Prevalence of Escherichia Coli in Some Selected Foods and Children Stools with Special Reference to Molecular Characterization of Enterohemorrhagic Strain

Corresponding Author: Farhan, R. Biotechnology Research Center, Suez Canal University, Egypt Email: salamaa_79@hotmail.com Abstract: Escherichia coli is one of the most important etiologic agents of childhood diarrhea that represents a major public health problem in developing countries and now is being recognized as emerging enteropathogens in the well developed countries. Among the Diarrhegenic Escherichia Coli (DEC) this work was focused on the Enterohemorrhagic Escherichia Coli (EHEC) that produce Shigatoxins (Stxs). The infection is mainly transmitted through food of bovine origin such as beef and dairy products that has been often associated with outbreaks which ranged from mild diarrhea to the life-threatening hemolytic uremic syndrome. The aim of this study was to evaluate the incidence of potentially virulent STEC isolates from minced meat, some selected dairy products and from children clinical cases. Out of 360 samples, a total of 115 E.coli isolates were recovered as following 35 (30.43%) isolate from meat, 21 (18.26%) from raw milk, 31 (26.96%) from cheese and 28 (24.35%) from children stool. The identification were based on Microscopical examination, Biochemical identification and Serotyping. Twenty eight isolates were identified serologically. O26:K60 serotype 6 (21.4%) was found to be the most prevalent serotype. Screening for virulence genes (Stx1, Stx2, eaeA, hlyA) was done using multiplex PCR, which revealed detection of the target genes in 10 out of 115 (8.7%) examined samples. About 7 (70%) samples possessed eaeA gene alone; while 2 (20%) samples contained Stx2 gene. Both Stx1 and eaeA genes were detected in 1(10%) sample only.

The infection is mainly food-borne, but it can also be acquired by person-to-person spread or direct contact with animals. 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). Moreover, an increasing number of episodes have been associated with consumption of fruits and vegetables fertilized with ruminants' manure or contaminated during harvesting or processing (Conedera et al., 2007).
Among the six recognized diarrhegenic categories of E. coli, ETEC is the most common, particularly in the developing countries (Qadri et al., 2005). EHEC is the most important recently emerged group of food-borne pathogens. It can cause severe gastrointestinal disease, including fatal infections and is being detected more frequently worldwide. EHEC strains not only produce potent cytotoxins (verotoxins) but have also acquired the ability to adhere to the intestinal mucosa in an intimate fashion (Fagan et al., 1999). Specific virulence factors such as enterotoxins and colonization factors differentiate ETEC from other categories of diarrheagenic E. coli (Qadri et al., 2005). Many assays for the detection of diarrheagenic E. coli are available, such as biochemical reactions, serotyping, phenotypic assays based on virulence characteristics and molecular detection methods (Nataro and Kaper, 1998;Nguyen et al., 2005). Among these, Polymerase Chain Reaction (PCR) is one of the molecular biology-based detection methods, is a commonly used method that gives rapid, reliable results and that also has a high sensitivity and a high specificity (Nguyen et al., 2005).
Recently various multiplex PCR methods have been developed for the simultaneous detection of several pathogenic genes in one PCR reaction. These methods showed high sensitivity and specificity for identification of human diarreheagenic E. coli (Arif and Salih, 2010).
The aim of this study was to evaluate the prevalence of E. coli in collected food of animal origin such as minced meat, raw milk, kareesh cheese and in fecal samples of clinical cases of children and to investigate the characteristics and efficacy of multiplex PCR to simultaneously detect diarrheagenic E. coli.

Collection and Sampling Process
Three hundred samples of minced meat, fresh raw milk and Kareesh cheese (one hundred of each) are randomly collected from butcheries, supermarkets, dairy stores and milk vendors. In addition, 60 random diarrheal stool samples were collected from different hospitals and private laboratories of children clinical cases. All samples were collected from different localities in Ismailia Governorate; the samples were transported to the laboratory without delay in a temperature-regulated ice box to be examined. (ISO 16654:2001;De Boer and Heuvelink, 2000) About 25 g. or mL of examined samples were added to 225 mL of modified Tryptone Soya Broth ([Difco) supplemented with novobiocin solution (20 mg L −1 ). The samples were placed in individual bags homogenized in a stomacher for 2 min and incubated for 24 h at 37°C. Each recovered sample was streaked onto modified Sorbitol MacConkey agar and incubated at 37°C for 24 h. Both of Cexifime and potassium tellurite were added to the SMAC to increase selectivity especially for heavily contaminated samples.

