1887

Abstract

Purpose. Mycobacteria are common causative agents of bacterial infections in many species of freshwater and marine fish. Identification of mycobacteria to the species level based on phenotypic tests is inappropriate and time consuming. Molecular methods such as partial or entire gene sequence determination in mycobacteria have been employed to resolve these problems. The objective of this study was to assess the use of sequence analysis of the mycobacterial 16S–23S internal transcribed spacer (ITS) region for the identification of different aquatic mycobacteria species.

Methodology. Using published primers, the ITS sequences of 64 field and reference strains were determined.

Results/Key findings. The identity of all isolates previously identified as Mycobacterium marinum by RFLP was confirmed as M. marinum by sequence analysis. With the exception of five rapidly growing mycobacteria isolates, all other mycobacteria were easily identified by sequencing of the ITS region. Using this spacer region, it was possible to differentiate between slowly growing and rapidly growing mycobacteria, even before sequence analysis, by the size of the PCR product, although species identification could not be made by size alone.

Conclusion. Overall, direct sequencing of this genetic element following PCR has been shown to be useful in the identification of aquatic mycobacteria species. With regard to the variability of the ITS region for different mycobacteria isolates, this may be a useful tool in epidemiological studies.

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2018-12-12
2020-01-29
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References

  1. Smole SC, McAleese F, Ngampasutadol J, von Reyn CF, Arbeit RD. Clinical and epidemiological correlates of genotypes within the Mycobacterium avium complex defined by restriction and sequence analysis of hsp65. J Clin Microbiol 2002;40:3374–3380 [CrossRef][PubMed]
    [Google Scholar]
  2. Lévy-Frébault VV, Portaels F. Proposed minimal standards for the genus Mycobacterium and for description of new slowly growing Mycobacterium species. Int J Syst Bacteriol 1992;42:315–323 [CrossRef][PubMed]
    [Google Scholar]
  3. Trujillo ME, Velázquez E, Kroppenstedt RM, Schumann P, Rivas R et al. Mycobacterium psychrotolerans sp. nov., isolated from pond water near a uranium mine. Int J Syst Evol Microbiol 2004;54:1459–1463 [CrossRef][PubMed]
    [Google Scholar]
  4. Whipps CM, Butler WR, Pourahmad F, Watral VG, Kent ML. Molecular systematics support the revival of Mycobacterium salmoniphilum (ex Ross 1960) sp. nov., nom. rev., a species closely related to Mycobacterium chelonae. Int J Syst Evol Microbiol 2007;57:2525–2531 [CrossRef][PubMed]
    [Google Scholar]
  5. Rhodes MW, Kator H, Kotob S, van Berkum P, Kaattari I et al. Mycobacterium shottsii sp. nov., a slowly growing species isolated from Chesapeake Bay striped bass (Morone saxatilis). Int J Syst Evol Microbiol 2003;53:421–424 [CrossRef][PubMed]
    [Google Scholar]
  6. Rhodes MW, Kator H, McNabb A, Deshayes C, Reyrat JM et al. Mycobacterium pseudoshottsii sp. nov., a slowly growing chromogenic species isolated from Chesapeake Bay striped bass (Morone saxatilis). Int J Syst Evol Microbiol 2005;55:1139–1147 [CrossRef][PubMed]
    [Google Scholar]
  7. Pourahmad F, Cervellione F, Thompson KD, Taggart JB, Adams A et al. Mycobacterium stomatepiae sp. nov., a slowly growing, non-chromogenic species isolated from fish. Int J Syst Evol Microbiol 2008;58:2821–2827 [CrossRef][PubMed]
    [Google Scholar]
  8. Pourahmad F, Pate M, Ocepek M, Borroni E, Cabibbe AM et al. Mycobacterium angelicum sp. nov., a non-chromogenic, slow-growing species isolated from fish and related to Mycobacterium szulgai. Int J Syst Evol Microbiol 2015;65:4724–4729 [CrossRef][PubMed]
    [Google Scholar]
  9. Patel JB, Leonard DG, Pan X, Musser JM, Berman RE et al. Sequence-based identification of Mycobacterium species using the MicroSeq 500 16S rDNA bacterial identification system. J Clin Microbiol 2000;38:246–251[PubMed]
    [Google Scholar]
  10. Kirschner P, Springer B, Vogel U, Meier A, Wrede A et al. Genotypic identification of mycobacteria by nucleic acid sequence determination: report of a 2-year experience in a clinical laboratory. J Clin Microbiol 1993;31:2882–2889[PubMed]
    [Google Scholar]
  11. Ringuet H, Akoua-Koffi C, Honore S, Varnerot A, Vincent V et al. hsp65 sequencing for identification of rapidly growing mycobacteria. J Clin Microbiol 1999;37:852–857[PubMed]
    [Google Scholar]
  12. Roth A, Fischer M, Hamid ME, Michalke S, Ludwig W et al. Differentiation of phylogenetically related slowly growing mycobacteria based on 16S-23S rRNA gene internal transcribed spacer sequences. J Clin Microbiol 1998;36:139–147[PubMed]
    [Google Scholar]
  13. Kim BJ, Lee SH, Lyu MA, Kim SJ, Bai GH et al. Identification of mycobacterial species by comparative sequence analysis of the RNA polymerase gene (rpoB). J Clin Microbiol 1999;37:1714–1720[PubMed]
