Immunodiffusion and lipid analyses in the classification of “Mycobacterium album” and the “aurantiaca” taxon

Zbl. Bakt. Hyg. A 259, 1-10 (1985)

Immunodiffusion and Lipid Analyses in the Classification of "Mycobacterium album" and the "aurantiaca" Taxon MALIN RIDELL\ MICHAEL GOODFELLOW 2 , and DAVID E. MINNIKIN 3 Department of Medical Microbiology, University of G6teborg, G6teborg, Sweden Departments of Microbiolgy', and Organic Chemistry:', The University, Newcastle upon Tyne, England, U. K. 1

2,3

Received June 22, 1984 . Accepted July 12, 1984

Abstract Comparative immunodiffusion studies were performed on representative strains of "Mycobacterium album", the "aurantiaca" taxon and an "aurantiaca"-like group of actinomycetes, using reference systems representing 25 taxa assigned to the genera Arthrobacter, Corynebacterium, Kurthia, Mycobacterium, Nocardia, Rhodococcus, Streptomyces and the "aurantiaca" taxon. Thin-layer chromatographic analyses of whole-organism methanolysates were carried out on single strains of "Mycobacterium album" and the "aurantiaca"-like taxon. Both the distribution of precipitinogens and the type of mycolic acids determined showed that the "Mycobacterium album" and "aurantiaca" strains were closely related to one another, but not to the "aurantiaca"-like strains. The aggregate "aurantiaca" taxon, containing "Mycobacterium album" and the "aurantiaca" taxon, can be distinguished from the established mycolic-acid containing taxa, Corynebacterium, Mycobacterium, Nocardia and Rhodococcus, using both serological and lipid data. Further studies on additional strains are needed to determine whether the aggregate "aurantiaca" taxon merits generic status.

Zusammenfassung Vergleichende Immundiffusionsstudien wurden mit reprasentativen Stammen von "Mycobaterium album" und dem "aurantiaca"-Taxon sowie mit einer "aurantiaca"-iihnlichen Gruppe von Aktinomyzeten durchgefiihrt, wobei Referenzsysteme von 25 Angehorigen der Gattungen Arthrobacter, Corynebacterium, Kurthia, Mycobacterium, Nocardia, Rhodococcus, Streptomyces und des "aurantiaca"-Taxon zur Anwendung kamen. Diinnschichtchromatographische Analysen von Ganz-Zell-Methanolysaten wurden mit Einzelstamrnen von "Mycobacterium album" und dem "aurantiaca"-iihnlichen Taxon durchgefiihrt. Sowohl die Verteilung der Prazipirinogene als auch die Mykolsauremuster zeigten, daiS die "Mycobacterium album"- und "aurantiaca"-Stiimme nahe miteinander verwandt waren, wahrend sich die "aurantiaca"-iihnlichen Isolate abweichend verhielten. Als Sammelbegriff umfaiSt das "aurantiaca"-Taxon "Mycobacterium album" und die "aurantiaca"-Stiimme 1 Zbl. Bakt. Hyg. A 259/1

2

M.Ridell, M.Goodfellow, and D.E.Minnikin

und laBtsich von den etablierten, Mykolsaure-haltigen Gattungen Corynebacterium, Mycobacterium, Nocardia und Rhodococcus anhand serologischer und lipidchemischer Daten abgrenzen. Weitere Untersuchungen an zusarzlichen Stammen sind allerdings erforderlich, urn festzustellen, ob das "a urantiaca"-Taxon im erweiterten Sinne Genusstatus verdient oder nicht.

