Aim: Shiga toxin-producing Escherichia coli (STEC) represent a severe public health issue worldwide, causing life-threatening diseases in the human gastrointestinal tract. This study aimed to determine the occurrence of virulent and antibiotic-resistant STEC in retail meat and milk products and human stool samples and to characterize the genes encoding for virulence and antibiotic resistance among the identified STEC isolates.

Materials and Methods: A total of 260 food samples were randomly collected from retail markets in different localities of El Giza Governorate, Egypt. 50 stool specimens were obtained from children that had diarrhea at Embaba Fever Hospital. All collected samples were initially subjected to bacteriological examination and serotyping, and then subsequently, the isolates were exposed to polymerase chain reaction application and sequencing for the identification of the virulence-related genes. Finally, the virulent STEC isolates were tested for antibiotic susceptibility.

Results: Serotyping of the 76 biochemically identified isolates showed that 18 were STEC with a predominance of non- O157 (16) while 2 O157:K-serotype was detected only in one food and one human isolate. Molecular identification of the virulence genes illustrated that the minced meat showed the highest prevalence of STEC (8%) as compared to the other food products. In the humans, the O157 was the only serotype that expresses the Shiga toxin-associated gene (eaeA). Antibiotic susceptibility test displayed that 13 of the 17 food and human isolates (76.47%) were resistant to cephalothin (KF30). 9 of the 13 cephalothin-resistant isolates harbor the β lactamase (bla_TEM)-resistant gene. All isolates were sensitive to chloramphenicol, ciprofloxacin, amikacin, and gentamicin. DNA sequencing and phylogenetic analysis of the stx2-positive minced meat isolate revealed a high genetic relatedness with beef minced meat from the USA and Australia.

Conclusion: This study showed the predominance of non-O157 among the identified isolates. Minced meat showed the highest prevalence of STEC as compared to the other food products, and this work illustrates the necessity to consider the food products as a potential source of the non-O157 STEC serotypes. DNA sequencing and phylogenetic analysis revealed a high genetic relatedness with beef minced meat from the USA and Australia. This highlights the high probability of worldwide spread of such serotypes, signifying the importance of the one world concept.

Keywords: antibiotic resistance bacteria, cheese, DNA sequencing, human stool, meat, polymerase chain reaction, serotyping, shiga toxin-producing Escherichia coli.

1. Malik, K. and Memona, H. (2010) Molecular and immunological studies of pathogenic Escherichia coli in meat samples collected from different localities of Lahore. Int. J. Cell Sci. Mol. B, 1: 218-224.

2. Jafari, A., Alani, M. and Bouzari, S. (2012) Escherichia coli: A brief review of diarrheagenic pathotypes and their role in diarrheal diseases in Iran. Int. J. Manage., 4(3): 102-117.

3. Etcheverria, A.I. and Padola, N.L. (2013) Shiga toxin-producing Escherichia coli factors involved in virulence and cattle colonization. Virulence J., 4: 366-372. [Crossref] [PubMed] [PMC]

4. Rahal, E.A., Fadlallah, S.M., Nassar, F.J., Kazzi, N. and Matar, G.M. (2015) Approaches to treatment of emerging Shiga toxin-producing Escherichia coli infections highlighting the O104:H4 serotype. Front Cell. Infect. Microbiol., 5: 24. [Crossref] [PubMed] [PMC]

5. Magwedere, K., Dang, A., Mills, E.W., Cutter, C.N., Roberts, E.L. and De Broy, C. (2013) Incidence of Shiga toxin-producing Escherichia coli strains in beef, pork, chicken, deer, boar, bison, and rabbit retail meat. J. Vet. Diagn. Invest., 25: 254-258. [Crossref]

