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Middle East Journal of Applied

ISSN 2077-4613

Volume : 05 | Issue : 02 | April-June | 2015

Pages: 431-438

Diarrheagenic, Enteroaggregative, Shiga Toxin-Producing Escherichia coli and

Enterobacteriaceae in Retailed Raw Meat

El Gamel M.S.,2El Dairouty R.,2M.A. El-Shenawy,2Sahar H. S. Mohamed and 2Samy M. Abd


Microbiology Dept., Faculty of Science, Al Azhar Univ, Cairo, Egypt.

Dairy Dept. National Research Centre, 33 El-Bohouth St., (former El- Tahrir St.,) Dokki, Giza, Egypt. Postal
Code: 12622.

Prevalence, serotypes and virulence shiga toxins producing diarrheagenic, Enteroaggregative Escherichia
coli and related Enterobacteriaceae, as foodborne bacteria were determined in 75 retailed meat samples, as raw
meat 25, poultry meat 25 and Liver 25 samples, in Cairo and Giza area. Identification of diarrheagenic
Enteroaggregative E. coli (EAEC) and Enterobacteriaceae were performed using the traditional biochemical
tests and kits of HiBio-IDTM and Hi25TM Enterobacteriaceae identification kits KB003. Serological tests for
Escherichia coli were carried out using Kits for O antisera and H Pools Kits. Clump formation, biofilm assay
and the HEp-2 cell adhesion assay were performed to confirm Enteroaggregative E. coli. Immuno -Card STAT
EHEC Kit was used to detect the shiga toxins producer strains. Four groups of diarrheagenic E.coli (, EHEC,
ETEC, EPEC and EAEC) representing six serovars as O142:HN, O157:H7, O126:H27, O126:H7, O111:H21
and O128:H7 were detected in 9.3% of the seventy five retailed meat samples. Also, EAEC was successfully
isolated from 4% of raw meat and 4% of poultry meat samples, while all liver samples were negative. One out
of the tow EAEC strains, as serovar O111:H21, isolated from raw Poultry meat sample showed no shiga toxins
(ST1 or ST2) production, while the other EAEC strain, as serovar O128:H7, from raw meat Sample showed ST2
production. Any of the other diarrheagenic E. coli strains were found to produce neither of ST1 nor ST2. The
two positive samples, of raw meat and poultry, for (EAEC) were further examined for the incidence and
frequency of other bacteriological criteria as indicated in total aerobic colony count (TACC), and
Enterobacteriaceae related bacteria as coliform bacteria (100%), Yersinia enterocolitica (50%), Citrobacter spp
(50%), Klibsiella spp (50%), Proteus spp (50%), Enterobacter spp (50%) and Salmonella spp (50%) the most
hazardous foodborne bacteria. Though, raw meat and raw poultry meat distributed in Cairo and Giza markets
represent a risk for public health threatens via bad hygiene in locals and cross contamination.
Key words: Diarrheagenic Enteroaggregative E. coli, Shiga toxins, Enterobacteriaceae and raw meat and

Escherichia coli are now considered as one of the common diarrheagenic bacteria within the family
Enterobacteriaceae. Strains of E. coli that cause diarrhea are of six major categories: Enterohemorrhagic
(EHEC), Enterotoxigenic (ETEC), Enteroinvasive (EIEC), Enteropathogenic (EPEC), Enteroaggregative
(EAEC) and diffuse-adherent (DAEC) (Torres et al. 2005). More than 170 O serogroups and more than 60 H
types are currently recognized. Each category of diarrheagenic E. coli comprises a separate set of O: H
serotypes, and has distinct virulence attributes resulting in distinctive clinical syndromes and characteristic
epidemiologic patterns (Kaper et al., 2004).
EAEC has been recognized as an important agent of acute and prolonged diarrhea especially in children,
travelers diarrhea, and increasingly as an agent of outbreaks in developed countries (Nataro 2005). EAEC
strains are a highly Heterogeneous group consisting of about 90 identified serotypes, the most common being
O15:H18, O44:H18, O77:H18, O111:H12, O125, and O126 (Okeke and Nataro 2001). EAEC are defined by
their aggregative adherence to intestinal epithelial cells in a characteristic stacked-brick pattern (Nataro and
Kaper 1998). During pathogenesis, EAEC have adhered to the intestinal mucosa by plasmid encoded
aggregative adherence fimbria (AAF/IAAF/III), increased production of a mucus biofilm, mucosal toxicity due
to inflammation, and cytokine release (Torres et al. 2005).Other virulence factors of EAEC have been plasmid
enterotoxin (PET) (Eslava et al., 1998), and enteroaggregative heat stable toxin 1 (EAST-1) (Okeke and Nataro
2001). Tozzoli et al. (2014) investigated the possibility that, besides STEC and EAggEC, other pathogenic E.
coli could be susceptible to infection with stx-phages. So they suggested that the stx2-phages used may not have
Corresponding Author: Samy M. Abd elhamid, 2Dairy Dept. National Research Centre, 33 El-Bohouth St., (former ElTahrir St.,) Dokki, Giza, Egypt. Postal Code: 12622.


