Escherichia coli is a Gram-negative, nonsporulating, rod shaped, flagellated, and facultative anaerobic bacteria belonging to the Enterobacteriaceae family (Bavaro, 2012), which considered a reliable indicator of contamination by manure, soil and contaminated water (Todar, 2008). Most E. coli are harmless, but some are known to be pathogenic bacteria, causing severe intestinal and extra intestinal diseases in man (Kaper et al., 2004). Based on its virulence, this species is classified as enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), attaching and effacing E. coli (AEEC) and Shiga toxinproducing E. coli (STEC) (Bavaro, 2012). The broad group of E. coli are known as enterohemorrhagic Escherichia coli (EHEC), including E. coli O157:H7 and non-O157 (Louie et al., 1993). EHEC refers to a subset of Shiga toxin-producing Escherichia coli (STEC) strains found to cause human and sometimes animal disease (Terrance et al., 2002). Shiga toxin-producing Escherichia coli (STEC) present two kinds of interactions with animals: (i) intestinal disease in newborn and (ii) carriage by healthy animals as a source of infection to humans (Paton and Paton, 1998b).

Cattle are considered the primary reservoir of both O157:H7 and non-O157 STEC bacteria (Terrance et al., 2002). Cattle frequently excrete the bacteria in their feces (Molina et al., 2003). Transmission of this food-borne pathogens occur through consumption of under cooked meat, unpasteurized dairy products, vegetables or water contaminated by ruminant feces. Contact with infected animal or human has also been documented (Jamshidi, 2008). Raw milk is a well-known good medium that supports the growth and multiplication of many kinds of microorganisms due to its complex biochemical composition and high water activity (Oliver et al., 2005). Many microorganisms can get access to raw milk and among these are Escherichia coli, which is an environmental pathogen found in the immediate surroundings of the cow such as the soil, grass, manure and the bedding of housed cows. It is therefore easy for the organism to be found in the udder of cow thereby gaining entrance to the milk. Majority of cases STEC outbreaks were associated with consumption of raw seafood products, traditional dairy products, unpasteurized milk, contaminated food with pollution sources such as feces, contaminated water, contaminated equipments, infected water and even infected chief, fast-food, contaminated plants’ food and finally raw or even undercooked foods (Madic et al., 2011). It produces toxins that destroy cell membrane and can directly damage milk-producing tissues which can lead to bovine mastitis (Jones et al., 1998). The presence of bacteria in milk has many undesirable effects on the quality and safety of milk and its products (Gruetzmacher and Bradle 1999). Milk contaminated by high levels of bacteria usually becomes unsuitable for further processing (Nanu et al., 2007). Therefore, insufficient heat-treatment of raw milk forms a potential infection risk (Betts, 2000), and the processing conditions for different milk products are very important, for the risk of survival of the bacterium.

Martin et al. (1986) reported two cases of hemolytic uraemic syndrome which provide evidence that raw milk may be a vehicle of transmission of E. coli O157: H7, both affected person consumed raw milk. Recovery of E. coli from food is an indicative of possible presence of enteropathogenic and/or toxigenic micro-organism which could constitute a public health hazard. They are linked to development of hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS) (Hussein, 2003) and thrombotic thrombocytopenic purpura (TTP), which requires hospitalization and intensive care (Riley,1983). STEC O157:H7 strains were first isolated from cattle in Argentina in 1977, although the strains were identified as such 10 years later (Bonardi, 1999).

Shiga toxin-producing Escherichia coli (STEC), also called Verotoxin-producing E. coli (VTEC), are a subgroup of Escherichia coli capable of producing potent toxins called two phage-encoded cytotoxins called Shiga toxin (Stx1, Stx2), Shiga toxin 1 (Ludwig et al., 2001) is 98% homologous to the Stx produced by Shigella dysenteriae type 1, while Stx2 is about 60% homologous with Stx1 and is antigenically different (Nataro & Kaper, 1998; Paton & Paton, 1998), and may also possess additional putative virulence factors such as Intimin which is responsible for intimate attachment of STEC to the intestinal epithelial cells, causing attaching and effacing (A/E) lesions in the intestinal mucosa (Paton&Paton, 1998. Karmali, 1989) and the plasmid-encoded enterohaemolysin or enterohaemorrhagic E. coli haemolysin (ehly) (Law, 2000). According to Nataro and Kaper, 1998, in addition to Stx, typical EHEC strains carry on their chromosomes the locus for enterocyte effacement (LEE), a large pathogenicity island that is shared with enteropathogenic E. coli (EPEC). The LEE is responsible for attaching and effacing (A/E) lesions on enterocytes. This pathotype is a major cause of gastroenteritis that may be complicated by hemorrhagic colitis (HC) or the hemolytic uremic syndrome (HUS), which is the main cause of acute renal failure in children (Nataro and Kaper, 1998). But if we administer antimicrobials early in the course of infection, may prevent disease progression to HUS (Fukuma et al., 1998; Ikeda et al., 1999).

