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Sensitivity to capreomycin and prothionamide in strains of Mycobacterium, Nocardia, Rhodococcus, and related taxa for taxonomical purposes
Zbl. Bakt. Hyg., 1. Abt. Orig. A 255, 309-316 (1983)
Sensitivity to Capreomycin and Prothionamide in Strains of Mycobacterium, Nocardia, Rhodococcus, and Related Taxa for Taxonomical Purposes Empfindlichkeit gegen Capreomycin und Protionamide in Stammen von Mycobacterium, Nocardia, Rhodococcus und verwandten Taxa fUr taxonomische Z wecke MALIN RIDELL
Department of Medical Microbiology, University of Goteborg, Goteborg, Sweden
Received February 18, 1983
Abstract Sensitivity to capreomycin and to prothionamide was analysed for 150 strains belonging to the genera Mycobacterium, Nocardia, Rhodococcus, and related taxa. The analyses showed, e.g., that strains of Mycobacterium chelonei and Nocardia brasiliensis were more resistant to capreomycin than the other strains tested and that M. farcinogenes differed from M. senegalense concerning sensitivity to this drug. The analysis showed, furthermore, that strains of Mycobacterium differ from those of Nocardia, Rhodococcus, "Mycobacterium album", and "Cardona aurantiaca" in being more sensitive to prothionamide than these organisms. Strains designated N. amarae were more sensitive to both drugs than were the other tested strains of Nocardia, which indicates that N. amarae diverges from the other species of this genus.
Zusammenfassung Fur 150 Stamme aus den Genera Mycobacterium, Nocardia, Rhodococcus und verwandte Taxa wurde die Sensibilitat gegen Capreomycin (Tab. 1) und Protionamid (Tab. 2 und 3) bestimmt. Die Tests wurden in Lowenstein-Jensen-Medium mit den Konzentrationen 10, 20, 40, 160 und 320 flg/ml durchgefuhrt. Die Methode wurde primar entwickelt, um einen einfachen Test fur taxonomische Zwecke zur Verfiigung zu haben; sie ist nicht zur Beurteilung der Sensibilitat fur therapeutische Zwecke geeignet. - Es wurde festgestellt, daB Stamme von Mycobacterium chelonei und Nocardia brasiliensis resistenter gegen Capreomycin sind als die ubrigen untersuchten Stamme und daB sich M. senegalense durch seine hohere Capreomycin-Resistenz von M. farcinogenes unterscheidet. Es wurde weiterhin nachgewiesen, daB die gepruften Mycobacterium-Stamme empfindlicher gegen Protionamide sind als die Stamme von Nocardia, Rhodococcus, "Mycobacterium album"
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M. Ridell
und "Cardona aurantiaca". Die als N. amarae bezeichneten Stamme waren gegen Capreomycin und Protionamid empfindlicher als aIle iibrigen Nocardia-Stiimme; dies weist darauf hin, daiS sich N. amarae von den anderen Species dieses Genus unterscheidet.
Introduction In recent years many taxonomical studies have been performed on Mycobacterium, Nocardia, Rhodococcus, and related taxa, and these studies have resulted in an improvement of the classification of these organisms (see, e.g., recent review articles (14, 18, 20)). A number of taxonomical problems remain, however, and good characters for classification and identification purposes are still needed. A large part of the taxonomical studies hitherto performed have, furthermore, been made by chemical, genetical or immunological methods which are too expensive, timeconsuming or complicated to be used for identification purposes in diagnostic laboratories. Additional methods are, therefore, needed. Such methods are, e.g., sensitivity tests which have shown to be good characters for classification and identification of the pertinent taxa (2, 7, 12, 15, 19,23,30,32). It is well known that many of the non tuberculous mycobacteria and several of the nocardiae are pathogenic. Recently, certain rhodococci have also been found to cause disease (5). Knowledge concerning the sensitivity of these organisms to drugs is, however, scattered. Some species are fairly well studied (1,4,9, 10, 11, 28), whereas information about most of these organisms is sparse or nonexistent. The aim of the present study was to test the sensitivity to capreomycin and prothionamide in strains referred to Mycobacterium, Nocardia, Rhodococcus, and related taxa.
Materials and Methods Strains. Eighty-seven strains of Mycobacterium, 38 of Nocardia, 9 of Rhodococcus, 9 designated "Cardona aurantiaca", and 7 designated "Mycobacterium album" were ex-
amined (see tables). Media. The tests were performed on Lowenstein-Jensen media containing capreomycin or prothionamide at the concentrations 10, 20, 40, 160 and 320 flg/ml. As controls Lowenstein-Jensen media without any drug were used. Inoculum. One loopful (the inner diameter of the loop was 4 mm) of bacterial mass from Lowenstein-Jensen cultures was suspended in 2 ml phosphate buffer solution and 0.1 ml of this suspension was used as inoculum. Sensitivity test. The cultures were incubated at 37°C for 3 weeks. Some slow-growing strains were incubated for 4 weeks. Registration of growth was performed in accordance with a 5-step scale: no colonies, 1-20 colonies, 20-100 colonies, more than 100 colonies, and confluent growth. Inhibition of growth was defined as a decrease in growth in relation to the control by 2 steps or more.
