Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

The Korean Society of Food Hygiene and Safety (한국식품위생안전성학회)
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Comparative Evaluation of the VITEK 2 System and Species-specific PCR Methods for the Detection of Vibrio Species Isolated from Shrimp

새우에서 분리된 Vibrio species 동정을 위한 VITEK 2 system방법과 species-specific PCR방법 비교 평가

  • Lee, Jeong-Min (Department of Food Science and Technology, Chung-Ang University) ;
  • Lee, Won-Jun (Department of Food Science and Technology, Chung-Ang University) ;
  • Kim, Min-Ju (Department of Food Science and Technology, Chung-Ang University) ;
  • Cho, Yong-Sun (Food Analysis Center, Korea Food Research Institute) ;
  • Lee, Jin-Sung (Department of Biological Sciences, Kyonggi University) ;
  • Lee, Hyun-Jin (Research and Development Center, DSLine Co., Ltd.) ;
  • Yoon, Sang-Woo (Research and Development Center, DSLine Co., Ltd.) ;
  • Kim, Keun-Sung (Department of Food Science and Technology, Chung-Ang University)
  • 이정민 (중앙대학교 식품공학과) ;
  • 이원준 (중앙대학교 식품공학과) ;
  • 김민주 (중앙대학교 식품공학과) ;
  • 조용선 (한국식품연구원 식품분석센터) ;
  • 이진성 (경기대학교 생명과학과) ;
  • 이현진 ((주)디에스라인 기업부설연구소) ;
  • 윤상우 ((주)디에스라인 기업부설연구소) ;
  • 김근성 (중앙대학교 식품공학과)
  • Received : 2015.06.05
  • Accepted : 2015.07.17
  • Published : 2015.09.30
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Abstract

Vibrio is a genus of Gram-negative, curved, halophilic, and non-spore-forming bacteria. Some of the Vibrio species, such as V. cholerae and V. parahaemolyticus, often contaminate seafood products and occasionally cause human diseases when the seafood products are ingested. A total of 24 Vibrio strains were isolated from shrimp samples on Thiosulphate citrate bile salt sucrose (TCBS) media in this study. All of the 24 isolates were confirmed to belong to the genus Vibrio by using 16S rRNA gene sequence analyses. Vitek 2 system and species-specific polymerase chain reaction (PCR) methods were used to further identify a total of 29 Vibrio strains at the species level, including the 24 shrimp Vibrio isolates and five Vibrio reference strains. The specificities of the two methods to identify Vibrio strains at the species level were compared in this study. The species-specific PCR method was designed to detect five different Vibrio species, such as Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, and Vibrio mimicus. From the 24 Vibrio shrimp isolates, the Vitek 2 system method could identify 15 (62.5%) strains as Vibrio species and 7 (29.2%) strains as non-Vibrio species, but could not identify the rest 2 (8.3%) strains. But species-specific PCR method could identify 16 (66.7%) strains as Vibrio species and could not identify the rest 8 (33.3%) strains. Among the 24 Vibrio shrimp strains, these two methods could unanimously identify 7 (7/24, 29.2%) strains (2 V. parahaemolyticus, 4 V. alginolyticus, and 1 V. mimicus). Considering that such different identification results were obtained using the two different methods in this study, identification method for Vibrio species must be carefully chosen.

Vibrio 속에 속한 세균에 의한 식중독은 오염된 해산물 식품의 섭취로 인하여 빈번하게 발생하고 있다. 그러므로 해산물을 날것으로 섭취하는 한국인의 특성을 고려할 때, 빠르고 정확한 Vibrio 검출기술은 식품위생 및 공중보건의 측면에서 매우 중요하다. 이와 관련하여 본 연구에서는 전통적인 배지를 이용한 동정방법의 단점을 보완할 수 있는 생화학적 방법인 Vitek 2 system방법과 분자생물학적 방법인 species-specific PCR 방법을 사용하여 얻은 동정결과를 비교 평가하고자 하였다. 본 연구에서는 5개의 Vibrio 표준균주와 16S rRNA gene sequencing 결과에 의하여 Vibrio 속으로 확인된 24개의 분리균주가 이용되었다. Vitek 2 system방법을 이용한 경우, 이와 같이 본 연구에 사용된 29개 균주 중 Vibrio 표준균주 2개(2/5, 40%), 16S rRNA gene sequencing 결과 Vibrio 속으로 확인된 분리균주 15개(15/24, 62.5%) 등의 총 17개 균주(17/29, 58.6%)가 Vibrio 종으로 동정되었다. 그리고 species-specific PCR방법을 이용한 경우, 위의 29개 균주 중 Vibrio 표준균주 5개(5/5, 100%), 16S rRNA gene sequencing 결과 Vibrio 속으로 확인된 분리균주 16개(16/24, 66.7%) 등의 총 21개 균주(21/29, 72.5%)가 Vibrio 종으로 동정되었다. Vitek 2 system방법과 species-specific PCR방법을 이용하여 동정된 결과를 비교하였을 때 표준균주 중 V. parahaemolyticus, V. mimicus 등의 2개(2/5, 40%), 새우분리균주 24개 중에서 16S rRNA gene sequencing 결과 Vibrio 속으로 확인된 분리균주 7개 (7/24, 29.2%) 등의 총 9개(9/29, 31%) 균주들에 대한 동정결과만이 일치하였다.

