Korean Journal of Food Science and Technology (한국식품과학회지)

Korean Society of Food Science and Technology (한국식품과학회)
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Rapid detection of shiga-toxin producing E. coli by bacteriophage amplification assay

박테리오파지 증폭 기법을 활용한 시가 독소 생성 병원성 대장균의 신속 검출

  • Baek, Da-Yun (Department of Food Science and Biotechnology, College of BioNano Technology, Gachon University) ;
  • Park, Jong-Hyun (Department of Food Science and Biotechnology, College of BioNano Technology, Gachon University) ;
  • Cho, Seok-Cheol (Department of Food Science and Engineering, Seowon University) ;
  • Lee, Young-Duck (Department of Food Science and Engineering, Seowon University)
  • 백다윤 (가천대학교 식품생물공학과) ;
  • 박종현 (가천대학교 식품생물공학과) ;
  • 조석철 (서원대학교 식품공학과) ;
  • 이영덕 (서원대학교 식품공학과)
  • Received : 2020.01.15
  • Accepted : 2020.02.05
  • Published : 2020.02.29
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Abstract

Shiga toxin-producing Escherichia coli (STEC) is an important pathogenic bacteria and can cause severe foodborne disease. For STEC detection, conventional culture methods have disadvantages in the fact that conventional culture takes a long time to detect and PCR can also detect dead bacteria. To overcome these problems, we suggest a bacteriophage amplification assay, which utilizes the ability of bacteriophages to infect living cells and their high specificity. We used a combination of six bacteriophages infecting E. coli to make the bacteriophage cocktail and added ferrous ammonium sulfate as a virucidal agent to remove free-bacteriophages. When cherry tomato and paprika were artificially inoculated with the cocktail at a final concentration of around 3 log CFU/mL and were enriched for at least 5 h in mTSB broth with Novobiocin, approximately 2-3 log PFU/mL were detected through the bacteriophage amplification assay. Therefore, bacteriophage amplification assay might be convenient and a useful method to detect STEC in a short period of time.

본 연구는 식품에서 문제가 되는 시가독소생성 대장균(STEC)을 박테리오파지 증폭 기법을 통해 검출하고자 시가독소 생성 대장균에 대한 박테리오파지를 분리하였고 분리된 4종의 파지와 기 분리된 2종의 박테리오파지를 혼합하여 사용하였다. 분리된 박테리오파지는 형태학적 특성 및 제한효소 절단 패턴 등을 통해서 동정하였다. 5종의 파지는 E. coli O157:H7 및 non-O157 시가독소 생성 대장균을 모두 저해하는 특징을 가지는 것으로 나타났다. 박테리오파지 증폭 기법에서 중요한 단계인 세균에 감염되지 않은 박테리오파지를 제거하기 위해 10% (v/v) ferrous ammonium sulfate (FAS)을 사용하였으며 약 7-9 log PFU/mL 수준의 박테리오파지를 10분 내로 제거하는 것을 확인하였다. 시가독소 생성 대장균인 E. coli NCCP 13937을 검출하기 위해서는 약 6 log PFU/mL 이상의 박테리오파지 혼합액의 농도 및 약 4-5 log CFU/mL 이상의 목표 균주가 필요한 것으로 나타났다. 이러한 조건을 바탕으로 실제 판매되고 있는 신선식품에서 시가독소생성 대장균을 검출한 결과, 5시간 이내에 증폭된 약 2-3 log PFU/mL의 plaque를 통해 검출이 가능한 것을 확인하였다. 따라서 본 연구를 통해 박테리오파지 혼합액을 이용한 증폭 기법을 통해 시가독소 생성 대장균의 오염 여부를 보다 효율적으로 확인할 수 있음을 보여주었고 이를 적용한 제품을 개발하여 검출 단계의 간편화가 가능할 것으로 판단된다.