6. Kawano, K., Ono, H., Iwashita, O., Kurogi, M., Haga, T., Maeda, K. and Goto, Y. (2012) Stx genotype and molecular epidemiological analyses of Shiga toxin-producing Escherichia coli O157: H7/H in human and cattle isolates. Eur. J. Clin. Microbiol. Infect. Dis., 31: 119-127. [Crossref] [PubMed]

7. Slanec, T., Fruth, A., Creuzburg, K. and Schmidt, H. (2009) Molecular analysis of virulence profiles and Shiga toxin genes in food-borne Shiga toxin-producing Escherichia coli. Appl. Environ. Microbiol., 75: 6187-6197. [Crossref] [PubMed] [PMC]

8. Hammerum, A.M. and Heuer, O.E. (2009), Human health hazards from antimicrobial resistant Escherichia coli of animal origin. Clin. Infect. D, 48: 916-921. [Crossref] [PubMed]

9. Alvarez-Fern-Andez, E., Cancelo, A., Diaz-Vega, C., Capita, R. and Alonso-Calleja, C. (2013) Antimicrobial resistance in E. coli isolates from conventionally and organically reared poultry: A comparison of agar disc diffusion and sensitivity test gram-negative methods. Food Control. 30: 227-234. [Crossref]

10. Bush, K. and Jacoby, G.A. (2010) Updated functional classification of beta-lactamases. Antimicrob. Agents Chemother., 54: 969-976. [Crossref] [PubMed] [PMC]

11. Sharma, J., Sharma, M. and Ray, P. (2010) Detection of TEM and SHV genes in Escherichia coli and Klebsiella pneumoniae isolates in a tertiary care hospital from Indian. J. Med. Res., 132: 332-336.

12. Chandra, V. and Goswami, P.N. (2014) Detection of TEM and SHV genes in extended spectrum beta lactamase (ESBL) producing E. colI and Klebsiella pneumonia isolated from a tertiary care cancer hospital. Natl. J. Med. Res., 4: 201-204.

13. Tuckman, M., Petersen, P.J., Howe, A.Y., Orlowski, M., Mullen, S., Chan, K., Bradford, P.A. and Jones, C.H. (2007) Occurrence of tetracycline resistance genes among Escherichia coli isolates from the phase 3 clinical trials for tetracyclines. Antimicrob Agents Chemother., 51: 3205-3211. [Crossref] [PubMed] [PMC]

14. Zhang, T., Wang, C.G., Lv, J.C., Wang, R.S. and Zhong, X.H. (2012) Survey on tetracycline resistance and antibiotic-resistant genotype of avian Escherichia coli in North China. Poult. Sci., 91: 2774-2777. [Crossref] [PubMed]

15. Slama, K.B., Jouini, A., Sallem, R.B., Somalo, S., Saenz, Y. and Estepa, V. (2010) Prevalence of broad-spectrum cephalosporin-resistant Escherichia coli isolates in food samples in Tunisia, and characterization of integrons and antimicrobial resistance mechanisms implicated. Int. J. Food Microbiol., 137: 281-286. [Crossref]

16. Zhang, X.Y., Ding, L.J. and Yue, J. (2009) Occurrence and characteristics of class 1 and class 2 integrons in resistant Escherichia coli isolates from animals and farm workers in northeastern China. Microb. Drug Resist., 15: 223-228. [Crossref] [PubMed]

17. de Boer, E. and Heuvelink, A.E. (2000) Methods for the detection and isolation of Shiga toxin producing Escherichia coli. J. Appl. Microbiol., 88: 133-143.

18. Edwards, P.R. and Ewing, W.H. (1972) Identification of Enterobacteriaceae. 3rd ed. Burgess Publ. Co., Minneapolis, Minnesota.

19. Clinical and Laboratory Standards Institute, CLSI. (2014) Performance Standards for Antimicrobial Susceptibility Testing, 24th Informational Supplement, Document M100-S24. CLSI, Wayne, P.A., USA.

