Skip to main content
PMC home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1996 Dec;34(12):3151–3159. doi: 10.1128/jcm.34.12.3151-3159.1996

Identification and subspecific differentiation of Mycobacterium scrofulaceum by automated sequencing of a region of the gene (hsp65) encoding a 65-kilodalton heat shock protein.

D S Swanson 1, X Pan 1, J M Musser 1
PMCID: PMC229474  PMID: 8940463

Abstract

Mycobacterium scrofulaceum is most commonly recovered from children with cervical lymphadenitis, although it also accounts for approximately 2% of the mycobacterial infections in AIDS patients. Species assignment of M. scrofulaceum isolated by conventional techniques can be difficult and time-consuming. To develop a strategy for rapid species assignment of these organisms, a 360-bp region of the gene (hsp65) encoding a 65-kDa heat shock protein in 37 isolates from diverse sources was sequenced. Eight hsp65 alleles were identified, and these sequences formed phylogenetic clusters and lineages largely distinct from other Mycobacterium species. There was incomplete correlation between serovar designation and hsp65 allele assignment. The hsp65 data correlated strongly with the results of sequence analysis of the gene coding for 16S rRNA. Automated DNA sequencing of a 360-bp region of the hsp65 gene provides a rapid and unambiguous method for species assignment of these acid-fast organisms for diagnostic purposes.

Full Text

The Full Text of this article is available as a PDF (241.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bosquée L., Böttger E. C., De Beenhouwer H., Fonteyne P. A., Hirschel B., Larsson L., Meyers W. M., Palomino J. C., Realini L., Rigouts L. Cervical lymphadenitis caused by a fastidious mycobacterium closely related to Mycobacterium genavense in an apparently immunocompetent woman: diagnosis by culture-free microbiological methods. J Clin Microbiol. 1995 Oct;33(10):2670–2674. doi: 10.1128/jcm.33.10.2670-2674.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brennan P. J., Heifets M., Ullom B. P. Thin-layer chromatography of lipid antigens as a means of identifying nontuberculous mycobacteria. J Clin Microbiol. 1982 Mar;15(3):447–455. doi: 10.1128/jcm.15.3.447-455.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brooks R. W., Parker B. C., Gruft H., Falkinham J. O., 3rd Epidemiology of infection by nontuberculous mycobacteria. V. Numbers in eastern United States soils and correlation with soil characteristics. Am Rev Respir Dis. 1984 Oct;130(4):630–633. doi: 10.1164/arrd.1984.130.4.630. [DOI] [PubMed] [Google Scholar]
  4. Butler W. R., Jost K. C., Jr, Kilburn J. O. Identification of mycobacteria by high-performance liquid chromatography. J Clin Microbiol. 1991 Nov;29(11):2468–2472. doi: 10.1128/jcm.29.11.2468-2472.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Böddinghaus B., Rogall T., Flohr T., Blöcker H., Böttger E. C. Detection and identification of mycobacteria by amplification of rRNA. J Clin Microbiol. 1990 Aug;28(8):1751–1759. doi: 10.1128/jcm.28.8.1751-1759.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Böddinghaus B., Wolters J., Heikens W., Böttger E. C. Phylogenetic analysis and identification of different serovars of Mycobacterium intracellulare at the molecular level. FEMS Microbiol Lett. 1990 Jul;58(2):197–203. doi: 10.1111/j.1574-6968.1990.tb13978.x. [DOI] [PubMed] [Google Scholar]
  7. Chapman J. S., Speight M. Isolation of atypical mycobacteria from pasteurized milk. Am Rev Respir Dis. 1968 Dec;98(6):1052–1054. doi: 10.1164/arrd.1968.98.6.1052. [DOI] [PubMed] [Google Scholar]
  8. Coyle M. B., Carlson L. C., Wallis C. K., Leonard R. B., Raisys V. A., Kilburn J. O., Samadpour M., Böttger E. C. Laboratory aspects of "Mycobacterium genavense," a proposed species isolated from AIDS patients. J Clin Microbiol. 1992 Dec;30(12):3206–3212. doi: 10.1128/jcm.30.12.3206-3212.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dunn B. L., Hodgson D. J. "Atypical' mycobacteria in milk. J Appl Bacteriol. 1982 Jun;52(3):373–376. doi: 10.1111/j.1365-2672.1982.tb05067.x. [DOI] [PubMed] [Google Scholar]
