Molecular characterization of diarrheagenic Escherichia coli isolated from meat products sold at Mansoura city, Egypt

Food Control 25 (2012) 159e164

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Molecular characterization of diarrheagenic Escherichia coli isolated from meat products sold at Mansoura city, Egypt Mahmoud Ahmed Mahmoud Mohammed* Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 July 2011 Received in revised form 3 October 2011 Accepted 12 October 2011

This study was carried out to determine the serological characteristics and virulence-associated genes of 32 Escherichia coli strains isolated from different meat products. Serotyping of somatic (O) and flagellar (H) antigens reveled that 3 strains were typed into 2 serogroups; O121:H19 (2 strains) and O148:H8 (1 strain), while the other 29 strains were not agglutinated with any serum. For molecular characterization, multiplex PCR has been performed by combining seven primer pairs specific for enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), and enteroinvasive E. coli (EIEC). The targets selected for each group were the genes encoding heat-labile (LT) and heat-stable (ST) toxins for ETEC isolates, eae, stx1 and stx2 for EHEC isolates, eae and bfpA for EPEC isolates, and ipaH for EIEC isolates. This facilitates simultaneous identification of the four different categories of diarrheagenic E. coli in a single reaction. Amongst the 32 E. coli strains tested, eleven (37.5%) were potentially diarrheagenic. Five of which (15.63%) were ETEC, three (9.38%) were EHEC, two (6.26%) were EPEC, and one strain (3.13%) was EIEC. The results appear to indicate that virulence gene-carrying E. coli strains are a normal part of intestinal bacterial populations that may be present among high numbers of E. coli contaminating meat products which do not necessarily correlate with disease. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Meat products Diarrheagenic E. coli Multiplex PCR

1. Introduction The Gram-negative bacterium, Escherichia coli is a prominent member of the bacterial microbiota of the environment and in the feces of many species of birds and mammals. It is estimated that 1e4% of all cultivable bacteria of the colon are E. coli bacteria, and up to 1010 CFU E. coli bacteria can be detected in 1 g of feces (Selander, Musser, Caugant, Gilmour, & Whittam, 1987). Nonpathogenic E. coli (commensal E. coli) strains are thought to maintain the physiological milieu of the gut and support digestion as well as defend against enteric pathogens. Other E. coli strains carry and express virulence genes that cause severe outbreaks of diarrhea (diarrheagenic E. coli) (Wieler et al., 2001). Virulence genes are often located on transmissible genetic elements and can thus be transmitted to receptive E. coli recipient strains. In addition to virulence factor typing, serotyping of pathogenic E. coli strains is routinely used to classify isolates and to determine associations between serotypes, virulence, and epidemiology (Dobrindt, Hentschel, Kaper, & Hacker, 2002).

* Tel.: þ20 502372593; fax: þ20 50 2379952. E-mail address: [email protected]. 0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2011.10.026

Cattle are considered the major reservoir of diarrheagenic E. coli especially EHEC and the main source for contamination of human food supply. Fecal contamination of meat and milk are important routes through which these pathogens enter the food chain, together with the contamination of vegetables with cattle manure (Karch, Bielaszewska, Bitzan, & Schmidt, 1999). There are many vectors that can be used to transfer E. coli on and/or into meat products. The feces of the animal can be transferred on the hides and carcass, the equipment can be contaminated, personnel might not use proper hygienic practices, airborne contamination, and rodents, insects, and other animals are all potential sources (Laury, Echeverry, & Brashears, 2009). Five categories of E. coli have been well associated with diarrhea in several epidemiological studies (Nataro & Kaper, 1998; Novicki, Daly, Mottice, & Carroll, 2000): enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and enterohemorrhagic E. coli (EHEC) which mostly regarded as Shiga toxin-producing E. coli (STEC). The virulence mechanisms that characterize these categories of E. coli are genetically encoded by chromosomal, plasmid, and bacteriophage DNAs and are represented by several virulent genes. These genes include eae (attaching and effacing lesions), bfpA (localized adherence), the gene encoding enteroaggregative adherence, ipaH

