Glob. J. Agric. Food Safety Sci., Vol.1: pp. 157 - 167 (2014)

Prevalence and significance of non O157 shiga toxin producing E. coli in milk and some dairy products

Walaa M. A. Elsherif

Department of Food Hyigene, Animal Health Research Institute, Assiut city, Egypt

ABSTRACT

This study was undertaken to isolate pathogenic E. coli non O157 from milk and some milk products and detection of virulence gene producing shiga toxin using PCR. One hundred and fifty samples of raw milk, talaga cheese and ice cream (50 each) were collected from milk vendors and retail shops. The Shiga toxin (Stx)-producing non O157E. coli (non O157 STEC) isolates were screened for the presence of toxogenic associated genes (A single set of primers was used to detect the genes stx1 and stx2 in the same reaction) by PCR. The incidence of non O157 STEC that identified biochemicaly was 66 % in the samples of milk, talaga cheese (40%), and ice cream (70%). Serological tests revealed that the incidence of non O157 STEC was 20%, 12% and 30% in raw milk, talaga cheese and ice cream, respectively. E. coli O26, O111, O125 and O127 isolates were positive for stx genes. Current study supports the finding that raw milk and various milk products can be regarded as critical source of pathogenic non O157 STEC. This explains the need of strict monitoring and surveillance for effective measures of hygiene and sanitary practice during production of milk and various milk products.

Keywords: E. coli non O157, shiga toxin, milk, milk products, talaga cheese, ice cream and PCR

1- INTRODUCTION

Huge numbers of microbes can get access to milk and various milk products including E. coli which is an indicator of fecal contamination, constituting a public health hazard (Virpari et al., 2013) while, the infectious dose of non O157 STEC strains must be as low as 10 bacteria (European Commission, 2003). Diarrheaogenic E. coli (DEC) are some of the most frequently detected pathogens worldwide. There are six pathotypes of DEC: enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteropathogenic E.coli (EPEC), enteroinvasive E. coli (EIEC), enterohaemorrhagic E. coli (EHEC) or verocytotoxin-producing E. coli and diffusely adherent E. coli (Bischoff et al., 2005 and Vernacchio et al., 2006).The most important causes of food borne diseases Enterohemorrhagic Escherichia coli (EHEC) or Verocytotoxin-producing Escherichia coli (VTEC) strains are a subset of Shiga toxin (Stx)-producing E. coli (STEC) strains

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Walaa M. A. Elsherif, (2014)

that are isolated from human patients and are responsible for severe clinical symptoms, such as hemorrhagic colitis (HC) and the potentially lethal hemolytic uremic syndrome (HUS) (Karch et al., 2005 and Karmali et al., 2010). Although most outbreaks of HC and HUS have been attributed to serotype O157:H7, an increasing number of human infections are caused by other serotypes, such as O26, O103:H2, O111 and O125 (Karch et al., 2005 and European Food Safety Authority, 2007). However, more than 250 serotypes of non-O157 STEC strains have been reported in animals and human and over 150 STEC serotypes have been associated with illness (Akiba et al., 2005 and Gould et al., 2009). Shiga toxins, the main virulence factors contributing to pathogenicity, consist of two major types, Stx1 and Stx2, each including several variants (Scheutz and Strockbine, 2005). Report indicate that consumption of raw milk and various milk products related with occurrence of 1-5% of food infections and among that 53 % of cases produced by enteropathogenic E. coli (EPEC) (Schrade and Yager,2001). In view of these particulars, the current study was undertaken to detect and characterize the E. coli non O157 from milk and various milk products.

