Japan. J. Microbiol. Vol. 15 (3), 229-236, 1971

Relationship between nonchromogenicum , Mycobacterium terrae, Mycobacterium novum and Subgroup "V" (Mycobacterium triviale)1

Michio TSUKAMURA The National Sanatorium, Chubu Chest Hospital , Aichi-Prefecture

(Received for publication, October 13, 1970)

ABSTRACT

Comparison was made among Mycobacterium nonchromogenicum, M. terrae, M. novum and subgroup "V" (M. triviale). These organisms together are differentiated from other mycobacteria of group II and group III by the following characters: (1) Sensitiveness to ethambutol; (2) Tolerance to nitrite; (3) Tolerance to Tween 80; (4) In- ability to utilize glucose and succinate in the presence of glutamate. To exclude the influence of growth rate, rough colony mutants (R-type mutants) were isolated from M . nonchromogenicum, M. terrae and M. novum and compared with each other and subgroup "V" that was originally of R-type. The R-type mutants of M. nonchromo- genicum were similar to those of M. terrae, with the exception of the intensity of nicotinamidase and pyrazinamidase activities. These two have been suggested to belong to the same taxon, appropriate name of which is M. nonchromogenicum. The R-type mutants of M. novum were similar to the subgroup "V", with the exception of the intensity of arylsulfatase activity. It has been considered that subgroup "V" is an R-type mutant of M. novum. Although M. nonchromogenicum and M. novum could be differ- entiated from each other by the intensity of the arylsulfatase, nicotinamidase and pyrazinamidase activities and requirement of nitrogen compounds, these two organisms are differentiated from other mycobacteria by the same characteristics and are therefore considered to be closely related organisms.

Mycobacterium nonchromogenicum Tsu- 18, 21], but much remains unknown. These kamura [8], Mycobacterium terrae Wayne organisms occur sporadically in the sputum [20], Mycobacterium novum Tsukamura of patients with pulmonary disease and are [12, 13] and subgroup "V" (Mycobacterium important for their necessity of differenti- triviale) Kubica et al. [2, 4] belong to the ation from pathogenic mycobacteria belong- Group III nonphotochromogens and resem- ing to the same group, Mycobacterium ble each other [22]. Relationship between intracellulare, Mycobacterium avium and these 4 organisms has been discussed [17, Mycobacterium xenopei. The purpose of the present paper is concerned with taxo- 1 An outline of this article was read at the annual meeting of the Committee of Bacteriology and nomic relationship between these organ- Immunology, the International Union against isms. Tuberculosis, held in New York in August-Septem- ber 1969.