Detection and isolation of STEC in foodstuffs by traditional culture methods is rather laborious and time-consuming and is complicated by the lack of common biochemical characteristics distinguishing most STEC from other E. coli strains. Development of rapid methods for the detection of the most pathogenic STEC strains is essential to ensure the safety of food products.

Materials and Method                                         

Isolation and Identification

A total of 50 samples were collected from local dairy farms and local markets in and around Proddatur town, YSR Kadapa district. The samples were collected in sterile containers and transferred under aseptic conditions (on icepack) to the labo­ratory for bacterial isolation, each sample was pre enriched in 90ml buffered peptone water (BPW). Then a loopful of  inoculum was transferred from each tube onto selective media plates (EMB agar plates) and incuba­ted at 37⁰C for 24 h. Colonies  with typical metallic sheen were taken and subjected for biochemical tests like IMViC (Indole, MethylRed, Voges- Prosverker, Citrate) tests, Triple sugar iron agar test, and urease test for confirmation of E. coli. For the detection of Shiga toxin producing Escherichia coli all the isolates were streaked on Sorbitol Mac Con key agar plates and incuba­ted at 37⁰C for 24 h.

DNA Isolation

1.5 ml of each enriched broths were transferred to eppendorf tubes then bacterial contents were pelleted by centrifugation at 8000 rpm for 10 min. After the supernatant was discarded. 50 μl of sterile distilled water was added to each tube and boiled at 100⁰C for 10 min in a water bath, then immediately snap chilled to release DNA. Each eppendorf tube was centrifuged at 13,000 rpm for 5 min then the supernatants were used as DNA templates for PCR analysis.

PCR Assay

Bacterial DNA amplification was done in 20μl reaction mixture containing 2 μl of 10x Taq DNA polymerase buffer (100mM tris PH 9.0, 500 mM KCl, 15 mM MgCl₂ and 1% triton X-100), 2 μl of 10 mM dNTPs mix, 0.9U/μl Taq DNA polymerase (Genei), 2 μl of each primer (4pmol/μl) and 5 μl bacterial DNA. Complete mixture volume to 20 μl using nuclease free water. Amplification was done in thermal cycler (Eppendorf, MC gradient) following standardized conditions. The amplified DNA fragments were resolved by agarose gel electrophoresis (B.Genei), stained with ethidium bromide (0.5 μg/ml) and visualized with an UV transilluminater (B.genei, MD-20, 312nm).

Cyclic Conditions

Results

Among the 50 raw milk samples 22(44%) isolates were found, Escherichia coli by culture method. Gram’s staining of the isolates revealed that they are Gram negative cocobacillary rods. All the 22 isolates were negative for Voges-Proskauer and Citrate utilization tests, whereas the same isolates produced bright red color in methyl red test and red color ring in Indole test. Further all the 22 isolates were subjected to urease test and triple sugar iron agar test. The results revealed that all the isolates were positive for urease test and triple sugar iron agar test as the isolates produced acid butt and slant and also gas. All the biochemical reactions confirmed the presence of Escherichia coli. For the detection of Shiga toxin producing Escherichia coli all the isolates were streaked on Sorbitol Mac Con key agar plates and the results revealed that all the isolates have shown pink colonies and none have shown colourless colonies. PCR assay of the isolates revealed that out of 22 samples 2(9.09%) were positive for STEC. Among the two positive samples two isolates carried stx₁ gene and one isolate carried stx₂ gene.

M           1              2                3            4         5          6

Lane: M (100 bp DNA size marker); Lane: 1 Positive control; Lane: 2 positive for stx1; Lane: 3 positive for stx2; Lane: 4 positive for stx1

Fig.: PCR assay Detection of STEC in milk samples using stx1 and stx2 gene primers pair, where the obtained amplicons sized 180 and 255 bp respectively.