2 - MATERIAL AND METHODS

2-1- Sample collection: One hundred and fifty milk and various milk products samples consisting of raw milk, talaga cheese and ice cream (50 each), were collected from milk vendors and retail shops. 2-2- Isolation and identification of non O157 E. coli: Samples were prepared to isolate the E. coli as per the standard Bacteriological Analytical Manual (BAM), U.S. Food and Drug Administration (USFDA) method (Kumar et al., 2008). The samples were enriched in modified vancomycin- trypticase soy broth (mvTSB) (Samadpour, et al. 1990), the loopful of culture inoculated into Sorbitol MacConkey (SMAC) agar plates. Suspected E. coli O157 colonies were sorbitol negative and appeared pale in colour as compared with bright pink sorbitol positive, these colonies produced by E. coli of non-O157 and other enteric pathogens (De Boer and Heuvelink, 2000). Various biochemical tests such as sugar fermentation specially sorbitol fermentation test, catalase test, Indole production, Methyl red, Voges proskauer, Simon's citrate agar, Urease production, Nitrate reduction etc. and microscopic test were done for the confirmation non O157 E. coli as proposed by A.P.H.A. (1992)2-3-Serodiagnosis of E. coli:

This part has been done in the Food Hygiene Lab in the Faculty of Veterinary Medicine of Moshtohor, Banha Univ., Egypt. The isolates were serologically identified according to Kok et al. (1996) by using rapid diagnostic E. coli antisera sets for diagnosis of the Enteropathogenic types. The technique applied as recommended by the manufacture (DENKA SEIKEN Co., Japan)

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2-4-Detection of toxigenic non-O157 isolates using PCR assay:This part has been done in the Food Hygiene Lab in the Faculty of Veterinary

Medicine of Moshtohor, Banha Univ., Egypt.Each serological type of non O157 STEC was screened for the presence of

virulence associated genes by using the PCR technique. The PCR was standardized for the detection of stx1 and stx2 genes following the methodology as described by Paneto et al. (2007). Standardization of PCR was done by using standard strain of E. coli. The reactions were performed in the thermal cycler (Applied Biosystem, Sweden) with pre-heated lid (Lid temp. 105 OC). For the confirmation of targeted PCR amplification, consisting of 1 μl of 6X gel loading buffer along with 5 μl of the PCR product, then electrophoresis was performed with use of DNA molecular weight marker (Gene Ruler, MBI Fermentas). Agarose gel (2%) along with ethidium bromide (at the rate of 0.5 μg/ml) was used. Electrophoresis was performed in 0.5X TBE buffer at 5V/cm for 60 min. Visualization of amplified product was done under ultraviolet light and was documented by gel documentation system (SynGene, Gene Genius BioImaging System, UK).

3- RESULTS

Table (1): Incidence of isolated non- O157 E. coli in the examined samples (biochemically and serologically).

Type of examined samples

No. ofexaminedsamples

presumptive strains identified biochemically

Isolated strain identified serologically

NO. of positive % NO. of positive %

* Raw Milk 50 33 66 10 20* Talaga Cheese 50 20 40 6 12* Ice Cream 50 35 70 15 30 Total 150 88 58.67 31 20.67

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Table (2): Incidence of different isolated non-O157 E. coli strains in the positive samples based on their serological identification.

Serotypes of theisolated strains

Raw Milk Talaga Cheese Ice CreamNO./50 % NO./50 % NO./50 %

EHEC*

O26 2 4 - - 4 8

O111:H4 - - 2 4 3 6

ETEC**O125:H21 1 2 - - 3 6O127:H6 - - 2 4 1 2

EPEC***O55:H7 - - - - 2 4O44:H18 - - 1 2 - -

O86 2 4 - - 1 2O114:H21 - - 1 2 - -O119:H6 3 6 - - - -

O78 - - - - 1 2EIEC****

O124 2 4 - - - -

* EHEC: - enterohaemorrhagic E. coli, ** ETEC: - enterotoxigenic E. coli*** EPEC: - enteropathogenic E.coli , **** EIEC: -enteroinvasive E. coli

Photo (1): PCR assay for detection of Shiga toxin producing E.coli (STEC).Photograph (1): Agarose gel electrophoresis of PCR amplification products used to detect the genes (stx1 and stx2) in the same reaction.Lane M: 200 bp ladder as molecular DNA marker.Lane 1: Control positive for Shiga toxin producing E.coli.Lanes 2 (O26), 6 (O111), 10 (O125) and 11 (O127): Positive STEC.Lanes 3 (O55), 4 (O78), 5 (O86), 7 (O114), 8 (O119), 9 (O124) and 12 (O44): Negative STEC.