Keywords

References

  1. Kim, S.M., Won, K.M., Woo, S.H., Li, H., Kim, E.J., Choi, K.J., Cho, M.Y., Kim, S.H. and Park, S.I.: Vibrios isolated from diseased marine culturing fishes in Korea. J. Fish Pathol. 18, 133-145 (2005).
  2. Diggles, B.K., Carson, J., Hine, P.M., Hickman, R.W. and Tait, M.J.: Vibrio species associated with mortalities in hatchery-reared turbot (Colistium nudipinnis) and brill (C. guntheri) in New Zealand. Aquaculture 183, 1-12 (2000). https://doi.org/10.1016/S0044-8486(99)00280-X
  3. Joseph, S.W., Cowell, R.R. and Kaper, J.B.: Vibrio parahaemolyticus and related halophilic vibrios. Crit. Rev. Microbiol. 10, 77-123 (1983).
  4. Su, Y.C. and Liu, C.: Vibrio parahaemolyticus: A concern of seafood safety. Food Microbiol. 24, 549-558 (2007). https://doi.org/10.1016/j.fm.2007.01.005
  5. Jung, S.H., Hur, M.J., Ju, J.H., Kim, K.A., Oh, S.S., Go, J.M., Kim, Y.H. and Im, J.S.: Microbiological evaluation of raw vegetables. J. Food Hyg. Safety 21, 250-257 (2006).
  6. Kim, M.H. and Shin, W.S.: Microbiological quality of raw and cooked foods in middle and high school food service establishments. J. Korean Soc. Food Sci. Nutr. 37, 1343-1356 (2008). https://doi.org/10.3746/jkfn.2008.37.10.1343
  7. Korea Food and Drug Administration.: Food & Drug Statistical Yearbook for 2009. Available from http://www.kfda.go.kr/index.kfda?mid=96&pageNo=1&seq=7181&cmd=v, Accessed Nov. 10 (2009).
  8. Lee, S.K., Wang, H.Z., Law, S.H., Wu, R.S. and Kong, R.Y.: Analysis of the 16S-23S rDNA intergenic spacers (IGSs) of marine vibrios for species-specific signature DNA sequences. Marine pollution Bulletin 44, 412-420 (2002). https://doi.org/10.1016/S0025-326X(01)00256-9
  9. Chon, J.W., Hyeon, J.Y., Hwang, I.G., Kwak, H.S., Han, J.A., Chung, Y.H., Song, K.Y. and Seo, K.H.: Comparison of the standard culture method and real-time PCR for the detection of Vibrio parahaemolyticus in seafoods and vegetables. Korean J. Food Sci. Technol. 42, 355-360 (2010).
  10. Miller, L.T.: Single derivatization method for routine analysis of bacterial whole-cell fatty acid methyl esters, including hydroxyl acids. J. Clin. Microbiol. 16, 584-586 (1982).
  11. Stager, C.E. and Davis, J.R.: Automated systems for identification of microorganisms. Clin. Microbiol. Rev. 5, 302-327 (1992). https://doi.org/10.1128/CMR.5.3.302
  12. Jill, K.W., Hanko, G., Stewart, R., Pape, J. and Nachamkin, I.: Comparison of three commercial systems for identification of yeasts commonly isolated in the clinical microbiology laboratory. J. Clin. Microbiol., 37, 1967-1970 (1999).
  13. Tang, Y.W., Ellis, N. M., Hopkins, M. K., Smith, D. H., Dodge, D. E. and Persing, D. H.: Comparison of phenotypic and genotypic techniques for identification of unusual aerobic pathogenic Gram-negative bacilli. J. Clin. Microbiol. 36, 3674-3679 (1998).
  14. Yeon S.H., Jeong W..J. and Park J.S.: The diversity of culturable organotrophic bacteria from local solar salterns. J. Microbiol. 43, 1-10 (2005).
