RAPIDLY GROWING, ACID FAST ' 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, 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 (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 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. For practical hours at 60 C. Two-thirds of the strains grew in reasons alone, however, the application of a 5 per cent NaCl broth. Gelatin and casein were specific name in the synonymy of Mycobacterium not attacked; nitrates were reduced to nitrites paratuberculosis Bergey et al. to a species of the by all strains; starch was hydrolyzed; citrate, rapidly growing strains would not be feasible. succinate, and malate were utilized; acid was The next oldest species represented was formed from glucose, rhamnose, xylose, arab- labeled M. fortuitum and described by Cruz in inose, mannose, galactose, mannitol,3 sorbitol, 1938. This name is tentatively adopted for the inositol, and dulcitol. Lactose and raffinose were species. It is difficult to believe, however, that not used as sources of carbon. such a widespread organism was not isolated and The tests used to supplement the species' characterized long before 1938. Should an description of M. phlei were also applied to all authentic strain bearing an earlier effectively the strains of M. smegmatis. published name (Buchanan et al., 1948) be found Decomposition of tyrosine. All cultures were to belong to this species, the name now applied negative. would have to be changed to the earlier one. Utilization of benzoate. All but four cultures Under the conditions of this examination, (nos. 375 to 377, 380) were positive. strains of Mycobacterium giae Darzins (1950) and Acid production from carbohydrates. Twenty- Mycobacterium minetti Penso et al. (1952) were four per cent of the cultures formed acid from identical with others of this taxon. Although some maltose; 100 per cent, from trehalose. discrepancies existed between the original The addition of the foregoing characters to descriptions of the two species and the one given the species' description of M. smegmatis and a here, they were believed due to differences in examinations resulted methods. It is recommended, therefore, that careful check of previous M. giae and M. minetti be placed in the synonymy ' Omitted by error from the previous list of of M. fortuitum. The similarity between NCTC carbohydrates hydrolyzed by M. smegmatis. strain no. N946 of M. friedmannii Holland and 1955] RAPIDLY GROWING, ACID FAST BACTERIA 505 those of M. fortuitum casts some doubt on the TABLE 3 previous assignment of M. friedmannii to the Strains identified as Mycobacterium fortuitum synonymy of M. smegmatis. This assignment was Cruz made because ATCC and CCTM strains of Laboratory Name and Source M. friedmannii were identical with those of Number M. smegmatis, and Friedmann's original de- scription (1903) was too general to establish N6926 Mycobacterium enteritidis Lehmann definitely the identity of his strain. Friedmann's and Neumann; NCTC (5026) account could be applied to a strain of M. 6841, 6842 Mycobacterium fortuitum Cruz; AT- smegmatis or to a strain of M. fortuitum. Until CC; J. C. Cruz (1, 2, human ab- more information can be obtained, it is suggested scess) N946 Mycobacteriumfriedmannii Holland; that M. friedmannii be left in the synonymy of NCTC M. amegmatis and NCTC strain no. N946 be re- 11440 Mycobacterium giae Darzins; ATCC; garded as possibly misnamed. E. Darzins (systemic, nodular in- The following description of M. fortuitum was fection of gias) based on the 28 strains listed in table 3: 480 Mycobacterium minetti Penso et al.; Microscopic appearance. CelLs grown on G. Penso, Istituto Superiore de glycerol agar for 72 hours at 28 C varied from Sanita, Rome (C70) 1.0 , to 3.0 A in length, the largest number rang- 48 Mycobacterium sp; W. A. Hagan, ing from 2.0,u to 2.2,u. After 5 days' incubation Cornell Univ. (soil) at 28 C on glycerol agar, 70 to 100 per cent of 374 Mycobacterium sp; J. Bentinck- Smith, Cornell Univ.; E. W. the cells of 17 of the 28 cultures were acid fast; Tucker (bovine mastitis) the remaining 9 cultures were 10 to 50 per cent 389 Mycobacterium sp; G. Alexander, acid fast. The cells varied from coccoid and Rutgers Univ. (B8, barnyard soil) short rods to long, slender rods, occasionally 394, 395 Mycobacterium spp; W. A. Hagan beaded or swollen by a nonacid fast, oval body (52-80, 52-79); P. Stuart (MS. at one end. 197.51, MS.144.51, bovine masti- Colonial morphology was similar to that of tis) M. phlei and M. smegnatis. After 3 or 4 days' 448, 490 Mycobacterium spp; CCTM (Kar- incubation, dense colonies with smooth edges linski 58, sable 73) and dense colonies with filaments 449 Mycobacterium sp; D. W. Bruner, fringed short Cornell Univ. (48, soil) and/or filamentous colonies were