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4- DISCUSSION

Although, E. coli O157 is the most renowned VTEC, other serogroups, including O26, O111, O103, O145 and O121 have the potential to cause serious human illness (Bonnet et al., 1998). A number of non-O157 VTEC infections in humans (mostly in children) have been reported in Ireland. Human cases attributed to O26 have already been documented in the North West region of Ireland (McMaster et al., 2001).

In present study, Table1 postulated that the detection rate of non O157 STEC using biochemical technique was 66% in raw milk, almost similar result was found (57%) by Soomro et al. (2002) and (60%) Virpari et al. (2013). However, lower incidence of non O157 STEC than present study was reported 31.6 % by Nanu et al. (2007), 26.43 % by Bandyopadhyay et al. (2011), 30.28 % by Farzan et al. (2012) and 33.96 % by Mohd et al. (2013). The presence of non O157 STEC in raw milk may be related to that ruminants, and especially cattle, are considered as a natural reservoir of STEC. Extended risks originate in domesticated cattle kept in close contact to humans. Furthermore, cattle-derived foods can be contaminated and represent an important source of infection as well as fecal shedding by dairy cattle, hygiene, husbandry and changes in housing or structure of the herd (Kuhnert et al., 2005 and Werber et al., 2007).

In the incidence of non O157 STEC was 40% in talaga cheese, the lower result 12.9% in cheese was reported by Singh and Prakash (2008), 29.2 % in cheese by Fadel and Ismail (2009), 16.6 % by Farzan et al. (2012) and 28% in cheese by Virpari et al. (2013). Higher incidence 96 % in cheese was reported by Paneto et al. (2007).

Regarding ice cream samples was 70% in the present study. In contrast, low incidence of non O157 STEC in ice cream (58%) was recorded by Amany and Marcel (2008), 31.8 % by Fadel and Ismail (2009), 16.6 % by Farzan et al. (2012) and 20 % by Virpari et al. (2013). This may attributed to the neglected sanitary control adopted during manufacturing, handling and distribution, also low income people use raw milk preparation of some products increasing the potential risk of infections spread among community elements.

As shown in Table 1, the results declared serologically, the highly contaminated product was the ice cream (30%) followed by raw milk (20%) and 12% of talaga cheese samples. Concerning, the different serotypes of E. coli non O157 and their distribution as described in Table 2; they proved to be belonged to 11different groups, in raw milk (3 strains) belong to O119:H6, 2 strains for each of O26, O86 and O124 and one strain of O125:H21. Elsewhere, 2 strains each of O111:H4, O127:H6 and one strain each of O44:H18 and O114:H21 groups were detected in talaga cheese. It is worthwhile to state that ice cream samples in this study were polluted with 15 strains of E. coli groups; 4 strains for O26, 3 strains each of O111:H4 and O125:H21, 2 strains of O55:H7 and One strain each of O127:H6, O86 and O78 (Table 2).

Nearly similar serotypes were reported by Auvray et al. (2009) and Jordan et al. (2011) but high than these results in other studies occurred by Vernozy-Rozand et

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al. (2005); Andrea et al. (2010) and Zweifel et al. (2010). Likewise, Abo-Zeid (1990); Farag (1991) and Possé et al. (2008) identified lower serotypes from dairy products than which present in this study. Other serotypes of E. coli non- O157 were discovered by Lih-Ching-Chiueh et al. (2002); Thabet (2003) and CDC (2009).

Table 2, verified serological phenotypic identification of different entero-toxigenic E.coli non-O157 isolated from all examined samples. The result represented that O26 and O111:H4 were identified as EHEC. The ETEC strains recognized in serogroups O125:H21 and O127:H6 while, EPEC represented in O55:H7, O44:H18, O114:H21, O86, O119:H6 and O78. O124 serogroup belonged to EIEC. Vilchez et al. (2009) tested five different diarrheaogenic E. coli pathotypes simultaneously. ETEC, EAEC, EPEC, EHEC and EIEC by using multiplex PCR. The findings cleared that 100% associated type with diarrhea were ETEC estA (the gene for shiga-toxin) and EHEC. ETEC continues to be an important agent associated with diarrhea in children from LeÓn, Nicaragua. Although not very frequent, in children with severe diarrhea, more than half had EAEC, ETEC or EPEC. In 2007 there was an outbreak of EHEC in which five children were infected by two serotypes (O145 and O26) from consumption of ice-cream produced from a Belgium farm and had features very similar to those of the German outbreak strain: EAEC aggregative adherence, and associated with HUS (De et al., 2008).