Introduction Nocard ioform actinom ycetes represent 'a n aggregate taxo n that encompasses mycolic acid-containing bacteria classified in the genera Caseobacter, Corynebacterium, My cobacterium, Noc ardia, Rh odococcus, and the "au rantiaca" taxon (11). Members of these taxa have man y biochemical, chemical and ph ysiological properties in common (6, 8, 10, 11, 12, 13, 17, 18) and there is evidence that the nocardiaform taxon constitutes a recognisable phylogenetic group (20,33). Comm on precipitinogens have been detected in representative strains of Corynebacterium, My cobacterium, Nocardia, and Rhodococcus (14, 15,28). It is also of interest that corynebacteria, mycobacteria, nocardiae, rhodococci and aura ntiaca strains have been recovered as distinct taxa in numerical phenetic surveys (7, 9). The taxonomic status of the "aurantiaca" taxon, however, is a matter of some controversy. Th e " aurantiaca" taxon accomm od ates strains, isolated from sputa of patients with pulmon ary disease, and previously classified as " Gordona" aurantiaca (34, 35). [Names that do not appear in the Approved Lists of Bacterial N ames (31) or subsequent validation lists are written in quo tation mark s.}Numerical taxon omic studies highlighted the equivocal position of this taxon within the genus "Go rdona" (35, 36). Further, the type strain fell out side the Rh odococcus cluster delineated by Goo dfellow and Alderson (3). In a more extensive study (4) aurantiaca strains formed a numericall y well-defined taxon equivalent in rank to phena corresponding to the genera Corynebacterium, My cobacterium, Noca rdia and Rhodococcus. The y were also shown to contai n characteristic mycolic acids and unsaturated menaquinones with nine isoprene unit s. Th ese findings are at variance with the view that the " aurantiaca" taxon be assigned to the genus Rhodococcus as Rhodococcus aurantiacus (37, 39). Rhodococcus aurantiacus was not cited in the Approved Lists of Bacterial Names (31) but is listed in Bergey's Manual of Determinative Bacteriology as a species incertae sedis (10). In preliminary reports (5, 8) of an extensive numeric al phenetic survey of mycolic acid-containing actinomycetes, cultures received as "My cobacterium album" and as "aurantiaca"-like strains were recovered as two homogeneous clusters in an aggregate cluster that also contained the "aurantiaca" taxon. "Mycobacterium album" (32) is listed as a species incertae sedis in Bergey's Manual of Determinative Bacteriology (30), and can be distinguished from most true mycobacteria by its greater resistance to isoniazid (21), mitomycin C (38) and prothionamide (27) and by the fact that it contains unsaturated menaquinones with nine isoprene units as the predominant isoprenologue (2). In the present investigat ion, representatives of "Myco bacterium album ", the "aurantiaca" taxo n and "aurantiaca"-like strains were the subject of compar ative immun odiffusion and lipid analyses designed to help esta blish the relationship of these taxa to one another and to related actinomycetes.

Classification of "Mycobacterium album"

3

Table 1. Designation and source of test strains GB031 GB032 GB205 GA973 GB202 GB460 GB461 GB035 GB036

"Mycobacterium album", R. E. Gordon, Rutgers University, New Brunswick, U.S.A., IMRU 1505; C. McDurmont, R161, M. Goodfellow, M337 "M. album", R. E. Gordon, IMRU 340; L. K. Georg, 44-328-45, M. Goodfellow, M340 "M. album", ATCC 25969° "aurantiaca" taxon, M. Tsukamura, 4465, M. Goodfellow, M293 "aurantiaca" taxon, NCTC 10741, M. Goodfellow, N663°* "aurantiaca" taxon, M. Tsuhamura, 4409, M. Goodfellow, M292 "aurantiaca" taxon, M. Tsukamura, 7972, M. Goodfellow, M294 "aurantiaca"-like taxon, R. E. Gordon, IMRU 469, R. L. Starkey, 213; soil, M. Goodfellow, N800 "aurantiaca"-like taxon, R. E. Gordon, IMRU 669, N. M. McClung, 33; soil, M. Goodfellow, N802