Discussion

In the present study out of 50 milk samples tested for the presence of E. coli, 22samples(44%) were identified to have E. coli by cultural method. These results were in agreement with the reports of Rasheed et al. (2014) (43.33%). Higher prevalence than the present study was reported by Neher et al. (2015), Ali & Abdelgadir (2011) Lingathurai & Vellathurai (2010) and Shahzad et al. (2013) as 58.82%, 63%, 70% and 76% respectively. Lower prevalence than the present study was reported by Bali et al. (2013) as 32.5%. The present study reported 9.09% of STEC among 22 E. coli isolates  by using PCR assay, which was lower than the reports of Vendramin et al. (2014) (31.1%), Neher et al. (2015)(23.33%) and Mohammadi et al. (2013)(17.47%). Almost similar results to the present study were reported by Rasheed et al. (2014) as 6.7%. Presence of even single STEC in food sample may lead to gastrointestinal disorder due to their multiplication in the body or the food itself during storage in poor condition (Gyles, 2007). Prevalence of STEC in milk may show seasonal variation. Mohammadi et al. (2013) reported prevalence of STEC as 17.47% in milk samples that were collected during summer months of the year (Paton et al., 1996). Numerous factors along with season that are likely to contribute to the variation observed are geographical location, farm size, number of animals in farm, hygiene, farm management practices, variation in sampling, variation in types of samples evaluated and differences in detection methodologies used (Jelacic et al., 2003).

In the present study out of 2 isolates of STEC that were subjected to PCR amplification for virulence factor (Stx1 and stx2), one is possessing stx1 gene (4.54%) and the other isolate is having stx2 gene (4.54%). Higher percentage of STEC with stx1 gene were reported by Shahzad et al. (2013), Mohammadi et al. (2013), Lingathurai & Vellathurai (2010), and  Neher et al. (2015) as 73%, 43.59%, 25.8% and  23.33% respectively. Lower percentage of STEC with stx1 gene than the present study was reported by Vendramin et al. (2014) (1.9%) and Parisi et al. (2010) (5.7%). STEC harboring stx1 and stx2 genes were reported to be typical cattle colonizers (Bretta et al., 2003, Vukhac & Cornick, 2008). Present study reported 4.54% of STEC possessing stx2 gene as virulent factor, which is lower than the reports of Mohammadi et al. (2013) (56.4%), Shahzad et al. (2013) (62%) and Vendramin et al. (2014) (28.3%). In humans epidemiologic data suggested that E. coli O157 strains that express stx2 are more important than stx1 in the development of Haemolyric Uraemic syndrome(HUS) and  that strains that express stx2 alone are more  likely to be  associated with the progression to HUS than strains that produce both stx1 and stx2(Griffin,  1995).

After doing several studies some authors have observed a significant association of stx1 and stx2 genes with bovine meat and milk products. They compared the properties of food borne STEC with published data on fecal STEC from food producing animals and found that virulence profiles and serotypes of STEC from food showed remarkable similarities to those of fecal STEC that were from the same animal species and they also pointed out that food producing animals represent the most important source for the entry of STEC in the food chain (Martin and Beutin, 2011).

Conclusion

The present study has investigated and reported the prevalence of Escherichia coli in the raw milk samples, which was may be due to fecal contamination of raw milk from different sources. This suggests that raw milk should be considered as a vehicle for the transmission of potentially pathogenic bacteria. So its better to follow strict hygienic practices before, during and after milking process and to create awareness among the people to avoid raw milk consumption.

Acknowledgement

Authors are thankful to Sri Venkateswara Veterinary University, Tirupathi.

