OCCURRENCE OF ESCHERICHIA COLI O157:H7 IN SOME DAIRY PRODUCTS AT PORT -SAID CITY MARKETS

Animal Health Research Institute, Dokki,

Port -Said Laboratory for Food Hygiene

Occurrence of Escherichia coli O157:H7 in some dairy Products at Port -Said City markets 

(With 4 Tables)

By

Amany M. Shalaby and Marcel F. Galab

(Received at 18/9/2008)

تواجد الميکروب القولوني  O157:H7فى بعض منتجات الألبان المتداولة

فى أسواق مدينة بورسعيد

أمانى محمود شلبى ، مارسيل فخرى جلاب

لا شک أن منتجات الألبان تلعب دورا هاما فى حياتنا. ويحتل الايس کريم شعبية کبيرة حيث  يقبل عليه الکبار والصغار خاصة فى فصل الصيف. أما الزبادي فتعتبر من أکثر منتجات الألبان استهلاکا وذلک لقيمته الغذائية والصحية. ولما کانت منتجات الألبان وسط جيد لنمو الکثير من الميکروبات, لذا فإنها تلعب دورا خطيرا فى نقل الکثير من الميکروبات للإنسان ومنها ميکروب الاشيرشيا کولاى: H7   O157حيث يعتبر هذا الميکروب من اخطر الميکروبات الممرضة وذلک لأنه يمکن إحداث العدوى بجرعة هى الأقل بين مثيلاتها من الميکروبات وقد تؤدى إلى حدوث فشل کلوى وخاصة فى الأطفال لذلک تهدف هذه الدراسة إلى عزل وتقييم انتشار الميکروب القولونى O157 : H7 إلى جانب العترات الأخرى من الميکروب القولونى فى عينات الايس کريم والزبادى التى تباع فى السوبر مارکت, وقد تم تجميع خمسين عينة من کل منتج. وقد أسفرت التحاليل عن وجود ميکروب الاشيرشيا کولاى  بنسب 58 ,42 % فى عينا ت الايس کريم والزبادى على التوالى. کذلک تم عزل وتصنيف العترة O157 : H7  من اجمالى عدد العينات الإيجابية لکل منتج. کما تم تصنيف عترات أخرى من العينات الايجابية وهى O112,O119, O111 , O26 ,O55  هذا وقد تم مناقشة خطورة وجود العترة O157:H7   الى جانب العترات الأخرى على الصحة العامة.

Summary

A total of one hundred random samples, fifty from ice- cream and 50 from yoghurt were collected from Port-Said markets. Samples were examined to isolate and evaluate the prevalence rate of E. coli O157:H7 and other serotypes. E. coli was detected in ice-cream and yoghurt samples at a rate of 58 and 42 %, respectively. 9(18 %) and 5(10 %) out of 50 ice- cream and yoghurt samples, respectively were found to be contaminated with E. coli O157: H7. A total of 66 E. coli isolates recovered from positive samples were identified to serogroups, O112(40.9%), O119(31.8%) O111(21.2%), O26(3 %) and O55(1.5%). The majority of E. coli serotypes recovered from the examined samples showed hemolytic activity. The public health significance of the isolated serogroups and consumer's safety were discussed.

Key words: Dairy products, ice-cream, yoghurt, E. coli

INTRODUCTION

Milk and dairy products are subjected to contamination with several types of microorganisms from different sources, such contaminants may render the milk and its products unsafe to use and expose the consumers to risk of infection. E. coli as a group of bacteria found in the intestines of human, animals and birds. Enterohaemorrhagic Escherichia coli (EHEC) was first identified as a human pathogen in 1982, when E. coli of serotype O157: H7 associated with two outbreaks of Haemorrhagic Colitis (HC) in the USA(Riley et al., 1983). Although, there are several strains and serogroups of E. coli have been identified as HC, E. coli serotype O157: H7 is the predominant cause of HC associated disease in many countries around the world(Thomas et al., 1993, Waters et al., 1994 and Sharpe et al., 1995).         