* Type strain ° Reference strain

Methods Comparative immunodiffusion analyses Strains. The designations and sources of the nine test strains and the 25 serological reference strains are given in Tables 1 and 2, respectively. "Mycobacterium album" GB205 and "aurantiaca" GB202 were included in the study both as test and reference strains. Antigen preparation. The test strains were grown on Sauton's agar supplemented with thiamine (0.1 gil). When good growth was obtained, the biomass was harvested, washed and disintegrated as described previously (29). The antigen preparation from the reference strains were either prepared in the same way or from culture filtrates (24). Antisera. The antigen preparations from the reference strains were inoculated into sheep or rabbits in order to obtain antisera (25, 29). Immunodiffusion technique. The analyses were carried out using a microplate modification (40) of the immunodiffusion technique of Ouchterlony (22, 23). Reference precipitation systems. The antigen preparations from the reference strains and their homologous antisera were used to prepare the reference precipitation systems (24,25, 26). Only precipitinogens identified by the reference systems were considered. Lipid analyses Biomass for the lipid analyses were prepared by growing the test strains, Mycobacterium album GB032 and "aurantiaca"-like strain GB036, in shake flasks of modified Sauton's medium (19) supplemented with thiamine (50 mg/l) for 3 to 5 days at 30°C. The strains were checked for purity at maximum growth, killed with formaldehyde (1 per cent, v/v), harvested by centrifugation, washed with distilled water and freeze-dried. Freeze-dried bacteria (50 mg) were degraded by acid methanolysis and hexane extracts examined for mycolic acids by thin-layer chromatography (TLC) as described previously (4, 16). Long chain components were isolated by preparative TLC on layers (1 mm) of Merck silica gel PF254+366' separated bands being detected with ultraviolet light (366 nm). The isoprenoid quinones were extracted and analysed as described by Collins et al. (1). Mass spectra of the purified menaquinones and mycolic esters were taken on an AEI MS9 instrument using a direct insertion probe, an ionizing voltage of 70eV and a temperature range of 190 to 220°C.

4

M.Ridell, M.Goodfellow, and D.E.Minnikin

Table 2. Designation and source of the serological reference strains GB242 GB246 GB243 GB252 GB244 GA009 GA001 GA923 GA023 GA120 GA010 GA924 GA029 GA681 GB144 GA761 GA875 GA873 GB245 GB011 GA766 GA785 GB285 GB205 GB202

Arthrobacter globiformis NCIB 8907, ATCC 8010* Corynebacterium bovis NCTC 3224* Corynebacterium glutamicum NCIB 10025 Corynebacterium "ulcerans" NCTC 7910 Kurthia zopfii NCIB 11155, ATCC 10538 Mycobacterium auium G. Penso Ceppo Faisan IV Mycobacterium bovis var BCG A. Lind Swedish substrain Mycobacterium farcinogenes NCTC 10955* Mycobacterium fortuitum G. Penso 456 Mycobacterium kansasii E. H. Runyon P16 Mycobacterium phlei NCTC 8151, ATCC 11758* Mycobacterium senegalense NCTC 10956* Mycobacterium smegmatis NCTC 8152 Mycobacterium vaccae ATCC 15483* Nocardia amarae M. Lechevalier Se 6, ATCC 27808* Nocardia asteroides I. Juhlin M-6 5006 Nocardia asteroides II ATCC 19247* Nocardia otitidis-caviarum ATCC 14629* Rhodococcus bronchialis NCTC 10667 Rhodococcus equi M. Goodfellow C7, NCTC 1621 * Rhodococcus ruber I. Juhlin 107 Rhodococcus rubropertinctus I. Juhlin M-6 5007 Streptomyces diastaticus ISP 5496 "Mycobacterium album" ATCC 25969 "aurantiaca" taxon NCTC 10741 *

* Type strain

Results Comparative immunodiffusion analyses

The number of identified precipitinogens found when the test strains were analysed with the "Mycobacterium album" (GB205) and "aurantiaca" (GB202) reference systems are shown in Table 3. The rwo reference strains had six identified precipitinogens in common. Most of these identified precipitinogens were also revealed in the remaining two "Mycobacterium album" strains and in two of the remaining three aurantiaca strains. In contrast the two strains of the "aurantiaca"-like taxon shared only one precipitinogen with the "Mycobacterium album" and "aurantiaca" reference systems. The 9 test strains were also analysed by 23 other serological reference systems representing Corynebacterium, Mycobacterium, Nocardia and Rhodococcus species together with some related taxa. None of the 9 test strains showed identifiable precipitinagens when the reference systems for Arthrobacter globiformis, Corynebacterium bovis, C. glutamicum, C. "ulcerans", Kurthia zop(ii, M. kansasii, M. smegmatis and R. bronchialis were used. Table 4 shows the results obtained when the test strains were analysed by the remaining 15 serological reference systems. It is clear that the "Mycobacterium album" and "aurantiaca" strains have few precipitinogens in common with any of these reference strains. This is also the case with the rwo "aurantiaca"-like

Classification of "Mycobacterium album"

5

Table 3. Number of precipitinogens revealed when the "Mycobacterium album", "aurantiaca" and "aurantiaca"-like strains were analysed using reference precipitation systems that represented "Mycobacterium album" and the "aurantiaca" taxon .