Enrichment and Recovery of E.coli
In parallel, a loopful from the incubated broth was streaked into Eosin Methylene Blue Agar. The inoculated plates were incubated at 37°C for 24 hr. Typical colonies of E. coli appear greenish, metallic and with dark purple center. Suspected colonies were purified and sub-cultured onto nutrient agar slopes and incubated at 37°C for 24 hr, then stored until further investigation was carried out.

Confirmation of the Isolates
Were carried out according to Edwards and Ewing's Identification of Enterobacteriaceae (Farmer et al., 1985).

Serotyping
The confirmed E. coli isolated strains were serological identified by Central Laboratories of Ministry of Health-Bacteriology Department-Cairo.

DNA Extraction
DNAs were extracted from reference strain that obtained from the Central Laboratories of the Ministry of Health Bacteriology Department-Cairo and isolated strains by the method described by (Yokoyama, 1993;Toma et al., 2003). The reference and isolated strains were cultured in 2 mL of Luria-Bertani broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl) and incubated overnight at 37°C with shaking. Thirty-six microliters of broth culture was added to 4 µL of 10x Tris-EDTA buffer (100 mM Tris-HCl, 10 mM EDTA, pH 8.3) and 60 µL of 2x proteinase K buffer (100 mM KCl, 20 mM Tris-HCl, 5 mM MgCl2, 1% Tween 20, 800 µg of proteinase K/mL, pH 8.3). After incubation for 90 min at 56°C and 10 min at 95°C, the sample was centrifuged at 10,000 × g for 1 min and the supernatant was used as DNA template (Fig. 1). The DNA quantity and purity were assessed spectrophotometrically at 260-280 nm, which was determined with NanoDrop ND-1000 full spectrum UV-Vis spectrophotometer (USA).  Molecular Characterization (Fagan et al., 1999) Multiplex PCR for detection of Stx 1 , Stx 2 , eaeA and EHEC hlyA gene sequences was performed with an eppendorf MasterCycler Gradient (Eppendorf-Netheler-Hamburg, Germany). Oligonucleotide primers were manufactured commercially (Metabion International AG, Germany). Primers and the predicted lengths of PCR amplification products are listed in Table 1. PCR assays were carried out in a 50 µL volume containing 2 µL of nucleic acid template prepared from isolated cultures (approximately 60 ng of DNA) or 1 µL (approximately 30 ng) of nucleic acid template prepared by using reference E. coli O157:H7 isolate which obtained from the Central Laboratories of the Ministry of Health Bacteriology Department-Cairo, 25 µL of Master Mix (Fermentas, USA). Temperature conditions consisted of an initial 95°C denaturation step for 3 min followed by 35 cycles of 95°C for 20 s, 58°C for 40 s and 72°C for 90 s. The final cycle was followed by 72°C incubation for 5 min (Fagan et al., 1999).
Ten microliters of PCR products were then electrophoresed on a 2% Agarose gel (Bioshop, Canada), stained with ethidium bromide (Sigma, USA) and visualized by UV transillumination. The buffer in the electrophoresis chamber and in the agarose gel was 1X TAE buffer. The amplicon was imaged with a gel documentation system LTF (Labortechnik, Germany).

Results
Percentage of isolated E. coli strains: A total of 115 E. coli strains were isolated regarding to the culture media and were confirmed by biochemical tests. About 35 (30.43%) strains were isolated from minced meat, 21 (18.26%); raw milk, 31 (26.96%); Kareesh cheese samples and 28 (24.35%) from the stool samples.

Antimicrobial Susceptibility Testing
All Identified serotypes were susceptible to different 8 antimicrobial agents. The results obtained in this study for each tested antimicrobial agent, have been reported according to (CLSI, 2011). The strains were classified as Sensitive (s), Intermediate (I) or Resistant (R) based on the inhibition zone diameter. Serological identification: All E. coli isolates were subjected to serological identification using 9 polyvalent antisera then we applied monovalent antisera to positive reactions   Table 3 results of antimicrobial susceptibility.

Multiplex PCR
The multiplex PCR detected targeted genes in 10 out of 115 (8.7%) collected samples. Seven samples possessed eaeA gene alone (70%); while 2 samples contained Stx 2 gene. Both Stx1 and eaeA genes were detected in one sample. Table 4 to 6 the illustrated results of multiplex PCR.