References

1. Adesiyun AA. 1994. Bacteriological quality and associated public health risk of pre-processed, bovine milk in Trinidad. Int. J. Food Microbiol., 21: 253-261.
2. Ali AA, Abdelgadir WS.2011. Incidence of Escherichia coli in Raw Cow’s Milk in Khartoum State. British Journal of Dairy Sciences. 2(1): 23-26.
3. Bali OS, Lajnef R, Felfoul I, Attia H, Ayadi M. 2013. Detection of Escherichia coli in unpasteurized raw. International Journal of Agricultural and Food Science. 3(2): 53-55.
4. Bavaro MF. 2012. coli O157:H7 and other toxigenic strains: the curse of global food distribution. Current Gastroenterology Reports, 2012; 14(4): 317-323.
5. Betts GD. 2000. Controlling coli O157:H7. Nutr. Food Sci, 30: 183-186.
6. Bonardi S, Maggi E, Bottarelli A, et al. 1999. Isolation of verocytotoxin-producing Escherichia coli O157:H7 from cattle at slaughter in Italy. Vet Microbiol., 67: 203-211.
7. Brett KN, Ramachandran V, Hornitzky MA, Bettelheim KA,Walker MJ, Djordjevic SP. 2003. Stx1c is the most common Shiga toxin 1 subtype among Shiga toxin-producing Escherichia coli isolates from sheep but not among isolates from cattle. Journal of Clinical Microbiology. 41(3): 926-936.
8. Fukushima H, Hashizume T, Morita Y, Tanaka J, Azuma K, Mizumoto Y, et al., 1999. Clinical experiences in Sakai City Hospital during the massive outbreak of enterohemorrhagic Escherichia coli O157 infections in Sakai City. Pediatrics International., 41: 213-217.
9. Griffin PM. Escherichia coli O157:H7 and other enterohemorrhagic Escherichia coli. 1995. In M. J. Blaser, P. D. Smith, J. I. Rovin, H. B. Greenberg & R. L. Guerrant (Eds.), Infections of the gastrointestinal tract (pp. 739-761). New York: Raven Press.
10. Gruetzmacher TJ, Bradle RL Jr. 1999. Identification and control of processing variables that affect the quality and safety of fluid milk. J Food Prot, 62: 625-631.
11. Gyles CL. 2007. Shigatoxin-producing Escherichia coli: an overview. Journal of Animal Science, 85(13): 45-62.
12. Hussein SH, Brandolyn HT, Hudson AG. 2003. Verotoxin- Producing Escherichia coli in sheep grazing an irrigat­ed pasture or arid rangeland forages. VTEC in Sheep, 2003; 4: 358-364.
13. Ikeda K, Ida O, Kimoto K, Takatorige T, Nakanishi N, Tatara K. 1999. Effect of early fosfomycin treatment on prevention of hemolytic–uremic syndrome accompany-ing Escherichia coli O157:H7 infection. Clin Nephrol., 52: 357-362.
14. Jamshidi A, Bassami MR, Rasooli M. 2008. Isolation of Escherichia coli O157:H7 from ground beef samples collected from beef markets, using conventional culture and polymerase chain reaction in Mashhad, northeastern Iran. Iran J Vet Res, Shiraz Univers 2008; 9: 72-76.
15. Jelacic JK, Damrow T, Chen GS, Jelacic S, Bielaszewska M, Ciol M et al., Shiga toxin-producing Escherichia coli in Montana: bacterial genotypes and clinical profiles. J. Infect. Dis. 188, 719–729.
16. Kaper JB, Nataro JP, Mobley HLT. 2004. Pathogenic Escherichia coli. Nat. Rev. Microbiol, 2: 123-140. .
17. Karmali MA. 1989 Infection by Verocytotoxin-producing Escherichia coli. Clin Microbiol Rev, 2: 5-38.
18. Law D.2000. Virulence factors of Escherichia coli O157 and other Shiga toxin- producing coli. Journal of Applied Microbiology, 88(5): 729–745.
19. Lingathurai, S, Vellathurai P. 2010. Bacteriological Quality and Safety of Raw Cow Milk in Madurai, South India. Webmed central.
20. Louie M, Azavedo J, Handelsman M, Clark C, Ally B, Dytoc M. 1993. Expression and characterization of the eaeA gene product of Escherichia coli serotype O157:H7. Infect Immun., 61: 4085–4092.
21. Ludwig K, Karmali MA, Sarkim V, Bobrowski C, Petric M, Karch H, et al., Antibody response to Shiga toxins Stx2 and Stx1 in children with enteropathic hemolytic-uremic syndrome. J Clin Microbiol., 39: 2272–2279.
22. Madic J, Vingadassalon N, Garam CP et al., Detection of Shiga toxin-producing Escherichia coli serotypes O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7 in raw-milk cheeses by using multiplex real-time PCR,” Applied and Environmental Microbiology, 77(6): 2035–2041.