Test strains

Reference systems "Mycobacterium album" "aurantiaca" taxon GB205 GB202

"Mycobacterium album" GB031 GB032 GB205

6 7 10*

6 8

"aurantiaca" taxon GA973 GB202 GB460 GB461

2 6 6 7

4 8* 5 8

"aurantiaca"-like taxon GB035 GB036

1 1

1

6

1

* Number of precipitinogens in the reference system

strains, although the latter did have between one and three identified precipitinogens in common with the Nocardia asteroides and N. otitidis-caviarum reference strains.

Lipid analyses Thin-layer chromatographic analysis of whole-organism methanolysates revealed the presence of mycolic acid methyl esters (RF 0.2 to 0.5) in each of the test strains. The mycolic ester from "Mycobacterium album" GB032 showed a similar mobility on TLC to the methyl mycolates of representatives of the "aurantiaca" taxon (4). In contrast, the mycolic ester from aurantiaca-like strain GB036 had a mobility similar to that expected of methyl mycolates from Nocardia strains (16). Methyl esters of mycolic acids fragment on mass spectrometry in several competing pathways depending on their overall size and structural features (18). A complex series of fragments corresponding to aldehydes were seen, on mass spectrometry of the mycolic esters of "Mycobacterium album" GB032, at the following m/z values (figures in parenthesis specify the number of C atoms and degree of unsaturation): 710 (CSO:3 )' 712 (C SO:2 ), 736 (C S2 :4 )' 738 (C52 :3 ), 762 (C S4 :S )' 764 (C S4:4), 790 (C s6:5l and 792 (CS8:6 )' Further, fragments at m/z 292,320,326 and 354 indicated the presence of C 20 and C 22 straight chain saturated and unsaturated fatty acid esters (4). In the case of aurantiaca-like strain GB036, anhydromycolate peaks at m/z 686, 714, 742, 768,770 and 796 corresponded to C 46 : h C SO: h C 52 :2 , CS2 :1 and CS4 :2 , respectively. Similarly, side chain peaks at mlz 242, 270, 271 and 299 correspond to C 14 and C 16 fatty acid esters (18). Extracts of each of the test strains contained menaquinones which co-chromatographed on TLC with vitamin K. The single peak from the extract of "Mycobacterium album" GB032 occurred at mlz 788 and corresponded to an unsaturated menaquinone

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Test strains

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"aurantiaca" taxon GA973 GB202 GB460 GB461

"aurantiaca"-like taxon GB035 GB036

Number of precipitinogens in the reference system

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Table 4. Number of precipitinogens revealed when the "Mycobacterium album", "aurantiaca" and "aurantiaca"-like strains were examined using reference precipitation systems representing the genera Mycobacterium, Nocardia, Rhodococcus and Streptomyces

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Classification of "Mycobacterium album"

7

with nine isoprene units. The corresponding peak in "aurantiaca"-like strain GB036 was at mlz 718 and indicated the presence of a dihydrogenated menaquinone with eight isoprene units.

