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Title: Detection and isolation of shiga toxin-producing Escherichia coli (STEC) O104 from sprouts

item BARANZONI, GIAN MARCO - University Of Bologna, Italy
item Fratamico, Pina
item RUBIO, FERNANDO - Abraxis, Llc
item GLAZE, GLAZE - Abraxis, Llc
item Bagi, Lori
item ALBONETTI, SABRINA - University Of Bologna, Italy

Submitted to: International Journal of Food Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/22/2013
Publication Date: 12/29/2013
Citation: Baranzoni, G., Fratamico, P.M., Rubio, F., Glaze, G., Bagi, L.K., Albonetti, S. 2013. Detection and isolation of shiga toxin-producing Escherichia coli (STEC) O104 from sprouts. International Journal of Food Microbiology. 173:99-104.

Interpretive Summary: Bacteria known as Shiga toxin-producing Escherichia coli (STEC) are food-borne pathogens that cause serious human illnesses, which can lead to death. In 2011, a very large outbreak due to STEC belonging to a specific serogroup known as O104 occurred in Europe in which there were close to 4000 cases of illness and over 50 deaths. The outbreak was linked to the consumption of sprouts contaminated with STEC O104. An outbreak and illnesses caused by STEC O104 also occurred in the United States. To prevent illnesses and outbreaks due to STEC O104, there is a critical need for methods to detect and isolate this pathogen from food. Thus, a methodology to detect and isolate STEC pathogens belonging to E. coli serogroup O104, based on the polymerase chain reaction in combination with the use of recently commercialized reagents necessary for efficient recovery of the pathogen from artificially contaminated sprouts was developed. The method was able to detect very low levels (less than one bacterium per gram of food) of STEC O104 in sprouts, and the pathogen was easily identified and recovered from the contaminated sprouts. Use of this methodology by regulatory agencies and the food industry will increase the ability to detect and isolate STEC O104 from sprouts and potentially other foods, as well, and thus will enhance food safety.

Technical Abstract: E. coli O157:H7 and non-O157 Shiga toxin-producing E. coli (STEC) are food-borne pathogens responsible for severe outbreaks of hemorrhagic colitis, which can lead to hemolytic uremic syndrome and/or death. STEC strains belonging to serogroup O104 have been associated with sporadic cases of illness and have caused outbreaks associated with milk and sprouts. An outbreak that occurred in Europe in 2011 linked to fenugreek sprouts was caused by E. coli O104:H4 that had characteristics of an enteroaggregative E. coli (EAEC) but carried the gene that encoded for Shiga toxin 2. Methods were developed for detection of this enteroaggregative STEC, as well as STEC O104 in sprouts. A multiplex PCR assay targeting O104:H4 targeted the stx2, aggR and wzx104 genes, and the multiplex PCR assay targeting STEC O104 targeted the stx1-2, ehxA and wzx104 genes. After incubating artificially contaminated sprouts at 4°C for 48 h and overnight enrichment in mBPWp, the pathogens were detected in all samples inoculated at a level of ca. 100 CFU/ 25g. Several samples inoculated a lower concentrations of ca. 10 CFU/25 g were negative by the PCR assays, and this could have been due to cells not surviving or not being able to recover after the stress treatment at 4°C for 48 h. For isolation of the pathogens, immunomagnetic separation (IMS) using magnetic beads coated with antibodies against O104 were employed, and this was followed by plating the beads onto mRBA and CHROMagar O104 for isolation of E. coli O104:H4 and mRBA and CHROMagar STEC for isolation of E. coli O104:H7. Presumptive colonies were confirmed by using latex agglutination using latex particles coated with antibodies against serogroup O104 and by the multiplex PCR assays. All colonies were confirmed as E. coli O104 by latex agglutination and by the PCR assays. The methodologies described in this study for enteroaggregative STEC O104:H4 and STEC O104 include the use of IMS and latex reagents for serogroup O104 and enhance the ability to detect and isolate these pathogens from sprouts and potentially other foods, as well.