23. Martin A, Beutin L. 2011. Characteristics of shiga toxinproducing Escherichia coli from meat and milk products of different origins and association with food producing animals as main contamination sources. International Journal of Food Microbiology, 146(1), 99-104.
24. Martin ML, Shipman LD, Potter ME, Wachsmuth LK, Wells JG, Hedberg K, et al., Isolation of Escherichia coli O157:H7 from dairy cattle associated with two cases of hemolytic uraemic syndrome. Lancet, 8514:1O43.
25. Mohammadi P, Abiri R, Rezaei M, Salmanzadeh-Ahrabi S. 2013. Isolation of Shiga toxin-producing Escherichia coli from raw milk in Kermanshah, Iran. Iranian J. Microb, 5(3): 233-238.
26. Molina PM, Parma AE, Sanz M. 2003. Survival in acidic and alcoholic medium of shiga toxin-producing Escheri­chia coli O157:H7 and non-O157:H7 isolates in Ar­gentina. BMC Microbiol., 13: 3-17.
27. Nanu E, Latha C, Sunil B, Prejit, Thomas M, et al. 2007. Quality assurance and public health safety of raw milk at the production point. American Journal of Food Technology, 2: 145-152.
28. Nataro JP, Kaper JB. 1998. Diarrheagenic Escherichia coli. Clin Microbiol Rev, 11: 142-201.
29. Neher S, Hazarika RK, Sharma L, Barkalita M, Bora P, Deka AK. et al. Detection of Shiga-Toxigenic Escherichia Coli in Milk Samples of Cattle by PCR. Journal of Agriculture and Veterinary Science., 8(5); PP 75-78.
30. Oliver SP, Jayarao BM, Almedia RA. 2005. Food borne pathogens in milk and the dairy environment food safety and public health implications. Food borne Pathogens and Disease, 2: 1115-1129.
31. Parisi A, Miccolupo A, Santagada G, Pedarra C, Dambrosio A, Normanno G. 2010. Detection of Verocytotoxin-producing Escherichia coli (VTEC) in minced beef and raw milk by colony blot hybridization. Food Control, 21: 770-773.
32. Paton AW, Ratcliff RM, Doyle RM, Seymour-Murray J, Davos D, Lanser JA et al., Molecular microbiological investigation of an outbreak of hemolytic-uremic syndrome caused by dry fermented sausage contaminated with Shiga-like-producing Escherichia coli. J Clin Microbiol, 34: 1622-1627.
33. Paton JC, Paton AW. 1998. Pathogenesis and diagnosis of 2. Shiga toxinproducing Escherichia coli Clin Microbiol Rev, 11: 450-479.
34. Paton JC, Paton AW.1998b Pathogenesis and diagnosis of Shiga toxin- producing Escherichia coli infections. Clinical Microbiology Reviews, 11: 450–479.
35. Rasheed MU, Jamil K, Thajuddin N, Pasupuleti M, Ahamed P, Muthukumaresan KP. Distribution of the stx1, stx2 and hlyA genes: Antibiotic profiling in Shiga-toxigenic E. coli strains isolated from food sources. Int.J.Curr.Microbiol.App.Sci, (5): 348-361.
36. Riley LW, Remis RS, Helgerson SD, McGee HB, Wells JG, Davis BR, et al. 1983. Hemorrhagic colitis associ­ated with a rare Escherichia coli serotype. New Eng J Med, 1983; 308: 681–685.
37. Shahzad KA, Muhammad K, Sheikh A. A,Yaqub T, Rabbani M, Hussain T et al, Isolation and Molecular Characterization of Shiga toxin producing E. coli O157. The Journal of Animal & Plant Sciences, 23(6): 1618-1621.
38. Terrance MA, Genevieve ABG, Mildred RB, Koohm­ M. 2002. Prevalence and Characterization of Non-O157 Shiga Toxin-Producing Escherichia coli on Carcassesin Commercial Beef Cattle Processing Plants. App En­viron Microbiol., 68: 4847–4852.
39. Todar K. Pathogenic E. coli. 2008. Available http://www.textbookofbacteriology. net/e.coli.html. Accessed on: 20th October, 2009.
40. Vendramin T, Kich DM, Molina RD, De souza CFV, Salvatori RU, Pozzobon A. Bustamante-filho IC. 2014. Molecular screening of bovine raw milk for the presence of Shiga toxin-producing Escherichia coli (STEC) on dairy farms. Food Sci. Technol, 34(3): 604-608.
41. Vu-Khac H, Cornick NA. 2008. Prevalence and genetic profiles of Shiga toxin-producing Escherichia coli strains isolated from buffaloes, cattle, and goats in central. Veterinary Microbiology., 126(4): 356-363.