Results The results of the capreomycin sensitivity tests are given in Table 1. Most mycobacteria were not inhibited by this drug at the concentration 10,Ltg/ml apart from
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Table 1. Sensitivity to capreomycin among strains of Mycobacterium, Nocardia, Rhodococcus and related taxa Species
BCG M. avium M. chelonei M. farcinogenes M. fortuitum M. gastri M. kansasii M. marinum M. microti M.phlei M. scrofulaceum M. senegalense M. smegmatis M. vaccae N. amarae N. asteroides N. brasiliensis N. farcinica N. otitidis-caviarum R. coprophilus R. corallinus R. ruber "G. aurantiaca" "M. album"
No. of strains inhibited by concentration in rlg/ml
No. of tested strains
10
20
40
160
320
1 5 10 15 15 3 3 3 2 3 3 15 6 3 6 14 4 10 4 2 3 4 9 7
0 0 0 14 0 2 0 0 1 3 1 1 1 0 5 2 0 0 1 1 0 1 2 0
0 1 0 15 1 3 1 3 2 3 1 6 2 1 5 6 0 0 4 2 3 4 4 1
0 3 0 15 6 3 2 3 2 3 3 10 6 3 5 8 0 5 4 2 3 4 6 6
1 5 1 15 9 3 3 3 2 3 3 12 6 3 6 12 0 9 4 2 3 4 6 7
1 5 3 15 12 3 3 3 2 3 3 15 6 3 6 12 0 10 4 2 3 4 6 7
14 out of 15 tested M. farcinogenes, 2 out of 3 tested M. gastri, all 3 tested M. ph lei and one strain of each of the four species M. microti, M. scrofulaceum, M. senegalense, and M. smegmatis. With increasing concentrations the number of sensitive strains rose and practically all mycobacteria were inhibited by 160 ,ug/ml, apart from 9 out of 10 strains of M. chelonei, 6 out of 15 strains of M. fortuitum, and 3 out of 15 strains of M. senegalense. Most M. chelonei (7 out of 10) and some M. fortuitum (3 out of 15) strains were not even inhibited by 320 ,ag/ml capreomycin. Five out of 6 N. amarae strains were inhibited by 10 ,ag/ml capreomycin, and all 4 tested N. otitidis-caviarum strains were inhibited by 20 flg/ml. In contrast, none of the N. brasiliensis strains was inhibited by 320 flg/ml. The N. asteroides strains varied strongly concerning their sensitivity to capreomycin. Two of them were inhibited by 10 flg/ml while 2 other strains were not inhibited by 320 flg/ml. The Rhodococcus strains were all inhibited by capreomycin at the concentration 20 flg/ml, whereas the strains designated "Cordona aurantiaca" varied in sensitivity. The results of the prothionamide sensitivity tests are given in Tables 2 and 3. At the concentration 10 flg/ml none but 2 of the tested mycobacteria were inhibited. Also at 20 ftg/ml most strains of this genus were not inhibited, apart from 8 strains representing the species M. gastri, M. kansasii, M. marinum, M. microti, or M.
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Table 2. Sensitivity to prothionamide among strains of Mycobacterium, Nocardia, Rhodococcus and related taxa Species
BeG M. avium M. che/onei M. farcinogenes M. fortuitum M. gastri M. kansasii M. mal'inum M. microti M. phlei M. scrofulaceum M. senegalense M. smegmatis M. vaccae N. amarae N. asteroides N. brasiliensis N. farcinica N. otitidis-caviarum R. coprophilus R. corallinus R. ruber "G. aurantiaca" "M. album"
No. of tested strains 1 5 10 15 15 3 3 3 2 3 3 15 6 3 6 14 4 10 4 2 3 4 9
7
No. of strains inhibited by concentration in pg/ml 20 40 160
10 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0
1
1 1 0 0 0 3 2 3 2 0 1 2 0 2 3 0 0 0 0 0 0 0 0 0
0 0 0 0 0 2 2 2 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0
320 1 5 10 15 13 3 3 3 2 3 3 15 6 3 6 2 4 0 0 2 0 0 4 4
5 5 13 5 3 3 3 2 2 3 12
2 3 6 0 0 0 0 1 0 0 0 0
Table 3. Sensitivity to prothionamide among strains of Mycobacterium, Nocardia, Rhodococcus and related organisms given as percentage of sensitive strains within each taxon Taxa
Mycobacterium Nocardia (except N. amarae) N. amarae Rhodococcus "Gordona aurantiaca" and "M. album"
Total no. of tested strains
% of strains sensitive 10
20
40
160
87 32
2 0
9 0
20 0
71
6 9 16
0 0 0
0 0 0
50 0 0
100
0
11 0
to
320 pg/ml 98 19 100 22 50
scrofulaceum. At 160,ug/ml, however, most mycobacterial strains (71 %, see Table 3) were inhibited. All but 2 M. fortuitum strains were inhibited by 320 ,ug/ml of
prothionamide (i. e. 98%). Hardly any of the remaining organisms tested, represent-
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313
ing Nocardia, Rhodococcus and related taxa, were inhibited by prothionamide at concentrations up to 160 ,Ltg/m!. Three out of the 6 tested N. amarae strains were, however, inhibited by 40,Ltg/ml, and all strains of these species were inhibited by 160 ,Ltg/m!. Furthermore, one strain of R. coprophilus was also inhibited by the latter concentration. At the highest test concentration of prothionamide, i. e. 320 ,Ltg/ml, 2 out of 14 tested strains of N. asteroides and all 4 tested N. brasiliensis strains were inhibited, while all tested N. farcinica and N. otitidis-caviarum strains grew well. Among the rhodococci 2 R. coprophilus strains were inhibited, but none of the R. corallinus and R. ruber strains. None of the 16 strains designated "G. aurantiaca" and "M. album" was inhibited by 160 flg/ml prothionamide, but 8 of them (50%) were inhibited by 320 flg/ml of this drug. Eight strains were included in triplicate at the analyses of both drugs. Test errors of about 7% were obtained.