Discussion The large number of precipitinogens common to the representatives of "Mycobacterium album" and the "aurantiaca" taxon underlines the close relationship between these taxa previously found in a numerical phenetic survey of mycolic acid-containing actinomycetes (5, 8). Indeed, the serological data suggest that these organisms might belong to the same species. In contrast, the "aurantiaca"-like strains had one antigen in common with the "Mycobacterium album" or "aurantiaca" reference strains though they did have up to three shared precipitinogens with the corresponding Nocardia asteroides and Nocardia otitidis-caviarum strains. The serological data support the separation of the aggregate "aurantiaca" taxon, containing "Mycobacterium album" and the "aurantiaca" cluster of Goodfellow et al. (4), from the established nocardioform genera Corynebacterium, Mycobacterium, Nocardia and Rhodococcus. The detection of major amounts of unsaturated menaquinones with nine isoprene units in "Mycobacterium album" GB032 is in good agreement with an earlier study that included a single strain of this taxon (2). The menaquinone data, together with the discovery that "Mycobacterium album" GB032 contained mycolic acids intermediate in size (C 68 to C 74 ) between those of mycobacteria, and nocardiae and rhodococci, further underscores the relationship with the "aurantiaca" taxon (4). Further, both the "Mycobacterium album" and the aurantiaca strains have mycolic acids with up to five double bonds and release C20 to Cl l saturated and monounsaturated esters upon fragmentation of the parent molecule. These characteristic mycolic acids and menaquinone profiles serve to distinguish members of the aggregate "aurantiaca" taxon from all of the established mycolic acid-containing taxa including the genus Rhodococcus (10, 11, 12, 18). The discovery of mycolic acids with between 46 and 54 carbon atoms in "aurantiaca"-like strain GB036 suggests a relationship with the genus Nocardia (11, 18). However, most nocardiae contain major amounts of tetrahydrogenated menaquinones with eight isoprene units (2, 18) but the aurantiaca-like strain possessed large amounts of dihydrogenated menaquinones with eight isoprene units. Large amounts of dihydrogenated menaquinones with eight isoprene units are also characteristic of some corynebacteria and certain rhodococci (2, 11, 18). Clearly, further comparative studies are required to unravel the taxonomic affinities of the aurantiaca-like strains. The results of this limited study clearly indicate that "Mycobacterium album" has properties inconsistent with its inclusion in the genus Mycobacterium (8, 13). Further, "Mycobacterium album" does appear to be homogeneous and is closely related to the "aurantiaca" taxon. Although additional comparative studies are required to determine the exact relationship between these two taxa, they can be assigned to a single aggregate group, which can be distinguished from the genera Corynebacterium, Mycobacterium, Nocardia and Rhodococcus on the basis of numerical phenetic, serological and lipid data (10, 11, 12). Further studies on additional strains are needed to determine whether the aggregate "aurantiaca" taxon merits generic rank.

8

M.Ri4eU, M.Goodfellow, and D.E.Minnikin

M. R. is grateful for support from the Swedish National Association against Heart and Chest Diseases and the Ellen, Walter and Lennart Hesselman Foundation (Sweden). M. G. and D. E. M. are grateful for support from the Medical Research Council (G974/522/S) (Great Britain). The authors are indebted to P. Kelly for mass spectroscopy and to V. Sundaeus, G. Wallerstrom and]. Parlett for valuable technical assistance.

