663-680 Issn 2077-4613

Middle East Journal of Applied Volume : 07 | Issue :04 |Oct.-Dec.| 2017 Sciences Pages: 663-680 ISSN 2077-4613 Diarrheagenic Escherichia coli, shiga toxin production, relevant virulence genes, and association with other foodborne Enterobacteriaceae in retailed milk and soft cheese 1S.M. Abdelhamid, 1A. Y. Okda, 2M. Shaheen, 1Fatma, A. Fathy and 1R.K. El Dairouty 1Lab of Microbiology, Dairy Science Department, Food Industry and Nutrition Research Division, National Research Centre, El Tahrir Street, Dokki, Cairo, Egypt. 2Environmental Virology laboratory, Water pollution Research Department, Environmental Research Division, National Research Center, Dokki, Cairo, Egypt. Received: 10 August 2017 / Accepted: 30 Sept. 2017 / Publication date: 15 Oct. 2017 ABSTRACT Raw milk and soft cheese can be a major source of potentially pathogenic E. coli to human. The objectives of this work were to investigate the occurrence of diarrheagenic E. coli (DEC) in raw milk and some kinds of soft cheese retailed in Egypt and to investigate the prevalence of shiga toxin producing enteroaggregative E. coli (EAEC), and other DEC subtypes of the isolates. Diarrheagenic E. coli (DEC) as enteroaggregative (EAEC), enterohemorrhagic (EHEC), enteropathogenic (EPEC), enteroinvasive (EIEC), and enterotoxigenic E. coli (ETEC) was tested in 100 samples of dairy products, namely, raw milk (25 samples) and soft cheeses (Tallaga 25 samples, Paramely 25, and Kariesh 25). The DEC strains were identified using serological analysis with O and H antisera by means of Escherichia coli kits (Bio-Rad, France). The isolated strains were also confirmed by polymerase chain reaction (PCR) analysis. The DEC types were found in 6% of the tested samples. For raw milk, EAEC, EPEC, and EHEC were detected in 8% of the samples each. In soft cheeses, EAEC was found only in 4% of samples of both of Kariesh and Paramely, while EPEC (8%), EHEC (8%), EIEC (4%) and EAEC (8%) were found in Tallaga cheese. The most common serotypes were O111:H2 for EAEC, O157 H7 for EHEC, O124:H30 for EIEC, and O145: HN for EPEC. Similar results for DEC (6%) were obtained by PCR detection of genes pCVD432 and aggR present in EAEC, genes eaeA and bfpA were used for confirmation of EPEC, ipaH for EIEC, ehxA and eaeA for EHEC, and estA and esltB for ETEC. Shiga toxin stx1, shiga toxin stx2, or both, were detected in four out of the six EAEC isolated strains (66.6%), representing 4% of all the samples, using the ImmunoCard STAT (serology detection), and shiga toxin genes were detected by PCR (molecular detection). Other bacteriological criteria such as total aerobic colony count (TACC), coliform bacteria, E. coli O157:H7, Yersinia enterocolitica, Enterobacter spp., Klebsiella spp., Citrobacter spp., Proteus spp. and salmonella spp. were analyzed in EAEC-containing samples. This work highlights that raw milk, Kariesh, Paramely, and Tallaga cheeses retailed in Egypt are highly contaminated with DEC including potentially pathogenic strains imposing a public health threat. Key words: Diarrheagenic Escherichia coli, shiga toxin, virulence gene, foodborne pathogens, raw milk, soft cheese. Introduction Uncontrollable marketing of raw milk and soft cheeses in the Egyptian market resulted in the spread of many diseases and many cases of diarrhea, particularly due to the intestinal bacteria E. coli (Rakha et al., 1990 and El Gamal et al., 2015a). Escherichia coli, as a member of the family Enterobacteriaceae, represents one of the microorganisms commonly found in the lower intestinal tract of healthy humans and animals. Strains of E. coli that can cause diarrhea (diarrheagenic E. coli, DEC) fall into six major categories: enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), and diffuse-adherent E. coli Corresponding Author: S.M. Abdelhamid, Lab of Microbiology, Dairy Science Department, Food Industry and Nutrition Research Division, National Research Centre, El Tahrir Street, Dokki, Cairo, Egypt, E-mail: [email protected] 663 Middle East J. Appl. Sci., 7(4): 663-680, 2017 ISSN 2077-4613 (DAEC) (Xiaodong, 2010). 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 manifestations and characteristic epidemiological patterns as reported by Nataro et al., (2004). Shiga toxin-producing E. coli (STEC), also known as verotoxin-producing E. coli (VTEC) or enterohemorrhagic E. coli (EHEC), are important emerging pathogens with a strong ability to cause foodborne infections and subsequently severe and potentially fatal illnesses in humans, e.g., hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) (Blanco et al., (2004). Recently, EAEC strains were a strong concern (Flores and Okhuysen, 2009 and Tozzoli et al., (2014). STEC/VTEC strains can produce two powerful cytotoxins called shiga toxins or verotoxins (Stx1/VT1 and Stx2/VT2), which are encoded in the genome of prophages. There are three subtypes of Stx1 (a, c, and d) and seven of Stx2 (a, b, c, d, e, f, and g). Among them, Stx2/VT2 is