Discussion The present method was primarily developed in order to obtain a simple method for taxonomical purposes, and the therapeutic aspects were considered as being of secondary interest. The method differs therefore considerably from the established methods used for testing drug sensitivity in mycobacteria, such as the well-known methods by Canetti et al. (6). For example, the inoculum is larger in the present study than in the mentioned analyses, which might give results in which some sensitive strains containing resistant mutants are reported as resistant. Capreomycin and prothionamide might, therefore, have therapeutic value also for some of the organisms here registered as resistant. The study demonstrates, however, clear differences in susceptibility to these drugs among the strains studied. The present method, which is simple to perform, is therefore of value in the classification and identification of these organisms. Among the mycobacteria, the species being most resistant to capreomycin was M. chelonei. Most tested strains of this species grew well in the presence of this drug at the concentration 320 ltg/ml in contrast to the other mycobacteria tested, including the closely related M. fortuitum - a characteristic that might be of value for differentiation and identification of M. chelonei. Swenson et al. (28) have also demonstrated that strains of M. chelonei are more resistant to capreomycin than those of M. fortuitum. The present results demonstrate that sensitivity to capreomycin can be used in order to differentiate M. farcinogenes from M. senegalense. These two species were proposed by Chamoiseau (8) for organisms earlier referred to one species: M. farcinogenes. Recent studies have demonstrated that M. farcinogenes and M. senegalense have the same serological pattern (25) but are phenetically distinguishable (26).
The Nocardia species tested demonstrated different patterns of sensitivity to the two drugs. For example, all strains of N. brasiliensis were far less sensitive to capreomycin and more resistant to prothionamide than were the strains of N. farcinica and N. otitidis-caviarum. The number of strains tested was, however, small and more strains have to be analysed before the differential value of the sensitivity tests can be evaluated.
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The strains of N. asteroides varied strongly concerning their sensitivity to capreomycin, in contrast to the strains of the other Nocardia species. This result is not surprising since several earlier studies based on genetical (21), numerical (22) and serological (24) methods have shown that N. asteroides is a heterogeneous species. Strains referred to the species N. amarae (17) differed concerning their sensitivity to capreomycin and prothionamide from other nocardiae, being more sensitive to these drugs. These findings are in agreement with results from lipid analyses (20), phage-typing (34) and immunodiffusion (24), indicating that N. amarae diverges from the species of Nocardia sensu stricto. The results of the sensitivity tests concerning prothionamide (Tables 2 and 3) suggest a clear difference between strains of the genus Mycobacterium on one hand and those of the genera Nocardia and Rhodococcus on the other, the latter being more resistant to this drug. This difference is more evident when N. amarae, with its doubtful taxonomical position, is excluded from the genus Nocardia. The species "M. album" is listed as a species incertae sedis in Bergey's Manual of Determinative Bacteriology (27), and organisms designated "Gordona aurantiaca" or the "aurantiaca" taxon also have an unsettled taxonomical position (20). Goodfellow et aI. (13) demonstrated that "G. aurantiaca" formed a distinct taxon different from mycobacteria, nocardiae and rhodococci, while Tsukamura et aI. (29, 31) placed them in the genus Rhodococcus. The present study demonstrates that "M. album" and "G. aurantiaca" were different from the majority of the mycobacteria tested concerning their sensitivity to prothionamide, being less sensitive to this drug. Some data from the present study might be of interest from a chemotherapeutic point of view. It is, e.g., worth mentioning that strains of M. farcinogenes, N. asteroides, N. otitidis-caviarum and strains of Rhodococcus were sensitive to the lower concentrations of capreomycin. M. farcinogenes and N. asteroides are well known pathogens but N. otitidis-caviarum and certain strains of Rhodococcus have also been shown to cause infections (3, 5, 16, 33). Prothionamide seems to be of little value for the treatment of infections caused by strains of Nocardia, Rhodococcus and many mycobacteria, since these organisms showed very high resistance to this drug. Acknowledgements. This investigation was supported by the Swedish National Association against Heart and Chest Diseases and the Ellen, Walter and Lennart Hesselman Foundation. The skillful technical assistance of Vivianne Sundaeus and Gun Wallerstrom is gratefully acknowledged.
