Isolation and Molecular Detection of some Virulence Genes (eaeA, bfpA) of Escherichia coli from Meat, Milk and Cheese.
Aim: The study was undertaken to isolate diarrheagenic E. coli from meat and various milk products, and detection of some virulence genes using Polymerase chain reaction (PCR) .
Materials and Methods: Altogether 300 meat and various milk products samples consisting of raw meat (100), cheese (100), milk (100), were collected from milk vendors, retail shops located in Thi-Qar, under aseptic precautions. For the enrichment of the organism from the collected samples, MacConkey broth was used and inoculation was carried out on MacConkey agar and EMB agar. Later, to confirm the isolates, various biochemical tests such as IMVC tests, Urease test were performed. Finally the E. coli isolates were screened for the presence of virulence associated genes by PCR .
Results: The prevalence of E. coli was observed in 18.33 % of the samples comprising of meat (25%), cheese (13%), milk (17%). In this study, out of 55 E. coli isolates, 18 isolates (32.72%) were positive for eaeA gene, of which 6 (33.3%) isolates from meat, while 5 (27.7%) isolates from milk and 7 (38.88%) isolates from cheese,while 8 isolates (14.5%) were positive for bfpA gene: of which 2(25%) were from each of meat, and 2(25%) for milk, and 4(50%) from cheese.
2- Albert MJ, Faruque SM, Faruque AS, et al. Controlled study of Escherichia coli diarrheal infections in Bangladeshi children. J Clin Microbiol. 1995;33(4):973-977.
3- Al-Gallasa, N., R. Ben Aissa Ra, T. Attia Annabib, O. Bahric and A. Boudabousd,. Isolation and characterization of shiga toxin-producing Escherichia coli from meat and dairy products. Food Microbial., 2002; 19: 389-398.
4- Ali AA, Abdelgadir SW . Incidence of Escherichia coli in raw cow's milk in Khartoum state Br J dairy Sci 2011;2(1):23-6.
5- 229- Gwida MM, EL-Gohary FA . Zoonotic bacterial pathogenic isolated from raw milk with special reference to Escherichia coli and Staphylococcus aureus in Dakahlia . Governorate , Egypt 2013;2(4):705.
6- Alikhani MY, Hashemi SH, Aslani MM, Farajnia S. Prevalence and antibiotic resistance patterns of diarrheagenic Escherichia coli isolated from adolescents and adults in Hamedan, West Iran. Iran J Microbiol. 2013;5(1):42-47.
7- Alikhani MY, Mirsalehian A, Aslani MM. Detection of typical and atypical enteropathogenic Escherichia coli (EPEC) in Iranian children with and without diarrhea. J Med Microbiol. 2006;55(Pt 9):1159-1163. doi:10.1099/jmm.0.46539-0.
8- Amisano G, Fornasero S, Migliaretti G, Caramello S, Tarasco V, Savino F. Diarrheagenic Escherichia coli in acute gastroenteritis in infants in North-West Italy. New Microbiol. 2011;34:45–51.
9- Balcha, E., Kumar, A. and Tassew H. Evaluation of Safety of Beef sold in and around Mekellle with speicla reference to Enterohemorrhagic Escherichia coli O157:H7. Global Veterinaria (2014); 12(4): 569-572 .
10- Betts R, Blackburn CW . Detecting pathogens in food. In: Foodborne pathogens: hazards, risk analysis and control, 2nd edn. Edited by: Blackburn CW, McClure PJ. Woodhead Publishing, Oxford, UK. pp. 2009; 17–65.
11- Bolton, D.J.; Doherty A.M.; and Sheridan J.J. Beef HACCP intervention and non intervention systems.int. J. Microbiol. 2001; 66:119-129.
12- Canizalez-Roman A, Gonzalez-Nuriez E, E. Vidal J., Flores-Villaserior H., Leon-Sicairos N. Prevalence and antibiotic resistance profiles of diarrheagenic Escherichia coli strains isolated from food items in northwestern Mexico. 2013
13- De Buyser , M.L., Dufour, B., Maire, M., Lafarge, V., Implication of milk and milk products in food-borne diseases in France and in different industerilised countries . Int. J. Food microbial . 2001; 67(1-2), 1-17.
14- Donnenberg, M.S., Kaper, J.B., Enteropathogenic Escherichia coli . Infect. Immun. 1992; 60(10), 3953-3961.
