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. 1997 Apr;35(4):817–822. doi: 10.1128/jcm.35.4.817-822.1997

Rapid identification of clinically significant species and taxa of aerobic actinomycetes, including Actinomadura, Gordona, Nocardia, Rhodococcus, Streptomyces, and Tsukamurella isolates, by DNA amplification and restriction endonuclease analysis.

V A Steingrube 1, R W Wilson 1, B A Brown 1, K C Jost Jr 1, Z Blacklock 1, J L Gibson 1, R J Wallace Jr 1
PMCID: PMC229682  PMID: 9157134

Abstract

A previously described PCR-restriction fragment length polymorphism (RFLP) identification schema for Nocardia that used an amplified 439-bp segment (amplicon) of the 65-kDa heat shock protein gene was evaluated for potential use with isolates of all clinically significant aerobic actinomycetes. The study included 28 reference (American Type Culture Collection) strains and 198 clinical isolates belonging to 20 taxonomic groups. Of these 198 isolates, 188 could be differentiated by this PCR-RFLP method. Amplicons from all aerobic actinomycete isolates lacked BstEII recognition sites, thereby distinguishing them from those of mycobacteria that contain one or more such sites. Of 29 restriction endonucleases, MspI plus HinfI produced RFLP patterns that differentiated 16 of the 20 taxa. A single RFLP pattern was observed for 15 of 20 taxa that included 65% of phenotypically clustered isolates. Multiple patterns were seen with Gordona bronchialis, Nocardia asteroides complex type VI, Nocardia otitidiscaviarum, Nocardia transvalensis, and Streptomyces spp. Streptomyces RFLP patterns were the most heterogeneous (five patterns among 19 isolates), but exhibited a unique HinfI fragment of > 320 bp. RFLP patterns that matched those from type strains of Streptomyces albus, Streptomyces griseus, or Streptomyces somaliensis were obtained from 14 of 19 Streptomyces isolates. Only 10 of 28 isolates of N. otitidiscaviarum failed to yield satisfactory amplicons, while only 6 of 188 (3.2%) clinical isolates exhibited patterns that failed to match one of the 21 defined RFLP patterns. These studies extended the feasibility of using PCR-RFLP analysis as a rapid method for the identification of all clinically significant species and taxa of aerobic actinomycetes.