References 1. Bach, M.e., L.D.Sabath, and M.Finland: Susceptibility of Nocardia asteroides to 45 antimicrobial agents in vitro. Antimicrob. Agents Chemother. 3 (1973) 1-8 2. Backelin, B., A. Lind, and M. Ridell: A new test method for demonstrating j3-lactamase activity among mycobacteria and nocardia. Tubercle 54 (1973) 297-307 3. Beaman, B. L., ]. Burnside, B. Edwards, and W. Causey: Nocardial infections in the United States, 1972-1974. ]. infect. Dis. 134 (1976) 286-289 4. Bradley, S. G.: Susceptibility of mycobacteria to antitubercular drugs. Develop. Industr. Microbiol. 17 (1976) 199-207 5. Broughton, R. A., H. D. Wilson, N. L. Goodman, and J. A. Hedrick: Septic arthritis and
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osteomyelitis caused by an organism of the genus Rhodococcus. J. Clin. Microbiol. 13 (1981) 209-213 6. Canetti, G., S. Froman, ]. Grosset, P. Hauduroy, M. Langerova, H. T. Mahler, G. Meissner, D. A. Mitchison, and S. Sula: Mycobacteria: laboratory methods for testing drug sensitivity and resistance. Bull. Wid Hlth Org. 29 (1963) 565-578 7. Casal, M.]. and F. C. Rodriques: Simple, new test for rapid differentiation of the Mycobacterium fortuitum complex. J. Clin. Microbiol. 13 (1981) 989-990 8. Chamoiseau, G.: Etiology of farcy in African bovines: nomenclature of the causal organisms Mycobacterium farcinogenes Chamoiseau and Mycobacterium senegalense (Chamoiseau) comb. nov. Int. J. system. Bact. 29 (1979) 407-410 9. Dalovisio, ]. R. and G. A. Pankey: Problems in diagnosis and therapy of Mycobacterium fortuitum infections. Am. Rev. Respir. Dis. 117 (1978) 625-630 10. Freerksen, E., E.H. Orlowski, and ].H. Thumin: Experimental and clinical evaluation of the tuberculostatics Capreomycin, Isoxyl. Myambutol and Rifampicin, in: Antibiotica et Chemotherapia, Vol. 16. S. Karger, Basel 1970 11. Gangadharam, P. R.]. and E. R. Candler: Activity of some anti leprosy compounds against Mycobacterium intracellulare in vitro. Amer. Rev. resp. Dis. 115 (1977) 705-708 12. Goodfellow, M. and V. A. Orchard: Antibiotic sensitivity of some nocardioform bacteria and its value as a criterion for taxonomy. J. gen. Microbiol. 83 (1974) 375-387 13. Goodfellow, M., P. A. Orlean, M. D. Collins, L. Aishamaony, and D. E. Minnikin: Chemical and numerical taxonomy of strains received as Gordona aurantiaca. J. gen. MicrobioI. 109 (1978) 57-68 14. Goodfellow, M. and D. E. Minnikin: Classification of nocardioform bacteria. Zbl. Bakt., LAbt., Suppl. 17 (1981) 7-16 15. Gordon, R. E. and D. A. Barnett: Resistance to Rifampin and Lysozyme of strains of some species of Mycobacterium and Nocardia as a taxonomic tool. Int. J. system. Bact. 27 (1977) 176-178 16. Handrick, W., P. Schneider, and C. Schonborn: Uber eine eitrige Perikarditis durch Mycobacterium rhodochrous bei einem Kleinkind. Paediatr. Paedol. 10 (1975) 276-281 17. Lechevalier, M. P. and H. A. Lechevalier: Nocardia amarae sp. nov. an actinomycete common in foaming activated studge. Int. J. system. Bact. 24 (1974) 278-288 18. Lind, A. and M. Ridell: Immunological classification: Immunodiffusion and immunoelectrophoresis, in: The Mycobacteria, A Source Book, ed. G. Kubica and L. G. Wayne. Marcel Dekker Inc., New York (in press) 19. Meissner, G. and Twenty-one Collaborators: A co-operative numerical analysis of nonscoto- and nonphotochromogenic slowly growing mycobacteria. J. gen. Microbiol. 83 (1974) 207-235 20. Minnikin, D. E. and M. Goodfellow: Lipid composition in the classification and identification of acid-fast bacteria, in: Microbiological Classification and Identification, ed. M. Goodfellow and R. C. Board, pp. 189-256. Academic Press, London (1980) 21. Mordarski, M., K.P.Schaal, K.Szyba, G.Pulverer, and A. Tkacz: Interrelation of Nocardia asteroides and related taxa as indicated by deoxyribonucleic acid reassociation. Int. J. system. Bact. 27 (1977) 66-70 22. Orchard, V. A. and M. Goodfellow: Numerical classification of some named strains of Nocardia asteroides and related isolates from soil. J. gen. Microbiol. 118 (1980) 295-312 23. 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. J. system. Bact. 28 (1978) 194-196 24. Ridell, M.: Immunodiffusion studies of some Nocardia strains. J. gen. Microbiol. 123 (1981) 69-74 25. Ridell, M.: Immunodiffusion analyses of Mycobacterium farcinogenes, Mycobacterium senegalense and some other mycobacteria. J. gen. Microbiol. 129 (1983) 613-619 26. Ridell, M. and M. Goodfellow: Numerical classification of Mycobacterium farcinogenes, Mycobacterium senegalense and related taxa. J. gen. Microbiol. 129 (1983) 599-611