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(enteroinvasive mechanism), the genes encoding heat-labile toxin (LT) and heat-stable toxin (ST), and stx1 and stx2 (Shiga toxins). To correctly identify diarrheagenic E. coli strains, these organisms must be differentiated from nonpathogenic members of the normal flora. Serotypic markers correlate, sometimes very closely, with specific categories of diarrheagenic E. coli; however, these markers are rarely sufficient to reliably identify a strain as diarrheagenic. Some 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 & Kaper, 1998). PCR is one of the molecular biology-based detection methods commonly used to give rapid, reliable results with a high sensitivity and high specificity (Stacy-Phipps, Mecca, & Weiss, 1995). In order to detect different categories of diarrheagenic E. coli, it is necessary to run several individual PCRs with different primer pairs which are very laborious and time consuming. Therefore, various multiplex PCR methods have been developed for the simultaneous detection of several pathogenic genes in one PCR reaction (Aranda, Fagundes-Neto, & Scaletsky, 2004; Pass, Odedra, & Batt, 2000; Paton & Paton, 1998). In this study, we used a multiplex PCR method (Aranda et al., 2004) by combining seven primer pairs in a single reaction to identify four categories of diarrheagenic E. coli, ETEC, EHEC, EPEC, and EIEC, isolated from meat products examined during a previous study (Mohammed, 2010).

of phenylalanine; malonate utilization; esculin hydrolysis; fermentation of arabinose, xylose, adonitol, rhamnose, cellobiose, melibiose, sucrose, trehalose, raffinose and glucose; production of indole; and production of acetoin; in addition to testing the strains for production of cytochrome oxidase. 2.3. Serotype identification Serotyping of O antigen (somatic lipopolysaccharide) and H antigen (flagellar) of motile strains was performed at the Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan, by conducting latex agglutination test (Presterl, Nadrchal, Wolf, Rotter, & Hirschl, 1999) with the use of E. coli antisera set 1 and set 2 (Denka Seiken, Tokyo, Japan). 2.4. Reference strains The reference strains used for PCR (listed in Table 1) are kindly provided by Prof. Trout Tobe (Laboratory of Infectious Disease Control, Department of Microbiology and Immunology, Graduate School of Medicine, Osaka University, Osaka, Japan). The reference strains are pathotypes of the ETEC, EHEC, EPEC, and EIEC. E. coli K12 DH5a was used as a negative control strain since it is a nonpathogenic strain and does not possess any virulent gene in its genome. 2.5. DNA extraction

2. Materials and methods 2.1. Bacterial strains The E. coli strains used in this study were isolated during our investigation of 100 samples of meat products (20 each of ground beef, beef sausage, beef burger, kofta, and beef luncheon) purchased from different supermarkets distributed in Mansoura city, Egypt. The samples were tested for the occurrence of E. coli O157:H7 according to the standard method reported by ISO (2001). Briefly, 25 g from each meat sample were blended with 225 ml of sterile modified tryptone soya broth (Oxoid CM0989) containing novobiocin (Sigma) (final concentration 20 mg/ml). The suspension was then incubated at 37  C for 18e24 h. Enriched culture was plated onto sorbitol MacConkey agar (Oxoid CM0813) supplemented with cefixime and potassium tellurite (Oxoid SR0172E) (CTSMAC). The plates were incubated at 37  C for 24 h then examined for typical E. coli O157:H7 colonies, i.e. colorless, smooth, circular and entire edge colonies with brown centers. Five typical presumptive E. coli O157:H7 colonies (sorbitol non-fermenting) and also red colored colonies (atypical E. coli O157:H7) from each plate were subcultured onto nutrient agar (Oxoid CM0003) and identified using a combination of biochemical tests and commercially available latex agglutination kits for O157 and H7 antigens (Wellcolex, Merseyside, UK). Out of 69 presumptive isolated stains, only 32 strains were identified as E. coli using standard microbiological techniques and non-O157:H7 by latex agglutination kits. These 32 strains were further subjected to serological and molecular characterization.