Middle East J. Appl. Sci.., 5(2): 431-438, 2015

specificity for E. coli adapted to the intestinal environment. On the other hand many different kinds of foods
have been identified as a potential source of STEC (Caprioli et al., 2005). Its usually involves in raw or
undercooked foodstuffs contaminated with E. coli either during primary production as slaughtering, milking or
further processing and handling and local cross contamination Gyles, (2007). Data on shiga toxin E. coli (STEC)
are reported annually on a mandatory basis by EU Member States to the European Commission and EFSA
(2011) based on Zoo noses Directive 2003/99/EC8. The most widely used analytical method only aimed to
detect STEC O157, whereas fewer investigations have been conducted with analytical methods aiming at
detecting all or selected serotypes of STEC. Also, due to EFSA (2011) reports during the years 2007-2009,
overall 0.3% - 2.3 % of fresh bovine meat samples and 0.7% - 3.2% for fresh sheep meat samples were found
positive for STEC, while no samples were positive for E.coli O157:H7.
Therefore, ruminants and in particular cattle, are considered to be the principal reservoir of E. coli, so
food of bovine origin such as beef and dairy products has often been associated with outbreaks (Perelle et al.,
2007. Shiga toxin-producing Escherichia coli (STEC) represents one of the least six different recognized
categories of diarrheagenic E. coli, Kaper et al., (2004). A large number of STEC serogroups as non-O157:H7,
O26: H11, O111: H2, O103: H2, O128 and O145:H28 have been associated with food borne illnesses and their
importance is increasing worldwide (Bettelheim 2007 and Conedera et al., 2007).

Materials and Methods

Collection of samples:
Seventy five total samples of raw meat (25), raw poultry meat (25) and liver samples (25) were randomly
collected from different areas of Cairo and Giza governorates. Collected samples were transferred in sterile
plastic bags in ice box to the laboratory as soon as possible to be examined.
Sample preparation
The methods described by FDA (2002) were followed as twenty five grams of each of meat, poultry or
Liver samples were homogenized with 225 ml sterilized buffer peptone water in sterile blender cups
Isolation and identification of E. coli:
Portions (10g/each) from center of each sample was extracted aseptically and homogenized with 90 ml
sterile enrichment broth (lactose broth ) incubated at 37 C for 24 hours. The enriched sample was cultured on
selective medium Levine Eosin Methylene Blue (EMB) agar (Becton Dickinson, Sparks, MD, USA). After
icubation typical colonies were taken into nutrient broth for further identification via biochemical analysis
(Priyanka and Alka 2008). Strains of E. coli isolates were stored in 5% glycerol supplemented broth at -70C
until use (Iseri, et al., (2011).
Rapid Identification of E. coli and Enterobacteriaceae:
Rapid biochemical identification of E. coli isolates and Enterobacteriaceae bacteria was carried out using
rapid test kits Hibio-ID and Hi25tm (Enterobacteriaceae identification kits KB003, address is HiMEDIA
laboratories Pvt. Limited, A-406 Bhaveshwar Plaza, Mumbai-400 086, India) in besides of the traditional
methods (FDA, 2002 and APHA, 1976).
Stereotyping of diarrheagenic and Enteroaggregative E. coli:
The biochemically confirmed E. coli strains (isolates) were submitted to the following serological typing:
A) Slide agglutination tests using O antiserum:
A slide agglutination test was followed using O antiserum for Escherichia coli Kits (BIORAD, France)
polyvalent and trivalent sera against E.coli serogroups. The test is based on agglutination, by specific sera of
bacteria possessing the corresponding antigens.
B) H agglutination of formalin killed culture:
Microtiter plate agglutination of formalin killed culture using H antisera (E. coli antiserum H Pool s
Kits) was used for serotyping H of the isolated E. coli strains according to the supplier instructions (SSI
Diagnostica, 2Herredsvejen Denmark) (3rd Edition, July 2011. 61289).
EAEC aggregative tests:
Biofilm assay:
Bacterial clump formation on liquid cultures was previously reported as a rapid test to EAEC. This
convenient test is also based on the biofilm formation of EAEC and useful for screening. Furthermore it may
overlook EAEC strains with weak biofilm formation (Albert et al., (1993).