20. Belanger, S.D., Boissinot, M., Menard, C., Picard, F.J. and Bergeron, M.G. (2002) Rapid detection of Shiga toxin-producing bacteria in feces by multiplex PCR with molecular beacons on the smart cycler. J. Clin. Microbiol., 40: 1436-1440. [Crossref]

21. Dipineto, L., Santaniell, O.A., Fontanella, M., Lagos, K., Fioretti, A. and Menna, L.F. (2006) Presence of Shiga toxin-producing Escherichia coli O157:H7 in living layer hens. Lett. Appl. Microbiol., 43: 293-295. [Crossref] [PubMed]

22. Bisi-Johnson, M.A., Obi, C.L., Vasaikar, S.D., Bab, A.K.A. and Hattori, T. (2011) Molecular basis of virulence in clinical isolates of Escherichia coli and Salmonella species from a tertiary hospital in the Eastern Cape, South Africa. Gut Pathog., 3: 3-9. [Crossref]

23. Piva, I.C., Pereira, A.L., Ferraz, L.R., Silva, R.S.N., Vieira, A.C., Blanco, J.E., Blanco, M., Blanco, J. and Giugliano, L.G. (2003) Virulence markers of enteroaggregative Escherichia coli isolated from children and adults with diarrhea in Brasilia, Brazil. J. Microbiol., 41: 1827-1832. [Crossref]

24. Colom, K., Perez, J., Alons, O.R., Fernandez-Aranguiz, A. and Cisterna, R. (2003) Simple and reliable multiplex PCR assay for detection of blatem, blaSHV and blaOXA-1 genes in Enterobacteriaceae. FEMS Microbiol. Lett., 223: 147-151. [Crossref]

25. Walker, R.A., Lindsay, E., Woodward, M.J., Ward, L.R. and Threlfall, E.J. (2001) Variation in clonality and antibiotic-resistance genes among multi-resistant Salmonella enterica serotype Typhimurium phage-type U302 (MR U302) from humans, animals, and foods. Microbiol. Res., 7: 13-21.

26. Randall, L.P., Cooles, S.W., Osborn, M.K., Piddock, L.J.V. and Woodward, M.J. (2004) Antibiotic resistance genes, integrons and multiple antibiotic resistance in thirty-five serotypes of Salmonella enterica isolated from humans and animals in the UK. J. Antimicrob. Chemother., 53: 208-216. [Crossref]

27. Kudakwashe, M., Huu, A.D., Edward, W.M., Catherine, N.C., Elisabeth, L.R. and Chitrita, D. (2013) Incidence of Shiga toxin-producing Escherichia coli strains in beef, pork, chicken, deer, boar, bison, and rabbit retail meat. J. Vet. Diagn. Invest., 25: 254-258. [Crossref] [PubMed]

28. Llorente, P., Laura, B., Kinue, I., Maria, V.R. and Adriana, B. (2014) Characterization of Shiga toxin-producing Escherichia coli isolated from ground beef collected in different socioeconomic strata markets in Buenos Aires, Argentina. Biomed. Res. Int., 2014: 9. [Crossref] [PubMed] [PMC]

29. Loukiadis, E., Callon, H., Mazuy-Cruchaudet, C., Vallet, V., Bidaud, C., Ferre, F., Giuliani, L., Bouteiller, L., Pihier, N. and Danan, C. (2012) Surveillance des E. coli producers de shigatoxines (STEC) dans les denrees alimentaires en France (2005-2011). Bull Epidemiol Hebd (Paris) 55:3-9.