All strains examined by PCR were grown overnight on MacConkey agar plates at 37  C. DNA was extracted from the bacterial strains according to the method of Choo et al. (2007). Briefly, one bacterial colony from each strain was resuspended in 50 ml of deionized water, followed by boiling the suspension for 5 min, and centrifuging it at 10,000  g for 1 min. The supernatant was then used as the DNA template for PCR. Stocks of each isolate were kept at 70  C for further characterization. 2.6. Application of multiplex PCR Primer sets for PCR amplification of the target genes of examined E. coli strains are listed in Table 2. The target genes selected for each category were the LT and ST for ETEC, eae, stx1 and stx2 for EHEC, eae and bfpA for EPEC isolates (detection of eae confirms the presence of typical and/or atypical EPEC strains, while bfpA confirms the presence of the bundle-forming pilus major subunit that is found only in typical EPEC strains), and ipaH for EIEC isolates. The multiplex PCR was performed according to the method reported by Aranda et al. (2004) with some modifications. The optimized protocol was carried out with a 50 ml mixture containing 10 mM TriseHCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, a 2 mM concentration of each deoxynucleoside triphosphate, 1.5 U of Taq DNA polymerase (Takara Bio Inc., Shiga, Japan), 2 ml of the DNA template, and 2 ml the PCR primers. The optimal concentration of each primer pair in the reaction mixture was determined empirically. The concentration for each primer pair used in the final reactions is given in Table 2. The PCR mixtures were then subjected

2.2. Biochemical characterization The strains were examined for biochemical properties using API Rapid 20E system (bio-Mérieux, Marcy l’Etoile, France) according to the manufacturer’s instructions. Twenty biochemical tests were performed in the API cupules. These tests include o-nitrophenyl-bD-galactosidase (ONPG test); lysine decarboxylase; ornithine decarboxylase; urease production; citrate utilization; deamination

Table 1 E. coli reference strains used in this study. Category

Serotype

Positive genes

ETEC EHEC EPEC EIEC

H10407 O157:H7 sakai B171-8 1438e1

LT, ST eae, stx1, stx2 eae, bfpA ipaH

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Table 2 Primer sets for PCR amplification of the target genes of examined E. coli strains. Target gene

Primer sequence (50 e30 )

Amplicon size (pb)

eae

Forward: 50 -CTGAACGGCGATTACGCGAA-30 Reverse: 50 -CCAGACGATACGATCCAG-30 Forward: 50 -AATGGTGCTTGCGCTTGCTGC-30 Reverse: 50 -GCCGCTTTATCCAACCTGGTA-30 Forward: 50 -GGCGACAGATTATACCGTGC-30 Reverse: 50 -CGGTCTCTATATTCCCTGTT-30 Forward: 50 -ATTTTTMTTTCTGTATTRTCTT-30 Reverse: 50 -CACCCGGTACARGCAGGATT-30 Forward: 50 -GTTCCTTGACCGCCTTTCCGATACCGTC-30 Reverse: 50 -GCCGGTCAGCCACCCTCTGAGAGTAC-30 Forward: 50 -ATAAATCGCCATTCGTTGACTAC-30 Reverse: 50 -AGAACGCCCACTGAGATCATC-30 Forward: 50 -GGCACTGTCTGAAACTGCTCC-30 Reverse: 50 -TCGCCAGTTATCTGACATTCTG-30