Middle East J. Appl. Sci.., 5(2): 431-438, 2015

HEp-2 cell adhesion:

Aggregative adhesion to HEp2-cells was examined as described by Jenkins et al. (2006).
Shiga Toxins Production test:
E. coli strains were tested for shiga toxin production using Immuno Card STAT EHEC Kit (Meridian
Bioscience, Inc. USA). This is a rapid test for detecting shiga toxin production by E.coli, not only for shiga
toxin but also to differentiate between the two types of shiga toxin (shiga toxin type 1, ST1 and shiga toxin type
2 ,ST2) in broth methods, according to the kit supplier instructions.
EAEC in association with Enterobacteriaceae and other bacteriological criteria:
Bacteriological criteria such as total aerobic colony count (TACC), and incidence and frequency
distribution of other EAEC related Gram- negative bacteria "Enterobacteriaceae" as coliform bacteria, Yersinia
enterocolitica, E. coli O157: H7, Salmonella spp., Klebsiella spp., proteus spp. and Enterobacter spp. were
studded in association with the positive(EAEC) samples as follow:
Total aerobic colony count (TACC):
Total aerobic colony count (TACC) was carried out as the conventional method (FDA, 2002) using plate
count agar (Oxoid).
Determination of coliform bacteria:
Coliform group was determined using solid medium method onto plates of violet red bile agar (VRBA)
according to the method reported by FDA (2002). Suspected colonies were transferred (loopful) into tubes of
MacConkey broth medium (Oxoid, England.). Positive acid and gas tubes were further transferred into EC
broth. Positive tubes were streaked onto MacConkey agar (Merck, Germany) and tested for IMViC test for
typical E.coli and other coliform bacteria according to APHA (1976).
Detection of Escherichia coli O157: H7:
Samples dilutions were spread onto plates of Sorbitol MacConkey (SMAC) agar medium (Oxoid,
England). After incubation, Typical E. coli O157:H7 were further biochemical and serological tested with
seroscreen tests (G1390) (Oxoid) according to kits supplier instructions and FDA (2002).
Isolation and identification of Salmonellae:
Aseptically 25ml or grams of each sample was mixed with 225 ml of sterile lactose broth, mixed well
and incubated at 35 C for 24h.One ml of culture was transferred to 10ml of selenite cystein broth (SC) (Oxoid)
and incubated at 37 C for 72h. Plates of Salmonella & Shigella ager (SS agar) (Oxoid) were streaked every day
(3days) and incubated at 37 C for 24h. Lactose negative suspected Salmonella or Shigella spp. were
biochemically and serologically identified according to FDA (2002) and APHA (1976).
Detection of Yersinia enterocolitica:
Aseptically weight 25ml or grams of each sample into 225 ml peptone sorbitol bile broth (PSBB) (Oxoid,
M120) homogenized 30s and incubated at 37 Co for 48h modified. Spreading portion of 0.l ml PSBB was spread
onto the surface of Yersinia selective agar medium (Oxoid code CM653) supplemented with Yersinia selective
supplement (Oxoid, SR 109), and incubated at 25Co for 24-48h, the method and media were recommended by
FDA (2002).
Isolation and identification of other Enterobacteriaceae bacteria:
Isolation and identification of Citrobacter spp, Klibsiella spp, Proteus spp, and Enterobacter spp. were
carried out according to the methods and media outlined by FDA (2002).
Statistical Analysis:
Statistical analyses were performed using the GLM procedure with SAS (2004) software. Duncans
multiple comparison procedure was used to compare the means. A probability to P0.5 was used establish the
statistical significance.