229 230 M. TSUKAMURA

of Wayne. MATERIALSAND METHODS Since it is known that the characters of Strains used. 93 strains of M. nonchro- are influenced by their growth rate mogenicum including strains NCTC 10424 [7, 21], it is necessary to pay particular at- (ATCC 19530; #317; type strain), ATCC tention to the growth rate in comparison 19531 (#1711), ATCC 19532 (#1721) and of organisms with different growth rates. ATCC 19533 (#1731); 15 strains of M. ter- Since M. nonchromogenicum, M. terrae and rae including ATCC 15755 (#W-45; type M. novum are of the S-type (smooth and strain); 26 strains of M. novum including moist) colonies and subgroup "V" is of the ATCC 19619 (#1945; type strain), ATCC R-type (rough and dry) colonies, it is de- 19620 (#1951), ATCC 19621 (#1952) and sired to compare these organisms under ATCC 19622 (#1954); 22 strains of sub- similar conditions. For this purpose, rough group "V" (M. triviale) including ATCC colony-mutants (R-type mutants) were iso- 19386 (C-5181) and ATCC 19387 (T-254-3); lated from M. nonchromogenicum, M. ter- 26 strains of M. avium; 8 strains of M. rae and M. novum. gastri; 76 strains of M. intracellulare; 26 The R-type mutants of M. nonchromo- strains of M. scrofulaceum; 49 strains of tap genicum were isolated by use of ultraviolet water scotochromogens; 4 strains of M. irradiation. Bacterial suspensions were ir- xenopei. The strains were purified by single radiated with ultraviolet light (GL-15-WC colony-isolation technique and maintained ultraviolet light; 0.3A, 100V, 60 cycles; dis- in Ogawa egg medium at-20C. tance 100cm; 10min) and inoculated to M. terrae and subgroup "V" were re- Ogawa egg medium. In order to obtain dis- ceived from Dr. L. G. Wayne and Dr. G. P. crete colonies, various concentrations of the Kubica, respectively. suspension were used for inoculation. M. nonchromogenicum was described by The R-type mutants of M. terrae and M. Tsukamura [8] in 1965. However, he novum could be obtained spontaneously. changed its name to "M. terrae" in 1966 [9], When M. terrae growing on Ogawa egg and several papers were published using medium was allowed to stand at room tem- this name, "M. terrae" Tsukamura [10-14], perature (at 25C to 37C), using a non- and the strains were deposited under this paraffinized cotton stopper, for one to two name in the National Collection of Type months, and the R-type colonies grew as Cultures, the American Type Culture Col- large colonies elevated from a basal mem- lection and the Lausanne Collection. Inde- braneous growth of S-type organism. The pendently from Tsukamura, Wayne [20] de- R-type mutants of M. novum could be iso- scribed M. terrae in 1966 and, thus, the lated by a heavy inoculation of the organ- change of the name by Tsukamura caused ism to Sauton agar. Though this organism confusion. Since, according to the Inter- originally showed no growth or scant national Code of Nomenclature of Bacteria growth on synthetic agar media, some mu- [3], the change of the name should not be tants could grow on synthetic agar media. made, the correct name for the organism of These mutants showed R-type colonies and Tsukamura should be M. nonchromogeni- were considered to be prototrophic mutants cum. Hence, in the present paper, the name of M. novum, which is originally auxo- M. nonchromogenicum was used for the trophic. organism of Tsukamura and the name M. The tests used are listed in a previous terrae was used correctly for the organism paper [16]. The following tests have newly M. NONCHROMOGENICUM, M. TERRAE, M. NOVUM AND SUBGROUP "V" 231 been added: Resistance to 5ƒÊg/ml etham- tolerance to nitrite was most characteristic butol in Ogawa egg medium [6, 15]; toler- for these organisms. Among slow-growing

ance to 0.1% and 0.2% sodium nitrite in mycobacteria, only these organisms could Sauton agar [1, 15, 19]; tolerance to 1% grow on the Sauton agar containing 0.1% Tween 80 in Sauton agar. The total num- and 0.2% sodium nitrite (Table 1).

ber of tests were 101 (97 plus the above 4). Together with M. gastri and tap water The tolerance to Tween 80 was tested on scotochromogens, the organisms were sensi-

the Sauton agar containing 1% Tween 80. tive to ethambutol, but the former were After inoculation with one loopful of the sensitive to Tween 80 and, by this, were

test organism, the growth was read after differentiated from the latter. incubation at 37C for 3 weeks in compari- Tolerances to nitrite and to Tween 80 son with the growth on control medium were not tested in M. novum, because this

(Sauton agar containing no agent). Positive organism did not grow on the Sauton agar. growth on the test medium was regarded as The R-type mutants of M. novum (M. positive tolerance. This test was introduced novum R) proved to be tolerant to both most recently and was not used for calcula- nitrite and Tween 80 so as the other 3 tion of S-values. Hence, S-values were calcu- organisms were tolerant (Table 1). lated based on the tests of 100 characters. The four organisms were differentiated "Hypothetical mean organism" (HMO) from the other slow-growing mycobacteria

[16] was used for description of a bacterial by the same characteristics and were sug- taxon. It is a modification of the hypotheti- gested to belong to the same taxon of some cal median organism of Liston et al. [5]. rank. Calculation of S-values was made as fol- lows: Comparison among M. nonchromogenicum,

S-value(%)={ns/(ns+nd)}•~100. M. terrae, M. novurn and Subgroup "V"

Here, ns is the number of characters in Comparison between the 4 organisms are

which the strains show similar code symbols shown in Table 2. The characters shown in (++ or --) and nd is the number of char- table are distinguishing ones, which are actersin which the strainsshow different useful for differentiation of one from an- code symbols (+-). other. The organisms showed similar char- acters in respect to the other characters. RESULTS AND DISCUSSION The following common features are shown: Differentiation of M. nonchromogenicum, Nonphotochromogenic; growth occurs at 7 M. terrae, M. novum and Subgroup to 14 days on egg media; growth occurs at "V" from the Other Mycobacterial 28C and 37C, but growth does not occur Species of Group II and Group III at 45C and 52C; not tolerant to 0.1% and M. nonchromogenicum, M. terrae, M. 0.2% picric acid in Sauton agar; amidases novum and their R-type mutants and sub- other than nicotinamidase and pyrazin- group "V" were clearly differentiated from amidase not demonstrated; no acid formed the other mycobacteria of group II and from various carbohydrates; carbohydrates group III by tolerance to nitrite, intoler- other than acetate, pyruvate and propanol ance to ethambutol, tolerance to Tween 80 not utilized as the sole source of carbon in and inability to utilize glucose and succi- the presence of ammoniacal nitrogen and nate in the presence of glutamate (Table 1). in the presence of glutamate nitrogen; nitro- Among these distinguishing characters, gen compounds other than those shown in 232 M. TSUKAMURA