  15. Hernandez, G. and Olmos, J.: Molecular identification of pathogenic and nonpathogenic strains of Vibrio harveyi using PCR and RAPD. Appl. Microbiol. Biotechnol. 63, 722-727 (2003).
  16. Rivas, R., Garcia-Fraile, P., Mateos, P. F., Martinez-Molina, E. and Velazquez, E.: Photobacterium halotolerans sp. Nov.,isolated from lake Martel in Spain. Inc. J. Syst. Evol. Microbiol. 56, 1067-1071 (2003).
  17. Thompson, F. L., Gevers, D., Thompson, C. C., Dawyndt, P., Naser, S., Hoste, B., Munn, C. B. and Swings, J.: Phylogeny and molecular identification of vibrios on the basis of multilocus sequence analysis. Appl. Microbiol. Biotechnol. 71, 5107-5115 (2005).
  18. Yoon, M., Kim, K.J., Shin, J.H, Seo, S.P. and Yang, D.U.: Identification of Vibrio vulnificus by the Vitek GNI+ card. Korean J. Clin. Pathol. 20, 314-319 (2000).
  19. Ryang, D. W., Koo, S. B., Shin, M. G., Shin, J. H. and Suh, S. P.: Molecular typing of Vibrio vulnificus isolated from clinical specimens by pulsed-field gel electrophoresis and random amplified polymorphic DNA analysis. Jpn. J. Infect. Dis. 52, 38-41 (1999).
  20. Yoon, Y.J., Kim, D.Y., Lee, C.H., Lee, U.Y., Koh, Y.H., Kim, S.K. and Kim, J.W.: Isolation and Identification of Vibrio species contaminated in imported frozen seafoods. J. Fd. Hyg. Safety 15, 128-136 (2000).
  21. O'Hara, C.M., Westbrook, G.L. and Miller, J.M.: Evaluation of Vitek GNI+ and Becton Dickinson microbiology systems Crystal E/NF identification systems for identification of members of the family Enterobacteriaceae and other gramnegative, glucose-fermenting and non-glucose-fermenting bacilli. J. Clin. Microbiol. 35, 3269-3273 (1997).
  22. Cheryl, L.T., Jayna, S.P., Nancy, D.P., Evangeline, G.S., Cheryl, A.B. and Nancy, A.S.: Identification of Vibrio isolates by a multiplex PCR assay and rpoB sequence determination. J. Clin. Microbiol., 45, 134 (2007). https://doi.org/10.1128/JCM.01544-06
  23. Angela, D.P., Giuseppina, C., Giuseppina, T., Domenico, C. and Vito, T.F.: A collagenase-targeted multiplex PCR assay for identification of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus. J. Food Prot. 68, 150-153 (2005). https://doi.org/10.4315/0362-028X-68.1.150
  24. Lee, S. H., Jang, S. J., Moon, D. S., Park, Y. J., Ahn, G. Y., Han, H. L., Chaulagain, B. P. and Jenog, O. Y.: Evaluation of the detectability of vitek II system for inducible clindamycin resistance in Staphylococci. Kor. J. Lab. Med. 25, 406-410 (2005).
  25. Eisner, A., Gorkiewicz, G., Feierl, G., Leitner, E., Kofer, J., Kessler, H. H. and Marth, E.: Identification of glycopeptideresistant Enterococci by Vitek 2 system and conventional and real-time polymerase chain reaction. Diagn. Microbiol. Infect. Dis. 53, 17-21 (2005). https://doi.org/10.1016/j.diagmicrobio.2005.04.001
  26. Thwe, S. M., Kobayashi, T., Luan, T., Shirai, T., Onodera, M. and Hamada-Sato N.: Isolation, characterization, and utilization of ${\gamma}$-aminobutyric acid (GABA)-producing lactic acid bacteria from Myanmar fishery products fermented with boiled rice. Fisheries Science 77, 279-288 (2011). https://doi.org/10.1007/s12562-011-0328-9