Escherichia coli O157: H7 emerged as one of the most important foodborne bacterial pathogens within the last 20 years. The organism has a low infective dose as cfu/ml or/gm (Doyle et al., 1997) and causes haemorrhagic colitis which is occasionally complicated by haemolytic uremic syndrome (Morrison et al., 1986, Neil et al., 1987 and Kelly et al., 1990). Moreover, the bacterium can produce a variety of clinical symptoms including mild to sever bloody diarrhea and thrombotic thrombocytopenic Purpura haemorrhagica (Dean-Nystrom et al., 1999, Todd and Dundas, 2001 and Meichtri et al., 2004). The main virulence factor of EHEC bacteria is the production of verotoxins either verotoxins (VT1) and/or (VT2) so it‘s called Verocytotoxic E. coli(Kaper, and O'Brien, 1998 and Normanno et al., 2004). E. coli O157: H7 can survive in the stomach at pH level of 3 for up to 5 hrs before passage to the intestinal tract (Price et al., 2004). These characteristics provide a competitive edge for E. coli O157: H7 in ready–to–eat foods. E. coli O157: H7can contaminate avariety of foods resulting in E. coli O157: H7associated illness(Foley et al., 2004). However many epidemiologic and microbiologic investigations on large epidemic outbreaks have implicated the consumption of raw milk (Benkerroum et al., 2004) and raw milk products including yoghurt and ice -cream (Cheng and Chou., 2001; Oksuz et al., 2004). The organism may get access to milk and milk products through fecal contamination as well as post pasteurization contamination (Murinda et al., 2002). In addition yoghurt and ice-cream are the most likely dairy products, which are prefered by adult and young. Ice- cream is a food commodity consumed widely during summer and is considerd the major dairy product thatdominates interest of large segments of population (Anuranjini et al., 2008). On the other hand yoghurt is consumed by a wide cross section of people throughout the world, has an established market as a functional therapeutic food. It is also considered more digestible than ordinary milk, it is usually eaten for prevention and treatment of several disorders and particularly recommended for sick and convalescent people (Gilliland, 2000).

World Health Organization (1988) emphasized the importance of examining the behavior of pathogens during preparation of food products as a first step to control their growth .Therefore the aim of the present study was to determine the prevalence of E. coli O157 and other serotypes in ice- cream and yoghurt sold in Port-Said markets.

MATERIALS and METHODS

A total of one hundred random samples, fifty from each of ice-cream and yoghurt were collected from Port-Said markets. Ice-cream samples were thawed in their original containers in a refrigerator at       2-5°C. Twenty five gm of each of ice- cream and yoghurt samples were transferred to 225 ml of tryptone phosphate broth as a pre-enrichment fluid and thoroughly mixed thenincubated for 4-6 hr. at37°C. (Mehlman and Lovett, 1984).Two Mossel’s enteric enrichment broth (E.E), tubes (10ml) were inoculated with 1ml from the pre-enrichment tryptone phosphate broth medium. One tube was incubated at 44°C for 24 hr, permit the growth of pathogenic E. coli, other than serovar O157:H7. The other tube was incubated at 37°C to permit the growth of E. coli O157and other serovars which are unable to grow at higher (44°C) temperatures. (Mehlman and Romero, 1982) Then the two tubes were incubated for 24 hr. One loopful from each of the selective enrichment cultures incubated at 37°C & 44°C was inoculated onto MacConkey -Sorbitol agar (MACS) and Eosin Methylene Blue agar (EMB), then incubated at 37°C for 24hr. Suspected colonies: white and colorless with smoky center 1-2mm diameter, sorbitol negative colonies on MACS and metallic green colored smooth sides colonies on EMB, were picked up and subcultured onto nutrient agar plates and incubated at 37°C for 24hr. The purified colonies were picked up and streaked onto slope nutrient agar for morphological and biochemical tests according to(Cruickshank et al., 1975; Quinn et al., 2002). The isolates were identified serologically by the slide agglutination test using diagnostic polyvalent and monovalent E. coli O157 antisera and H7 antisera. Escherichia coliantisera (Denka Seikenco., Ltd, Tokyo, Japan), following the manufacturer’s specification.

Detection of haemolysin: (Beutin et al., 1989).

Isolated E. coli were inoculated onto blood agar containing sheep blood 5℅ and incubated at 37°C for 24 hr. Positive haemolysin production was detected.

RESULTS

Table 1: Prevalence of E .coli in the examined samples (n=50 of each)                                  

Table 2: Prevalence of E. coli O157:H7 among the examined samples (n=50 of each).                          

Table 3: Serovars of E. coli isolates (other than O157: H7) recovered from the examined milk products samples.

Table 4: Haemolytic activity of E. coli isolates recovered from the     examined milk products samples.                 

DISCUSSION

E. coli strains are important cause of diarrheal diseases in the world and remain one of the major public health problems of children and young infants (Levine et al., 1986). Several well-documented outbreaks due to enterohaemorrhagic E. coli (EHEC) infection have been linked to consumption of milk and its products(Champman and Wright, 1993; Upton and Coia, 1994).Although more than 60 E .coli serotypes produce shiga-like toxins, (SLTS) serotype O157: H7, is the predominant pathogen in the EHEC group and the one associated most frequently with human infection worldwide (Karmali, 1989).                 