Discussion
E. coli, which are normal flora of the human and animal intestine, have been identified as a leading cause of food borne illness all over the world. E. coli strains have previously been isolated from meat samples and have also been implicated in the contamination of vegetables such as lettuce (Hilborn et al., 1999;Enabulele and Uraih, 2009). In our study, the incidence of E. coli was 35% in minced meat and 26% in raw milk and kareesh cheese. This was lower than that reported by other authors (Al-Gallasa et al., 2002;Hussein and Sakuma, 2005;Temelli et al., 2012); while it was higher than that reported by (Abdul-Raouf et al., 1996;Heuvelink et al., 1998;Dontorou et al., 2003). There are many factors affecting the differences in prevalence rates among studies; these differences are mainly related to samples and sampling (type, source/location and initial bacterial load), environmental and seasonal factors and the detection methodology used (Temelli et al., 2012). Usually high incidence is attributing to behavior of slaughtering and manipulation of food before reaching the market. Additionally, in this study E. coli incidence in diarrheal samples was 24.35% which almost the same reported in other studies (Nguyen et al., 2005;Bueris et al., 2007) and lower than that reported by (Keskimäki et al., 2000;Amisano et al., 2011). Regarding to Serotyping, the most identified isolates in our study were EHEC strains. 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 (Fagan et al., 1999;Perelle et al., 2007). Of the typeable E. coli isolates found, O26:K60 was the most common type with a percentage of 21.4 (6 isolates). Among the identified serotypes, O124:K72 was representing 14.3% which was higher than that reported in Kalyobia governorate, Egypt (Hassanien, 2004). In addition O44:K74, O114:K90 and O25:K11 are also found in 10.7%. In our study, no E. coli O157:H7 was isolated and that similar to that reported in other studies (Dontorou et al., 2003;Fantelli and Stephan, 2001).
Concerning the antibiotic susceptibility of the seroidentified strains. All strains were resistant to one or more tested antibiotics. This was similar to that conducted by (Radu et al., 2001). Twenty two out of 28 (78.57%) were resistant to at least one antibiotic and 6 strains (21.43%) were resistant to 3 or more antibiotics. These results show higher resistance profiles than that reported by (Dontorou et al., 2003). No antimicrobial resistance was observed against Amikacin (Aminoglycoside), while other studies showed resistance pattern that reach up to 10% (Rigobelo et al., 2010;Aly et al., 2012). Meanwhile resistance towards Erythromycin was 96.43%; which higher than that reported in other studies (Harakeha et al., 2005). This high percentage of Erythromycin resistance refers to its frequent use in our country. Such resistance may not only be a direct concern to human health, but also, it is important because it could be transferred to other important pathogenic serotypes (Dzidic and Bedeković, 2003).
By using the multiplex PCR for the detection of Stx 1 , Stx 2 , eaeA and EHEC hlyA virulence genes out of 115 isolates which were identifies as E. coli by biochemical tests, 10 samples showed positive results for the target genes. In the present study, eaeA positive E. coli isolates that were negative for other virulence genes were found to be predominant (70%). This result was higher than that reported in other studies (Dhanashree and Mallya, 2008;Sharifi-Yazdi, 2011). EHEC hlyA gene was not found in any of examined isolates; which contrary to what found in other studies (Paton and Paton, 1998;Dastmalchi and Ayremlou, 2012). Two isolates (20%) carried Stx 2 gene which lower than that reported by Sharifi-Yazdi et al. (2011;Dastmalchi and Ayremlou, 2012). One isolate only (10%) showed association between stx 1 and eaeA genes.

Conclusion
This study revealed that minced meat, raw milk and kareesh cheese were contaminated by E. coli and represented a public health hazard, therefore measures should be taken to avoid such contamination and the product should be heat treated during manufacturing or before consuming. There is an increasing demand for improved diagnostic procedures for the detection of Shiga Toxigenic Escherichia Coli (STEC) in fecal samples and, in foods of animal origin because their ability to cause life-threatening diseases. However, considering the difficulty of performing phenotypic assay in some laboratories, the multiplex PCR can be used as a replacement of such conventional methods in detection of E. coli strains particularly the emerging ones.

Author's Contributions
All authors equally contributed in this work.

Ethics
This article is original and contains unpublished material. The corresponding author confirms that all of the other authors have read and approved the manuscript and no ethical issues involved.