Journal List > Korean J Clin Microbiol > v.13(1) > 1038208

Park, Shin, Kim, Roh, Yum, Yong, Jeong, and Lee: Identification of Bacterial and Fungal Isolates by Sequence Analysis of 16S rRNA and Internal Transcribed Spacer

Abstract

Background

Accurate and rapid identification of pathogens is one of the most important tasks of the clinical microbiology laboratory, and, in cases of rare pathogens, the identification is difficult and time-consuming upon the use of conventional methods alone. Herein, we will report our molecular work involving the identification of bacteria and fungi.

Methods

Sixty bacterial isolates had been collected from November 2004 to May 2007, and 15 fungal isolates had been collected from September 2005 to May 2007. Species identifications were performed using sequence analyses of the 16S rRNA region of bacteria and the internal transcribed spacer (ITS) region of fungi. The data were compared with those of GenBank (http://www.ncbi.nlm.nih.gov/) or EMBL (http://www.ebi.ac.uk/embl/).

Results

Sixty bacterial isolates included: 23 isolates with genus information (group 1), 17 isolates (group 2) that were too fastidious for genus or species identification, 16 isolates (group 3) with results from identification kits having low confidence, and 4 isolates (group 4) with odd antibiograms according to the species. In 58 of 60 isolates, identification of the genus or species could be obtained using molecular genetic methods. Thirty-eight isolates (63%) and 20 (33%) of 58 isolates could be identified at the species and genus levels, repectively. Among the total of 15 fungal isolates, 11 (73%) and 4 (27%) isolates were identified at the species and genus levels, respectively.

Conclusion

16S rRNA and ITS sequencing analyses are very useful for identifying the species or genus of a pathogenic microorganism in the clinical microbiology laboratory.

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Table 1.
Summary of bacterial identification by 16S rRNA sequence analysis
Group p Reasons for 16S rRNA sequencing No. of tests (%) Results of 16S rRNA sequencing No. . of isolates
1 To identify bacterial species 23 (38) Species identified 10
      Genus identified 13
2 Sequencing without presumptive identification due to difficulties in identifying organisms by conventional methods 17 (28) Species identified 14
      Genus identified Unidentified 1
      Unidentified 2
3 Low confidence in presumptive identification results 16 (27) Inconsistent with presumptive results 9
      Consistent with presumptive results 7
4 Inappropriate antibiogram with presumptive identification results 4 (7) Inconsistent with presumptive results 0
      Consistent with presumptive results 4
Table 2.
Bacterial identifications of grou up 1
Identification level Results (No. isolates) of
Phenotypic methods 16S rRNA gene sequencing
Species Enterococcus spp. E. faecium
  Staphylococcus spp. (3) S. haemolyticus
    S. hominis
    S. sciuri
  Streptococcus spp. S. intermedius
  Bacillus spp. B. cereus
  Corynebacterium spp. C. pseudogenitalium
  Bordetella spp. B. bronchiseptica
  Pseudomonas spp. P. otitidis
  Mycobacterium spp. Segniliparus rugosus
Genus Lactobacillus spp. (3) Corynebacterium spp. (2)
    Lactobacillus spp.
  Mycobacterium spp. (2) Mycobacterium spp. (2)
  Brucella spp. (3) Brucella spp. (3)
  Salmonella spp. (3) Salmonella spp. (3)
  Acinetobacter spp. (2) Acinetobacter spp. (2)
Table 3.
Summary of fun ngal identification by ITS sequence analysis
Type Specimen types Identification by ITS region gene sequencing Identity (%)
Yeast Blood Pichia ohmeri 100
    Pichia guilliermondii 100
  Body fluid Cryptococcus neoformans 100
  Tissue C. neoformans 100
  Wound C. neoformans 100
Mould Body fluid Cladosporium spp. 100
  Wound Phaeoacremonium aleophilum 100
    Pseudozyma spp. 99
    Trichosporon debeurumannianum 100
  Eye Alternaria spp. 99
    Alternaria spp. 98
    Aspergillus fumigatus 99
    Pseudallescheria boydii 99
  Environment Paecilomyces variotii 100
Dimorphic Wound Coccidioides immitis 100

Abbreviation: ITS, internal transcribed spacer region

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