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27. Runyon, E.H., L. G. Wayne, and G.Kubica: Mycobacteriaceae, in: Bergey's Manual of Determinative Bacteriology, ed. R.E.Buchanan and N.E.Gibbons, pp.681-701. The William and Wilkins Company, Baltimore (1974) 28. Swenson, ]. M., C. Thornsberry, and V. A. Silcox: Rapidly growing mycobacteria: testing of susceptibility to 34 antimicrobial agents by broth microdilution. Antimicrob. Agents Chemother. 22 (1982) 186-192 29. Tsukamura, M.: Numerical classification of Rhodococcus (formerly Gordona) organisms recently isolated from sputa of patients: description of Rhodococcus sputi Tsukamura sp. nov. Int. ]. system. Bact. 28 (1978) 169-181 30. Tsukamura, M.: Differentiation between the genera Rhodococcus and Nocardia and between species of the genus Mycobacterium by susceptibility to Bleomycin. ]. gen. Microbiol. 128 (1982) 2385-2388 31. Tsukamura, M., S.Mizuno, S. Tsukamura, and]. Tsukamura: Comprehensive numerical classification of 369 strains of Mycobacterium, Rhodococcus, and Nocardia. Int. ]. system. Bact. 29 (1979) 110-129 32. Wayne, L. G. and Fourteen Collaborators: A co-operative numerical analysis of scotochromogenic slowly growing mycobacteria. ]. gen. Microbiol. 66 (1971) 255-271 33. Williams, D.M., ].A.Krick, and ].S.Remington: Pulmonary infection in the compromised host. Amer. Rev. resp. Dis. 114 (1976) 359-386 34. Williams, S. T., E. M. H. Wellington, and L. S. Tipler: The taxonomical implications of the reactions of representative Nocardia strains to Actinophage. ]. gen. Microbiol. 119 (1980) 173-178 Dr. Malin Ridell, Department of Medical Microbiology, University of Goteborg, Guldhedsgatan 10, 5-41346 Goteborg, Sweden
Sensitivity to Capreomycin and Prothionamide in Strains of Mycobacterium, Nocardia, Rhodococcus, and Related Taxa for Taxonomical Purposes Empfindlichkeit gegen Capreomycin und Protionamide in Stammen von Mycobacterium, Nocardia, Rhodococcus und verwandten Taxa fUr taxonomische Z wecke MALIN RIDELL
Department of Medical Microbiology, University of Goteborg, Goteborg, Sweden
Received February 18, 1983
Abstract Sensitivity to capreomycin and to prothionamide was analysed for 150 strains belonging to the genera Mycobacterium, Nocardia, Rhodococcus, and related taxa. The analyses showed, e.g., that strains of Mycobacterium chelonei and Nocardia brasiliensis were more resistant to capreomycin than the other strains tested and that M. farcinogenes differed from M. senegalense concerning sensitivity to this drug. The analysis showed, furthermore, that strains of Mycobacterium differ from those of Nocardia, Rhodococcus, "Mycobacterium album", and "Cardona aurantiaca" in being more sensitive to prothionamide than these organisms. Strains designated N. amarae were more sensitive to both drugs than were the other tested strains of Nocardia, which indicates that N. amarae diverges from the other species of this genus.
Zusammenfassung Fur 150 Stamme aus den Genera Mycobacterium, Nocardia, Rhodococcus und verwandte Taxa wurde die Sensibilitat gegen Capreomycin (Tab. 1) und Protionamid (Tab. 2 und 3) bestimmt. Die Tests wurden in Lowenstein-Jensen-Medium mit den Konzentrationen 10, 20, 40, 160 und 320 flg/ml durchgefuhrt. Die Methode wurde primar entwickelt, um einen einfachen Test fur taxonomische Zwecke zur Verfiigung zu haben; sie ist nicht zur Beurteilung der Sensibilitat fur therapeutische Zwecke geeignet. - Es wurde festgestellt, daB Stamme von Mycobacterium chelonei und Nocardia brasiliensis resistenter gegen Capreomycin sind als die ubrigen untersuchten Stamme und daB sich M. senegalense durch seine hohere Capreomycin-Resistenz von M. farcinogenes unterscheidet. Es wurde weiterhin nachgewiesen, daB die gepruften Mycobacterium-Stamme empfindlicher gegen Protionamide sind als die Stamme von Nocardia, Rhodococcus, "Mycobacterium album"
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und "Cardona aurantiaca". Die als N. amarae bezeichneten Stamme waren gegen Capreomycin und Protionamid empfindlicher als aIle iibrigen Nocardia-Stiimme; dies weist darauf hin, daiS sich N. amarae von den anderen Species dieses Genus unterscheidet.