Keywords

References

  1. Ackermann HW. Bacteriophage observations and evolution. Res. Microbiol. 154: 245-251 (2003) https://doi.org/10.1016/S0923-2508(03)00067-6
  2. Brooks JT, Sowers EG, Wells JG, Greene KD, Griffin PM, Hoekstra RM, Strockbine NA. Non-O157 Shiga toxin-producing Escherichia coli infections in the United States, 1983-2002. J. Infect. Dis. 192: 1422-1429 (2005) https://doi.org/10.1086/466536
  3. de Siqueira R, Dodd C, Rees C. Evaluation of the natural virucidal activity of teas for use in the phage amplification assay. Int. J. Food Microbiol. 111: 259-262 (2006) https://doi.org/10.1016/j.ijfoodmicro.2006.04.047
  4. Derda R, Lockett MR, Tang SK, Fuller RC, Maxwell EJ, Breiten B, Cuddemi CA, Ozdogan A, Whitesides GM. Filter-based assay for Escherichia coli in aqueous samples using bacteriophage-based amplification. Anal. Chem. 85: 7213-7220 (2013) https://doi.org/10.1021/ac400961b
  5. Dini C, Urraza PJ. Isolation and selection of coliphages as potential biocontrol agents of enterohemorrhagic and Shiga toxin-producing E. coli (EHEC and STEC) in cattle. J. Appl. Microbiol. 109: 873-887 (2010) https://doi.org/10.1111/j.1365-2672.2010.04714.x
  6. Elhariry HM. Attachment strength and biofilm forming ability of Bacillus cereus on green-leafy vegetables: cabbage and lettuce. Food microbiol. 28: 1266-1274 (2011) https://doi.org/10.1016/j.fm.2011.05.004
  7. Favrin SJ, Jassim SA, Griffiths MW. Application of a novel immunomagnetic separation-bacteriophage assay for the detection of Salmonella enteritidis and Escherichia coli O157: H7 in food. Int. J. food microbiol. 85: 63-71 (2003) https://doi.org/10.1016/S0168-1605(02)00483-X
  8. Garrido-Maestu A, Fucios P, Azinheiro S, Carvalho C, Carvalho J, Prado M. Specific detection of viable Salmonella Enteritidis by phage amplification combined with qPCR (PAA-qPCR) in spiked chicken meat samples. Food control. 99: 79-83 (2019) https://doi.org/10.1016/j.foodcont.2018.12.038
  9. Gould LH, Mody RK, Ong KL, Clogher P, Cronquist AB, Garman KN, Lathrop S, Medus C, Spina NL, Webb TH. Increased recognition of non-O157 Shiga toxin-producing Escherichia coli infections in the United States during 2000-2010: epidemiologic features and comparison with E. coli O157 infections. Foodborne Pathog. Dis. 10: 453-460 (2013) https://doi.org/10.1089/fpd.2012.1401
  10. Guglielmotti DM, Mercanti DJ, Reinheimer JA, Quiberoni ADL. Efficiency of physical and chemical treatments on the inactivation of dairy bacteriophages. Front. Microbiol. 2: 282 (2012) https://doi.org/10.3389/fmicb.2011.00282
  11. Hendrix RW. Bacteriophage genomics. Curr. Opin. Microbiol. 6: 506-511 (2003) https://doi.org/10.1016/j.mib.2003.09.004
  12. Hughes JM, Wilson ME, Johnson KE, Thorpe CM, Sears CL. The emerging clinical importance of non-O157 Shiga toxin-producing Escherichia coli. Clin. Infect. Dis. 43: 1587-1595 (2006) https://doi.org/10.1086/509573
  13. Jassim S, Griffiths M. Evaluation of a rapid microbial detection method via phage lytic amplification assay coupled with Live/Dead fluorochromic stains. Lett. Appl. Microbiol. 44: 673-678 (2007) https://doi.org/10.1111/j.1472-765X.2007.02115.x
  14. Kaper JB, Nataro JP, Mobley HL. Pathogenic Escherichia coli. Nat. Rev. Microbiol. 2: 123-140 (2004) https://doi.org/10.1038/nrmicro818
  15. Kim EJ, Lee H, Lee JH, Ryu S, Park JH. Morphological features and lipopolysaccharide attachment of coliphages specific to Escherichia coli O157: H7 and to a broad range of E. coli hosts. Appl. Biol. Chem. 59: 109-116 (2016) https://doi.org/10.1007/s13765-015-0130-y
  16. Law D. Virulence factors of Escherichia coli O157 and other Shiga toxin-producing E. coli. J. Appl. Microbiol. 88: 729-745 (2000) https://doi.org/10.1046/j.1365-2672.2000.01031.x
  17. Lee YD, Park JH. Characterization and application of phages isolated from sewage for reduction of Escherichia coli O157: H7 in biofilm. LWT-Food Sci. Technol. 60: 571-577 (2015) https://doi.org/10.1016/j.lwt.2014.09.017