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Selected References

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  1. Avaniss-Aghajani E., Jones K., Holtzman A., Aronson T., Glover N., Boian M., Froman S., Brunk C. F. Molecular technique for rapid identification of mycobacteria. J Clin Microbiol. 1996 Jan;34(1):98–102. doi: 10.1128/jcm.34.1.98-102.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Butler W. R., Ahearn D. G., Kilburn J. O. High-performance liquid chromatography of mycolic acids as a tool in the identification of Corynebacterium, Nocardia, Rhodococcus, and Mycobacterium species. J Clin Microbiol. 1986 Jan;23(1):182–185. doi: 10.1128/jcm.23.1.182-185.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Butler W. R., Kilburn J. O. High-performance liquid chromatography patterns of mycolic acids as criteria for identification of Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium smegmatis. J Clin Microbiol. 1990 Sep;28(9):2094–2098. doi: 10.1128/jcm.28.9.2094-2098.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Butler W. R., Kilburn J. O., Kubica G. P. High-performance liquid chromatography analysis of mycolic acids as an aid in laboratory identification of Rhodococcus and Nocardia species. J Clin Microbiol. 1987 Nov;25(11):2126–2131. doi: 10.1128/jcm.25.11.2126-2131.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chun J., Goodfellow M. A phylogenetic analysis of the genus Nocardia with 16S rRNA gene sequences. Int J Syst Bacteriol. 1995 Apr;45(2):240–245. doi: 10.1099/00207713-45-2-240. [DOI] [PubMed] [Google Scholar]
  7. Cook S. M., Bartos R. E., Pierson C. L., Frank T. S. Detection and characterization of atypical mycobacteria by the polymerase chain reaction. Diagn Mol Pathol. 1994 Mar;3(1):53–58. doi: 10.1097/00019606-199403010-00009. [DOI] [PubMed] [Google Scholar]
  8. Inyaku K., Hiyama K., Ishioka S., Inamizu T., Yamakido M. Rapid detection and identification of mycobacteria in sputum samples by nested polymerase chain reaction and restriction fragment length polymorphisms of dnaJ heat shock protein gene. Hiroshima J Med Sci. 1993 Mar;42(1):21–31. [PubMed] [Google Scholar]
  9. Kirschner P., Kiekenbeck M., Meissner D., Wolters J., Böttger E. C. Genetic heterogeneity within Mycobacterium fortuitum complex species: genotypic criteria for identification. J Clin Microbiol. 1992 Nov;30(11):2772–2775. doi: 10.1128/jcm.30.11.2772-2775.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kox L. F., van Leeuwen J., Knijper S., Jansen H. M., Kolk A. H. PCR assay based on DNA coding for 16S rRNA for detection and identification of mycobacteria in clinical samples. J Clin Microbiol. 1995 Dec;33(12):3225–3233. doi: 10.1128/jcm.33.12.3225-3233.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lungu O., Della Latta P., Weitzman I., Silverstein S. Differentiation of Nocardia from rapidly growing Mycobacterium species by PCR-RFLP analysis. Diagn Microbiol Infect Dis. 1994 Jan;18(1):13–18. doi: 10.1016/0732-8893(94)90128-7. [DOI] [PubMed] [Google Scholar]
  12. Mishra S. K., Gordon R. E., Barnett D. A. Identification of nocardiae and streptomycetes of medical importance. J Clin Microbiol. 1980 Jun;11(6):728–736. doi: 10.1128/jcm.11.6.728-736.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Plikaytis B. B., Plikaytis B. D., Yakrus M. A., Butler W. R., Woodley C. L., Silcox V. A., Shinnick T. M. Differentiation of slowly growing Mycobacterium species, including Mycobacterium tuberculosis, by gene amplification and restriction fragment length polymorphism analysis. J Clin Microbiol. 1992 Jul;30(7):1815–1822. doi: 10.1128/jcm.30.7.1815-1822.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ruimy R., Riegel P., Carlotti A., Boiron P., Bernardin G., Monteil H., Wallace R. J., Jr, Christen R. Nocardia pseudobrasiliensis sp. nov., a new species of Nocardia which groups bacterial strains previously identified as Nocardia brasiliensis and associated with invasive diseases. Int J Syst Bacteriol. 1996 Jan;46(1):259–264. doi: 10.1099/00207713-46-1-259. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Springer B., Böttger E. C., Kirschner P., Wallace R. J., Jr Phylogeny of the Mycobacterium chelonae-like organism based on partial sequencing of the 16S rRNA gene and proposal of Mycobacterium mucogenicum sp. nov. Int J Syst Bacteriol. 1995 Apr;45(2):262–267. doi: 10.1099/00207713-45-2-262. [DOI] [PubMed] [Google Scholar]
  17. Steingrube V. A., Brown B. A., Gibson J. L., Wilson R. W., Brown J., Blacklock Z., Jost K., Locke S., Ulrich R. F., Wallace R. J., Jr DNA amplification and restriction endonuclease analysis for differentiation of 12 species and taxa of Nocardia, including recognition of four new taxa within the Nocardia asteroides complex. J Clin Microbiol. 1995 Dec;33(12):3096–3101. doi: 10.1128/jcm.33.12.3096-3101.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Steingrube V. A., Gibson J. L., Brown B. A., Zhang Y., Wilson R. W., Rajagopalan M., Wallace R. J., Jr PCR amplification and restriction endonuclease analysis of a 65-kilodalton heat shock protein gene sequence for taxonomic separation of rapidly growing mycobacteria. J Clin Microbiol. 1995 Jan;33(1):149–153. doi: 10.1128/jcm.33.1.149-153.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Vaneechoutte M., De Beenhouwer H., Claeys G., Verschraegen G., De Rouck A., Paepe N., Elaichouni A., Portaels F. Identification of Mycobacterium species by using amplified ribosomal DNA restriction analysis. J Clin Microbiol. 1993 Aug;31(8):2061–2065. doi: 10.1128/jcm.31.8.2061-2065.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wallace R. J., Jr, Brown B. A., Blacklock Z., Ulrich R., Jost K., Brown J. M., McNeil M. M., Onyi G., Steingrube V. A., Gibson J. New Nocardia taxon among isolates of Nocardia brasiliensis associated with invasive disease. J Clin Microbiol. 1995 Jun;33(6):1528–1533. doi: 10.1128/jcm.33.6.1528-1533.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wallace R. J., Jr, Brown B. A., Tsukamura M., Brown J. M., Onyi G. O. Clinical and laboratory features of Nocardia nova. J Clin Microbiol. 1991 Nov;29(11):2407–2411. doi: 10.1128/jcm.29.11.2407-2411.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wallace R. J., Jr, Steele L. C., Sumter G., Smith J. M. Antimicrobial susceptibility patterns of Nocardia asteroides. Antimicrob Agents Chemother. 1988 Dec;32(12):1776–1779. doi: 10.1128/aac.32.12.1776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wallace R. J., Jr, Steele L. C. Susceptibility testing of Nocardia species for the clinical laboratory. Diagn Microbiol Infect Dis. 1988 Mar;9(3):155–166. doi: 10.1016/0732-8893(88)90025-9. [DOI] [PubMed] [Google Scholar]
  26. Wallace R. J., Jr, Tsukamura M., Brown B. A., Brown J., Steingrube V. A., Zhang Y. S., Nash D. R. Cefotaxime-resistant Nocardia asteroides strains are isolates of the controversial species Nocardia farcinica. J Clin Microbiol. 1990 Dec;28(12):2726–2732. doi: 10.1128/jcm.28.12.2726-2732.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

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