INTERNATIONALJOURNAL OF SY~TEMATICBACTERIOLOGY, Apr. 1979, p. 110-129 Vol. 29, No. 2 Oo20-7713/79/O4~0110/20$02.Oo/O Comprehensive Numerical Classification of 369 Strains of Mycobacterium, Rhodococcus, and Nocardia MICHIO TSUKAMURA,’ SHOJI MIZUNO,] SUMIO TSUKAMURA,~ AND JUNKO TSUKAMURA~ The National Chubu Hospital, Obu,’ and Tsukamura Clinic, Obu (KYOWU),~Aichi 474, Japan A numerical analysis of 369 strains received as members of the genus Myco- bacterium, Rhodococcus (formerly Gordona), Nocardia, or Corynebacterium was carried out using 88 characters. Of the strains tested, 345 mycobacteria, rhodococci, and nocardiae formed four distinct clusters at a similarity level of 85%.The first cluster was composed of slowly growing mycobacteria; the second cluster, of strains of Mycobacterium flavescens and M. thermoresistibile; the third cluster, of strains of rapidly growing mycobacteria; and the fourth cluster, of strains of Rhodococcus and Nocardia. Strains of three species of rapidly growing mycobacteria, M. agri, M. smegmatis, and M. vaccae, were located outside of these clusters. The fourth cluster could be divided into three subclus- ters, one consisting of rhodococci plus some strains of Nocardia asteroides, a second consisting of the remaining strains of Nocardia, and a third consisting of strains of Rhodococcus aurantiacus (Gordona aurantiaca) and Jensenia cani- cruria. Mycobacterium, Rhodococcus (formerly Gor- The finding that “M. ” rhodochrous differs from dona), and Nocardia are closely related taxa both Mycobacterium and Nocardia was con- belonging to the order Actinomycetales (21). fmed by an international cooperative study (5). In 1966, Tsukamura (13) reported that myco- Goodfellow and Alderson (3) also proposed the bacteria can be divided into two subgroups, name Rhodococcus for the “M.” rhodochrous which roughly correspond to slowly growing and complex, in which they included the genus Gor- rapidly growing mycobacteria, by numerical dona. These authors also included several new classification. These two subgroups differed species in the genus Rhodococcus. from each other not only in growth rate, but also The taxonomic relationships of the mycobac- in several biological characters. Subsequently, teria, rhodococci, and nocardiae were studied by Tsukamura and Mizuno (14, 24) proposed the Tsukamura (21),who included hypothetical me- subgeneric names Mycobacterium and Myco- dian organisms of representative species of these mycobacterium for these subgroups. Later, after taxa in his study. Tsukamura proposed that the having compared mycobacteria and nocardiae, slowly growing mycobacteria, the rapidly grow- Tsukamura (17) recommended that the subge- ing mycobacteria, Gordona (now Rhodococcus), nera Myco bacterium (slowly growing mycobac- and Nocardia be regarded as distinct taxa. He teria) and Mycomycobacterium (rapidly growing noted that the slowly growing mycobacteria mycobacteria) be elevated to genus status, i.e., seemed to be only distantly related to the other taxa equivalent in status to the genus Nocardia. three groups, which showed a close relationship In 1971, Tsukamura (18) proposed a new ge- with each other. nus, Gordona, for slightly acid-fast organisms The purpose of the present study was to con- considered to be intermediate between Myco- duct a numerical taxonomy analysis of a large bacterium and Nocardia and placed it in the number of strains belonging to these taxa. A family Actinomycetaceae, to which the genus total of 369 strains of Mycobacterium, Rhodo- Nocardia belongs. It was later shown by Tsu- coccus, and Nocardia and 4 strains of Coryne- kamura that the genus Gordona contained bacterium were investigated. strain ATCC 13808 of Rhodococcus rhodoch- rous (19), and it was stated that the generic MATERIALS AND METHODS name Gordona should be changed to Rhodococ- cus (20), this is the first name used for an Strains. A total of 365 strains received as members as of Mycobacterium, Rhodococcus, or Nocardia and 4 organism belonging to the genus proposed. In- strains received as members of Corynebacterium were dependently, Goodfellow (2) observed that “My- studied (see Table 1). cobacterium” rhodochrous differs from nocar- Tests. A total of 88 characters were used in the diae, and Goodfellow et al. (4) reported that it numerical analysis. These characters were selected also differs from rapidly growing mycobacteria. from 118 characters routinely determined in this 110 VOL. 29, 1979 MYCOBACTERIUM, RHODOCOCCUS, AND NOCARDIA 111 TABLE1. Strains used in this study Received as: Serial Labora- Sourcea Habitat no. tory no. Name Strain 1 E6383 Mycobacterium tuberculosis TC1 Humans 2 E6384 Myco bacterium tuberculosis TC5 Humans 3 E6543 Myco bacterium tuberculosis TC32 Humans 4 E6548 Mycobacterium