Introduction In recent years many taxonomical studies have been performed on Mycobacterium, Nocardia, Rhodococcus, and related taxa, and these studies have resulted in an improvement of the classification of these organisms (see, e.g., recent review articles (14, 18, 20)). A number of taxonomical problems remain, however, and good characters for classification and identification purposes are still needed. A large part of the taxonomical studies hitherto performed have, furthermore, been made by chemical, genetical or immunological methods which are too expensive, timeconsuming or complicated to be used for identification purposes in diagnostic laboratories. Additional methods are, therefore, needed. Such methods are, e.g., sensitivity tests which have shown to be good characters for classification and identification of the pertinent taxa (2, 7, 12, 15, 19,23,30,32). It is well known that many of the non tuberculous mycobacteria and several of the nocardiae are pathogenic. Recently, certain rhodococci have also been found to cause disease (5). Knowledge concerning the sensitivity of these organisms to drugs is, however, scattered. Some species are fairly well studied (1,4,9, 10, 11, 28), whereas information about most of these organisms is sparse or nonexistent. The aim of the present study was to test the sensitivity to capreomycin and prothionamide in strains referred to Mycobacterium, Nocardia, Rhodococcus, and related taxa.
Materials and Methods Strains. Eighty-seven strains of Mycobacterium, 38 of Nocardia, 9 of Rhodococcus, 9 designated "Cardona aurantiaca", and 7 designated "Mycobacterium album" were ex-
amined (see tables). Media. The tests were performed on Lowenstein-Jensen media containing capreomycin or prothionamide at the concentrations 10, 20, 40, 160 and 320 flg/ml. As controls Lowenstein-Jensen media without any drug were used. Inoculum. One loopful (the inner diameter of the loop was 4 mm) of bacterial mass from Lowenstein-Jensen cultures was suspended in 2 ml phosphate buffer solution and 0.1 ml of this suspension was used as inoculum. Sensitivity test. The cultures were incubated at 37°C for 3 weeks. Some slow-growing strains were incubated for 4 weeks. Registration of growth was performed in accordance with a 5-step scale: no colonies, 1-20 colonies, 20-100 colonies, more than 100 colonies, and confluent growth. Inhibition of growth was defined as a decrease in growth in relation to the control by 2 steps or more.
Results The results of the capreomycin sensitivity tests are given in Table 1. Most mycobacteria were not inhibited by this drug at the concentration 10,Ltg/ml apart from
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Table 1. Sensitivity to capreomycin among strains of Mycobacterium, Nocardia, Rhodococcus and related taxa Species
BCG M. avium M. chelonei M. farcinogenes M. fortuitum M. gastri M. kansasii M. marinum M. microti M.phlei M. scrofulaceum M. senegalense M. smegmatis M. vaccae N. amarae N. asteroides N. brasiliensis N. farcinica N. otitidis-caviarum R. coprophilus R. corallinus R. ruber "G. aurantiaca" "M. album"
No. of strains inhibited by concentration in rlg/ml
No. of tested strains
10
20
40
160
320
1 5 10 15 15 3 3 3 2 3 3 15 6 3 6 14 4 10 4 2 3 4 9 7
0 0 0 14 0 2 0 0 1 3 1 1 1 0 5 2 0 0 1 1 0 1 2 0
0 1 0 15 1 3 1 3 2 3 1 6 2 1 5 6 0 0 4 2 3 4 4 1
0 3 0 15 6 3 2 3 2 3 3 10 6 3 5 8 0 5 4 2 3 4 6 6
1 5 1 15 9 3 3 3 2 3 3 12 6 3 6 12 0 9 4 2 3 4 6 7
1 5 3 15 12 3 3 3 2 3 3 15 6 3 6 12 0 10 4 2 3 4 6 7
14 out of 15 tested M. farcinogenes, 2 out of 3 tested M. gastri, all 3 tested M. ph lei and one strain of each of the four species M. microti, M. scrofulaceum, M. senegalense, and M. smegmatis. With increasing concentrations the number of sensitive strains rose and practically all mycobacteria were inhibited by 160 ,ug/ml, apart from 9 out of 10 strains of M. chelonei, 6 out of 15 strains of M. fortuitum, and 3 out of 15 strains of M. senegalense. Most M. chelonei (7 out of 10) and some M. fortuitum (3 out of 15) strains were not even inhibited by 320 ,ag/ml capreomycin. Five out of 6 N. amarae strains were inhibited by 10 ,ag/ml capreomycin, and all 4 tested N. otitidis-caviarum strains were inhibited by 20 flg/ml. In contrast, none of the N. brasiliensis strains was inhibited by 320 flg/ml. The N. asteroides strains varied strongly concerning their sensitivity to capreomycin. Two of them were inhibited by 10 flg/ml while 2 other strains were not inhibited by 320 flg/ml. The Rhodococcus strains were all inhibited by capreomycin at the concentration 20 flg/ml, whereas the strains designated "Cordona aurantiaca" varied in sensitivity. The results of the prothionamide sensitivity tests are given in Tables 2 and 3. At the concentration 10 flg/ml none but 2 of the tested mycobacteria were inhibited. Also at 20 ftg/ml most strains of this genus were not inhibited, apart from 8 strains representing the species M. gastri, M. kansasii, M. marinum, M. microti, or M.