15- 238- Vallance , B.A., Finlay, B.B., Exploitation of host cells by enteropathogenic Escherichia coli . Proc. Natl. Acad. Sci. 2000; 97(16), 8799-8806.
16- Dontorou, C., C. Papadopoulou, G. Filioussis, V. Economou and I. Apostolou et al., Isolation of Escherichia coli O157:H7 from foods in Greece. Int. J. Food Microbiol., 2003; 82: 273-279.
17- Fadel, H. M. and Ismail, J. Prevalence and Significance of Staphylococcus aureus and Enterobacteri- aceae species in Selected Dairy Products and Handlers. Int. J. Dairy science. 2009; 4(3): 100-108.
18- FAO (Food and Agriculture Organization), Food Outlook, Global Market Analysis 17 October 2011, pp. 43–44.
19- Farzan, R., Rahimi, E. and Momtaz, H. Virulence properties of Shiga Toxin-Producing Escherichia coli isolated from Iranian raw milk and dairy products. Slov. Vet . Res. 2012; 49(4): 159-66.
20- Feng P . Rapid methods for the detection of foodborne pathogens: current and next-generation technologies. In: Food microbiology, fundamentals and frontiers, 3rd edn. Edited by: Doyle MP, Beuchat LR. ASM Press, Washington, D.C. pp. 2007; 911–934.
21- Gwida MM, EL-Gohary FA. Zoonotic bacterial pathogenic isolated from raw milk with special reference to Escherichia coli and Staphylococcus aureus in Dakahlia . Governorate , Egypt 2013; 2(4): 705.
22- Heuvelink, A.E., F.L.A.M. Van Den Biggelaar, J.T.M. Zwartkruis-Nahuis, R.G. Herbes and R. Huyben et al., Occurrence of Verocytotoxin-Producing Escherichia coli O157 on Dutch Dairy Farms. J. Clin. Microbial., 1998; 36: 3480-3487.
23- Heuvelink, A.E., J.T. Zwartkruis-Nahuis, R.R. Beumer and E. De Boer, Occurrence and survival of verocytotoxin-producing Escherichia coli O157 in meats obtained from retail outlets in The Netherlands. J. Food Prot., 1999; 62: 1115-1122.
24- Hussein, H.S. and T. Sakuma,. Invited review: Prevalence of Shiga toxin-producing Escherichia coli in dairy cattle and their products. J. Dairy Sci., 2005; 88: 450-465.
25- Jantzen MM, Navas J, Corujo A, Moreno R, Lopez V, Martinez-Suarez JV . Review. Speciﬁ c detection of Listeria monocytogenes in foods using commercial methods: from chromogenic media to real-time PCR. Span J Agric Res 2006a ; 4:235–247.
26- Jasson V, Jacxsens L, Luning P, Rajkovic A, Uyttendaele M . Review. Alternative microbial methods: An overview and selection criteria. Food Microbiol 2010; 27:710–730.
27- Jelacic , J.K., Damrow , T., Chen , G.S., Jelacic, S., Bielaszewska, M., ciol, M., Carvalho, H.M., Melton-Celsa, A.R., O'Brien, A.D., Tarr, P.I., Shiga toxin-producing Escherichia coli in Montana: bacterial genotypes and clinical profiles . J. Infect. Dis. 2003; 188, 719-729.
28- Kaper, J.B., Defining EPEC. Review of Microbiology 1996; 27, 130-133.
29- Kivaria FM , Noordhuizen JPTM , Japaga AM. Evaluation of the hygienic quality and associated public health . hazards of raw milk marketed by smallholder dairy producers in the Dares Salaam region , Tanzania . Trop Anim Health prod 2006;38(3):185-94.
30- Koneman, E.W; Allen, S.D; Janda, W.M; Schreckenberger, P.C and Winn, W.C.J. Color Atlas and Textbook Of Diagnostic Microbiology. (4th) ed. J.B.Lippincott Company. Philadelphia. 1992
31- Kumar, R., Surendran, P. K. and Thampuran, N. Evaluation of culture, ELISA and PCR assays for the detection of Salmonella in seafood. Letters in Applied Microbiol. 2008; 46(2): 221-226.
32- Levine, M.M., . Escherichia coli that cause diarrhea : enterotoxigenic , enteropathogenic , enteroinvasive , enterohemorrhagic ,and enteroadherent. J. Infect. Dis. 1987; 155(3), 377-389.