tuberculosis TC40 Humans 5 05001B Mycobacterium tuberculosis H37Rv Humans 6 05002 Myco bacterium tuberculosis Aoyama-B Humans 7 E6542 Mycobacterium tuberculosis TC22 Humans 8 E7949 Myco bacterium tuberculosis Sputa, humans 9 E6165 Myco bacterium tuberculosis Sputa, humans 10 E6385 Mycobacterium tuberculosis TC6 Humans 11 E6387 Mycobacterium tuberculosis TC9 Humans 12 E6401 Mycobacterium tuberculosis TC29 Humans 13 E666 1 Mycobacterium tuberculosis TC13 Humans 14 E6662 Myco bacterium tuberculosis TC15 Humans 15 E7193 Mycobacterium tuberculosis Sputa, humans 16 E7966 Mycobacterium tuberculosis Sputa, humans 17 E7950 Mycobacterium tuberculosis Sputa, humans 18 05001A Mycobacterium tuberculosis H37Rv Humans 19 E6394 Mycobacterium tuberculosis TC21 HUlnans 20 E6549 Mycobacterium africanum TC41 Humans 21 E6677 Mycobacterium microti TC62 ? 22 E6567 Mycobacterium africanum TC93 Humans 23 E6689 Myco bacterium africanum TC84 Humans 24 E6386 Myco bacterium bovis TC7 Milk 25 E6537 Myco bacterium africanum TC3 Humans 26 E6412 Myco bacterium bovis TC92 Dogs 27 E6558 Myco bacterium africanum TC67 Humans 28 E6565 Mycobacterium africanum TC88 Humans 29 E6683 Mycobacterium africanum TC74 Humans 30 E6544 Myco bacterium bovds TC34 cows 31 E6554 Myco bacterium africanum TC57 Humans 32 E6560 Myco bacterium africanum TC73 Humans 33 E6664 Mycobacterium africanum TC37 Humans 34 E6678 Mycobacterium bouis TC65 Deer 35 E6692 Myco bacterium africanum TC8 Humans 36 E6393 Mycobacterium africanum TC20 Humans 37 E6396 Myco bacterium bovis TC24 cows 38 E6671 Mycobacterium bovis TC51 Bison 39 E6402 Mycobacterium bovis TC30 cows 40 06001A Myco bacterium bovis Ravenel cows 41 06002 Myco bacterium bovis Denken cows 42 06001B Mycobacterium bouis Ravenel cows 43 E6675 Myco bacterium bovis TC58 cows 44 E6388 Mycobacterium bovds TCll BCG (Czech strain) 45 E6691 Mycobacterium microti TC89 Llamas 46 E6392 Mycobacterium microti TC 19 ? 47 E6398 Mycobacterium microti TC26 Voles 48 E6400 Mycobacterium microti TC28 ? 49 E6668 Mycobacterium microti TC48 Cats 50 E6688 Myco bacterium bovis TC83 Minks 51 E7743 Myco bacterium intracellulare 13444 Humans 52 E7744 Mycobacterium intracellulare 18730 Humans 53 E7747 Mycobacterium intracellulare 25356 Humans 54 E7748 Mycobacterium intracellulare 28659 Sewage 55 E7741 Myco bacterium intracellulare 4130 Humans 56 E7745 Mycobacterium intracellulare 23774 Humans 57 E7749 Mycobacterium intracellulare 28660 ? 58 E7750 Mycobacterium intracellulare 28661 Birds 59 E7742 Mycobacterium intracellulare 10499 Humans 60 E7746 Mycobacterium intracellulare 24657 Humans 112 TSUKAMURA ET AL. INT. J. SYST.BACTERIOL. TABLE1 (continued) Received as: Serial Labora- Source" Habitat no. tory no. Name Strain 61 13022 Mycobacterium intracellulare NJ-16 Humans 62 13039 Mycobacterium intracellulare NJ-17 Humans 63 E6111 Mycobacterium intracellulare Sputa, humans 64 E6172 Mycobacterium intracellulare Sputa, humans 65 13014 Mycobacterium intracellulare Sputa, humans 66 13015 Mycobacterium intracellulare Sputa, humans 67 13023 Mycobacterium intracellulare NJ-7 Sputa, man 68 E7959 Mycobacterium intracellulare TS-P Swine, lymph nodes 69 E7961 Mycobacterium intracellulare No. 23 Swine, lymph nodes 70 13024 Mycobacterium intracellulare NJ-8 Humans 71 13032 Mycobacterium intracellulare NJ-10 Humans 72 13038 Mycobacterium intracellulare NJ-6 Humans 73 13936 Mycobacterium intracellulare Humans 74 E7957 Mycobacterium intracellulare TS-H Swine, lymph nodes 75 E7963 Mycobacterium intracellulare Sputa, humans 76 13543 Mycobacterium intracellulare ATCC 19177 Hens 77 E7958 Mycobacterium intracellulare TS-L Swine 78 11016 Mycobacterium avium ATCC 15769 Hens 79 11021 Myco bacterium avium ATCC 17942 9 80 11018 Mycobacterium avium ATCC 17941 ? 81 11017 Mycobacterium avium ATCC 19075 Humans, lymph nodes 82 11019 Mycobacterium avium ATCC 17944 ? 83 11020 Myco bacterium avium ATCC 15773 ? 84 E7682 Mycobacterium intracellulare Sputa, humans 85 E7954 Mycobacterium intracellulare A- 1 Swine, lymph nodes 86 11025 Mycobacterium avium? ATCC 17940 ? 87 E7960 Myco bacterium intracellulare No. 6 Swine, lymph nodes 88 13013 Mycobacterium intracellulare NJ-1 Humans 89 13016 Mycobacterium intracellulare NJ-2 Humans 90 13019 Mycobacterium intracellulare NJ-18 Humans 91 13542 ? ATCC 19076 Humans 92 13935 Myco bacterium intracellulare Sputa, humans 93 12004 Myco bacterium scrofulaceum SJ-8 Humans 94 12008 Mycobacterium scrofulaceum SJ-9 Humans 95 12014 Mycobacterium scrofulaceum SJ-1 Humans 96 12007A Mycobacterium scrofulaceum SJ-5 Humans 97 12017 Mycobacterium scrofulaceum SJ-12 Humans 98 12007B Myco bacterium scrofulaceum SJ-5 Humans 99 12016A Myco bacterium scrofulaceum SJ-14 Humans 100 12012 Myco bacterium scrofulaceum SJ-3 Humans 101 12019 Myco bacterium scrofilaceum SJ-15 Humans 102