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Table 2. Sensitivity to prothionamide among strains of Mycobacterium, Nocardia, Rhodococcus and related taxa Species
BeG M. avium M. che/onei M. farcinogenes M. fortuitum M. gastri M. kansasii M. mal'inum M. microti M. phlei M. scrofulaceum M. senegalense M. smegmatis M. vaccae N. amarae N. asteroides N. brasiliensis N. farcinica N. otitidis-caviarum R. coprophilus R. corallinus R. ruber "G. aurantiaca" "M. album"
No. of tested strains 1 5 10 15 15 3 3 3 2 3 3 15 6 3 6 14 4 10 4 2 3 4 9
7
No. of strains inhibited by concentration in pg/ml 20 40 160
10 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0
1
1 1 0 0 0 3 2 3 2 0 1 2 0 2 3 0 0 0 0 0 0 0 0 0
0 0 0 0 0 2 2 2 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0
320 1 5 10 15 13 3 3 3 2 3 3 15 6 3 6 2 4 0 0 2 0 0 4 4
5 5 13 5 3 3 3 2 2 3 12
2 3 6 0 0 0 0 1 0 0 0 0
Table 3. Sensitivity to prothionamide among strains of Mycobacterium, Nocardia, Rhodococcus and related organisms given as percentage of sensitive strains within each taxon Taxa
Mycobacterium Nocardia (except N. amarae) N. amarae Rhodococcus "Gordona aurantiaca" and "M. album"
Total no. of tested strains
% of strains sensitive 10
20
40
160
87 32
2 0
9 0
20 0
71
6 9 16
0 0 0
0 0 0
50 0 0
100
0
11 0
to
320 pg/ml 98 19 100 22 50
scrofulaceum. At 160,ug/ml, however, most mycobacterial strains (71 %, see Table 3) were inhibited. All but 2 M. fortuitum strains were inhibited by 320 ,ug/ml of
prothionamide (i. e. 98%). Hardly any of the remaining organisms tested, represent-
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313
ing Nocardia, Rhodococcus and related taxa, were inhibited by prothionamide at concentrations up to 160 ,Ltg/m!. Three out of the 6 tested N. amarae strains were, however, inhibited by 40,Ltg/ml, and all strains of these species were inhibited by 160 ,Ltg/m!. Furthermore, one strain of R. coprophilus was also inhibited by the latter concentration. At the highest test concentration of prothionamide, i. e. 320 ,Ltg/ml, 2 out of 14 tested strains of N. asteroides and all 4 tested N. brasiliensis strains were inhibited, while all tested N. farcinica and N. otitidis-caviarum strains grew well. Among the rhodococci 2 R. coprophilus strains were inhibited, but none of the R. corallinus and R. ruber strains. None of the 16 strains designated "G. aurantiaca" and "M. album" was inhibited by 160 flg/ml prothionamide, but 8 of them (50%) were inhibited by 320 flg/ml of this drug. Eight strains were included in triplicate at the analyses of both drugs. Test errors of about 7% were obtained.
Discussion The present method was primarily developed in order to obtain a simple method for taxonomical purposes, and the therapeutic aspects were considered as being of secondary interest. The method differs therefore considerably from the established methods used for testing drug sensitivity in mycobacteria, such as the well-known methods by Canetti et al. (6). For example, the inoculum is larger in the present study than in the mentioned analyses, which might give results in which some sensitive strains containing resistant mutants are reported as resistant. Capreomycin and prothionamide might, therefore, have therapeutic value also for some of the organisms here registered as resistant. The study demonstrates, however, clear differences in susceptibility to these drugs among the strains studied. The present method, which is simple to perform, is therefore of value in the classification and identification of these organisms. Among the mycobacteria, the species being most resistant to capreomycin was M. chelonei. Most tested strains of this species grew well in the presence of this drug at the concentration 320 ltg/ml in contrast to the other mycobacteria tested, including the closely related M. fortuitum - a characteristic that might be of value for differentiation and identification of M. chelonei. Swenson et al. (28) have also demonstrated that strains of M. chelonei are more resistant to capreomycin than those of M. fortuitum. The present results demonstrate that sensitivity to capreomycin can be used in order to differentiate M. farcinogenes from M. senegalense. These two species were proposed by Chamoiseau (8) for organisms earlier referred to one species: M. farcinogenes. Recent studies have demonstrated that M. farcinogenes and M. senegalense have the same serological pattern (25) but are phenetically distinguishable (26).
