Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Rapid Detection of Enterobacter sakazakii Using TaqMan Real-Time PCR Assay

  • Published : 2007.03.31
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Abstract

Enterobacter sakazakii is an emerging food pathogen, which induces severe meningitis and sepsis in neonates and infants, with a high fatality rate. The disease is generally associated with the ingestion of contaminated infant formula. In this study, we describe the development of a real-time PCR protocol to identify E. sakazakii using a TaqMan probe, predicated on the nucleotide sequence data of the 168 rRNA gene obtained from a variety of pathogens. To detect E. sakazakii, four primer sets and one probe were designed. Five strains of E. sakazakii and 28 non-E. sakazakii bacterial strains were used in order to ensure the accuracy of detection. The PCR protocol successfully identified all of the E. sakazakii strains, whereas the 28 non-E. sakazakii strains were not detected by this method. The detection limits of this method for E. sakazakii cells and purified genomic DNA were 2.3 CFU/ assay and 100 fg/assay, respectively. These findings suggest that our newly developed TaqMan real-time PCR method should prove to be a rapid, sensitive, and quantitative method for the detection of E. sakazakii.

Keywords

References

  1. Acinas, S. G., L. A. Marcelino, V. Klepac-Ceraj, and M. F. Polz. 2004. Divergence and redundancy of 16S rRNA sequences in genomes with multiple rrn operons. J. Bacteriol. 186: 2629-2635 https://doi.org/10.1128/JB.186.9.2629-2635.2004
  2. Biering, G., S. Karlsson, N. C. Clark, K. E. Jonsdottir, P. Ludvigsson, and O. Steingrimsson. 1989. Three cases of neonatal meningitis caused by Enterobacter sakazakii in powdered milk. J. Clin. Microbiol. 9: 2054-2056
  3. Bischoff, C., J. Luthy, M. Altwegg, and F. Baggi. 2005. Rapid detection of diarrheagenic E. coli by real-time PCR. J. Microbiol. Methods 61: 335-341 https://doi.org/10.1016/j.mimet.2004.12.007
  4. Gurtler, J. B., J. L. Kornacki, and L. R. Beuchat. 2005. Enterobacter sakazakii: A coliform of increased concern to infant health. Int. J. Food Microbiol. 104: 1-34 https://doi.org/10.1016/j.ijfoodmicro.2005.02.013
  5. Hawkins, R. E., C. R. Lissner, and J. P. Sanford. 1991. Enterobacter sakazakii bacteremia in an adult. South Med. J. 84: 793-795 https://doi.org/10.1097/00007611-199106000-00033
  6. Iversen, C., M. Waddington, S. L. On, and S. Forsythe. 2004. Identification and phylogeny of Enterobacter sakazakii relative to Enterobacter and Citrobacter species. J. Clin. Microbiol. 42: 5368-5370 https://doi.org/10.1128/JCM.42.11.5368-5370.2004
  7. Iversen, C. and S. Forsythe. 2004. Isolation of Enterobacter sakazakii and other Enterobacteriaceae from powdered infant formula milk and related products. Food Microbiol. 21: 771-777 https://doi.org/10.1016/j.fm.2004.01.009
  8. Jung, S. J., H. J. Kim, and H. Y. Kim. 2005. Quantitative detection of Salmonella typhimurium contamination in milk, using real-time PCR. J. Microbiol. Biotechnol. 15: 1353-1358
  9. Kandhai, M. C., M. W. Reij, K. van Puyvelde, O. Guillaume-Gentil, R. R. Beumer, and M. van Schothorst. 2004. A new protocol for the detection of Enterobacter sakazakii applied to environmental samples. J. Food Prot. 67: 1267-1270 https://doi.org/10.4315/0362-028X-67.6.1267
  10. Kandhai, M. C., M. W. Reij, L. Gorris, O. Guillaume-Gentil, and M. van Schothorst. 2004. Occurrence of Enterobacter sakazakii in food production environments and households. Lancet 363: 39-40 https://doi.org/10.1016/S0140-6736(03)15169-0