Results and Discussion

Incidence and frequency distribution of diarrheagenic, Enteroaggregative E. coli in retailed raw meat samples.
Results in Table (1) reveal that two strains of Enteroaggregative E. coli (EAEC) were isolated from 2
samples, one from Poultry meat sample (4%), serotype O111:H21 and one from raw meat Sample (4%) serotype
O126:H7, while EAEC could not be isolated from any of Liver samples. The obtained results exhibted E. coli


Middle East J. Appl. Sci.., 5(2): 431-438, 2015

incidence than obtained by Maysa et al. (2014) who found (EAEC) E. coli, serotype O111:K58, in 20% beef,
20% beef luncheon and 10% chicken luncheon ( 40 samples/each) retailed in Cairo area markets. Also, similar
prevalence of diarrheagenic E. coli in beef products was recorded in Giza, Egypt by AL-Mutairi et al. (2011)
who detected diarrheagenic E. coli in 20% out of 75 beef meat samples. Moreover, high prevalence rate (30.8%)
of E. coli in beef meat products was recorded in Sharkia, Egypt by Mohamed et al. (2009). On the contrary,
almost similar prevalence (2%) of EAEC in meat and meat products were recorded by Hassanien, (2004) in
Qalyobia, Egypt.
Table 1: Incidence and frequency distribution of diarrheagenic E. coli serovar strains in retailed raw meat, poultry meat and
liver meat samples.
Raw meat
Poultry meat
2/75 2.7
*Total N=75 as n=25 for each of meat, poultry and liver samples ** non motile serovar







Similar Escherichia coli (EAEC) serovars were isolated from meat products by Gobran (1985) isolate E.
coli strain O126; O26. Also, EL-Mossalami et al. (1989) isolated O26 and O111; O128 from meat products
samples. Al-Mutairi et al. (2011) found that 80% of Escherichia coli isolates were enteropathogenic serovars,
O166, O78, O55, O26, O20, O25: K11, O119, O125: K70, O146, and O126.However, 6 diarrheagenic E. coli
serotypes as O142:HN, O157:H7 (EHEC), O126:H27 (EPEC), O126:H7, O111:H21 (EAEC) and O128:H7
(ETEC) were found in the retailed raw meat samples in the current study (Table, 1).
Results in Table (1), also revealed that 8% of the raw meat samples, 16% of the poultry meat and 4% of
the liver retailed meat samples contained diarrheagenic E. coli, representing 9.3% of the seventy five total
samples. On the other hand, EHEC group was found only in 2 poultry samples (2.7%) serotype O142:HN and
O157:H7, respectively. while EPEC group was found in 2 samples (2.7%) in both of raw meat and poultry meat,
one sample (4%) each, as serotype O126:H27. Furthermore, only one (1.3%), (ETEC) Enterotoxigenic E. coli
serovar O128:H7 was isolated from liver samples. Our results were matched with Maysa et al. (2014) results,
who isolated non-O157 (STEC) O26:K60, O111:K58 and O128:K67 from beef and 3 isolates of O55:K59 and
O111:K58 from chicken meat (10% out of 40 samples each). Also, El-Nawawi et al. (2012) isolated the serovar
O44 from chicken meat products in Egypt. Lower prevalence (2%) of diarrheagenic E. coli was recorded by
Zahran (2004) in foods of animal origin in Qalyobia, Egypt. On the contrary, higher prevalence of diarrheagenic
E. coli in chicken meat (25%) was recorded by Sharaf and Sabra (2012). Alonso et al. (2012) in Argentina
reported a predominance of EPEC contamination in chicken meat and chicken meat products. Al-Mutairi (2011)
found 12 Serotype out of 15 retrieved diarrheagenic E. coli isolates were Enteropathogenic (EPEC) with an
incidence of 80% as serovars O166, O78, O126, O55, O26, O20, O25: K11, O119, O125: K70, O146, and
O126. Similar E. coli serovars were isolated from meat products as reported by Gobran (1985), for O126; O26
and O26; EL-Mossalami et al. (1989) for O26 and O111; O128; Niazi and Refai (1988) for O119; O26; O128
and O111; Taha (1989) for O26; O111; O119; O127 and O26; Fahem (1993) for O111; O126; O125 and O26
and Saad et al. (1994) for O128. The public health importance of isolated Enterotoxigenic (ETEC) serovars had
been attributed to its enterotoxin, which is implicated in causing gastroenteritis, diarrhea in children as well as
food poisoning (Bryan, 1980). Although, E. coli O157:H7 was the most common EHEC strain in many regions
of the world; serotypes O5, O26, O91, O103, O111, O113 and O145 were also recognized as a serious threat to
public health and have been recovered from infected patients Cliver, (1990) and Perelle et al., (2007).
However, diarrheagenic and Enteroaggregative E.coli prevalence rates among studies; differ due to
several factors such as kind of samples and sampling (type, source/location and initial bacterial load),
environmental and seasonal variations and the detection methodology used (Temelli et al., 2012). Usually high
incidence is attributing to behavior of slaughtering and manipulation of food before and after reaching the
market. Moreover, Sharmin Zaman et al. (2015) reported that E. coli and Salmonella spp contaminated water for
washing; rinsing and food preparation was a high risk of contamination of the final food products.
Shiga toxins producing strains of diarrheagenic Enteroaggregative E. coli in raw meat and raw poultry meat
Results as shown in Table (2) revealed that out of the 6 diarrheagenic E. coli serotypes isolated from raw
meat, poultry and liver samples only one (4% of the raw meat samples and 1.3% of the total samples) was
producing shiga toxin2 (ST2). The shiga toxin producing strain was (EAEC) serotype O126:H7 that was
isolated from raw meat samples, contrarily the other EAEC strain O111:H21 was found non producer of any of
shiga toxins (STs). However, not any serotype of the other groups EHEC, EPEC or ETEC were not found to
produce shiga toxins.