  10. Fourche J., Capdepuy M., Maugein J., Le Moigne F. Analysis of cellular fatty acids and proteins by capillary gas chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis to differentiate Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium scrofulaceum (MAIS) complex species. J Chromatogr. 1990 Nov 16;532(2):209–216. doi: 10.1016/s0378-4347(00)83772-x. [DOI] [PubMed] [Google Scholar]
  11. Frothingham R., Wilson K. H. Molecular phylogeny of the Mycobacterium avium complex demonstrates clinically meaningful divisions. J Infect Dis. 1994 Feb;169(2):305–312. doi: 10.1093/infdis/169.2.305. [DOI] [PubMed] [Google Scholar]
  12. Frothingham R., Wilson K. H. Sequence-based differentiation of strains in the Mycobacterium avium complex. J Bacteriol. 1993 May;175(10):2818–2825. doi: 10.1128/jb.175.10.2818-2825.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Good R. C. From the Center for Disease Control. Isolation of nontuberculous mycobacteria in the United States, 1979. J Infect Dis. 1980 Nov;142(5):779–783. doi: 10.1093/infdis/142.5.779. [DOI] [PubMed] [Google Scholar]
  14. Good R. C. Opportunistic pathogens in the genus Mycobacterium. Annu Rev Microbiol. 1985;39:347–369. doi: 10.1146/annurev.mi.39.100185.002023. [DOI] [PubMed] [Google Scholar]
  15. Good R. C., Snider D. E., Jr Isolation of nontuberculous mycobacteria in the United States, 1980. J Infect Dis. 1982 Dec;146(6):829–833. doi: 10.1093/infdis/146.6.829. [DOI] [PubMed] [Google Scholar]
  16. Goslee S., Wolinsky E. Water as a source of potentially pathogenic mycobacteria. Am Rev Respir Dis. 1976 Mar;113(3):287–292. doi: 10.1164/arrd.1976.113.3.287. [DOI] [PubMed] [Google Scholar]
  17. Gracey D. R., Byrd R. B. Scotochromogens and pulmonary disease. Five years' experience at a pulmonary disease center with report of a case. Am Rev Respir Dis. 1970 Jun;101(6):959–963. doi: 10.1164/arrd.1970.101.6.959. [DOI] [PubMed] [Google Scholar]
  18. Hawkins J. E. Scotochromogenic mycobacteria which appear intermediate between Mycobacterium avium-intracellulare and Mycobacterium scrofulaceum. Am Rev Respir Dis. 1977 Nov;116(5):963–964. doi: 10.1164/arrd.1977.116.5.963. [DOI] [PubMed] [Google Scholar]
  19. Hoffner S. E., Petrini B., Brennan P. J., Tsang A. Y., Källenius G. AIDS and Mycobacterium avium serovars in Sweden. Lancet. 1989 Aug 5;2(8658):336–337. doi: 10.1016/s0140-6736(89)90525-4. [DOI] [PubMed] [Google Scholar]
  20. Hosty T. S., McDurmont C. I. Isolation of acid-fast organisms from milk and oysters. Health Lab Sci. 1975 Jan;12(1):16–19. [PubMed] [Google Scholar]
  21. Hsueh P. R., Hsiue T. R., Jarn J. J., Ho S. W., Hsieh W. C. Disseminated infection due to Mycobacterium scrofulaceum in an immunocompetent host. Clin Infect Dis. 1996 Jan;22(1):159–161. doi: 10.1093/clinids/22.1.159. [DOI] [PubMed] [Google Scholar]
  22. Ichiyama S., Shimokata K., Tsukamura M. The isolation of Mycobacterium avium complex from soil, water, and dusts. Microbiol Immunol. 1988;32(7):733–739. doi: 10.1111/j.1348-0421.1988.tb01434.x. [DOI] [PubMed] [Google Scholar]
  23. Jin B. W., Saito H., Yoshii Z. Environmental mycobacteria in Korea. I. Distribution of the organisms. Microbiol Immunol. 1984;28(6):667–677. doi: 10.1111/j.1348-0421.1984.tb00721.x. [DOI] [PubMed] [Google Scholar]
  24. Jones R. J., Jenkins D. E., Hsu K. H. Raw milk as a source of Mycobacteria. Can J Microbiol. 1966 Oct;12(5):979–984. doi: 10.1139/m66-132. [DOI] [PubMed] [Google Scholar]
  25. Jucker M. T., Falkinham J. O., 3rd Epidemiology of infection by nontuberculous mycobacteria IX. Evidence for two DNA homology groups among small plasmids in Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum. Am Rev Respir Dis. 1990 Oct;142(4):858–862. doi: 10.1164/ajrccm/142.4.858. [DOI] [PubMed] [Google Scholar]