30. Hessain, A.M., Al-Arfaj, A.A., Zakri, A.M., El-Jakee, J.K., Al-Zogibi, O.G. and Hemeg, H.A. (2015) Molecular characterization of Escherichia coli O157:H7 recovered from meat and meat products relevant to human health in Riyadh, Saudi Arabia. Saudi J. Biol. Sci., 22: 725-729. [Crossref]

31. Mohammed, M.A.M. (2012) Molecular characterization of diarrheagenic Escherichia coli isolated from meat products sold at Mansoura city, Egypt. Food Control., 25: 159-164. [Crossref]

32. Bardasi, L., Taddei, R., Nocera, L., Ricchi, M. and Merialdi, G. (2015) Shiga-toxin-producing Escherichia coli in meat and vegetable products in Emilia Romagna Region, years 2012-2013. Ital. J. Food Saf., 4: 33-35. [Crossref]

33. Agaogu, S., Yavuz, M., Berktas, M. and Guiducouglu, H. (2000) Detection of E. coli O157:H7 in retail ground Beef, raw ground beef patties and raw meat balls solid in van. Eur. J. Mark., 5(2): 73-75.

34. Sethulekshmi, C., Latha, C. and Sunil, B. (2016) Occurrence of enterohaemorrhagic E. coli in raw meat samples in Kerala. Int. J. Adv. Res. Biol. Sci., 3: 220-222.

35. Todaro, A.A. and Omar, O.H. (2013) History, processing and quality enhancement of traditional Egyptian kareish cheese: A review. J. Food Sci. Technol., 1: 1-6.

36. Centers for Disease Control and Prevention, CDC. (2007) Escherichia coli O157:H7 infection associated with drinking raw milk-Washington and Oregon, November-December 2005. Morb. Mortal. Wkly. Rep., 56: 165-167. [PubMed]

37. Abd El-Atty, N.S. and Meshref, A.M.S. (2007) Prevalence of Salmonella and E. coli O157 in Some Foods, Beni-Suef Veterinary Medical Journal, 5th Scientific Conference. p73-78.

38. Zidan, S.A., Tarabees, R. and Hassanin, Z.H. (2014) Comparison of RIDA^®QUICK verotoxin/O157 test, verocytotoxicty assay and PCR in detection of Shiga toxins in cultures of human stool, animal faeces and meat products. Assiut Vet. Med. J., 60: 142.

39. Silveira, N.T., Silva, N., Contreras, C., Mitagusku, L., Baccin, M.L., Koono, E. and Beraquet, Y. (1999) Occurrence of Escherichia coli O157:H7 in hamburgers in Brazil. J. Food Prot., 62: 1333-1335. [Crossref] [PubMed]

40. Naidu, K.G., Goud, N.R., Gaddad, S.M. and Shivannavar, C.T. (2011), Detection of Shiga toxin genes (stx1 and stx2) and molecular characterization of Shiga toxigenic isolated from diverse sources in Gulbarga region, India. Pharmacophore, 2: 253-265.

41. Dambrosio, A., Lorusso, V., Quaglia, N.C., Parisi, A., La Salandra, G., Virgilio, S., Mula, G., Lucifora, G., Celano, V. and Normanno, G. (2007) Escherichia coli O26 in minced beef: Prevalence, characterization and antimicrobial resistance pattern. Int. J. Food. Microbiol., 118: 218-222. [Crossref] [PubMed]

42. Elhadidy, M. and Mohammed, M.A. (2012) Shiga toxin-producing Escherichia coli from raw milk cheese in Egypt: Prevalence, molecular characterization and survival to stress conditions. Lett. Appl. Microbiol., 56: 120-127. [Crossref] [PubMed]

43. Abd El-Tawab, A.A., El-Hofy, I.F., Nada, M.S. and Deiab, A.A.R. (2015) Detection of virulence genes of enterohaemorrhagic E. coli isolated from some meat products by polymerase chain reaction. Benha Vet. Med. J., 29: 45-52.

44. Khatib, A., Olama, Z. and Khawaja, G. (2015) Shiga toxin-producing E. coli (STEC) associated with Lebanese fresh produce. Int. J. Curr. Microbiol. Appl. Sci., 4: 481-496.