Middle East J. Appl. Sci.., 5(2): 431-438, 2015

The obtained results were similar to that found by Hussein and Bollinger, (2005) for shiga toxin
producing strains in beef meat products. However, our results showed lower incidence for STs Producer strains
than those found by Farhan et al. (2014) who used the multiplex PCR for the detection of Stx1, Stx2, eaeA and
EHEC hlyA virulence genes and found that 8.7% out of 115 isolates of E. coli were positive for the target genes
of shiga toxin production. Controversially, there are several investigations reported the production of STs by
E.coli (Dhanashree and Mallya, 2008; Sharifi-Yazdi, 2011) and others did not (Dastmalchi and Ayremlou,
2012). Also, several reports have documented the isolation and serotyping of E. coli in beef meat products ALMutairi, (2011) and chicken meat products EL-Nawawi et al. 2012). However, limited informations are
available on the genotypic characterizations of Stx1 and Stx2 producing E. coli. Such studies are important
because STEC cause life threatening infections when transmitted to man through contaminated food of animal
Table 2: Incidence of serotypes shiga toxin producing E. coli strains in the retailed raw meat samples:
Types of
Toxin Incidence
Total toxin incidence
Shiga toxins
Within the types
Raw meat
Poultry meat
Liver samples
* O142:HN, O157:H7 (EHEC), O126:H27 (EPEC), O126H7, O111:H21 (EAEC) and O128H7 (ETEC).//** non motile E .coli.
Types of
Meat samples


Enterobacteriaceae in association with Enteroaggregative E. coli in raw meat samples:

Results in Table (3) also, show Enterobacteriaceae, and other related bacteria in association with the
incidence of Enteroaggregative E. coli in the 2 positive (EAEC) raw meat and poultry meat samples. Statistical
analysis of the obtained data, as shown in the table, revealed that means with the same letters are not
significantly different (p 0.05), while values are means standard error .One sample from twenty five poultry
meat sample (4%) positive for Enteroaggregative E. coli O111:H21 yielded TACC 6.47 log cfu/g, and positive
for coliform bacteria 4.47 log cfu/g. Also it was positive for Proteus spp, Enterobacter and Y. enterocolitica,
yielded 3.0, 2.69 and 2.17/ log cfu/g, respectively. Moreover, Citrobacter spp was detected in the sample
yielded 2.3 log cfu/g. On the other hand, one raw meat sample (4%) positive or EAEC O126:H7 showed lower
bacterial counts than poultry, yielded TACC 2.9 log cfu/g and infected with Klibsiella spp, coliform bacteria,
yielded 2.0 and 2.3 log cfu/g, respectively. The worsted the sample that was positive for Salmonella spp, as the
most hazardous bacteria of Enterobacteriaceae. Generally, Enterobacteriaceae as E.coli associated bacteria were
detected as coliform bacteria (100%), Yersinia enterocolitica (50%), Citrobacter spp (50%), Klibsiella spp
(50%), Proteus spp (50%), Enterobacter spp (50%) and Salmonella spp (50%).
Enterobacteriaceae group has an epidemiological interest and have the most important pathogens
associated with meat products as Salmonella spp., Campylobacter spp. and Escherichia coli (Borch et al., 1996).
Our results similar to those reported by Al-Mutairi (2011) who found that two Salmonella spp isolates were
retrieved from a total of 25 sausage samples with an incidence of 8%, only one Salmonella isolate was retrieved
from a total of 25 shawerma chicken samples with an incidence of 4%. Moreover, Salmonella spp were
recovered from meat products in nearly similar incidence to the reported results by many investigators, such as
Abd El-Aziz (1987) (10%); Ahmed (1988) (8%); El Mossalami et al. (1989) (6%) and El-Mossalami (2003)
(5%); Torky (2004) (5%); Siriken et al. (2006) (7%). Contrarily, other investigators reported that they not detect
Salmonella in meat and meat products as El-Khateib (1994), Vazgecer et al. (2004) and Ismail (2008). Similar
investigation was carried out by Sharmin Zaman et al. (2015) who studied the association between the incidence
of E. coli and total aerobic colony cunt (TACC), total coliform count and the presence of E. coli O157:H7 and
Salmonella spp in 13 food categories.
Proteus spp in association with EAEC was present in our poultry meat samples (50%), similar
observations were recorded by Ahmed (1988), Gobran (1985) and Lofti et al. (1990) who had isolated different
species as Proteus rettegeri (20%), Proteus morganii (4%) and Proteus vulgaris (16%). Moreover, Ammar
(2005) found Proteus spp (42.7%) of the examined meat product samples. Yet, E. coli in association with
proteus spp still considered as an indicator of contamination of meat product during any of the processing,
handling, or storage stages. Also, Klibsiella spp were isolated in the current study from raw meat samples
(50%), similar to that was obtained by El-Khateib (1982), 47.7%. Contrarily, the incidence of Klibsiella spp in
our study were much lower than that reported by Al Mutairi (2011), Roushdy (1971), Gobran (1985) and Lofti
et al. (1990) in meat and meat products, from 4 to 20%. While, Ammar (2005) isolated 24% of Klibsiella
oxytoca in meat and meat products.


Middle East J. Appl. Sci.., 5(2): 431-438, 2015

However, undercooked meat, poultry and their products have caused many food poisoning outbreaks
associated with E. coli and Enterobacteriaceae group of bacteria which is present in the feces, intestines and hide
of healthy cattle and poultry from where it can potentially contaminate the raw meat and poultry during the
slaughtering process Duffy et al. (2003).
Table 3: Enteroaggregative E. coli in association with other pathogenic microorganisms, Enterobacteriaceae, in raw meat
and some meat products collected from Cairo and Giza markets.
No. of
Sample Type

Food borne microorganisms from Enterobacteriaceae


Log cfu/



E. coli

Log cfu/ gm

Log cfu/




Log cfu/




Row meat


























No. of total
(%) of positive


Log cfu/





Poultry meat




2.9 0b


ND (Not detected), + (Positive).

Means with the same letters are not significantly different (p 0.05). Values are means standard error

In conclusion, this study revealed that raw meat and raw poultry meat were contaminated by pathogenic
E. coli strains and represented a public health hazard, therefore measures should be taken to avoid such
contamination, particularly through cross contamination in locals. Moreover, there is an increasing demand for
improving diagnostic procedures for the detection of Shiga Toxic Escherichia Coli (STEC) in foods of animal
origin because their ability to cause life-threatening diseases.

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