917

5

Reid, Betting, & Whittam, 1999

326

5

Gunzburg et al., 1995

450

5

Stacy-Phipps et al., 1995

190

10

Stacy-Phipps et al., 1995

600

8

Sethabutr et al., 1993

180

4

Paton & Paton, 1998

255

3

Paton & Paton, 1998

bfpA LT ST ipaH stx1 stx2

to the following cycling conditions: 50  C (2 min, 1 cycle); 95  C (5 min, 1 cycle); 40 cycles of 95  C (40 s), 55  C (1 min), and 72  C (2 min); and a final extension step at 72  C (7 min, 1 cycle); in a thermal cycler (GeneAmpÒ PCR System 9700, Applied Biosystems, Foster City, USA). PCR products (10 ml) were visualized after electrophoresis in 2% agarose gel in Trisborate-EDTA buffer and ethidium bromide staining. In all experiments, the DNA mixture from the prototype EPEC, ETEC, EHEC, and EIEC strains served as the positive control, while E. coli K12 DH5a was the negative control. If the result was negative, the sample was considered negative for diarrheagenic E. coli. If the multiplex PCR was positive, the sizes of the bands on the gel were compared with those of the marker bands in order to identify certain kinds of diarrheagenic E. coli strains. Each positive strain was independently tested by PCR with a primer specific for a suspected diarrheagenic E. coli isolate from the multiplex PCR. 3. Results and discussion The sixty nine strains isolated were allowed to grow on SMAC to cover the plate with a sorbitol non-fermenting (colorless) and sorbitol fermenting (pink) colonies. Each colony was individually taken for subsequent testing. Only 32 isolates were Gram-negative rod-shaped bacteria identified following staining. All of these 32 strains showed negative reaction for VogeseProskaur, hydrogen sulfide, citrate, oxidase and urease production, while they were positive for indole, and hence, they considered as E. coli strains, but none of them were O157:H7 by using latex agglutination kits. The identified 32 different E. coli strains were taken for conducting this study. 3.1. Biochemical characterization The isolates were confirmed as E. coli following analysis with API Rapid 20E. The API Rapid 20E profile of 29 (90.6%) tested strains was 7045251, very good identification with identity % ¼ 99.5%. However, three (9.4%) strains revealed atypical phenotype and showed the same results except for a single biochemical test. Of these 3 strains, one showed negative reaction for lysine decarboxylase, the second exhibited a negative reaction for rhamnose fermentation, while the last revealed a negative reaction for xylose fermentation. 3.2. E. coli serotyping Differences between strains of E. coli lie in the combination of different antigens they possess. There are three types of antigens:

Primer conc. (pmol)

Reference

the somatic lipopolysaccharide antigen (O), the flagellar antigens (H), and the capsular antigens (K). There are approximately 174 Oantigens, 56 H-antigens, and 103 antigens that have been identified and newly emerged serotypes are still recognized (Toldra, 2009). In the present work, only 3 E. coli strains could be identified. Two strains (isolated from ground beef and beef burger) were serotyped as O121:H19 and the third (isolated from ground beef) is serotyped as O148:H8. The other 29 strains could not be serotyped (ONT:HNT) using the current available antisera (Table 3). 3.3. Multiplex PCR for verification of diarrheagenic E. coli E. coli serotyping is an important technique for making the proper diagnosis and epidemiological investigations during foodborne outbreaks. However, novel E. coli serotypes frequently emerge as intestinal pathogens and, when recognized, are included in a specific category of diarrheagenic E. coli. Thus serotyping alone cannot be relied on for categorizing a strain of E. coli, and the identification of specific virulence characteristics/genes must also be performed (Barlow, Hirst, Norton, Asshhurst-Smith, & Bettelheim, 1999). PCR is a powerful molecular biology technique for the detection of target DNA of many kinds of pathogens in various clinical specimens and food samples. It is not only highly sensitive and specific, but it also provides rapid and reliable results. It can help to distinguish diarrheagenic E. coli isolates from those of the normal flora. The identification of diarrheagenic E. coli strains through detection of virulence factors which affect the host cells encoded by virulence genes have been extensively studied and several PCR methods have developed (Aranda et al., 2004; Gunzburg, Tornieporth, & Riley, 1995; Kimata et al., 2005). In this study, the multiplex PCR used for identification of tested E. coli strains was highly specific with the primers chosen for the detection of four categories of diarrheagenic E. coli. It showed positive results for the diarrheagenic E. coli strains tested and negative results for all non-diarrheagenic E. coli strains, indicating the high degree of specificity of the assay.