References 1. Collins, M. D., T. Pirouz, M. Goodfellow, and D. E. Minnikin: Distribution of menaquinones in actinomycetes and corynebacteria.]. gen. Microbio!. 100 (1977) 221-230 2. Collins, M. D., M. Goodfellow, D. E. Minnikin, and G. Alderson: Menaquinone composition of mycolic acid-containing actinomycetes and some sporoactinomycetes. J. app!. Bact. in press 3. Goodfellow, M. and G. Alderson: The actinomycete-genus Rhodococcus: a home for the "rbodochrous" complex.]. gen. Microbio!. 100 (1977) 99-122 4. Goodfellow, M., P. A. B. Orlean, M. D. Collins, L. Alshamaony, and D. E. Minnikin: Chemical and numerical taxonomy of strains received as Gordona aurantiaca. J. gen. Microbiol. 109 (1978) 57-68 5. Goodfellow, M. and D. E. Minnikin: Definition of the genus Mycobacterium vis-a-vis other allied taxa. In: Twenty-Five Years of Mycobacterial Taxonomy, pp. 115-129, Eds. G. P. Kubica, L. G. Wayne, and L. S. Good U. S. Department of Health and Human Services, Atlanta (1980) 6. Goodfellow, M. and D. E. Minnikin: The genera Nocardia and Rhodococcus. In: The Prokaryotes, Volume II, pp. 2016-2027, Eds. M. P. Starr, H. Stolp, H. G. Triiper, A. Balows, and H. G. Schlegel. Springer Verlag, Berlin (1981) 7. Goodfellow, M., D. E. Minnikin, C. Todd, G. Alderson, S. M. Minnikin, and M. D. Collins: Numerical and chemical classification of Nocardia amarae. ]. gen. Microbio!. 128 (1982) 1283-1297 8. Goodfellow, M. and L. G. Wayne: Taxonomy and nomenclature. In: The Biology ofthe Mycobacteria, Volume 1, pp. 471-521, Eds. C. Ratledge and]. Stanford. Academic Press, London (1982) 9. Goodfellow, M., C. R. Weaver, and D. E. Minnikin: Numerical classification of some rhodococci, corynebacteria and related organisms. ]. gen. Microbio!. 128 (1982) 731-745 10. Goodfellow, M: Rhodococcus. In: Bergey's Manual of Determinative Bacteriology, Ninth Edition, Ed. P. H. A. Sneath. The Williams and Wilkins Co., Baltimore (1985) 11. Goodfellow, M. and T. Cross: Classification. In: The Biology of the Actinomycetes, pp. 7-164, Eds. M. Goodfellow, M. Mordarski, and S. T. Williams, Academic Press, London (1984) 12. Goodfellow, M. and M. P. Lechevalier: Nocardia. In: Bergey's Manual of Determinative Bacteriology, Ninth Edition, Ed. P. H. A. Sneath. The Williams and Wilkins Co., Baltimore (1985), in press 13. Goodfellow, M. and D. E. Minnikin: Circumscription of the genus. In: The Mycobacteria: A Sourcebook, Volume 1, The Mycobacteria, pp. 1-24, Eds. G. P. Kubica and L. G. Wayne. Marcel Dekker, New York (1984) 14. Lind, A. and M. Ridell: Serological relationship between Nocardia, Mycobacterium, Corynbacterium and the "rhodochrous" taxon. In: The Biology of the Nocardiae, pp. 220-235, Eds. M. Goodfellow, G. H. Brownell, and]. A. Serrano. Academic Press, London (1976) 15. Lind, A., 6. Ouchterlony, and M. Ridell: Mycobacterial antigens. In: Infektionskrankheiten und ihre Erreger: Mykobakterien und mykobakterielle Krankheiten, Bd. 4/1, pp. 245-303, Eds. G. Meissner and A. Schmiedel. VEB Gustav Fischer Verlag, Jena (1980)

Classification of "Mycobacterium album"