  18. Lienemann T, Kyyhkynen A, Halkilahti J, Haukka K, Siitonen A. Characterization of Salmonella Typhimurium isolates from domestically acquired infections in Finland by phage typing, antimicrobial susceptibility testing, PFGE and MLVA. BMC Microbiol. 15: 131 (2015) https://doi.org/10.1186/s12866-015-0467-8
  19. Lim GY, Park DW, Lee YD, Park JH. Isolation and characterization of bacteriophages for the control of Shiga Toxin-producing E. coli. Korean J. Food Sci. Technol. 50: 594-600 (2018) https://doi.org/10.9721/KJFST.2018.50.6.594
  20. Ly-Chatain MH, Moussaoui S, Vera A, Rigobello V, Demarigny Y. Antiviral effect of cationic compounds on bacteriophages. Front. Microbiol. 4: 46 (2013) https://doi.org/10.3389/fmicb.2013.00046
  21. Majowicz SE, Scallan E, Jones-Bitton A, Sargeant JM, Stapleton J, Angulo FJ, Yeung DH, Kirk MD. Global incidence of human Shiga toxinproducing Escherichia coli infections and deaths: a systematic review and knowledge synthesis. Foodborne Pathog. Dis. 11: 447-455 (2014) https://doi.org/10.1089/fpd.2013.1704
  22. McNerney R, Wilson S, Sidhu A, Harley Va, Al Suwaidi Z, Nye P, Parish T, Stoker N. Inactivation of mycobacteriophage D29 using ferrous ammonium sulphate as a tool for the detection of viable Mycobacterium smegmatis and M. tuberculosis. Res. Microbiol. 149: 487-495 (1998) https://doi.org/10.1016/S0923-2508(98)80003-X
  23. Nakao H, Kataoka C, Kiyokawa N, Fujimoto J, Yamasaki S, Takeda T. Monoclonal antibody to Shiga toxin 1, which blocks receptor binding and neutralizes cytotoxicity. Microbiol. immunol. 46: 777-780 (2002) https://doi.org/10.1111/j.1348-0421.2002.tb02764.x
  24. Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11: 142-201 (1998) https://doi.org/10.1128/CMR.11.1.142
  25. Oliveira A, Sillankorva S, Quinta R, Henriques A, Sereno R, Azeredo J. Isolation and characterization of bacteriophages for avian pathogenic E. coli strains. J. Appl. Microbiol. 106: 1919-1927 (2009) https://doi.org/10.1111/j.1365-2672.2009.04145.x
  26. Oliveira I, Almeida RCdC, Hofer E, Almeida PF. Bacteriophage amplification assay for detection of Listeria spp. using virucidal laser treatment. Braz. J. Microbiol. 43: 1128-1136 (2012) https://doi.org/10.1590/S1517-83822012000300040
  27. Park DJ, Drobniewski F, Meyer A, Wilson S. Use of a phage-based assay for phenotypic detection of mycobacteria directly from sputum. J. Clin. Microbiol. 41: 680-688 (2003) https://doi.org/10.1128/JCM.41.2.680-688.2003
  28. Park WJ, Lim GY, Park JH. Enumeration of Weissella cibaria phage with cytometry, epifluorescence microscopy, and plaque assay. Korean J. Food Sci. Technol. 50: 244-247 (2018) https://doi.org/10.9721/KJFST.2018.50.2.244
  29. Patel J, Sharma M. Differences in attachment of Salmonella enterica serovars to cabbage and lettuce leaves. Int. J Food Microbiol. 139: 41-47 (2010) https://doi.org/10.1016/j.ijfoodmicro.2010.02.005
  30. Raya RR, Varey P, Oot RA, Dyen MR, Callaway TR, Edrington TS, Kutter EM, Brabban AD. Isolation and characterization of a new T-even bacteriophage, CEV1, and determination of its potential to reduce Escherichia coli O157: H7 levels in sheep. Appl. Environ. Microbiol. 72: 6405-6410 (2006) https://doi.org/10.1128/AEM.03011-05
  31. Smith JL, Fratamico PM, Gunther IV NW. Shiga toxin-producing Escherichia coli. Adv. Appl. Microbiol. 86: 145-197 (2014) https://doi.org/10.1016/B978-0-12-800262-9.00003-2
  32. Stewart GS, Jassim SA, Denyer SP, Newby P, Linley K, Dhir VK. The specific and sensitive detection of bacterial pathogens within 4 h using bacteriophage amplification. J. Appl. Microbiol. 84: 777-783 (1998) https://doi.org/10.1046/j.1365-2672.1998.00408.x
  33. Sulakvelidze A, Alavidze Z, Morris JG. Bacteriophage therapy. Antimicrob. Agents. Chemother. 45: 649-659 (2001) https://doi.org/10.1128/AAC.45.3.649-659.2001
  34. Torres AG, Amaral MM, Bentancor L, Galli L, Goldstein J, Krger A, Rojas-Lopez M. Recent advances in shiga toxin-producing Escherichia coli research in Latin America. Microorganisms 6: 100-118 (2018) https://doi.org/10.3390/microorganisms6040100
  35. Tzipilevich E, Habusha M, Ben-Yehuda S. Acquisition of phage sensitivity by bacteria through exchange of phage receptors. Cell 168: 186-199 (2017) https://doi.org/10.1016/j.cell.2016.12.003