Table 3 Identification of diarrheagenic E. coli strains. Pathotype

Serotype

Number

Positive genes

ETEC EHEC

Ont:Hnt O121:H19 O148:H8 Ont:Hnt Ont:Hnt Ont:Hnt Ont:Hnt

5 2 1 1 1 1 21

LT eae, stx1, stx2 stx2 eae, bfpA eae ipaH e

EPEC EIEC Nonpathogenic

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In order to evaluate the multiplex PCR system, the multiplex PCR profile of the reference strains was tested for detection of the 7 virulence genes selected (Fig. 1). ETEC strain show specific bands of LT (450 bp) and ST (190 bp), while EHEC give three bands for eae (917 bp), stx2 (255 bp) and stx1 (180 bp), whereas EPEC strain was positive to eae (917 bp) and bfpA (326 bp), and finally EIEC give only one band specific for ipaH (600 bp). Eleven (37.5%) of the 32 tested strains in the present study were potentially diarrheagenic strains of E. coli. The frequency of isolation of the different diarrheagenic strains revealed that five strains (15.63%) were ETEC, three (9.38%) were EHEC, two (6.26%) were EPEC, and one strain (3.13%) was EIEC (Fig. 2). None of the other 21 E. coli strains had containing any of the target genes. Of the ETEC strains isolated, all the five positive ETEC strains produced heatlabile (LT) toxin only. One EPEC strains isolated was typical (positive eae and bfpA) and one was atypical (positive eae only). Multiplex PCR profile of positive diarrheagenic E. coli strains (Fig. 3) revealed the presence of LT gene in ETEC, eae, stx1, and stx2 genes in EHEC (O121:H19), eae and bfpA genes in EPEC, ipaH gene in EIEC, and stx2 gene in EHEC (O148:H8). To verify the existence of these virulent genes, single PCR of each positive strain was independently conducted with a primer specific for a suspected diarrheagenic E. coli isolate from the multiplex PCR (Fig. 4). Our results confirm the possibility that multiple types of diarrheagenic E. coli strains can be detected in food samples. Genes encoding for virulence factors were found in food that were both associated and nonassociated with the occurrence of cases. This illustrates the difficulty of determining whether a strain of diarrheagenic E. coli isolated from a food or animal sample is pathogenic or not, and also shows that the conclusion cannot be based exclusively on the presence of genes coding for virulence factors, or on the number of different genes found. The molecular subtyping is an essential complement to epidemiological investigations in order to identify the source of an outbreak of non-O157 diarrheagenic E. coli infections (Fantelli & Stephan, 2001). All the five ETEC strains identified were LT positive. ETEC is defined as E. coli strains that produce at least one of the two defined groups of enterotoxins: LT (heat-labile) and ST (heat-stable) enterotoxins and its detection has long relied on detection of these enterotoxins (Levine, 1987). This pathogen is responsible for one-

Fig. 1. Multiplex PCR amplification of E. coli reference strains from pure cultures. Lane M, 100-bp DNA ladder marker (BioLabs Inc., New England, USA); lane 1, ETEC, H10407 (LT, ST); lane 2, EHEC, O157:H7 sakai (eae, stx1, stx2); lane 3, EPEC, B171-8 (eae, bfpA); lane 4, EIEC, 1438-1 (ipaH); lane 5, mixed DNA of all reference strains showing all of the target genes, (eae, ipaH, LT, bfpA, stx2, ST, stx1, the lowest band refers to both ST and stx1); lane 6, E. coli K12 DH5a as a negative control.

