Article history: Received: 26-08-2017, Accepted: 05-12-2017, Published online: 03-02-2018
Corresponding author: C. Sethulekshmi
E-mail: firstname.lastname@example.orgCitation: Sethulekshmi C, Latha C, Anu CJ (2018) Occurrence and quantification of Shiga toxin-producing Escherichia coli from food matrices, Veterinary World, 11(2): 104-111.
Aim: The objective of the study was to detect Shiga toxin-producing Escherichia coli (STEC) and develop a quantitative polymerase chain reaction (qPCR) assay to quantify the bacterial DNA present in different food matrices.
Materials and Methods: A total of 758 samples were collected during a period from January 2015 to December 2016 from Kozhikode, Thrissur, and Alappuzha districts of Kerala. The samples consisted of raw milk (135), pasteurized milk (100), beef (132), buffalo meat (130), chevon (104), beef kheema (115), and beef sausage (42). All the samples collected were subjected to isolation and identification of STEC by conventional culture technique. Confirmation of virulence genes was carried out using PCR. For the quantification of STEC in different food matrices, a qPCR was standardized against stx1 gene of STEC by the construction of standard curve using SYBR green chemistry.
Results: The overall occurrence of STEC in raw milk (n=135), beef (n=132), buffalo meat (n=130), chevon (n=104), and beef kheema (n=115) samples collected from Kozhikode, Thrissur, and Alappuzha districts of Kerala was 19.26%, 41.6%, 16.92%, 28.85%, and 41.74%, respectively. PCR revealed the presence of stx1 and stx2 genes in 88.46 and 83.64 and 30.77 and 40.00% of STEC isolates from raw milk and beef samples, respectively, while 100% of the STEC isolates from buffalo beef and beef kheema samples carried stx1 gene. Real-time qPCR assay was used to quantify the bacterial cells present in different food matrices. The standard curve was developed, and the slopes, intercept, and R2 of linear regression curves were -3.10, 34.24, and 0.99, respectively.
Conclusion: The considerably high occurrence of STEC in the study confirms the importance of foods of animal origin as a vehicle of infection to humans. In the present study, on comparing the overall occurrence of STEC, the highest percentage of occurrence was reported in beef kheema samples. The study shows the need for rigid food safety measures to combat the potential pathogenic effects of harmful bacteria throughout the production chain from production to consumption.
Keywords: food matrices, occurrence, polymerase chain reaction, real-time quantitative polymerase chain reaction, Shiga toxigenic Escherichia coli.
1. Yamasaki, E., Watahiki, M., Isobe, J., Sata, T., Nair, G.B. and Kurazono, H. (2015) Quantitative detection of shiga toxins directly from stool specimens of patients associated with an outbreak of enterohemorrhagic Escherichia coli in Japan-quantitative shiga toxin detection from stool during EHEC outbreak. Toxins, 7(10): 4381-4389. [Crossref] [PubMed] [PMC]
2. Hiraka, T., Kanoto, M., Sugai, Y., Honma, T., Makino, N., Ueno, Y. and Hosoya, T. (2015) Computed tomographic findings of enterohemorrhagic Escherichia coli O157 infection: An analysis of a 7-case regional outbreak. J. Comput. Assist. Tomo.,39(3): 406-408. [Crossref]
3. Kiranmayi, C.B. and Krishnaiah, N. (2010) Detection of Escherichia coli O157: H7 prevalence in foods of animal origin by cultural methods and PCR technique.Vet. World, 3(1): 13-16.
4. Dhama, K., Rajagunalan, S., Chakraborty, S., Verma, A.K., Kumar, A., Tiwari, R. and Kapoor, S. (2013) Food-borne pathogens of animal origin-diagnosis, prevention, control and their zoonotic significance: A review. Pak. J. Biol. Sci.,16(20): 1076-1085. [Crossref]
5. Suzuki, H. and Okada, Y. (2014) Enterohemorrhagic Escherichia coli (EHEC) Infection and Beef Consumption in Japan. In: Proceeding of 2nd AFSSA Conference on Food Safety and Food Security. pp79-82.