observed. Upon 456 to 461 Mycobacterium spp; E. W. Tucker, further incubation the filaments usually frag- Cornell Univ. (1595, 84/2 cow mented into short rods. 1705, 21/2 cow 155, 89/3 cow 1351, Macroscopic appearance. Growth on glycerol 72/2 cow 1555, 76/3 cow 1422, agar after 2 or 3 days at 28 C was scant to fair, bovine mastitis) (Tucker, 1953) soft and butyrous, or waxy and nodular, off-white 9820 Mycobacterium sp; ATCC; NCTC to cream colored. After 2 weeks, growth of the (2014); KrAl Collection (fish) cultures was 9821 Mycobacterium sp; ATCC; NCTC good to abundant, spreading, (2291); A. S. Griffith (roe of hali- butyrous and glistening or dull, rough, and but) waxy, often very nodular, off-white to cream to 9822 Mycobacterium sp; ATCC; NCTC beige in color. On yeast extract agar growth was (2006); G. S. Wilson (terrapin) moderate to heavy, spreading, butyrous or dry N2291 Mycobacterium sp; NCTC (halibut) and waxy, usually nodular, sometimes finely N3630 Mycobacterium sp; NCTC (Cummin's M bacillus) wrinkled, off-white to cream to beige. Growth on N8573 Mycobacterium sp; NCTC; E. Aguis glucose-asparagine agar at 2 weeks was moderate, (Malta, cervical adenitis) spreading, soft and glistening or dull and waxy, N8697 Mycobacterium sp; NCTC (human off-white to cream color. abscess) Temperatures of growth. The 28 strains grew 435 Nocardia sp; Isabel Christison, from 28 C to 35 C; all but 6 (nos. 48, 448, 449, Duke Univ. (2229); N. F. Conant; 9822, 11440, N946) grew at 40 C; and approxi- E. Palik (sputum) 506 RUTH E. GORDON AND MILDRED M. SMITH [VOL1. 69 mately one-half grew at 10 C. Growth, if any, Hydrolysis of casein. All cultures were negative. was very scant at 45 C. Utilization of citrate, succinate, and malate. Survival of 60 C. None of the cultures grew Only one culture (no. 395) failed to utilize citrate; after 4 hours in the water bath at 60 C. all utilized succinate and malate. Hydrolysis of starch. All cultures were positive. Reduction of nitrate to nitrite. All but four Acid productionfrom carbohydrates. All cultures i cultures (nos. 395, 448, 9822, N946) reduced formed acid from glucose, mannose, and trehalose; nitrate to nitrite. a few (nos. 48, 449, 490, 11440, N2291) utilized NaCl tolerance. Growth, if any, was very scant mannitol. Acid production from rhamnose, in 7 per cent NaCl and usually positive in 5 per xylose, arabinose, galactose, sorbitol, inositol, cent. dulcitol, lactose, and raffinose was negative; with Deconposition of tyrosine. All cultures were one exception (no. 9822), maltose was not negative. hydrolyzed. Utilization of benzoate. All cultures were nega- Hydrolysis ofgelatin. All cultures were negative. tive. Portions of the descriptions of M. phlei, M. TABLE 4 smegmatis, and M. fortuitum are arranged for Comparison of reactions of three 8pecies of Myco- comparative purposes in table 4. Maximum bacterium temperatures of growth, survival of 60 C, utiliza- and acid from Myceb"Cterium tion of benzoate, production Reactions rhamnose, xylose, arabinose, galactose, sorbitol, pkles sme amisflferSuum inositol, and dulcitol provided combinations of reactions separating the three species one from Decomposition of: the other. Although the other reactions listed in gelatin 4 did casein table not differentiate the three species, tyrosine they were, on the whole, stable and of value in Growth at: the recognition of the three species. Some of them 52 C also gave promise of usefulness in the delineation 45C of the other species, at present partially repre- 40C sented by the remaining 32 per cent of the 195 Survival 60 C, 4 hr + strains in this collection. Nitrite from nitrate + Utilization of: + citrate + succinate The collection of rapidly growing, acid fast malate + bacteria was increased from 124 to 195 by the benzoate - examination of 71 more strains. The new strains, Hydrolysis of starch + identified as Mycobacterium phlei Lehmann and Acid from: + Neumann and Mycbacterium smegmatis C(re- glucose + + Lehmann and were rhamnose visan) Neumann, listed and xylose + a few supplementary characters were added to arabinose + the descriptions of these two species. mannose A third species, Mycobacteriumfortuitum Cruz, galactose was characterized and compared with M. phlei mannitol + and M. megmatis. Mycobacterium giae Darzins sorbitol and Mycobacterum mineti Penso et al. were inositol listed in the synonymy of M. fortuitum. dulcitol lactose REFERENCES raffinose maltose BUCHANAN, R. E., ST. JOHN-BROOKS, R., AND trehalose BREED, R. S. 1948 International bacterio- logical code of nomenclature. J. Bacteriol., * Majority of strains negative. 55, 287-306. t Majority of strains positive. CRUZ, J. C. 1938 Mycobacterium fortuitum um 1955] RAPIDLY GROWING, ACID FAST BACTERIA 507

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