Fig. 2. Frequency of different diarrheagenic E. coli types among the 32 selected E. coli strains.

fifth of all severe diarrheal illnesses and results in about 600 million cases of diarrhea worldwide annually, with an estimated 800,000 deaths in children younger than 5 years of age (WHO, 1999). In this study we could identify a variety of non-O157 STEC serotypes, including 2 strains of serotype O121:H19 and one O148:H8. Both strains of E. coli O121:H19 were positive for eaeA, stx1, and stx2 whereas E. coli O148:H8 was positive only for stx2. The Shiga toxin-producing E. coli O121 strains are classified as enterohemorrhagic E. coli (EHEC), since they have been isolated from patients with hemorrhagic colitis or HUS (Cornu et al., 1999; Novicki et al., 2000; Stock, Scott, Davis, Pierson, & Dummer, 2001; Yatsuyanagi et al., 1999). There was an outbreak of STEC O121 infections among school children in Chiba Prefecture, Japan (Akiba et al., 2005). The findings of Seto, Taguchi, Kobayashi, and Kozaki et al. (2007) are consistent with a transient local beach contamination in mid-July, with E coli O121:H19, which seems to be able to cause severe illness. Moreover, E. coli O121:H19 was implicated as the cause of a waterborne outbreak of HC in Connecticut in 1999

Fig. 3. Multiplex PCR amplification of positive diarrheagenic E. coli strains from pure cultures. Lane M, 100-bp DNA ladder marker (BioLabs Inc., New England, USA); lane 1, ETEC (LT); lane 2, EHEC, O121:H19 (eae, stx1, stx2); lane 3, EPEC (eae, bfpA); lane 4, EIEC (ipaH); lane 5, EHEC, O148:H8 (stx2); lane 6, E. coli K12 DH5a (negative control).

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Fig. 4. Single PCRs of each positive strain independently tested with a primer specific for a suspected diarrheagenic E. coli isolate from the multiplex PCR. A, ETEC strains (LT); B, EHEC strains, O121:H19 (eae, stx1, stx2) and O148:H8 (stx2); C, EPEC strains, typical EPEC (eae, bfpA) and atypical EPEC (eae); D, EIEC strain (ipaH); Lane M, 100-bp DNA ladder marker (BioLabs Inc., New England, USA); E. coli K12 DH5a is the negative control.

(McCarthy et al., 2001). The molecular data and the recent increase in the incidence of E. coli O121:H19 infections suggest that this clone is an emerging pathogen, and surveillance methods should be able to detect this as well as other non-O157 STEC serotypes. Fantelli and Stephan (2001) could isolate STEC O148:H8 from ground beef. Also, an E. coli O148:H8 strain (stx2d-positive and eaenegative) was isolated from a case of bloody diarrhea in Germany (Beutin, Krause, Zimmermann, Kaulfuss, & Gleier, 2004). In the present study, the E. coli O148:H8 strain isolated possessed the stx2 gene, which confirms its characterization as an STEC strain. An outbreak of STEC O148:H8 infection occurred among wedding attendees in France in June 2002 (Espié et al., 2006). E. coli O157:H7 is found most frequently during sporadic infections or outbreaks, but other non-O157 serogroups (O26, O103, O111, O121, O145, O153, etc.) have also been implicated in STEC infections and hemolytic uremic syndrome (HUS) (Caprioli et al., 1994; Gerber, Karch, Allerberger, Verweyen, & Zimmerhackl, 2002; Tarr, 1995). However, the actual frequency of non-O157 serogroups is difficult to estimate, and is probably underestimated because of unsuitable detection methods or a failure to search for non-0157 serogroups (Griffin & Tauxe, 1991). Clinical symptoms observed in STEC infections are associated primarily with the production of stx2 has both been associated with increased virulence of STEC (Boerlin et al., 1999). The eae gene, when present in STEC, produces intimin, which is associated significantly with bloody diarrhea and HUS (Ethelberg et al., 2004), but may not be essential for the development of HUS in adults (Bonnet et al., 1998). Most outbreaks and sporadic cases of hemorrhagic colitis (HC) and HUS have been attributed to O157:H7 VTEC strains. However, especially in Europe, infections with nonO157 strains, such as O26:H11 or O26:H, O91:H, O103:H2, O111:H, O113:H21, O117:H7, O118:H16, O121:H19, O128:H2 or O128:H, O145:H, and O146:H21 are frequently associated with severe illness in humans (ICMSF, 2005). Recently, a deadly outbreak of severe foodborne illness characterized by bloody diarrhea, and HUS caused by a novel strain of E. coli O104:H4 have been reported in Germany and several European countries. This serious outbreak was focused in Germany in May through June 2011, where 3785 cases and 45 deaths had been reported as of 27 July 2011 (http://en. wikipedia.org/wiki/2011_E._coli_O104:H4_outbreak). The two EPEC strains isolated in the present study were PCR positive for eae gene. One of them was also positive for bfpA gene, while the other was bfpA-negative. E. coli strains that harbor both eae and bfpA are classified as “typical EPEC”, while strains posses only the eae gene (bfpA) are classified as “atypical EPEC”. Atypical EPEC were found to be serologically more diverse and are isolated

