Vibrio 임상 분리주의 균종 동정을 위한 dnaJ 및 16S rDNA의 서열 분석법 비교 = Comparison of the Utility of dnaJ and 16S rDNA Sequences for Identification of Clinical Isolates of Vibrio Species

국문 초록 (Abstract)

배경: Vibrio 종에는 치명적인 패혈증을 일으키는 균종들도 포함되어 있어서 정확한 균종 동정이 매우 중요하다. 일부 비전형적인표현형을 보이는 균종이 있어 정확한 동정을 위해 적절한 분자 진단법의 도입이 필요하다.
방법: 본 연구에서는 Vibrio 임상 분리주 53주와 표준균주 8주가이용되었다. 분자 진단법으로는 dnaJ 유전자 서열 분석, 16S rDNA 서열 분석, gyrB V. vulnificus–특이적 PCR 서열 분석, gyrB V. navarrensis– 특이적 PCR 서열 분석, V. vulnificus hemolysin 유전자(vvh) PCR (V. vulnificus-특이적 PCR)를 시행하였다. 또한 16S rDNA 와 dnaJ 유전자, gyrB 유전자의 서열분석 결과를 토대로 계통수분석을 실시하였으며 상기 검사 결과를 종합하여 균의 최종 동정명이 결정되었다. 16S rDNA 서열 분석과 dnaJ 유전자 서열 분석 간의 일치율 분석은 카이 제곱 검정을 이용하였다.
결과: 61주의 Vibrio 균주의 최종 균종명 분포를 내림차순으로 배열하면 다음과 같다: V. vulnificus (78.69%), V. parahaemolyticus (6.56%), V. navarrensis (4.92%), V. mimicus (1.64%), V. cholera (1.64%), V. furnissii (1.64%), V. alginolyticus (1.64%), Grimontia hollisae (1.64%). dnaJ 유전자 서열 분석의 정확도는 91.80%였고16S rDNA 서열 분석의 정확도는 86.89%였다. dnaJ 유전자 서열 분석법과 16S rDNA 서열 분석법 간의 일치도는 0.45로서, 중등도의일치도였다.
결론: 본 연구는 dnaJ 유전자 서열 분석법이 서로 밀접히 관련된Vibrio 종 간의 정확한 균 종정에 유용한 방법이라는 것을 보여주었다.

다국어 초록 (Multilingual Abstract)

Background: Among the many Vibrio species that can cause infections in humans, several species can cause a fatal outcome. Therefore, accurate identification of Vibrio species is very important. Since some species show atypical phenotypic features, selecting an appropriate molecular method is necessary to avoid misdiagnosis.
Methods: Vibrio clinical isolates (N=53) and reference strains (N=8) were used in this study. We analyzed the following sequences for identification: dnaJ gene, 16S rDNA, gyrase B (gyrB) V. vulnificus–specific sequence, gyrB V. navarrensis–specific sequence, and V. vulnificus hemolysin gene PCR (Vvh PCR). We performed phylogenetic analysis of the 16S rDNA, dnaJ, and gyrB sequences. Final identification was based on the combined results of all tests described above. Concordance of the 16S rDNA and dnaJ sequence analysis was measured using the Chi-square test.
Results: The 61 Vibrio strains were identified as follows, in descending order: V. vulnificus (78.69%), V. parahaemolyticus (6.56%), V. navarrensis (4.92%), V. mimicus (1.64%), V. cholera (1.64%), V. furnissii (1.64%), V. alginolyticus (1.64%), and Grimontia hollisae (1.64%). The accuracy rates of the dnaJ gene and 16S rDNA sequence for identification were 91.80% and 86.89%, respectively. The 16S rDNA and dnaJ sequences showed a concordance rate of 0.45, which indicates moderate agreement.
Conclusions: Our results suggest that analysis of the dnaJ sequence may be a useful method for the identification of clinical isolates of Vibrio species, especially for distinguishing between closely related Vibrio species.

참고문헌 (Reference)

1 김성중, "Virulence Characteristics of Sucrose-fermenting Vibrio vulnificus Strains" 대한진단검사의학회 30 (30): 507-510, 2010

2 Erler R, "VibrioBase: A MALDI-TOF MS database for fast identi cation of Vibrio spp. that are potentially pathogenic in humans" 38 : 16-25, 2015

3 Nhung PH, "Use of the novel phylogenetic marker dnaJ and DNA-DNA hybridization to clarify interrelationships within the genus Aeromonas" 57 : 1232-1237, 2007

4 Nhung PH, "The dnaJ gene as a novel phylogenetic marker for identi cation of Vibrio species" 30 : 309-315, 2007

5 Kong KA, "Statistical methods: reliability assessment and method comparison" 40 : 9-16, 2017

6 Miyoshi T, "Phylogenetic characterization of 16S rRNA gene clones from deep-groundwater microorganisms that pass through 0.2-micrometer-pore-size lters" 71 : 1084-1088, 2005

7 Gladney LM, "Molecular and phenotypic characterization of Vibrio navarrensis isolates associated with human illness" 52 : 4070-4074, 2014

8 Ragon M, "Microbial diversity in the deep-subsurface hydrothermal aquifer feeding the giant gypsum crystal-bearing Naica Mine, Mexico" 4 : 37-, 2013

9 Tarr CL, "Manual of clinical microbiology" ASM Press 765-, 2015

10 Colodner R, "Identifiation of the emerging pathogen Vibrio vulni cus biotype 3 by commercially available phenotypic methods" 42 : 4137-4140, 2004

11 Brennt CE, "Growth of Vibrio vulnificus in serum from alcoholics: association with high transferrin iron saturation" 164 : 1030-1032, 1991

12 Kalia VC, "Genome wide analysis for rapid identi cation of Vibrio species" 55 : 375-383, 2015

13 Chun J, "EzTaxon: a webbased tool for the identi cation of prokaryotes based on 16S ribosomal RNA gene sequences" 57 : 2259-2261, 2007

14 Park G, "Evaluation of four methods of assigning species and genus to medically important bacteria using 16S rRNA gene sequence analysis" 59 : 285-298, 2015

15 Kim HS, "Application of rpoB and zinc protease gene for use in molecular discrimination of Fusobacterium nucleatum subspecies" 48 : 545-553, 2010

16 Daniels NA, "A review of pathogenic Vibrio infections for clinicians" 17 : 665-685, 2000