the most potent toxin usually associated with more serious infections (Gyles, 2007). Moreover, PCR technology allows for simultaneous amplification and detection of specific gene fragments providing immediate identification of pathogenic E. coli in contaminated milk and meat products. This approach is convenient when a researcher needs to use rapid and accurate methods for detection and to establish appropriate control measures to get rid of such microorganisms (Reinoso et al., (2004). On the other hand, Pradel et al., (2008) indicated that a few dairy products other than cheeses, including cream, ice cream, yoghurt, and butter, were found to contain STEC. Nonetheless, some studies have shown that STEC isolates from dairy products differ from the classical pathogenic genotypes by harboring stx1 rather than stx2. Recently, in Egypt, El Gamel et al., (2015a and b) found STEC and Enterobacteriaceae in hospitalized diarrheal children and in retailed raw meat, respectively. The presence of foodborne pathogens in raw milk may increase the risk of both pathogen transmission and ingestion of harmful toxins (Oliver et al., 2005 and Amal, 2014). Therefore, the aim of this work was to study the prevalence of DEC and EAEC shiga toxin- producing strains in particular, in raw milk and milk products, namely Kariesh, Paramely, and Tallaga cheeses retailed in Cairo and Giza areas, as well as the association with other Enterobacteriaceae. In addition, we conducted analyses of the prevalence of EAEC, shiga toxin-producing EAEC, and other DEC subtypes in milk and dairy products by biochemical, serological, and genotypic methods. Materials and Methods Collection of samples: A total of 100 dairy samples including raw milk (25 samples), Kariesh (25 samples), Paramely (25), and Tallaga (25) cheeses were randomly collected during 1 year from different localities and markets of Cairo and Giza areas. Sample preparation: The methods described by the FDA (2002) were followed for sample preparation as follows: -Raw-milk samples: From each raw-milk sample, 25 mL were mixed with 225 mL of enrichment broth: buffered peptone water (BPW). One milliliter from each raw-milk sample was diluted in 9 mL of sterilized saline. -Cheese samples: From each cheese sample, 25 g were ground in a sterile mortar for 2 min with 225 mL of enrichment broth (BPW). Dilution 1:10 was prepared, and then serial dilution in sterile saline was carried out. Isolation and identification of E. coli: Enrichment broth samples were incubated at 37 °C for 24 h and streaked onto a selective medium: Levine eosin methylene blue (EMB) agar (Becton Dickinson, Sparks, MD, USA). Suspected 664 Middle East J. Appl. Sci., 7(4): 663-680, 2017 ISSN 2077-4613 E. coli colonies were streaked onto MacConkey agar (Becton Dickinson) after 24 to 48 h of incubation at 37 °C. Putative E. coli colonies were picked and tested by means of IMVic tests, as traditional methods, for identification and confirmation (Edwards and Ewing, 1972 and FDA, 2002). The E. coli isolates were stored in broth supplemented with 5% glycerol at 70 °C until use for further assays (Iseri et al., 2011). Biofilm assay: Bacterial clump formation in liquid cultures was examined as a rapid test for EAEC. This convenient test is based on the biofilm formation by EAEC. To assess biofilm formation by the isolated strains, the method, reagents, and screening in 96-well flat-bottom microtiter polystyrene plates were the same as those described elsewhere (Albert et al., 1993 and Kawano et al., 1998) Rapid biochemical identification of E. coli and Enterobacteriaceae: Biochemical identification of E. coli isolates and other Enterobacteriaceae was also carried out using rapid test kits, Hibio-ID and Hi25tm (Enterobacteriaceae identification kits KB003, the vendor is HiMEDIA laboratories Pvt. Ltd., A-406 Bhaveshwar Plaza, Mumbai-400 086, India) instead of the traditional methods. Serotyping of EAEC and other DEC : The biochemically identified E. coli strains (isolates) were confirmed by serological typing using O and H antisera. A slide agglutination test was performed using an O antiserum and Escherichia coli kits (Bio-Rad, France): polyvalent and trivalent sera against E. coli serogroups. The test is based on agglutination (by specific antisera) of bacteria possessing the corresponding antigens. H agglutination of formalin-killed culture was tested as follows: agglutination in a microtiter plate of formalin-killed culture using H antisera (E. coli antiserum H Pool 5 Kits) was used for detection of serotype H of the isolated E. coli strains according to the supplier’s instructions (SSI Diagnostica, 2Herredsvejen – Denmark, 3rd Edition). PCR identification of EAEC, shiga toxin detection, and identification of other DEC during screening for virulence genes: The PCR methods (Schmidt et al., 1995; Pal et al., 1999; Yu and Kaper, 1992 and Sethabutr et al., 1993) were used to screen isolates of E.

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