Fluorogenic and Chromogenic Assay for Rapid Detection of Escherichia coli and Total Coliform Bacteria

효소발색법을 이용한 대장균 및 총대장균군 신속 검사

  • 이근헌 ((주)휴마스 부설연구소) ;
  • 김훈수 ((주)휴마스 부설연구소) ;
  • 김병렬 ((주)휴마스 부설연구소) ;
  • 이승희 (한국생명공학연구원 바이오산업화 공정개발센터) ;
  • 인치경 (충남보건환경연구원 폐기물분석과) ;
  • 박경량 (한남대학교 생명공학과)
  • Received : 2008.11.28
  • Accepted : 2009.01.29
  • Published : 2009.05.30

Abstract

We developed the Eco medium for Escherichia coli and total coliforms, which was modified by Violet Red Bile (VRB) medium, and derived the standard curve of exponential phase at $OD_{410}$ by using type strains such as E. coli ATCC11303, Enterobacter cloacae KCTC2361, Klebsiella pneumoniae KCTC2241, and Citrobacter freundii KCTC2359. Also, we used total 93 samples of spring and stream water to compare the detection ability of total coliforms between the method using Eco medium and such as most probable number (MPN), and plate count methods. As a result, the qualitative analysis of E. coli and total coliforms using Eco medium contained ortho-nitrophenyl-$\beta$-galactoside (ONPG) and 4-methylumbelliferyl-$\beta$-D-glucuronide (MUG) was same as those of Korean standard methods (Colilert kit). And the colony forming unit (CFU) detected in Eco medium was similar to those of result from MPN and plate count methods. Moreover, the agreement, sensitivity, and specificity of the developed kit was more than 97.5% in comparison with Colilert kit for 350 samples. Thus, the Eco medium can be used both qualitative and quantitative analysis of E. coli and total coliforms.

Keywords

Acknowledgement

Supported by : 환경부

References

  1. Atlas, R. M. (2004). Handbook of Microbiological Media (3rd. ed.). CRC press
  2. Chao, K. K., Chao, C. C., and Chao, W. L. (2004). Evaluation of Colilert-18 for Detection of Coliforms and Escherichia coli in Subtropical Freshwater. Appl. Environ. Microbiol., 70(2), pp. 1242-1244 https://doi.org/10.1128/AEM.70.2.1242-1244.2004
  3. Clark, D. L., Milner, B. B., Stewart, M. H., Wolfe, R. L., and Olson, B. H. (1991). Comparative study of commercial 4-methyl-$\beta$-D-glucuronide preparations with the Standard Methods membrane filtration fecal coliform test for the detection of Escherichia coli in water samples. Appl. Environ. Microbiol., 57, pp. 1528-1534
  4. Eckner, K. (1998). Comparison of membrane filtration and multiple-tube fermentation by the Colilert and Enterobacter methods for detection of waterborne coliforms bacteria, Escherichia coli, and enterococci used in drinking and bathing water quality monitoring in Southern Sweden. Appl. Environ. Microbiol., 64, pp. 3079-3083
  5. Edberg, S. C. and Kontnick, C. M. (1986). Comparison of $\beta$-glucuronidase-based substrate systems for identification of Escherichia coli., J. Clin. Microbiol., 24, pp. 368-371
  6. Edberg, S. C., Allen, M. J., and Smith, D. B. (1988). National field evaluation of a defined substrate method for the simultaneous detection of total coliforms and Escherichia coli from drinking water: comparison with the standard multiple tube fermentation methods. Appl. Environ. Microbiol.54, pp. 1595-1601
  7. Edberg, S. C., Allen, M. J., and Smith, D. B. (1989). National field evaluation of a defined substrate method for the simultaneous detection of total coliforms and Escherichia coli from drinking water: comparison with presence-absence techniques. Appl. Environ. Microbiol., 55, pp. 1003-1008
  8. Edberg, S. C., Allen, M. J., Smith, D. B., and Kriz, N. J. (1990). Enumeration of total coliforms and Escherichia coli from source water by the defined substrate technology. Appl. Environ. Microbiol., 56, pp. 366-369
  9. Feng, P. C. S. and Hartman, P. A. (1982). Fluorogenic assay for immediate confirmation of Escherichia coli., Appl. Environ. Microbiol., 43, pp. 1320-1329
  10. Fricker, E. J., Illingworth, K. S., and Fricker, C. R. (1997). Use of two formulations of Colilert and QuantitryTM for assessment of the bacteriological quality of water. Water Res., 31, pp. 2495-2499 https://doi.org/10.1016/S0043-1354(96)00342-9
  11. Kilian, M. and Bulow, P. (1976). Rapid diagnosis of Enterobacteriaceae I. Detection of bacterial glycosidases. Acta Pathol. Microbiol. Scand. Sect. 84, pp. 245-251 https://doi.org/10.1111/j.1699-0463.1976.tb01933.x
  12. Koburger, J. A. and Miller, M. L. (1985). Evaluation of a fluorogenic MPN procedure for determining Escherichia coli in oysters. J. Food Prot., 48, pp. 244-245
  13. Le Minor, L. (1979). Tetrathionate-reductase, $\beta$-glucuronidase and ONPG test in the genus Salmonella. Zentralbl. Bakteriol. Parasitenkd. Infektionskr, 243, pp. 321-325
  14. Moberg, L. J., Wagner, M. K., and Kellen, L. A. (1988). Fluorogenic assays for immediate confirmation of Escherichia coli in chilled and frozen food: collaborative study. J. Assoc. Off. Anal. Chem., 71, pp. 589-602
  15. Park, S. J., Lee, E., Lee, D., Lee, S., and Kim, S. (1995). Spectrofluorometric assay for rapid detection of total and fecal coliforms from surface water. Appl. Environ. Microbiol., 61, pp. 2027-2029
  16. Poelma, P. L., Wilson, C. R., and Andrews, W. H. (1987). Rapid fluorogenic enumeration of Escherichia coli in selected, naturally contaminated high moisture food. J. Assoc. Off. Anal. Chem., 70, pp. 991-993
  17. Pope, M. L., Bussen, M., Feige, M. A., Shadix, L., Gonder, S., Rodgers, C., Chambers, Y., Pulz, J., Miller, K., Connell, K., and Standridge, J. (2003). Assessment of the Effects of Holding Time and Temperature on Escherichia coli Densities in Surface Water Samples. Appl. Environ. Microbiol., 69, pp. 6201-6207 https://doi.org/10.1128/AEM.69.10.6201-6207.2003
  18. Rice, E. W., Allen, M. J., Brenner, D. J., and Edberg, S. C. (1991). Assay for β-glucuronidase in species of the genus Escherichia and its applications for drinking water analysis. Appl. Environ. Microbiol., 57, pp. 592-593
  19. Venkateswaran, K., Murakoshi, A., and Satake, M. (1996). Comparison of commercially available kits with standard methods for detection of Coliforms and Escherichia coli in foods. Appl. Environ. Microbiol., 62, pp. 2236-2243