The Nocardia species tested demonstrated different patterns of sensitivity to the two drugs. For example, all strains of N. brasiliensis were far less sensitive to capreomycin and more resistant to prothionamide than were the strains of N. farcinica and N. otitidis-caviarum. The number of strains tested was, however, small and more strains have to be analysed before the differential value of the sensitivity tests can be evaluated.
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The strains of N. asteroides varied strongly concerning their sensitivity to capreomycin, in contrast to the strains of the other Nocardia species. This result is not surprising since several earlier studies based on genetical (21), numerical (22) and serological (24) methods have shown that N. asteroides is a heterogeneous species. Strains referred to the species N. amarae (17) differed concerning their sensitivity to capreomycin and prothionamide from other nocardiae, being more sensitive to these drugs. These findings are in agreement with results from lipid analyses (20), phage-typing (34) and immunodiffusion (24), indicating that N. amarae diverges from the species of Nocardia sensu stricto. The results of the sensitivity tests concerning prothionamide (Tables 2 and 3) suggest a clear difference between strains of the genus Mycobacterium on one hand and those of the genera Nocardia and Rhodococcus on the other, the latter being more resistant to this drug. This difference is more evident when N. amarae, with its doubtful taxonomical position, is excluded from the genus Nocardia. The species "M. album" is listed as a species incertae sedis in Bergey's Manual of Determinative Bacteriology (27), and organisms designated "Gordona aurantiaca" or the "aurantiaca" taxon also have an unsettled taxonomical position (20). Goodfellow et aI. (13) demonstrated that "G. aurantiaca" formed a distinct taxon different from mycobacteria, nocardiae and rhodococci, while Tsukamura et aI. (29, 31) placed them in the genus Rhodococcus. The present study demonstrates that "M. album" and "G. aurantiaca" were different from the majority of the mycobacteria tested concerning their sensitivity to prothionamide, being less sensitive to this drug. Some data from the present study might be of interest from a chemotherapeutic point of view. It is, e.g., worth mentioning that strains of M. farcinogenes, N. asteroides, N. otitidis-caviarum and strains of Rhodococcus were sensitive to the lower concentrations of capreomycin. M. farcinogenes and N. asteroides are well known pathogens but N. otitidis-caviarum and certain strains of Rhodococcus have also been shown to cause infections (3, 5, 16, 33). Prothionamide seems to be of little value for the treatment of infections caused by strains of Nocardia, Rhodococcus and many mycobacteria, since these organisms showed very high resistance to this drug. Acknowledgements. This investigation was supported by the Swedish National Association against Heart and Chest Diseases and the Ellen, Walter and Lennart Hesselman Foundation. The skillful technical assistance of Vivianne Sundaeus and Gun Wallerstrom is gratefully acknowledged.
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27. Runyon, E.H., L. G. Wayne, and G.Kubica: Mycobacteriaceae, in: Bergey's Manual of Determinative Bacteriology, ed. R.E.Buchanan and N.E.Gibbons, pp.681-701. The William and Wilkins Company, Baltimore (1974) 28. Swenson, ]. M., C. Thornsberry, and V. A. Silcox: Rapidly growing mycobacteria: testing of susceptibility to 34 antimicrobial agents by broth microdilution. Antimicrob. Agents Chemother. 22 (1982) 186-192 29. Tsukamura, M.: Numerical classification of Rhodococcus (formerly Gordona) organisms recently isolated from sputa of patients: description of Rhodococcus sputi Tsukamura sp. nov. Int. ]. system. Bact. 28 (1978) 169-181 30. Tsukamura, M.: Differentiation between the genera Rhodococcus and Nocardia and between species of the genus Mycobacterium by susceptibility to Bleomycin. ]. gen. Microbiol. 128 (1982) 2385-2388 31. Tsukamura, M., S.Mizuno, S. Tsukamura, and]. Tsukamura: Comprehensive numerical classification of 369 strains of Mycobacterium, Rhodococcus, and Nocardia. Int. ]. system. Bact. 29 (1979) 110-129 32. Wayne, L. G. and Fourteen Collaborators: A co-operative numerical analysis of scotochromogenic slowly growing mycobacteria. ]. gen. Microbiol. 66 (1971) 255-271 33. Williams, D.M., ].A.Krick, and ].S.Remington: Pulmonary infection in the compromised host. Amer. Rev. resp. Dis. 114 (1976) 359-386 34. Williams, S. T., E. M. H. Wellington, and L. S. Tipler: The taxonomical implications of the reactions of representative Nocardia strains to Actinophage. ]. gen. Microbiol. 119 (1980) 173-178 Dr. Malin Ridell, Department of Medical Microbiology, University of Goteborg, Guldhedsgatan 10, 5-41346 Goteborg, Sweden