  26. Kirschner P., Springer B., Vogel U., Meier A., Wrede A., Kiekenbeck M., Bange F. C., Böttger E. C. Genotypic identification of mycobacteria by nucleic acid sequence determination: report of a 2-year experience in a clinical laboratory. J Clin Microbiol. 1993 Nov;31(11):2882–2889. doi: 10.1128/jcm.31.11.2882-2889.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Krajnack M. A., Dowda H. Non-tuberculous Mycobacteria in South Carolina, 1971 to 1980. J S C Med Assoc. 1981 Nov;77(11):551–555. [PubMed] [Google Scholar]
  28. Lincoln E. M., Gilbert L. A. Disease in children due to mycobacteria other than Mycobacterium tuberculosis. Am Rev Respir Dis. 1972 May;105(5):683–714. doi: 10.1164/arrd.1972.105.5.683. [DOI] [PubMed] [Google Scholar]
  29. MASSON A. M., PRISSICK F. H. Cervical lymphadenitis in children caused by chromogenic Mycobacteria. Can Med Assoc J. 1956 Nov 15;75(10):798–803. [PMC free article] [PubMed] [Google Scholar]
  30. Matthews P. R., Collins P., Jones P. W. Isolation of mycobacteria from dairy creamery effluent sludge. J Hyg (Lond) 1976 Jun;76(3):407–413. doi: 10.1017/s0022172400055339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. McNutt D. R., Fudenberg H. H. Disseminated scotochromogen infection and unusual myeloproliferative disorder. Report of a case and review of the literature. Ann Intern Med. 1971 Nov;75(5):737–744. doi: 10.7326/0003-4819-75-5-737. [DOI] [PubMed] [Google Scholar]
  32. Meissner P. S., Falkinham J. O., 3rd Plasmid DNA profiles as epidemiological markers for clinical and environmental isolates of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum. J Infect Dis. 1986 Feb;153(2):325–331. doi: 10.1093/infdis/153.2.325. [DOI] [PubMed] [Google Scholar]
  33. Musial C. E., Tice L. S., Stockman L., Roberts G. D. Identification of mycobacteria from culture by using the Gen-Probe Rapid Diagnostic System for Mycobacterium avium complex and Mycobacterium tuberculosis complex. J Clin Microbiol. 1988 Oct;26(10):2120–2123. doi: 10.1128/jcm.26.10.2120-2123.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Olson N. R. Atypical mycobacterial infections of the neck. Laryngoscope. 1967 Aug;77(8):1376–1389. doi: 10.1288/00005537-196708000-00013. [DOI] [PubMed] [Google Scholar]
  35. PRISSICK F. H., MASSON A. M. Yellow-pigmented pathogenic mycobacteria from cervical lymphadenitis. Can J Microbiol. 1957 Feb;3(1):91–100. doi: 10.1139/m57-011. [DOI] [PubMed] [Google Scholar]
  36. Patel K. M. Granulomatous hepatitis due to Mycobacterium scrofulaceum: report of a case. Gastroenterology. 1981 Jul;81(1):156–158. [PubMed] [Google Scholar]
  37. Portaels F. Difficulties encountered in identification of M. avium-M. intracellulare, M. scrofulaceum, and related strains. Am Rev Respir Dis. 1978 Nov;118(5):969–969. doi: 10.1164/arrd.1978.118.5.969. [DOI] [PubMed] [Google Scholar]
  38. Rogall T., Flohr T., Böttger E. C. Differentiation of Mycobacterium species by direct sequencing of amplified DNA. J Gen Microbiol. 1990 Sep;136(9):1915–1920. doi: 10.1099/00221287-136-9-1915. [DOI] [PubMed] [Google Scholar]
  39. Rogall T., Wolters J., Flohr T., Böttger E. C. Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium. Int J Syst Bacteriol. 1990 Oct;40(4):323–330. doi: 10.1099/00207713-40-4-323. [DOI] [PubMed] [Google Scholar]
  40. Saito H., Tomioka H., Sato K., Tasaka H., Dawson D. J. Identification of various serovar strains of Mycobacterium avium complex by using DNA probes specific for Mycobacterium avium and Mycobacterium intracellulare. J Clin Microbiol. 1990 Aug;28(8):1694–1697. doi: 10.1128/jcm.28.8.1694-1697.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shinnick T. M. The 65-kilodalton antigen of Mycobacterium tuberculosis. J Bacteriol. 1987 Mar;169(3):1080–1088. doi: 10.1128/jb.169.3.1080-1088.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Smid I., Salfinger M. Mycobacterial identification by computer-aided gas-liquid chromatography. Diagn Microbiol Infect Dis. 1994 Jun;19(2):81–88. doi: 10.1016/0732-8893(94)90117-1. [DOI] [PubMed] [Google Scholar]