more frequently from human patients and asymptomatic controls than typical EPEC strains (Kozub-Witkowski et al., 2008; ReguaMangia et al., 2004). We report here one strain ipaH positive as the predominant EIEC pathotype identified from our isolates. EIEC is known to cause shigellosis-like symptoms in both adults and children. A potential contributor to the lack of attention to the epidemiology of EIEC is that it is often observed to be an infrequent cause of diarrhea relative to other diarrhea-causing E. coli. In a Medline search of studies testing for the presence of EIEC, Vieira et al. (2007) identified 42 articles which are widely distributed geographically, including Europe, Central and South America, the Middle East, western Africa, and southeastern Asia. 4. Conclusion In conclusion, the multiplex PCR assays presented in this study showed very high specificity for detecting the corresponding diarrheagenic E. coli virulence genes in all strains tested and there was a complete conformity between the results of single and multiplex PCRs for all strains tested. This data highlights the need for increased monitoring for the presence of non-O157 diarrheagenic E. coli specially STEC strains in meat products in order to ascertain the potential public health risk of these emerging strains. Acknowledgments The author would like to thank Prof. Takeshi Honda and Prof. Nakaba Sugimoto, Osaka University, Japan who gave me the chance to work in their laboratories, in addition to Prof. Trout Tobe, Osaka University, Japan for providing the reference E. coli strains. Thanks are also provided to Prof. Khalid Sallam, Mansoura University, Egypt for critical reading of the manuscript and suggestions. References Akiba, Y., Kimura, T., Takagi, M., Akimoto, T., Mitsui, Y., Ogasawara, Y., et al. (2005). Outbreak of enterohemorrhagic Escherichia coli O121 among school children exposed to cattle in a ranch for public education on dairy farming. Japanese Journal of Infectious Diseases, 58, 190e192. Aranda, K. R. S., Fagundes-Neto, U., & Scaletsky, I. C. A. (2004). Evaluation of multiplex PCRs for diagnosis of infection with diarrheagenic Escherichia coli and Shigella spp. Journal of Clinical Microbiology, 42, 5849e5853. Barlow, R. S., Hirst, R. G., Norton, R. E., Asshhurst-Smith, C., & Bettelheim, K. A. (1999). Novel serotype of enteropathogenic Escherichia coli (EPEC) as a major pathogen in an outbreak of infantile diarrhoea. Journal of Medical Microbiology, 48, 1123e1125. Beutin, L., Krause, G., Zimmermann, S., Kaulfuss, S., & Gleier, K. (2004). Characterization of Shiga toxin-producing Escherichia coli strains from human patients in Germany over a 3-year period. Journal of Medical Microbiology, 42, 1099e1108.

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