VTEC strains isolated from cattle, food, and other animal sources have various virulence profiles; and to assess the potential virulence of VTEC isolates from these sources, it is important to examine them for the presence of virulence genes (Cobbold and Desmarchelier, 2000). Immunoassays, PCR methods, and molecular analytical methods, developed in the past five years. PCR methods using single primer sets have been reported elsewhere (Osek, 2001), in present study each of different serotypes group of non O157 STEC isolates were detected for stx1 and stx2 genes. Out of 11 serotypes, 4 serotypes group (36.36%) (O26, O111, O125 and O127) were positive for stx genes (Photo 1); similar results were obtained by Mansouri-Najand and Khalili (2007), Stephan et al. (2008), Islam et al. (2010), Ebrahim et al. (2011), Farzan et al. (2012), Mohd et al. (2013) and Virpari et al. (2013). On the other hand, high per cent of stx positive isolates were reported by Martin and Beutin (2011) and Njage et al. (2012).

The majority of raw milk and various milk products were found to be contaminated or carried E. coli infections, which require strict management for effective measures for hygiene and sanitary practice. PCR based molecular epidemiological studies are required for detection of all types of pathogenic as well as zoonotic potential strains of E. coli isolates for future research.

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القولونية االشريكية عترات تواجد المنتجة أهميةغير توكسين منتجات O157للشيجا وبعض اللبن هفى

الشريف على محمود والء , , مصر اسيوط الحيوان صحة بحوث معهد االغذية صحة قسم

اال أ لعزل الدراسة هذة غير يجريت كوالى الخام O157شريشيا اللبن منبواقع كريم وااليس الى 150والجبن مقسمة . 50عينة تجميع تم منهم لكل عينة

. على العينات زرع وتم االلبان بيع محالت من -modified vancomycinالعيناتtrypticase soy broth (mvTSB) خاص مستنبت على نقلها تم ثم الميكروب لتنمية

وهو الميكروبات. Sorbitol MacConkey (SMAC) agarبها تصنيف تم كماعن وللكشف وسيرولوجيا كيميائيا توكسين العتراتالمعزولة للشيجا المفرزة

stx1 و stx2) المتسلسل البلمرة تفاعل اختبار استخدام النتائج). PCRتم اثبتتان الكيميائية االختبارات بإستخدام الخام , 66االولية اللبن عينات من من ٪40 ٪

و الغير 70الجبن كوالى االشريشيا بميكروب ملوثة كريم االيس عينات من ٪ O157 (non O157 STEC) .كالتالى النتائج فكانت سيرولوجيا و 12٪, 20اما ٪30٪

. وب التوالى على كريم وااليس والجبن الخام اللبن من تفاعل إلكل ستخدامالمتسلسل ( كانت) PCRالبلمرة سيرولوجيا المصنفة ، O26 المعزوالتللعتراتO111 ، O125, O127 ال هى الشيجا فقط إفراز عن المسؤله للجينات إيجابية

التى. المختلفة االلبان ومنتجات الخام اللبن خطورة الدراسة ناقشت توكسينلالمراض المسببة الميكروبات هذة مثل لنقل حيويا مصدرا nonO157تعتبر

STEC . و النظافة من الفعالة و الصارمة الرقابة إلى الحاجة يفسر ما هذا . نوصي كما المختلفة األلبان ومنتجات اللبن إنتاج أثناء الصحية الممارسات

تناوله قبل اللبن وبسترة غلي إالمن. وتصفيتةبضرورة كريم االيس شراء عدملضمان الكبيرة كالشركات فيها موثوق والجودة مصادر اليتعرض السالمة لكي. الميكروبات من النوع هذا لخطورة المستهلك

الرئيسية ومنتجات O157كوالي : الكلمات والحليب توكسين شيجا ، غيرو كريم واآليس ، والجبن ، PCRالحليب

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