    [Google Scholar]
  14. Clarridge JE. Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clin Microbiol Rev 2004;17:840–862 [CrossRef][PubMed]
    [Google Scholar]
  15. Dobner P, Feldmann K, Rifai M, Löscher T, Rinder H. Rapid identification of mycobacterial species by PCR amplification of hypervariable 16S rRNA gene promoter region. J Clin Microbiol 1996;34:866–869[PubMed]
    [Google Scholar]
  16. Gürtler V, Harford C, Bywater J, Mayall BC. Direct identification of slowly growing Mycobacterium species by analysis of the intergenic 16S-23S rDNA spacer region (ISR) using a GelCompar II database containing sequence based optimization for restriction fragment site polymorphisms (RFLPs) for 12 enzymes. J Microbiol Methods 2006;64:185–199 [CrossRef][PubMed]
    [Google Scholar]
  17. Mohamed AM, Kuyper DJ, Iwen PC, Ali HH, Bastola DR et al. Computational approach involving use of the internal transcribed spacer 1 region for identification of Mycobacterium species. J Clin Microbiol 2005;43:3811–3817 [CrossRef][PubMed]
    [Google Scholar]
  18. Pourahmad F, Richards RH. Use of restriction enzyme fragment length polymorphism (RFLP) of the 16S–23S rRNA internal transcribed spacer region (ITS) for identification of fish mycobacteria. Aquaculture 2013;411:410–184–189
    [Google Scholar]
  19. Pourahmad F, Thompson KD, Taggart JB, Adams A, Richards RH. Evaluation of the INNO-LiPA mycobacteria v2 assay for identification of aquatic mycobacteria. J Fish Dis 2008;31:931–940 [CrossRef][PubMed]
    [Google Scholar]
  20. Roth A, Reischl U, Streubel A, Naumann L, Kroppenstedt RM et al. Novel diagnostic algorithm for identification of mycobacteria using genus-specific amplification of the 16S-23S rRNA gene spacer and restriction endonucleases. J Clin Microbiol 2000;38:1094–1104[PubMed]
    [Google Scholar]
  21. Pourahmad F, Thompson KD, Adams A, Richards RH. Detection and identification of aquatic mycobacteria in formalin-fixed, paraffin-embedded fish tissues. J Fish Dis 2009;32:409–419 [CrossRef][PubMed]
    [Google Scholar]
  22. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 2013;30:2725–2729 [CrossRef][PubMed]
    [Google Scholar]
  23. Novi C, Rindi L, Lari N, Garzelli C. Molecular typing of Mycobacterium avium isolates by sequencing of the 16S-23S rDNA internal transcribed spacer and comparison with IS1245-based fingerprinting.. J Med Microbiol 2000;49:1091–1095
    [Google Scholar]
  24. Hamid ME, Roth A, Landt O, Kroppenstedt RM, Goodfellow M et al. Differentiation between Mycobacterium farcinogenes and Mycobacterium senegalense strains based on 16S-23S ribosomal DNA internal transcribed spacer sequences. J Clin Microbiol 2002;40:707–711 [CrossRef][PubMed]
    [Google Scholar]
  25. Khan IU, Selvaraju SB, Yadav JS. Method for rapid identification and differentiation of the species of the Mycobacterium chelonae complex based on 16S-23S rRNA gene internal transcribed spacer PCR-restriction analysis. J Clin Microbiol 2005;43:4466–4472 [CrossRef][PubMed]
    [Google Scholar]
  26. Lappayawichit P, Rienthong S, Rienthong D, Chuchottaworn C, Chaiprasert A et al. Differentiation of Mycobacterium species by restriction enzyme analysis of amplified 16S-23S ribosomal DNA spacer sequences. Tuber Lung Dis 1996;77:257–263 [CrossRef][PubMed]
    [Google Scholar]
  27. Xiong L, Kong F, Yang Y, Cheng J, Gilbert GL. Use of PCR and reverse line blot hybridization macroarray based on 16S-23S rRNA gene internal transcribed spacer sequences for rapid identification of 34 mycobacterium species. J Clin Microbiol 2006;44:3544–3550 [CrossRef][PubMed]
    [Google Scholar]
  28. Cloud JL, Meyer JJ, Pounder JI, Jost KC, Sweeney A et al. Mycobacterium arupense sp. nov., a non-chromogenic bacterium isolated from clinical specimens. Int J Syst Evol Microbiol 2006;56:1413–1418 [CrossRef][PubMed]
    [Google Scholar]
  29. Ucko M, Colorni A, Kvitt H, Diamant A, Zlotkin A et al. Strain variation in Mycobacterium marinum fish isolates. Appl Environ Microbiol 2002;68:5281–5287 [CrossRef][PubMed]
    [Google Scholar]
  30. Devulder G, Pérouse de Montclos M, Flandrois JP. A multigene approach to phylogenetic analysis using the genus Mycobacterium as a model. Int J Syst Evol Microbiol 2005;55:293–302 [CrossRef][PubMed]
    [Google Scholar]
  31. Pourahmad F, Richards RH. Comparative evaluation of sequence analysis of 16S rRNA and rpoB genes for identification of aquatic mycobacteria. Turk J Vet Anim Sci 2016;40:406–416 [CrossRef]
    [Google Scholar]
  32. Stackebrandt E, Goebel BM. Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Evol Microbiol 1994;44:846–849 [CrossRef]
    [Google Scholar]
  33. Leclerc MC, Haddad N, Moreau R, Thorel MF. Molecular characterization of environmental mycobacterium strains by PCR-restriction fragment length polymorphism of hsp65 and by sequencing of hsp65, and of 16S and ITS1 rDNA. Res Microbiol 2000;151:629–638 [CrossRef][PubMed]
    [Google Scholar]
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