45. Welinder-Olsson, C. and Kaijser, B. (2005) Review: Enterohemorrhagic Escherichia coli (EHEC). Scand. J. Infect. Dis., 37: 405-416. [Crossref] [PubMed]

46. Bettelheim, K.A. (2007) The non-O157 Shiga-toxigenic (verocytotoxigenic) Escherichia coli; under-rated pathogens. Crit. Rev. Microbiol., 33: 67-87. [Crossref] [PubMed]

47. Smith, J.L., Fratamico, P.M. and Gunther, N.W. (2014) Shigatoxin-producing Escherichia coli. In: Sima, S. and Geoffrey, M.G., editor. Advances in Applied Microbiology. Academic Press, Burlington, US. p14597.

48. Lim, K., Yasin, R., Yeo, C., Puthucheary, S. and Thong, K. (2009) Characterization of multi-drug resistant ESBL-producing Escherichia coli isolates from hospitals in Malaysia. J. Biomed. Biotechnol., 2009: 10. [Crossref] [PubMed] [PMC]

49. Parveen, S., Murphree, R.L., Edmiston, L., Kaspar, C.W., Portier, K.M. and Tamplin, M.L. (1997) Association of multiple antibiotic resistance profiles with point and nonpoint sources of Escherichia coli in Apalachicola bay. Appl. Environ. Microbiol., 63: 2607-2612. [PubMed] [PMC]

50. Schlegelova, J., Babak, V., Klimova, E., Lukasova, J., Navritalova, P., Sustackova, A., Sediva, I. and Rysanek, D. (2002) Prevalence of and resistance to anti-microbial drugs in selected microbial species isolated from bulk milk samples. J. Vet. Med. Educ., 49: 216-225.

51. Schroeder, C.M., Zhao, C., DebRoy, C., Torcolini, J. and Zhao, S., White, D.G., Wagner, D.D., McDermott, P.F., Walker, R.D. and Meng, J. (2002) Antimicrobial resistance Escherichia coli O157 isolated from humans, cattle, swine, and food. Appl. Environ. Microbiol., 68: 576-581. [Crossref] [PubMed] [PMC]

52. Adenaike, O., Olonitola, O.S., Ameh, J.B. and Whong, C.M.Z. (2013) Incidence of extended spectrum β-lactamase producing bacteria and multidrug resistance strains from processed meat 'Suya' sold in a university community. Int. J. Eng. Sci., 2: 12.

53. Colello, R., Etcheverria, A.I., Conza, J.A.D., Gutkind, G.O. and Padola, N.L. (2015) Antibiotic resistance and integrons in Shiga toxin-producing Escherichia coli (STEC). Braz. J. Microbiol., 46: 1-5. [Crossref] [PubMed] [PMC]

54. Jiao, S.C., Fami, R.M.L., Pedernal, V.A.D. and Cabrera, E.C. (2007) Prevalence of multiple drug-resistant Escherichia coli from chicken, Pig and Nile Tilapia (Oreochromis nilotica) intestines sold in wet markets in Manila and the conjugative transferability of the resistance antimicrobial resistant Escherichia coli from livestock. Philipp. JAS, 90: 64-70.

55. Jeyasanta, K.I., Aiyamperumal, V. and Patterson, J. (2012) Prevalence of antibiotic resistant Escherichia coli in sea foods of tuticorin coast, southeastern India. Adv. Biomed. Res., 6: 70-77.

56. Kehrenberg, C., Boudewijn, C., Haesebrouck, F., de Kruif, A. and Schwarz, S. (2005) Novel spectinomycin/streptomycin resistance gene, aadA14, from Pasteurella multocida. Antimicrob Agents Chemother., 49: 3046-3049. [Crossref] [PubMed] [PMC]

57. Sharada, R., Wilfred, R.S. and Thiyageerswaran, M. (2010) Isolation, characterization and antibiotic resistance pattern of Escherichia coli isolated from poultry. Am. Eurasian J. Sci. Res., 5: 18-22.