Table 1. Differentiation of M. nonchromogenicum, M. terrae, M, novum and subgroup "V" from other mycobacteria of groups II and III

a) R means R-type mutant (rough colony-mutant) of the corresponding species. b) Utilization for growth as the sole source of carbon in the presence of glutamate-nitrogen.c ) Not tested (M. novum did not grow on control medium).

Table 2. Comparison between M. nonchromogenicum, M. terrae, M. novum. and subgroup "V"

a) Only distinguishing characters are shown. As to the other characters, test organisms showed

almost similar reactions. The numbers in table show the numbers of strains showing the positive reaction. M. NONCHROMOGENICUM, M. TERRAE, M. NOVUM AND SUBGROUP "V" 233

Table 2 not utilized as the sole source of seemed to be in the intensity of nicotin- nitrogen; nitrogen compounds not utilized amidase and pyrazinamidase activities . as simultaneous nitrogen and carbon However, these seemed to be not distinct, sources; niacin-negative; tolerant to thio- as about one third of the M. terrae strains phen-2-carboxylic acid hydrazide; tolerant showed a positive activity in these enzymes, to 0.5mg/ml and 1.0mg/ml sodium salicyl- when the time of incubation for measure- ate in Ogawa egg medium; tolerant to ment of the enzyme activity was extended 2mg/ml sodium p-aminosalicylate (PAS) in to 24hr and the concentration of bacterial Ogawa egg medium; PAS not degraded; suspensions was increased 1.5 times. It was salicylate not degraded; nitrate not reduced noted that the type strain of M. terrae, or slightly reduced to nitrite (as to the ATCC 15755 showed characters almost above characters, refer to reference 14). similar to the characters of the type strain Since M. novum is auxotrophic, it was of M. nonchromogenicum, NCTC 10424. desired to compare prototrophic mutants of Major differences between the type strains M. novum with other organisms. Further- were as follows: (i) The former was less more, since subgroup "V" shows the R-type active in respect to the nicotinamidase and colony-morphology, it was desired to com- pyrazinamidase activities; (ii) The former pare the R-type mutants of the other organ- did not utilize glutamate as simultaneous isms with subgroup "V." Comparison nitrogen and carbon sources, but the latter among the R-type mutants of M. nonchro- utilized it. However, the second character mogenicum, M. terrae and M. novum and is not a common feature of M. nonchromo- subgroup "V" is shown in Tables 2 and 3. genicum. Only 5 of 93 strains of this organ- The characters of the R-type mutants of ism showed this ability [10, 14]. M. terrae were almost similar to those of These results suggest that M. nonchromo- the R-type mutants of M. nonchromogeni- genicum and M. terrae are probably the cum. Major differences between these same. Since the former was described in

Table 3. Comparison between R-type mutants of M. nonchrornogenicum, M. terrae, M. novum and subgroup "V"

a) Distinguishing characters only . The characters are expressed as the hypothetical mean organism. 234 M. TSUKAMURA

1965 and the latter in 1966, the name for differentiated from the other mycobacteria this taxon is M. nonchromogenicum. by the same characteristics. This fact sug- On the other hand, the R-type mutants gests that these are very closely related to of M. novum were similar to the strains of each other. Furthermore, the strains of subgroup "V." Major differences between M. terrae seem to show an intermediate these seem to be in the intensity of aryl- feature between M. nonchromogenicum and sulfatase activity (Tables 2 and 3). It is sug- M. novum (Table 2). These findings sug- gested that the subgroup "V" is an R-type gest that a clear-cut differentiation between mutant of M. novum, and hence, subgroup these organisms was not always easy. It is "V" or M . triviale is a synonym of M. possible that M. novum may be an auxo- novum. trophic mutant of M. nonchromogenicum , Differences between M. nonchromogeni- the enzyme activity of the mutant being cum and M. novum are as follows: (i) M. decreased by mutation to auxotrophy. nonchromogenicum shows a more intensive activity in arylsulfatase, nicotinamidase and Numerical Considerations on the Taxono- pyrazinamidase than shows M. novum (but my of M. nonchromogenicum, M. ter- subgroup "V" shows an intensive arylsul- rae, M, novum and Subgroup "V" fatase activity); (ii) M. nonchromogenicum Mean S-values to the hypothetical mean is able to utilize more compounds as the organism (HMO) of various taxons were sole source of nitrogen than does M. novum. calculated for the R-type mutants of M. However, as shown in Table 1, both nonchromogenicum, M. terrae and M. M. nonchromogenicum and M. novum, are novum and subgroup "V" (Table 4).

