identity of M. phlei was again derneath and around the growth. The agar con- evidenced by the correct identification of the tained peptone, 5 g; beef extract, 3 g; agar, 15 g; three named strains and the absence of mis- tyrosine, 5 g; distilled water, 1,000 ml; pH 7.0. named strains and of strains bearing specific The medium was thoroughly mixed during each names in synonymy. step in its preparation to provide a final, even Cultures of the six strains grown on glycerol distribution of the insoluble tyrosine throughout agar for 5 days at 28 C were 25 to 80 per cent the agar. acid fast. The cells varied from coccoid forms Utilization of benzoate. The cultures were in- to short filaments, and some had granular oculated and incubated at 28 C for 4 weeks on protoplasm. The colonial morphology, macro- scopic appearance, temperature relationships, and 1 This investigation was supported in part by a research grant, no. E 157-E 157 (C2), from the 2 The writers wish to express their sincere ap- National Institutes of Health, Public Health preciation to the investigators who kindly sup- Service. plied the cultures for this study. 502 1955] RAPIDLY GROWING, ACID FAST BACTERIA 503 TABLE 1 no. 465 of Mycobacterium butyricum and strain Strains identified as Mycobacterium phlei Lehmann no. 446 of Mycobacterium friedmannii were in and Neumann agreement with others bearing the same labels and previously assigned to the synonymy of Laboratory Name and Source Number M. smegmati. The three strains received as 464 Mycobacterium phlei Lehmann and Mycobacterium ranae also conformed to those Neumann; W. Steenken, Jr., Tru- examined earlier and were identical with strains deau Lab. 468 Mycobacterium phlei; G. Penso, Isti- TABLE 2 tuto Superiore de Sanith, Rome Strains identified as Mycobacterium smegmatis (Timoteo 2X) (Trevisan) Lehmann and Neumann 500 Mycobacterium phlei; A. Abrams, Army Med. Services Grad. School Laboratory Name and Source (52-R-1) Number 451 Mycobacterium sp; D. W. Bruner, Cor- 447 Mycobacterium aquae Maie; CCTM nell Univ. (57); C. A. Frey (soil) (7) 479 Mycobacterium sp; P. Stuart, Minis- 465 Mycobacterium butyricum Bergey et try of Agr. and Fisheries, England al.; W. Steenken, Jr., Trudeau (MS. 247.52, bovine mastitis) Lab. 501 Mycobacterium sp; A. Abrams (52-B-1, 446 Mycobacteriumfriedmannii Holland; associated with leprous lesions) CCTM (178) 540 to 542 Mycobacterium ranae (Kuster) Ber- gey et al.; J. Hsie, Still College (1, other reactions agreed with those of the pre- original wild type; 2, isoniazid viously described strains of M. phlei. All grew resistant, slow growing; 5, isonia- from 28 C to 52 C inclusive; none, at 10 C; and zid resistant, fast growing) all survived 60 C for 4 hours. Only one culture 108, 114M Mycobacterium spp; E. B. McKin- (no. 451) hydrolyzed gelatin to a limited extent; ley, George Washington Univ. none decomposed casein or grew in broth con- 422 Mycobacterium sp; Isabel Christi- taining 5 per cent NaCl. The six strmins utilized son, Duke Univ. (317); D. K. citrate, succinate, and malate; reduced nitrate Miller to 450 Mycobacterium sp; D. W. Bruner, nitrite; hydrolyzed starch; and formed acid Cornell Univ. (50); C. A. Frey from glucose, xylose, arabinose, mannose, galac- (soil) tose, mannitol, and sorbitol. Acid formation 454 Mycobacterium sp; D. W. Bruner from rhamnose, inositol, dulcitol, lactose, and (135); W. Willie (soil) raffinose was negative. 455 Mycobacterium sp; D. W. Bruner The following reactions of the 27 strains in this (166); Ruth E. Gordon (soil) collection identified as M. phlei were deter- 470 to 472 Mycobacterium spp; P. Stuart, Min- mined and used to supplement the description istry of Agr. and Fisheries, Eng- of the species: land (MS.231.52"S", MS.231.52 bovine mas- Decomposition of tyrosine. All cultures were "R", MS.104.51"S", titis) (Stuart and Harvey, 1951) negative. 474 to 477 Mycobacterium spp; P. Stuart (MS. Utilization of benzoate. All cultures were nega- 302.51"S", MS.302.51"R", MS. tive. 48.52"S", MS.48.52"R", bovine Acid production from carbohydrates. Two mastitis) cultures (nos. N8151 and M354, of the 27 were 497, 498 Mycobacterium spp; M. Panisset, able to form acid from maltose; 18 formed acid Univ. of Montreal (7A, 1OA); G. from trehalose. Penso 573, 574 Mycobacterium spp; S. R. Kham- Mycobacterium smegmadis (Trevisan) Lehmann bata, St. Xavier's College, India and Neumann (O1, 012, intestinal flora of earth- Eighteen of the 24 additional strains assigned worm) 4242 C. W. to this species were unidentified isolations, and Mycobacterium sp; ATCC; Duval (Levy-Kedrowski 115, as- the remaining six were received as representa- sociated with tives of four different species (see table 2). Strain leprous lesions) 5045RUTH E. GORDON AND MILDRED M. SMITH [voL. 69 of M. megmatis. Because of their disagreement in the separation from the species of two strains, with the original account of M. mranae (KEster, nos. 283 and 4236 (Gordon and Smith, 1953, p. 1905), they had to be regarded as misnamed. 45), previously assigned to M. smegmatis. Varia- Strain no. 447 was the first obtained under the tion from the normal in one or two reactions of name Mycobacterium aquae. Galli-Valerio (1912) the species' pattern was considered of no taxo- mentioned acid fast organisms present in scrap- nomic significance. Strains nos. 283 and 4236, ings from water faucets but gave them no however, differed in too many respects from the description or name. Later, Maie (1922) de- enlarged series of characteristics to be accepted scribed an acid fast strain from the water pipes of as belonging to the species and were again placed Lausanne and referred to it as M. aquae Galli- with the unclassified strains of the collection. Valerio. In spite of a careful search, no paper Except for a few, uncorrelated, minor variations, by Galli-Valerio naming and characterizing the the remaining 54 strains previously identified as species has been found to precede 1922. It is M. smegmatis and the 24 listed in table 2 con- tentatively assumed that Maie is the proper formed to the proposed species' description. authority for the species and that he attributed Mycobacterium fortuitum Cruz the specific name to Galli-Valerio as a matter of courtesy. MaWi's account and a later one by The strains comprising the third taxon of Galli-Valerio and Bournand (1927) were very rapidly growing acid fast bacteria were isolated general but did not prohibit the acceptance of from soil and from infections of humans, cattle, strain no. 447 as authentic and the listing of and cold-blooded animals (table 3). The oldest M. aquae in the synonymy of M. smegmatis. species represented in the group was designated The microscopic aspect, acid fastness, colonial Mycobacterium enteritidis, Lehmann and Neu- morphology, and macroscopic appearance of the mann's name (1927) for the Johne's bacillus. 24 strains conformed to those of the other strains On the basis of a single strain, no conclusion previously classified as M. smegmotis. The 24 can be drawn on the relationship between the strains grew from 28 C to 45 C inclusive; few grew slowly growing, fastidious Johne's bacillus and at 10 C; none, at 52 C; and none survived 4 these rapidly growing strains.
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