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16. Minnikin, D. E., L. Alshamaony, and M. Goodfellow: Differentiation of Mycobacterium, Nocardia and related taxa by thin-layer chromatographic analyses of wholeorganism methanolysates. J. gen. Microbiol. 88 (1975) 200-204 17. Minnikin, D. E., M. Goodfellow, and M. D. Collins: Lipid composition in the classification and identification of coryneform and related taxa. In: Coryneform Bacteria, pp. 85-160, Eds, I. J. Bousfield and A. G. Callely. Academic Press, London (1978) 18. Minnikin, D. E. and M. Goodfellow: Lipid composition in the classification and identification of acid-fast bacteria. In: Microbiological Classification and Identification, pp. 189-256, Eds. M. Goodfellow and R. G. Board. Academic Press, London (1980) 19. Mordarska, H., M. Mordarski, and M. Goodfellow: Chemotaxonomic characters and classification of some nocardioform bacteria. J. gen. Microbiol. 71 (1972) 77-86 20. Mordarski, M., M. Goodfellow, A. Tkacz, G. Pulverer, and K. P. Schaal: Ribosomal ribonucleic acid similarities in the classification of Rhodococcus and related taxa. ]. gen. Microbiol. 118 (1980) 313-319 21. Orlean, P. A. B., M. Goodfellow, and D. E. Minnikin: Isoniazid susceptibility as a criterion for the differentiation of mycobacterial species from other mycolic acid-containing taxa. Int. ]. system. Bact. 28 (1978) 194-196 22. Ouchterlony, 0.: Diffusion-in-gel methods for immunological analysis. I. Progr. Allergy 5 (1958) 1-78 23. Ouchterlony, 0.: Diffusion-in-gel methods for immunological analysis. II. Progr. Allergy 6 (1962) 30-154 24. Ridell, M. and M. Norlin: Serological study of Nocardia by using mycobacterial precipitation reference systems. J. Bact. 113 (1973) 1-7 25. Ridell, M.: Taxonomic study of Nocardia farcinica using serological and physiological characters. Int.]. system. Bact. 25 (1975) 124-132 26. Ridell, M., R. Baker, A. Lind, and O. Ouchterlony: Immunodiffusion studies of ribosomes in classification of mycobacteria and related taxa. Int. Arch. Allergy appl. Immunol. 59 (1979) 162-172 27. Ridell, M.: Sensitivity to capreomycin and prothionamide in strains of Mycobacterium, Nocardia, Rhodococcus, and related taxa for taxonomical purposes. Zbl. Bakt. Hyg., I. Abt. Orig-A 255 (1983) 309-316 28. Ridell, M.: Cross-reactivity between Mycobacterium tuberculosis H37Rv and various actinomycetes and related taxa. Tubercle 64 (1983) 211-216 29. Ridell, M. and S. T. Williams: Serotaxonomical analyses of some Streptomyces and related organisms, J. gen. Microbiol. 129 (1983) 2857-2861 30. Runyon, E. H., L. G. Wayne, and G. P. Kubica: Mycobacterium. In: Bergey's Manual of Determinative Bacteriology, Eight Edition, pp. 681-701, Eds. R. E. Buchanan and N. E. Gibbons. The Williams and Wilkins Co., Baltimore (1974) 31. Skerman, V. B. D., V. McGowan, and P. H. A. Sneath: Approved lists of bacterial names. Int. J. system. Bact. 30 (1980) 225-420 32. Sohngen, N. L.: Benzin, Petroleum, Paraffinol und Paraffin als Kohlenstoff- und Energiequelle fiir Mikroben. Zbl. Bakt., II. Abt, 37 (1913) 595-609 33. Stackebrandt, E. and C.R. Woese: The evolution of prokaryotes. In: Molecular and Cellular Aspects of Microbial Evolution, pp. 1-31, Eds. M. j. Carlile, j. F. Collins, and B. E. B. Moseley. University Press, Cambridge (1981) 34. Tsukamura, M. and S. Mizuno: A new species Gordona aurantiaca occurring in sputa of patients with pulmonary disease. Kekkaku 46 (1971) 93-98 35. Tsukamura, M.: A further numerical taxonomic study of the rhodochrous group. Jap.]. Microbiol. 18 (1974) 37-44 36. Tsukamura, M.: Numerical analysis of the relationship between Mycobacterium, rhodochrous group and Nocardia by use of hypothetical median organisms. Int. J. system. Bact. 25 (1975) 329-335 37. Tsukamura, M.: Numerical classification of Rhodococcus (formerly Gordona) orga-

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M.Ridell, M.Goodfellow, and D.E.Minnikin

nisms recently isolated from the sputa of patients: Description of Rhodococcus sputi Tsukamura sp. nov. Int.]. system. Bact. 28 (1978) 169-181 38. Tsukamura, M.: Test for susceptibility to mitomycin C as aids for differentiating the genus Rhodococcus from the genus Nocardia, and for differentiating Mycobacterium fortuitum and Mycobacterium chelonei from other rapidly growing mycobacteria. Microbiol. Immunol. 25 (1981) 1197-1199 39. Tsukamura, M.: Numerical analysis of the taxonomy of the nocardiae and rhodococci. Microbial. Immunol. 26 (1982) 1101-1119 40. Wadsworth, c.: A microplate technique employing a gel chamber compared with other micro- and macroplate techniques for immune diffusion. Int. Arch. Allergy app!. Immuno!. 21 (1962) 131-137 Dr. Malin Ridell, Department of Medical Microbiology, University of Goteborg, Guldhedsgatan 10, S-413 46 Goteborg, Sweden