Table 4. Inter-taxon mean S-values for M. nonchromogenicum R, M. terrae R, M. novum R, and subgroup "V" to the hypothetical mean organism (HMO) of various mycobacterial species of group II and group III

The values in table show (inter-taxon mean S-value)•}(standard deviation). a) The number of strains-tested .b ) (Intra-taxon mean S-value)•}(standard deviation).c

) The highest S-value significantly higher than the others.

d) S-value not significantly different from the highest. M. NONCHROMOGENICUM, M. TERRAE, M. NOVUM AND SUBGROUP "V" 235

Among the inter-taxon mean S-values for S-value of 91.3% to the HMO of M. non- the strains of the R-type M. nonchromo- chromogenicum, the parent of M. non- genicum (M. nonchromogenicum R), the chromogenicum R. On the other hand, S-value to the HMO of M. terrae R was the though not shown in Table, the intra-taxon highest and was significantly higher than mean S-value for the strains of M. non- the others (P less than 5%). This result indi- chromogenicum was 96.0•}2.69% (the num- cates that M. nonchromogenicum most re- ber of strains tested is 93). This value is sembles M. terrae, and that M. novum and significantly higher than the inter-taxon subgroup "V" are differentiated from M. mean S-value for the strains of M. non- nonchromogenicum and M. terrae. chromogenicum R to the HMO of M. non- Among the inter-taxon mean S-values for chromogenicum, 91.3•}1.11% (the number the strains of M. terrae R, the S-values to of strains tested is 13). Since M. nonchromo-

the HMOs of M. nonchromogenicum, M. genicum R is only mutant of M. nonchro- nonchromogenicum R, M. novum R and mogenicum, the S-values should not differ subgroup "V" were significantly higher than significantly from each other.

the S-values to the other HMOs. No sig- However, the S-values differed, in fact,

nificant differences were found among the significantly and the R-type mutants be- S-values of the above organisms. haved in the numerical as if they Among the inter-taxon mean S-values for were of a different taxon. Of course, this is the strains of M. novum R, no significant due to the fact that the changes in the R- difference was found among the S-values of type caused simultaneously the changes of the HMO of subgroup "V," M. terrae and other characters. The change in several M. terrae R. These results seem to indicate characters occurred at one step. This change that M. novum R might be the intermedi- may be interpreted as produced by a muta- ate feature of M. terrae. tion of a factor controlling growth rate or Among the inter-taxon mean S-values for a factor controlling requirement of nitrogen the strains of subgroup "V," the S-value to compounds. The phenomenon supports the the HMO of M. novum R was the highest importance of the concept of "Vigour" pub- and was significantly higher than the others. lished recently by Sneath [7]. It should be This finding indicates that subgroup "V" emphasized that the comparison shown in is the most similar to M. novum R, and Table 4 has been made excluding the influ- subgroup "V" is suggested to be an R-type ence of the Vigour factor, as the comparison mutant of M. novum. has been made by synchronizing the growth In view of the above results, M. non- rate of the 4 organisms. chromogenicum and M. novum are con- In conclusion, M. nonchromogenicum sidered to be different taxons. M. nonchro- and M. terrae are probably the same and mogenicum and M. terrae are very similar the name proposed for this taxon is M. non- to each other, and M. novum and subgroup chromogenicum. Subgroup "V" (M. tri- "V" also are similar to each other . viale) is probably an R-type mutant of M. The results shown in Table 4 seem to novum. M. nonchromogenicum and M. present a serious question to the numerical novum are closely related to each other and taxonomy and the conventional taxonomy. are differentiated from other mycobacteria For example, the strains of M. nonchromo- by the same characteristics. genicum R showed the intra-taxon mean S-value of 97.5%, and the inter-taxon mean 236 M. TSUKAMURA