6. Diercke, M., Kirchner, M., Claussen, K., Mayr, E., Strotmann, I., Frangenberg, J., Schiffmann, A., Bettge-Weller, G., Arvand, M. and Uphoff, H. (2014) Transmission of shiga toxin-producing Escherichia coli O104: H4 at a family party possibly due to contamination by a food handler, Germany 2011. Epidemiol. Infect., 142(1): 99-106. [PubMed]
7. Lozinak, K.A., Jani, N., Gangiredla, J., Patel, I., Elkins, C.A., Hu, Z., Kassim, P.A., Myers, R.A. and Laksanalamai, P. (2016) Investigation of potential shiga toxin producing Escherichia coli (STEC) associated with a local foodborne outbreak using multidisciplinary approaches. Food Sci., 5(3): 163-168. [Crossref]
8. Conrad, C., Stanford, K., McAllister, T., Thomas, J. and Reuter, T. (2016) Shiga toxin-producing and current trends in diagnostics. Anim. Frontiers., 6(2): 37-43. [Crossref]
9. Verhaegen, B., De Reu, K., Heyndrickx, M. and De Zutter, L. (2015) comparison of six chromogenic agar media for the isolation of a broad variety of non-O157 shigatoxin-producing Escherichia coli (STEC) serogroups. Int. J. Environ. Res. Public. Health.,12(6): 6965-6978. [Crossref] [PubMed] [PMC]
11. Noll, L.W., Shridhar, P.B., Shi, X., An, B., Cernicchiaro, N., Renter, D.G., Nagaraja, T.G. and Bai, J. (2015) A four-plex real-time PCR assay, based on rfb E, stx 1, stx 2, and eae genes, for the detection and quantification of shiga toxin-producing Escherichia coli O157 in cattle faeces. Foodborne Pathog. Dis., 12(9): 787-794. [Crossref] [PubMed]
12. Taylor, M.T., Sofos, J.N., Bodnaruk, P. and Acuff, G.R. (2015) Sampling plans, sample collection, shipment and preparation for analysis. In: Downes FP, Ito K. 5th ed. Compendium of Methods for the Microbiological Examination of Foods. Washington DC: American Public Health Association. [Crossref]
13. Yang, X., Cheng, H.W., Chen, L., Zhao, J., Chang, H.T., Wang, X.W., Liu, H.Y., Yao, H.X., Zhang, L.X. and Wang, C.Q. (2013) A duplex SYBR Green I real-time quantitative PCR assay for detecting Escherichia coli O157: H7. Genet. Mol. Res.12(4): 4836. [Crossref]
14. Meng, J., Zhao, S., Doyle, M.P., Mitchell, S.E. and Kresovich, S. (2001) A multiplex PCR for identifying Shiga toxin producing E. coli. Lett. Appl. Microbiol., 24: 172-176. [Crossref]
15. Fujisawa, T., Sata, S., Aikawa, K., Takahashi, T., Yamai, S. and Shimada, T. (2000) Modification of sorbitol mac conkey medium containing cefixime and tellurite for isolation of Escherichia coli O157: H7 from radish sprouts. Appl. Environ. Microbiol.,66: 3117-3118. [Crossref]
16. Paton, A.W. and Paton, J.C. (1998) Detection and characterization of shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx 2, eae A, enter haemorrhagic E. coli hly A, rfb O111, and rfbO157. J. Clin. Microbiol., 36: 598- 602. [PubMed] [PMC]
17. Adam, P.S. (2006) Data analysis and reporting. In: Dorak MT, editors. Real time PCR. Abingdon: Taylor & Francis Group. pp39-59.
18. Aranda, K.R., Fagundes-Neto, U. and Scaletsky, I.C.A. (2004) Evaluation of multiplex PCR for diagnosis of infection with diarrheagenic Escherichia coli and Shigellaspp. J. Clin. Microbiol.,42: 5849-5853. [Crossref] [PubMed] [PMC]
19. Anu, P.J. (2015) Identification of Critical Control Points in Beef Processing Line with Special Reference to Enterohaemorrhagic Escherichia coli. M.V.Sc Thesis, Kerala Veterinary and Animal Sciences University, Pookode. p86.
20. Mohammadi, P., Abiri, R., Rezaei, M. and Salmanzadeh-Ahrabi, S. (2013) Isolation of shiga toxin-producing Escherichia coli from raw milk in Kermanshah, Iran. Iran. J. Microbiol., 5(3): 233-238. [PubMed] [PMC]
21. Junior, L.B.R., Oliveira, P.M., Silva, F.J.M., Gomes, A.P., Martins, M.L., Santos, M.T., Moraes, C.A. and Junior, A.S. (2014) Occurrence of shiga toxin-producing Escherichia coli (STEC) in bovine faeces, feed, water, raw milk, pasteurized milk, Minas Frescal cheese and ground beef samples collected in Minas Gerais, Brazil. Int. Food Res. J., 21(6): 2481-2486.
22. Hoffmann, S.A., Pieretti, G.G., Fiorini, A., Patussi, E.V., Cardoso, R.F. and Mikcha, J.M.G. (2014) Shiga-Toxin genes and genetic diversity of Escherichia coli isolated from pasteurized cow milk in Brazil. J. Food Sci.,79(6): 1175-1180. [Crossref] [PubMed]
23. Temelli, S., Eyigor, A. and Anar, S. (2012) Prevalence of Escherichia coli O157 in red meat and meat products determined by VIDAS ECPT and light cycler PCR. Turk.J. Vet. Anim. Sci., 36: 305-310.