  11. Kim, S. B., H. J. Lim, W. K. Lee, I. G. Hwang, G. J. Woo, and S. R. Ryu. 2006. PCR-based detection and molecular genotyping of enterotoxigenic Clostridium perfringens isolates from swine diarrhea in Korea. J. Microbiol. Biotechnol. 16: 291-294
  12. Klappenbach, J. A., P. R. Saxman, J. R. Cole, and T. M. Schmidt. 2001. rrndb: The ribosomal RNA operon copy number database. Nucleic Acids Res. 29: 181-184 https://doi.org/10.1093/nar/29.1.181
  13. Klein, D. 2002. Quantification using real-time PCR technology: Applications and limitations. Trends Mol. Med. 8: 257-260 https://doi.org/10.1016/S1471-4914(02)02355-9
  14. Koh, Y. H., M. D. Kim, N. S. Han, and J. H. Seo. 2002. Quantitative analysis of Leuconostoc mesenteroides and Lactobacillus plantarum populations by a competitive polymerase chain reaction. J. Microbiol. Biotechnol. 12: 801-806
  15. Lai, K. K. 2001. Enterobacter sakazakii infections among neonates, infants, children, and adult. Medicine 80: 113-122 https://doi.org/10.1097/00005792-200103000-00004
  16. Liu, Y., X. Cai, X. Zhang, Q. Gao, X. Yang, Z. Zheng, M. Luo, and X. Huang. 2006. Real time PCR using TaqMan and SYBR Green for detection of Enterobacter sakazakii in infant formula. J. Microbiol. Methods 65: 21-31 https://doi.org/10.1016/j.mimet.2005.06.007
  17. Lukinmaa, S., U. M. Nakari, M. Eklund, and A. Siitonen. 2004. Application of molecular genetic methods in diagnostics and epidemiology of food-borne bacterial pathogens. APMIS 112: 908-929 https://doi.org/10.1111/j.1600-0463.2004.apm11211-1213.x
  18. Matsuki, T., K. Watanabe, J. Fujimoto, T. Takada, and R. Tanaka. 2004. Use of 16S rRNA gene-targeted groupspecific primers for real-time PCR analysis of predominant bacteria in human feces. Appl. Environ. Microbiol. 70: 7220-7228 https://doi.org/10.1128/AEM.70.12.7220-7228.2004
  19. Muytjens, H. L., H. Roelofs-Willemse, and G. H. Jaspar. 1988. Quality of powdered substitutes for breast milk with regard to members of the family Enterobacteriacea. J. Clin. Microbiol. 26: 743-746
  20. Nazarowec-White, M., R. C. Mckellar, and P. Piyasena. 1999. Predictive modelling of Enterobacter sakazakii inactivation in bovine milk during high-temperature short-time pasteurization. Food Res. Int. 32: 375-379 https://doi.org/10.1016/S0963-9969(99)00100-3
  21. Park, S. H., H. J. Kim, and H. Y. Kim. 2006. Simultaneous detection of Yersinia enterocolitica, Staphylococcus aureus, and Shigella spp. in lettuce using multiplex PCR method. J. Microbiol. Biotechnol. 16: 1301-1305
  22. Poms R. E., E. Anklam, and M. Kuhn. 2004. Polymerase chain reaction techniques for food allergen detection. J. AOAC Int. 87: 1391-1397
  23. Seo, K. H. and R. E. Brackett. 2005. Rapid, specific detection of Enterobacter sakazakii in infant formula using a real-time PCR assay. J. Food Prot. 68: 59-63 https://doi.org/10.4315/0362-028X-68.1.59
  24. Simmons, B. P., M. S. Gelfand, M. Hass, L. Metts, and J. Ferguson. 1989. Enterobacter sakazakii infections in neonates associated with intrinsic contamination of a powdered infant formula. Infect. Control Hosp. Epidemiol. 10: 398-401 https://doi.org/10.1086/646060
  25. Van Acker, J., F. De Smet, G. Muyldermans, A. Bougatef, A. Naessens, and S. Lauwers. 2001. Outbreak of necrotizing enterocolitis associated with Enterobacter sakazakii in powdered milk formula. J. Clin. Microbiol. 39: 293-297 https://doi.org/10.1128/JCM.39.1.293-297.2001
  26. Warsen, A. E., M. J. Krig, S. LaFrentz, D. R. Stanek, F. J. Long, and D. R. Call. 2004. Simultaneous discrimination between 15 fish pathogens by using 16S ribosomal DNA PCR and DNA microarrays. Appl. Environ. Microbiol. 70: 4216-4221 https://doi.org/10.1128/AEM.70.7.4216-4221.2004