REFERENCES [11] Tsukamura, M. 1966. Adansonian classifica- tion of mycobacteria. J. Gen. Microbiol. [1] Abbott, V. D., Kilburn, J. O., Harrison, W., 45: 253-273. and Kubica, G. P. 1968. Differential iden- [12] Tsukamura, M. 1966. Two species of slowly tification of mycobacteria. IV. The nitrite growing, nonphotochromogenic soil myco- inhibition test. Amer. Rev. Resp. Dis. 97: bacteria obtainable by mouse body-passage 931-934. method. Med. Biol. (Tokyo) 73: 244-247. [2] Jones, W. D., Jr., Abbott, V. D., Vestal, A. L., (in Japanese) and Kubica, G. P. 1966. A hitherto un- [13] Tsukamura, M. 1967. Two types of slowly described group of nonchromogenic myco- growing, nonphotochromogenic mycobac- bacteria. Amer. Rev. Resp. Dis. 94: 790- teria obtained from soil by the mouse 795. passage method: Mycobacterium, terrae and [3] Judical Commission of the International Mycobacterium novum. Japan. J. Micro- Committee on Nomenclature of Bacteria. biol. 11: 163-172. 1966. International code of nomenclature [14] Tsukamura, M. 1967. Identification of myco- of bacteria. Internat. J. Syst. Bacteriol. 16: bacteria. Tubercle (London) 48: 311-338. 459-490. [15] Tsukamura, M. 1969. Identification of group [4] Kestle, D. G., Abbott, V. D., and Kubica, G. II scotochromogens and group III non- P. 1967. Differential identification of photochromogens of mycobacteria. Tuber- mycobacteria. II. Subgroups of group II cle (London) 50: 51-60. and group III (Runyon) with different [16] Tsukamura, M., and Mizuno, S. 1968. "Hypo- clinical significance. Amer. Rev. Resp. Dis. thetical mean organisms" of mycobacteria. 95: 1041-1052. A study of classification of mycobacteria. [5] Liston, J., Wiebe, W., and Colwell, R. R. Japan. J. Microbiol. 12: 371-384. 1963. Quantitative approach to the study [17] Tsukamura, M., and Mizuno, S. 1969. Taxo- of bacterial species. J. Bacteriol. 85: 1061- nomy of subgroup "V" of the Group III 1070. nonphotochromogenic mycobacteria. Kek- [6] Mizuno, S., Toyama, H., and Tsukamura, M. kaku 44: 13-17. (in Japanese) 1966. Susceptibility of various mycobac- [18] Tsukamura, M., and Tsukamura, S. 1967. teria to ethambutol. Differentiation be- Further observations on Mycobacterium tween M. avium and M. terrae. Japan. J. terrae. A method for isolating slowly grow- Bacteriol. 21: 672-674. (in Japanese) ing, nonphotochromogenic mycobacteria [7] Sneath, P. H. A. 1968. Vigour and pattern from soil sources. Amer. Rev. Resp. Dis. in taxonomy. J. Gen. Microbiol. 54: 1-11. 96: 299-304. [8] Tsukamura, M. 1965. A group of mycobac- [19] Tsukamura, M., and Tsukamura, S. 1968. teria from soil sources resembling non- Differentiation of mycobacteria by suscep- photochromogens (group III). A descrip- tibility to nitrite and propylene glycol. tion of Mycobacterium nonchrornogenicum. Amer. Rev. Resp. Dis. 98: 505-506. Med. Biol. (Tokyo) 71: 110-113. (in Japa- [20] Wayne, L, G. 1966. Classification and iden- nese) tification of mycobacteria. III. Species with- [9] Tsukamura, M. 1966. Classification of myco- in group III. Amer. Rev. Resp. Dis. 93: bacteria. I. Adansonian classification of 919-928. slowly growing mycobacteria. Med. Biol. [21] Wayne, L. G. 1967. Selection of characters (Tokyo) 72: 75-78. (in Japanese) for an Adansonian analysis of mycobacteri- [10] Tsukamura, M. 1966. Further notes on al taxonomy. J. Bacteriol. 93: 1382-1391. Mycobacterium terrae, a new species. A [22] Wayne, L. G., Runyon, E. H., and Kubica, method for isolating slowly growing myco- G. P. 1969. Mycobacteria: A guide to bacteria from soil. Med. Biol. (Tokyo) 72: nomenclatural usage. Amer. Rev. Resp. 292-295. (in Japanese) Dis. 100: 732-734.