e.coli non o157 and incidence of it in milk and some milk product

1. 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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2. 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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3. Prevalence and significance of non O157 shiga toxin producing E. coli in milk and some …..
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 O
C). 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. of
examined
samples
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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4. Walaa M. A. Elsherif, (2014(
Table (2): Incidence of different isolated non-O157 E. coli strains in the
positive samples based on their serological identification.
Serotypes of the
isolated strains
Raw Milk Talaga Cheese Ice Cream
NO./50 % NO./50 % NO./50 %
EHEC*
O26 2 4 - - 4 8
O111:H4 - - 2 4 3 6
ETEC**
O125:H21 1 2 - - 3 6
O127:H6 - - 2 4 1 2
EPEC***
O55:H7 - - - - 2 4
O44:H18 - - 1 2 - -
O86 2 4 - - 1 2
O114:H21 - - 1 2 - -
O119:H6 3 6 - - - -
O78 - - - - 1 2
EIEC****
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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5. Prevalence and significance of non O157 shiga toxin producing E. coli in milk and some …..
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
161

6. Walaa M. A. Elsherif, (2014(
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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7. Prevalence and significance of non O157 shiga toxin producing E. coli in milk and some …..
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11. Prevalence and significance of non O157 shiga toxin producing E. coli in milk and some …..
‫غير‬ ‫توكسين‬ ‫للشيجا‬ ‫المنتجة‬ ‫القولونية‬ ‫الرشريكية‬ ‫عترات‬ ‫تواجد‬ ‫أهمية‬O157
‫منتجاته‬ ‫وبعض‬ ‫اللبن‬ ‫فى‬
‫الشريف‬ ‫على‬ ‫محمود‬ ‫ول ء‬
‫مصر‬ ,‫اسيوط‬ ‫الحيوان‬ ‫صحة‬ ‫بحوث‬ ‫معهد‬ ,‫الذغذية‬ ‫صحة‬ ‫قسم‬
‫أ‬‫جريت‬‫هذة‬‫الدراسة‬‫لعزل‬‫اليشريشيا‬‫كول ى‬‫ذغير‬O157‫من‬‫اللبن‬‫الخام‬‫والجبن‬‫واليس‬
‫كريم‬‫بواقع‬150‫عينة‬‫مقسمة‬‫الى‬50‫عينة‬‫لكل‬‫تم‬ .‫منهم‬‫تجميع‬‫العينات‬‫من‬‫محلت‬‫بيع‬‫وتم‬ .‫اللبان‬
‫زرع‬‫العينات‬‫على‬modified vancomycin- trypticase soy broth (mvTSB‫لتنمية‬ (
‫الميكروب‬‫ثم‬‫تم‬‫نقلها‬‫على‬‫مستنبت‬‫خاص‬‫بها‬‫وهو‬Sorbitol MacConkey (SMAC) agar‫كما‬ .
‫تم‬‫تصنيف‬‫الميكروبات‬‫المعزولة‬‫كيميائيا‬‫وسيرولوجيا‬‫وللكشف‬‫عن‬‫العترات‬‫المفرزة‬‫للشيجا‬‫توكسين‬
stx1‫و‬stx2‫تم‬‫استخدام‬‫اختبار‬‫تفاعل‬‫البلمرة‬‫المتسلسل‬)PCR‫اثبتت‬ .(‫النتائج‬‫الولية‬‫بإستخدام‬
‫الختبارات‬‫الكيميائية‬‫ان‬66٪‫من‬‫عينات‬‫اللبن‬‫الخام‬,40٪‫من‬‫الجبن‬‫و‬70٪‫من‬‫عينات‬‫اليس‬‫كريم‬
‫ملوثة‬‫بميكروب‬‫الرشريشيا‬‫كول ى‬‫الغير‬O157 (non O157 STEC‫اما‬ .(‫سيرولوجيا‬‫فكانت‬‫النتائج‬
‫كالتالى‬20,٪12٪‫و‬30٪‫لكل‬‫من‬‫اللبن‬‫الخام‬‫والجبن‬‫واليس‬‫كريم‬‫على‬‫وب‬ .‫التوالى‬‫إ‬‫ستخدام‬‫تفاعل‬
‫البلمرة‬‫المتسلسل‬)PCR‫للعترات‬ (‫المصنفة‬‫سيرولوجيا‬‫كانت‬‫المعزولت‬O26 ، O111، O125,
O127‫فقط‬‫هى‬‫ال‬‫إيجابية‬‫للجينات‬‫المسؤله‬‫عن‬‫إفراز‬‫الشيجا‬‫ناقشت‬ .‫توكسين‬‫الدراسة‬‫خطورة‬‫اللبن‬
‫الخام‬‫ومنتجات‬‫اللبان‬‫المختلفة‬‫التى‬‫تعتبر‬‫مصدرا‬‫حيويا‬‫لنقل‬‫مثل‬‫هذة‬‫الميكروبات‬‫المسببة‬‫للمراض‬
nonO157 STEC‫هذا‬ .‫ما‬‫يفسر‬‫الحاجة‬‫إلى‬‫الرقابة‬‫الصارمة‬‫و‬‫الفعالة‬‫من‬‫النظافة‬‫و‬‫الممارسات‬
‫الصحية‬‫أثناء‬‫إنتاج‬‫اللبن‬‫ومنتجات‬‫اللبان‬‫كما‬ .‫المختلفة‬‫نوصي‬‫بضرورة‬‫ذغلي‬‫وبسترة‬‫اللبن‬‫قبل‬‫تناوله‬
‫عدم‬ .‫وتصفيتة‬‫رشراء‬‫اليس‬‫كريم‬‫إلمن‬‫مصادر‬‫موثوق‬‫فيها‬‫كالشركات‬‫الكبيرة‬‫لضمان‬‫السلمة‬
‫والجودة‬‫لكي‬‫ليتعرض‬‫المستهلك‬‫لخطورة‬‫هذا‬‫النوع‬‫من‬.‫الميكروبات‬
‫الكلمات‬‫الرئيسية‬‫كول ي‬ :O157‫ذغير‬،‫رشيجا‬‫توكسين‬‫والحليب‬‫ومنتجات‬‫الحليب‬،‫والجبن‬،‫واليس‬
‫كريم‬‫و‬PCR
167