The present investigation was carried out to evaluate the prevalence of E. coli O157: H7 and other E. coli serotypes among selected types of milk products. The overall incidence of E. coli in ice-cream and yoghurt samples was recorded in Table 1; E. coli was recovered from ice-cream samples at a rate of 58%. El-Essawy and Riad (1990) and Anuranjini et al., (2008) could detect E. coli in 8.16 and 21.1% respectively from the examined ice-cream samples. In comparative, higher isolation rate of E. coli in this study may be due to contamination during handling as well as in efficiency and bad sanitary conditions during storage (Anuranjini et al., 2008). On the other hand, 42% of the yoghurt samples showed positive results. These results are nearly in agreement with that recorded by Moursy (1969) who detected E. coli in 55% of the examined yoghurt samples. While Moustafa et al. (1988) isolated 25(62.5%) E. coli strain out of the examined 40 yoghurt samples. This contamination rate of the examined yoghurt samples indicated unhygienic practices, the fact that the major route of E. coli transmission is through the consumption of contaminated food, water, person-to-person and animal to person intact (Heuvelink et al., 1995; Leyer et al., 1995; Reilly 1998). Moreover, milk and milk products are good media for the growth of a number of pathogens. (Erskine et al., 1988).

Results of biochemical and serological identifications of sorbitol negative E. coli isolates, revealed that 9 (18%) out of the 50 examined ice-cream samples were found to be contaminated with E. coli O157:H7 (Table 2).In this respect Wilson et al. (1997) isolated E. coli O157 from 18% of the examined ice-cream samples. On the other hand, Josefa et al., (2005) recorded  one  case of  outbreak due to consuming  ice-cream in the United States .The fact  that ice-cream is a good source for microbial growth due to its nutrient content ,neutral pH and long storage time  even though, it is stored in a frozen state. (Kanbakna et al., 2004). The results in Table 2 showed that 5(10%) out of the 50 examined yoghurt samples were found to be contaminated with E. coli O157. (Abdel -Hakiem et al., 1988) could isolate E. coli O157: H7 from yoghurt samples. EL-Gaml, (2000) and Mohamed et al., (2005) explained that yoghurt can serve as conveyors of E. coli O157: H7 infection despite of it is acidity because the pathogen have the ability to grow during processing and could survive for long period during cold storage. E. coli O157: H7 has the ability to tolerate acidic conditions of yoghurt (Lederberg, 2000) since milk fermentation usually produce anaerobic conditions within the fermented dairy products, it is thought that anaerobic growth of E. coli O157: H7in an acidic medium, like yoghurt, results in the development of acid tolerance (Cheng and Kaspar, 1998). This may be due to production of RPoS-regulated protein or arginine decarboxylase, which is also induced during anaerobic growth at acidic pH (Auger et al., 1989; Lin et al., 1996).                                                                                 

Regarding other serogroups of isolated E. coli as shown in Table 3 O112and O119 were the most prevalent serotypes recovered from the examined samples of ice-cream and yoghurt followed by O111.O26 which could be detected in ice-cream samples only. Two isolates of the serovar O55 were isolated from both ice-cream and yoghurt samples .Most of the isolated serotypes are usually associated with many cases of food borne outbreaks and multiple sporadic cases in different parts of the world. Anathan and Subramanian (1995) could isolate E. coli belonging to serotypes from cases of perisistent diarrhea in young children. Although E. coli O157was the main focus of attention, there is increasing evidence that verotoxigenic E. coli (VTEC) serotypes other than O157 are the cause of haemolytic uraemic syndromes (Goldwater and Bettelheim, 1995). Furthermore, Saridakis et al. (1997) recorded that E. coli of serogroup O26 was commonly isolated from infants and calves affected with diarrhea and has been considered as one of the most important enteropathogenic E. coli (EPEC) and SLT producers.

Blood haemolysis is one of character of virulent E. coli. (Stephen et al., 1985). Fourteen isolates identified serologically as E. coli     O157: H7 were tested for haemolysis production using sheep blood agar. As shown in Table 4, all the tested isolates were haemolytic. Furthermore, the majority of E. coli isolates other than O157: H7 were isolated from the examined samples showed haemolytic activity. In this respect Adesiyun et al., (1997) concluded that from E. coli isolates tested for haemolysis 13.8% were haemolytic  and this could be used as phenotypic marker or virulence factors of serotypes. Meanwhile Gad EL-Said et al., (2005) reported that 81.25% of isolated E. coli recovered from milk samples showed haemolytic activity. E. coli strains were found to be haemolytic is an indication contaminating of E. coli may result in problems for consumers. since haemolysin production has been associated with pathogenicity of E. coli strains.                                                                                                 

The results of this study showed that, ice-cream and yoghurt can serve as conveyors of E. coli O157: H7 and some other serotypes             (Cheng and Kaspar, 1998) of E. coli which pose a health hazard to consumers, therefore efficient heat treatment of raw milk and prevention of post-treatment contamination during processing ,storage and handling should be strictly adopted.                                      

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