33- Mohammed, O., Shimelis, D., Admasu P.and Feyera, T. Prevalence and Antimicrobial Susceptibility Pattern of E. coli isolates from raw meat samples obtained from Abattoirs in Dire Dawa City, Eastern Ethiopia. International Journal of Microbiological Research 2014; 5 (1): 35-39 .
34- Murphy SC, Boor KJ. Trouble –Shooting sources and causes of high bacterial counts in raw milk . Dairy food Environ Sani . 2000;606-11.
35- Nguyen, R.N., Taylor, L.S., Tauschek, M., Robins-Brown, R.M., Atypical enteropathogenic Escherichia coli infection and prolonged diarrhea in children. Emerging Infectious Diseases 2006; 12, 597-603.
36- Nunes Mdo, R., Magalhaes, P.P., Macedo Ada, S., Franco, R.T., Penna, F.J., Mendes, E.N., Attaching and effacing Escherichia coli and Shiga toxin-producing E. coli in children with acute diarrhea and controls in Teresina/PI, Brazil. Transactions of the Royal Society of Tropical Medicine and Hygiene 2012; 106, 43-47.
37- Oliver, S. P., Jayarao, B. M. and Almeida, R. A. Foodborne pathogens in milk and the dairy farm environment: food safety and public health implications. Food borne Pathogenic Dis. 2005; 2(2): 115-129.
38- Paneto, B. R., Schocken-Iturrino, R. P., Macedo, C., Santo,E. and Marin, J. M. Occurrence of toxigenic Escherichia coli in raw milk cheese in Brazil. Arq. Bras . Med. Vet. Zootec. 2007; 59: 508-512.
39- Rajeev K , Amit P . Detection of E. coli and Staphylococcus in milk and milk products in and around pantnagar .Vet World 2010;3(11):495-6.
40- Ryser ET , Public health concerns . In : Marth EH, Steele JL , editors . Applied dairy microbiology . New York : Marcel Dekker , Inc ;1998 . P. 263 – 403.
41- Samira Badri, Ingrid Filliol, Isabelle Carle, Mohammed Hassar, Aziz Fassouane, Nozha Cohen. Prevalence of virulence genes in Escherichia coli isolated from food in Casablanca (Morocco). 2008.
42- Singh, P. and Prakash, A. Isolation of Escherichia coli, Staphylococcus aureus and Listeria monocytogenes from milk products sold under market conditions at agra region. Acta. Agriculturae Slovenica. 2008; 92(1): 83–88.
43- Srinu, B., Vijaya Kumar, A., Shashi Kumar, M., Narayana, B. V. L. and Madhava Rao T. Assessment of microbiological quality and associated health risks of raw milk sold in and around Hyderabad city. Int. J. Pharm. Bio. Sci. 2012; 3(4): 609-614.
44- Taye, M, Berhanu. T, Berhanu .Y, Tamiru,F. and Terefe. D: Studyon Carcass Contaminating Escherichia coli in Apparently Healthy SlaughteredCattle in Haramaya University Slaughter House with Special Emphasison Escherichia coliO157:H7. Ethiopia. J Veterinar Sci Technol. 2013; 4: 132.
45- Torkar, K. G. and Teger S. G. The Microbiological quality of raw milk after introducting the two day's milk collecting system. Acta Agri. Slovenica. 2008; 92(1): 61–74.
46- Trabulsi LR, Keller R, Gomes TAT. Typical and atypical enteropathogenic Escherichia coli. Emerg Infect Dis 2002; 8: 508-513.
47- Usein CR, Tatu-Chitoiu D, Ciontea S, Condei M. Damian M. Escherichia coli pathotypes associated with diarrhea in Romanian children younger than 5 years of age. Jpn J Infect Dis. 2009;62(4):289-293.
48- Vallance, B.A., Finlay, B.B., Exploitation of host cells by enteropathogenic Escherichia coli . Proc. Natl. Acad. Sci. 2000; 97(16), 8799-8806.
49- Yatsuyanagi, J., Saito, S., Miyajima, Y., Amano, K., Enomoto, K.,. Characterization of atypical enteropathogenic Escherichia coli strains harboring the astA gene that were associated with a waterborne outbreak of diarrhea in Japan. Journal of Clinical Microbiology 2003; 41, 2033-2039.