  43. Soini H., Böttger E. C., Viljanen M. K. Identification of mycobacteria by PCR-based sequence determination of the 32-kilodalton protein gene. J Clin Microbiol. 1994 Dec;32(12):2944–2947. doi: 10.1128/jcm.32.12.2944-2947.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Soini H., Eerola E., Viljanen M. K. Genetic diversity among Mycobacterium avium complex AccuProbe-positive isolates. J Clin Microbiol. 1996 Jan;34(1):55–57. doi: 10.1128/jcm.34.1.55-57.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Springer B., Stockman L., Teschner K., Roberts G. D., Böttger E. C. Two-laboratory collaborative study on identification of mycobacteria: molecular versus phenotypic methods. J Clin Microbiol. 1996 Feb;34(2):296–303. doi: 10.1128/jcm.34.2.296-303.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Stone B. B., Nietupski R. M., Breton G. L., Weisburg W. G. Comparison of Mycobacterium 23S rRNA sequences by high-temperature reverse transcription and PCR. Int J Syst Bacteriol. 1995 Oct;45(4):811–819. doi: 10.1099/00207713-45-4-811. [DOI] [PubMed] [Google Scholar]
  47. Telenti A., Marchesi F., Balz M., Bally F., Böttger E. C., Bodmer T. Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J Clin Microbiol. 1993 Feb;31(2):175–178. doi: 10.1128/jcm.31.2.175-178.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Thibert L., Lapierre S. Routine application of high-performance liquid chromatography for identification of mycobacteria. J Clin Microbiol. 1993 Jul;31(7):1759–1763. doi: 10.1128/jcm.31.7.1759-1763.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tisdall P. A., Roberts G. D., Anhalt J. P. Identification of clinical isolates of mycobacteria with gas-liquid chromatography alone. J Clin Microbiol. 1979 Oct;10(4):506–514. doi: 10.1128/jcm.10.4.506-514.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tomioka H., Saito H., Sato K., Tasaka H., Dawson D. J. Identification of Mycobacterium avium complex strains belonging to serovars 21-28 by three commercial DNA probe tests. Tuber Lung Dis. 1993 Apr;74(2):91–95. doi: 10.1016/0962-8479(93)90033-T. [DOI] [PubMed] [Google Scholar]
  51. Tsukamura M., Mizuno S. Studies on thin-layer chromatography after incubation with [35S]methionine as an aid to identification of mycobacteria. Microbiol Immunol. 1981;25(3):215–227. doi: 10.1111/j.1348-0421.1981.tb00025.x. [DOI] [PubMed] [Google Scholar]
  52. Wayne L. G., Good R. C., Böttger E. C., Butler R., Dorsch M., Ezaki T., Gross W., Jonas V., Kilburn J., Kirschner P. Semantide- and chemotaxonomy-based analyses of some problematic phenotypic clusters of slowly growing mycobacteria, a cooperative study of the International Working Group on Mycobacterial Taxonomy. Int J Syst Bacteriol. 1996 Jan;46(1):280–297. doi: 10.1099/00207713-46-1-280. [DOI] [PubMed] [Google Scholar]
  53. Wayne L. G., Good R. C., Krichevsky M. I., Blacklock Z., David H. L., Dawson D., Gross W., Hawkins J., Levy-Frebault V. V., McManus C. Fourth report of the cooperative, open-ended study of slowly growing mycobacteria by the International Working Group on Mycobacterial Taxonomy. Int J Syst Bacteriol. 1991 Oct;41(4):463–472. doi: 10.1099/00207713-41-4-463. [DOI] [PubMed] [Google Scholar]
  54. Wayne L. G., Good R. C., Tsang A., Butler R., Dawson D., Groothuis D., Gross W., Hawkins J., Kilburn J., Kubin M. Serovar determination and molecular taxonomic correlation in Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum: a cooperative study of the International Working Group on Mycobacterial Taxonomy. Int J Syst Bacteriol. 1993 Jul;43(3):482–489. doi: 10.1099/00207713-43-3-482. [DOI] [PubMed] [Google Scholar]
  55. Wolinsky E. Mycobacterial lymphadenitis in children: a prospective study of 105 nontuberculous cases with long-term follow-up. Clin Infect Dis. 1995 Apr;20(4):954–963. doi: 10.1093/clinids/20.4.954. [DOI] [PubMed] [Google Scholar]
  56. Wolinsky E. Nontuberculous mycobacteria and associated diseases. Am Rev Respir Dis. 1979 Jan;119(1):107–159. doi: 10.1164/arrd.1979.119.1.107. [DOI] [PubMed] [Google Scholar]
  57. Yamamoto M., Sudo K., Taga M., Hibino S. A study of diseases caused by atypical mycobacteria in Japan. Am Rev Respir Dis. 1967 Oct;96(4):779–787. doi: 10.1164/arrd.1967.96.4.779. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES