A STUDY ON THE OCCURRENCE OF ESCHERICHIA COLI IN SOME BEEF PRODUCTS WITH SPECIAL REFERENCE TO ESCHERICHIA COLI O157:H7.

Assiut Vet. Med. J. Vol. 52 No. 110 July 2006

Animal Health Research Institute, Port Said Laboratory for Food Hygiene.

A STUDY ON THE OCCURRENCE OF ESCHERICHIA COLI IN SOME BEEF PRODUCTS WITH SPECIAL REFERENCE TO ESCHERICHIA COLI O157:H7.

(With 4 Tables)

By ZIENAB I. SOLIMAN and AZZA A. EL-TABIY

(Received at 1/6/2006)

دراسة عن وجود میکروب الإشيرشيا کولاى في بعض منتجات اللحوم

مع الإشارة إلى عترة

O157 :H7 زینب إبراهيم سليمان،عزه على حسين التابعي تعتبر منتجات اللحوم المصنعة من أهم المنتجات التي يقبل عليها المستهلک. وقد تتعرض لحوم الذبائح للتلوث بالميکروب القولوني أثناء الذبح وتختلط هذه البکتريا جيدا باللحم عند فرم وتجهيز اللحم لإعداد الأنواع المختلفة من منتجات اللحوم کالبرجر والسجق إلى غير ذلک من منتجات اللحوم المصنعة. ومع أن معظم عترات ميکروب الإشيرشيا کولای غیر ضارة إلا أن هناک بعض العترات قد تشکل خطرا على صحة الإنسان. فعلى سبيل المثال العترةH7:0157 تذکر دائما مرتبطة بحالات شديدة من التسمم الغذائي وخصوصا في منتجات اللحوم الغير مطهية جيدا وقد تؤدي العدوى إلى حدوث فشل کلوي وخاصة في الأطفال. لذلک تهدف الدراسة الحالية إلى عزل وتقييم انتشار الميکروب القولوني العترة7

O157 :H إلى جانب العترات الأخرى في بعض منتجات اللحوم المصنعة التي تباع في السوبر مارکت. تم تجميع خمسة وعشرون عينة من کل من البرجر،الفرانکفورتر، الکفتة، اللحم المفروم، والسجق. وقد أسفرت التحاليل عن وجود میکروب الإشيرشيا کولای بنسب56 % ،40% ،%۹۲،%۹۸،

%۷۲في عينات البرجر،الفرانکفورتر، الکفتة، اللحم المفروم، والسجق على التوالي. کذلک تم عزل وتصنيف العترة7:H 0157 من6 (4٫۸%) من اجمالي عدد العينات التي تم فحصها. کما تم تصنيف عترات أخرى من العينات الإيجابية وهي068 0119 ,0113 ,0111 ,055،0126. هذا وقد تم مناقشة خطورة وجود العترة7 H: 0157 إلى جانب العترات الأخرى على الصحة العامة وکذلک أهم التوصيات بالنسبة لمستهلکي منتجات اللحوم المفرومة المصنعة والتي تترکز في الطهي الجيد وضمان وصول الحرارة إلى کافة الأجزاء الداخلية وليس السطح الخارجي وذلک للقضاء على الميکروب.

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SUMMARY

A total of one hundred and twenty five samples, twenty five of each beef burger, frankfurter, kofta, minced meat and sausage were collected from Port-Said markets. Samples were examined to isolate and evaluate the prevalence rate of E. coli O157:H7 and other E. coli serotypes. E. coli was detected in burger, frankfurter, kofta, minced meat and sausage samples at a rate of 56, 40, 92 68 and 72%, respectively. Six (4.8%) out of all 125 tested meat products samples were found to be contaminated with E. coli O157:H7, ten isolates of E. coli O157:H7 could be recovered. A total of 50 E. coli isolates recovered from positive samples were identified to serogroups, 055 (30%), 0111 (22%), 0113 (22%), 0119 (16%), 068 (6%) and 0126 (4%). The majority of E.coli serotypes recovered from the examined samples showed hemolytic activity. The public health significant of the isolated serogroups and consumer's safety were discussed.

Key words: Escherichia coli, beef products, E.coli 0157:H7.

INTRODUCTION

Many people enjoy beef burgers, sausages and other meat products, especially during the summer months. However, raw and improperly handled or cooked sausages and beef burgers can harbour harmful bacteria including Escherichia coli. The bacteria constituting the species E. coli are bacteria that normally live in the intestines of humans and animals. Although most strains are harmless, several are known to produce toxins that can cause diarrhea. The pathogenic groups includes enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC). Of these, only the first 4 groups have been implicated in food or water borne illness(Levine, 1987 and Nataro and Kaper. 1998).

In recent years, it has become apparent that one can contract a rather serious bacterial gastro-enteritis by consuming undercooked ground beef. Scientists have identified a rare but dangerous type of Escherichia coli, E. coli O157:H7 that is responsible for this foodborne illness. Scientists believed that E. coli O157:H7 is a mutant strain that was created when a virus infected benign E.coli and gave it a string of DNA from Shigella, a bacterium that causes severe bloody diarrhea. In both Shigella and E. coli O157:H7, as few as 10 germs can cause illness;

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by comparison, it takes about a billion Salmonella bacteria to make sick (Wong et al., 2000). E. coli O157:H7 was first recognized as a cause of illness in 1982 during an outbreak of severe bloody diarrhea; the outbreak was traced to under cooked burgers served from a fast food chains (Riley et al., 1983). Since 1983, an increasing in number of E. coli O157:H7 have been reported in association with consumption of improperly cooked ground beef (Cohen and Giannella, 1991 and Siegler et al., 1993).

The organism colonizes in the large intestine and produces one or more of the potent cytotoxins referred to as Shiga-like toxins (SLTS) (O'Brien and Kaper 1998). Although more than 60 E. coli serotypes produce SLTs, serotype 0157:H7 is the predominant pathogen in the EHEC group and the one associated most frequently with human infections worldwide (Karmali, 1989). These toxins are responsible for severe hemorrhagic colitis in humans. In some persons, particularly children under 5 years of age and the elderly, the infection can also cause a complication called hemolytic uremic syndrome (HUS), in which the red blood cells are destroyed and the kidneys fail, about 2% 7% of infections lead to this complication (Doyle, 1991).

In a view of the importance of E. coli O157:H7 from a food safety stand point, this study was planned to investigate the presence of this agent and other pathogenic E. coli serotypes among some selected meat products. The public health significant and consumer's safety were

discussed.

MATERIALS and METHODS

One hundred and twenty five samples, twenty-five of each beef burger, frankfurter, minced meat, kofta and sausage were collected from Port-Said markets.

The frozen samples were thawed in their original containers in a refrigerator at 2-5°C. Twenty-five grams of each sample were homogenized with 225 ml of tryptone phosphate broth as a pre enrichment fluid then incubated for 4-6 hours at 37 C. (Mehlman and Lovett, 1984). Two Mossel's enteric enrichment broth tubes (10 ml) were inoculated each by 1 ml from the pre-enrichment medium. One tube was incubated at 44°C for 24 hours to permit the growth of pathogenic E.coli, other than serovar 0157. The other tube was incubated at 37°C for 24 hours to permit the growth of E. coli O157:H7 as well as other serovars unable to grow at high (44°C) temperature (Mehlman and Romero, 1982). Dilutions of culture in tryptone phosphate broth with

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peptone water (0.1%) to 106 were prepared. About 0.1 ml obtained from appropriate dilution were inoculated in MacConkey Sorbitol agar (MACS) and Eosin methylene blue agar (EMB) as double parallel by using spread -plating. The plates were incubated at 37°C for 24 hours. Randomly selected white and colorless sorbitol negative colonies were picked from MACS and streaked separately onto MACS supplemented with cefixime- tellurite (CT, Difco) (CT- MACS) and onto EMB to purify the colonies. The plates were incubated at 37°C for 24 hours (FDA, 2002). Morphological and biochemical tests were applied to colorless or neutral /gray with smoky center and 1-2 mm diameter sorbitol negative colonies on CT- MACS and to metallic green colored, smooth sided colonies on EMB according to Quinn et al. (2002). The isolates were identified serologically by the slide agglutination test using diagnostic polyvalent and monovalent E.coli O antisera and H 7 antisera (Escherichia coli antisera, Denka Seiken Co., Ltd, Tokyo, Japan)

following the manufacturer's specification. Hemolysin production (Beutin et al., 1989)

E. coli isolates were inoculated onto blood agar plates containing sheep blood (5%) and incubated at 37°C for 24 hours. The plates were examined for the presence of haemolysis.

RESULTS

Table 1: Prevalence rate of Escherichia coli in the examined meat

products samples (n=25 of each).

Meat products Positive samples % of Positive samples Beef burger

14 Frankfurter

10 Kofta

23

92 Minced meat

Sausage

56

40

17

68

18

72

Table 2: Prevalence of E. coli O157: H7 among the examined meat

products (n=25 of each).

Meat products

Positive samples for serovar 0157:H7

No.

Beef burger Frankfurter Kofta Minced meat Sausage Total

4.8

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Table 3: Serovars of E. coli isolates recovered from the examined meat

products samples.

Serovar 055

0111

0113

0119

068

0126

-

source

No | % No % No % No % No Beef burger 4 8 3 6 4 8 3 6 1 Frankfurter 1 2 2 4 1 2 0 0 1 Kofta 15 | 10 4 8 3 6 3 160 Minced meat 3 6 2 4 1 2 1 2 1 Sausage 2 4 T0 0 2 4 1 2 0 Total

15 30 11 22 11 22 8 16 3 NB: Percentage was calculated according to the total number of the isolates (50)

% No 1 2 2 4

2 0 0 TOTO TO 2 0 0

0 0 0 6 2 4

Table 4: Hemolytic activity of E. coli isolates recovered from the

examined meat products samples.

E. coli serovars

No. of isolates

%

10

15

11

0157 : H7 055 0111 0113 0119 068 0126

Total

Hemolytic activity No. 10

100 15

100 81.8 63.6

100 0

NW00

0

60

49

81.7

DISCUSSION

Most enteropathogenic E. coli outbreaks have been blamed on ground beef and other meat products such as beef burger, and hot dog (Desmarchelier and Grau, 1997). The present investigation was carried out to evaluate the prevalence of E. coli O157: H7 and other E. coli serotypes among selected types of meat products.

The overall incidence of E. coli in different samples was recorded in Table (1), E. coli were recovered from burger, Frankfurter, kofta, minced meat and sausage samples at a rate of 56, 40, 92, 68 and 72%, respectively. In this concern, prevalence of E. coli from meat and meat products ranging from 30% to76% have been reported by Doyle and Schoeni, (1987), Gallas et al., (2002) and Gad El-Said et al., (2005). This contamination rate of the present samples indicates unhygienic

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practices prevailed in slaughter. Cattle are a major reservoir of these groups of bacteria and ground beef have been the major vehicle of E. coli transmission. During slaughter process, meat may become contaminated by fecal contamination during evisceration and through skin or hide during dressing (Desmarchelier, 1997 and Rice et al., 1997). When the meat is ground, fecal organisms on the outside of the meat are mixed throughout the ground beef. Also contamination of meat probably occurs during processing. In this respect, Read et al. (1990) reported that ground beef meat- processing plants were heavily contaminated with verocytotoxin E.coli. In addition, E.coli is an indicator of food safety for dehydrated, frozen and refrigerated food, as E. coli does not survive well under such condition (Mossel et al.,1979). Therefore, its presence might indicate poor temperature control.

Escherichia coli O157: H7, predominantly originated from beef, is a significant pathogen to the public health and thus, need to be vigorously surveyed in meat products. Lake of sorbitol fermentation within 24 hours has been considered a stable phenotypic character of E. coli O157: H7 therefore; MACS was used for differentiation of E. coli 0157: H7 from other enteric bacteria (March and Ratnam, 1986).

Results of biochemical and serological identification of sorbitol negative E. coli isolates revealed that six (4.8%) out of all 125 meat products examined were found to be contaminated with E. coli O157:H7 (Table 2), three (8%) out of 25 minced meat samples, two (8%) out of 25 burger samples and one (4%) out of 25 kofta samples. The exact contamination rate may be higher than stated here due to the low isolation rate of the culture methods compared to other immunological and genetical methods. Considerably higher isolation rates of E. coli 0157:H7 than in this study have been reported elsewhere. In South Africa, it was isolated from a total of 74.5% and in Malaysia from 36% of beef samples (Vorster et al., 1994 and Radu et al., 1998). On the other hand, in some studies beef and beef samples have found to be free (Junghannss et al., 1996, Simmons, 1997 and Uhitil et al., 2001). In another studies it was isolated at low contaminated rate, Pai et al., (1984) reported the presence of E. coli O157:H7 in 5 out of 17 beef samples. In USA, E. coli O157:H7 was isolated from six (3.7%) out of 164 beef samples (Doyle and Schoeni, 1987), in India, Dutta and Deb, (2000) isolated E. coli O157:H7 from two (9%) out of 22 minced beef samples. Also in turkey, Baran and Gulmez, (2005) isolated E. coli 0157:H7 from three (6%) of ground beef samples. Positive isolation of E. coli O157:H7 from beef samples in Egypt was reported by Tanios et

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al., (2002) from two (6.7%) of minced meat samples and by Gad El-Said et al., (2005) with a rate of 3.95% from meat samples.

Enterohemorrahagic Escherichia coli O157: H7 is an important foodborne pathogen, its presence even at low rate (4.8%) in the presen study may constitutes dangerous beef products. The ability of E. coli 0157:H7 to withstand the acidic conditions encountered in various foods have generally suggested that passage through the stomach would be insufficient to inactivate the pathogen. (Naim et al., 2003). In addition, the organism always enters the digestive system within a food matrix, Waterman and Small (1998) postulated that high protein content in food (such as ground beef and boiled egg white) might protect enteric bacteria against the killing effect of gastric acids. The data from epidemiological investigations indicated that as few as 10 to 100 cells of E. coli O157:H7 per g of raw ground beef were sufficient to cause illness (Abdul-Raouf et al., 1993). Moreover, Wong et al. (2000) believed that treatment with antibiotics is contraindicated for E. coli O157 poisoning, since it is when the bacteria die, they release the toxins which produce hemolytic uremic syndrome (HUS), for which there is no cure.

Symptoms of E. coli O157 infection include bloody and nonbloody diarrhea, vomiting, and abdominal cramps. Illness resolves typically within 7-10 days .A subset of patients, particularly the young and the elderly, will develop HUS, characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal failure (Russell et al., 2000). In the United States, hemolytic uremic syndrome is the principal cause of acute kidney failure in children, and E. coli O157: H7 causes most cases of hemolytic uremic syndrome (Besser et al., 1999). An estimated 73,480 people a year are infected with E. coli O157:H7 and about 600 of those cases are fatal, according to the federal Centers for Disease Control and Prevention (Wong et al., 2000). These illnesses and deaths were factors that began changing policy towards foodborne disease. The Food Safety and Inspection Service, declared that raw ground beef contaminated with E. coli O157 is adulterated and must be further processed to kill the pathogen or be destroyed (FDA., 2000).

While E. coli O157: H7 is the most renowned Shiga toxin producing E. coli (STEC), over 200 different types of STEC have been documented in meat and animals, at least 60 of which have been linked with human disease. A number of studies have suggested that non-0157 STEC are associated with clinical disease, and non-0157 STEC are present in the food supply (Acheson, 2000).

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Regarding to other serogroups, as shown in Table (3), 055, 0111, 0113 and 0119 were the most prevalent serotypes recovered from the examined samples with an incidence of 30, 22, 22, and 16% respectively, followed by 068 (6%) ,0126 (4%). Most of the isolated serotypes are usually associated with many cases of foodborne outbreaks and multiple sporadic cases in different part of the world. In this concern, Anathan and Subramaniam, (1995) isolated E. coli belonging to serotypes 0111 from cases of persistent diarrhea in young children. Enteropathogenic E. coli belonging to serotypes 0111, 0103 and 055 were isolated from patients suffering from bloody diarrhea, which may be accompanied by HUS (Desmarchelier, 1997). Non-0157 STEC, such as 0111 has caused large outbreaks and HUS in the United States and other countries. (Acheson, 2000). Moreover, Hussein and Omaye, (2003) found that the serogroups belonging to 026, 0113, 0111, 0119 and 0166 have caused approximately 30% of the hemolytic uremic syndrome (HUS) in US.

Blood haemolysis is one of character of virulent E. coli (Stephen et al., 1985).

Ten isolates identified serologically as E. coli O157: H7 were tested for hemolysis production using sheep blood. All tested isolates were haemolytic. Moreover, the majority of E. coli isolates other than 0157: H7 isolated from the examined samples showed haemolytic activity (Table 4). In this respect, Adesiyun et al., (1997) reported that from 94 E. coli isolates tested for haemolysis 13.8 % were haemolytic. Meanwhile, Gad El-Said et al., (2005) stated that 81.58 % of E. coli isolates recovered from meat samples showed haemolytic activity.

The productions of haemolysin have a potential role in virulence of hemolytic E. coli. Therefore, contamination of meat products with E. coli O157:H7 and other E. coli serotypes may results in problems for consumers. There is a close association between enterohaemolysin production and SLT production (Beutin et al., 1998). Moreover, the genes involved in enterohaemolysin production were carried on the EHEC plasmid (Scotland et al., 1990).

The risk of contamination of raw meat products with E. coli 0157:H7 and other pathogens constitute a major problem for human. The low infective dose E. coli O157:H7 present a major threat. Hemolytic uremic syndrome, a disease caused mostly by E. coli 0157:H7 may cause sever kidney diseases and/or failure among children. The main means of combating this organism are good food hygiene covering activities on farm, in abattoir and minced beef

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industries. However, until E. coli can be eliminated from meat processing systems, consumers should protect themselves by using safe food practices and advice for those who eat ground beef. Frozen ground beef should be thawed in the refrigerator rather than at room temperature. While thawing and preparing ground beef, raw meat must be separated from ready-to-eat foods. It is not enough to merely brown the outside of a burger, and other meat products Ground beef should be cooked thoroughly to an internal temperature of at least 160 F (71 C), food safety experts recommends that consumers use a meat thermometer to cook ground beef to ensure that internal temperatures are high enough to kill bacteria. To reduce the risk for cross-contamination, consumers should use soap and hot water to wash hands, utensils, and other surfaces that might have been exposed to raw or undercooked ground beef and other meat products. In addition, consumers should be aware from under cooked burgers and other meat products served from fast food restaurants.

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