24. Fantelli, K. and Stephan, R. (2001) Prevalence and characteristics of shigatoxin-producing Escherichia coli and Listeria monocytogenes strains isolated from minced meat in Switzerland. Int. J. Food Microbiol.,70(1): 63-69. [Crossref]
25. Maktabi, S., Zarei, M. and Mohammadpour, H. (2016) Isolation and molecular characterization of non-sorbitol fermenting Escherichia coli isolated from fresh ground beef Jundishapur. J. Health. Sci., 8(1): 20-24.
26. Arthur, T.M., Barkocy-Gallagher, G.A., Rivera-Betancourt, M. and Koohmaraie, M. (2002) Prevalence and characterization of non-O157 Shiga toxin-producing Escherichia coli on carcasses in commercial beef cattle processing plants. Appl. Environ. Microbiol.,68: 4847-4852. [Crossref] [PMC]
27. Rahimi, E., Kazemeini, H.R. and Salajegheh, M. (2012) Escherichia coli O157: H7/ NM prevalence in raw beef, camel, sheep, goat and water buffalo meat in Fars and Khuzestan provinces, Iran. Vet. Res. Forum., 3: 13-17.
28. Sinha, N., Kshirsagar, D.P., Brahmbhatt, M.N. and Nayak, J.B. (2015) Detection and virulence gene characterization of shiga toxigenic E. coli from buffalo meat samples. Indian J. Anim. Res., 49(5): 694-698. [Crossref]
29. Barlow, R.S., Gobius, K.S. and Desmarchelier, P. (2006) Shiga toxin producing Escherichia coli in ground beef and lamb cuts: results of a one-year study. Int. J. Food Microbiol., 111: 1-5. [Crossref] [PubMed]
30. Miko, A., Pries, K., Haby, S., Steege, K., Albrecht, N., Krause, G. and Beutin, L. (2009) Assessment of shiga toxin-producing Escherichia coli isolates from wildlife meat as potential pathogens for humans. Appl. Environ. Microbiol., 75: 6462-6470. [Crossref] [PubMed] [PMC]
31. Ojo, O.E., Ajuwape, A.T.P., Otesile, E.B., Owoade, A.A., Oyekunle, M.A. and Adetosoye, A.I. (2010) Potentially zoonotic Shiga toxin-producing Escherichia coliserogroups in the faeces and meat of food-producing animals in Ibadan, Nigeria. Int. J. Food Microbiol.,142: 214-221. [Crossref] [PubMed]
32. Momtaz, H., Dehkordi, F.S., Rahimi, E., Ezadi, H. and Arab, R. (2013) Incidence of shiga toxin-producing Escherichia coliserogroups in ruminant's meat. Meat Sci.,95(2): 381-388. [Crossref] [PubMed]
33. Rashid, M., Kotwal, S.K., Malik, M.A. and Singh, M. (2013) Prevalence, genetic profile of virulence determinants and multidrug resistance of Escherichia coli isolates from foods of animal origin. Vet. World, 6: 139-142. [Crossref]
34. Ranjbar, R., Masoudimanesh, M., Dehkordi, F.S., Jonaidi-Jafari, N. and Rahimi, E. (2017) Shiga (Vero)-toxin producing Escherichia coli isolated from the hospital foods; Virulence factors, o-serogroups and antimicrobial resistance properties. Antimicrob Resist Infect Ctrol.,6(1): 4-15. [Crossref] [PubMed] [PMC]
35. Ibekwe, A.M., Watt, P.M., Grieve, C.M., Sharma, V.K. and Lyons, S.R. (2002) Multiplex fluorogenic real time PCR for detection and quantification of Escherichia coli O157: H7 in dairy waste water wetlands. Appl. Environ. Microbiol.,68: 4853-4862. [Crossref] [PMC]
36. Bellin, T., Pulz, M., Matussek, A., Hempen, H.G. and Gunzer, F. (2001) Rapid detection of enterohemorrhagic Escherichia coli by real-time PCR with fluorescent hybridization probes. J. Clin. Microbiol., 39: 370-374. [Crossref] [PubMed] [PMC]
37. Hara-Kudo, Y., Konishi, N., Ohtsuka, K., Iwabuchi, K., Kikuchi, R., Isobe, J., Yamazaki, T., Suzuki, F., Nagai, Y., Yamada, H. and Tanouchi, A. (2016) An interlaboratory study on efficient detection of Shiga toxin-producing Escherichia coli O26, O103, O111, O121, O145, and O157 in food using real-time PCR assay and chromogenic agar. Int. J. Food Microbiol.,230: 81-88. [Crossref] [PubMed]
38. Fukushima, H. and Seki, R. (2004) High numbers of Shiga toxin-producing Escherichia coli found in bovine faeces collected at slaughter in Japan. FEMS Microbiol. Lett.,238(1): 189-197. [PubMed]39. Bono, J.L., Keen, J.E. and Miller, L.C. (2004) Evaluation of a real-time PCR kit for detecting Escherichia coli O157 in bovine fecal samples. Appl. Environ. Microbiol., 70: 1855-1857. [Crossref] [PubMed] [PMC]