title: Detection and isolation of Shiga toxin-producing Escherichia coli (STEC) O104 from sprouts.
abstract: Shiga toxin-producing Escherichia coli (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. In this study, methods were developed for detection of this enteroaggregative STEC O104, as well as STEC O104 in sprouts. Multiplex PCR assays for enteroaggregative STEC O104:H4 targeted the stx2, aggR, and wzx104 genes, and for 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 modified buffered peptone water with pyruvate supplemented with three antibiotics (mBPWp), the pathogens were detected in all samples inoculated at a level of ca. 100CFU/25 g. Several samples inoculated at lower concentrations of ca. 10CFU/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 STEC 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 agglutination using latex particles attached to antibodies against serogroup O104 and by the multiplex PCR assays. The methodologies described in this study for detection of enteroaggregative STEC O104:H4 and STEC O104 include the use of IMS and latex reagents for serogroup O104, and they enhance the ability to detect and isolate these pathogens from sprouts and potentially other foods, as well.
authors.affiliation: Department of Veterinary Medical Science, University of Bologna, 50 via Tolara di Sopra, Ozzano dell'Emilia (BO), Italy.
authors.forename: Gian Marco
authors.initials: GM
authors.lastname: Baranzoni
authors.affiliation: USDA, Agricultural Research Service, Eastern Regional Research Center, 600 E. Mermaid Lane, Wyndmoor, PA, United States. Electronic address: pina.fratamico@ars.usda.gov.
authors.forename: Pina M
authors.initials: PM
authors.lastname: Fratamico
authors.affiliation: Abraxis, LLC, 54 Steamwhistle Drive, Warminster, PA, United States.
authors.forename: Fernando
authors.initials: F
authors.lastname: Rubio
authors.affiliation: Abraxis, LLC, 54 Steamwhistle Drive, Warminster, PA, United States.
authors.forename: Thomas
authors.initials: T
authors.lastname: Glaze
authors.affiliation: USDA, Agricultural Research Service, Eastern Regional Research Center, 600 E. Mermaid Lane, Wyndmoor, PA, United States.
authors.forename: Lori K
authors.initials: LK
authors.lastname: Bagi
authors.affiliation: Department of Veterinary Medical Science, University of Bologna, 50 via Tolara di Sopra, Ozzano dell'Emilia (BO), Italy.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Albonetti
mesh_terms: D028524:Anethum graveolens / Q000382:microbiology*; D000818:Animals; D005060:Europe; D005516:Food Microbiology; D005798:Genes, Bacterial / Q000235:genetics; D018189:Immunomagnetic Separation; D000455:Medicago sativa / Q000382:microbiology*; D060885:Multiplex Polymerase Chain Reaction; D054323:Shiga-Toxigenic Escherichia coli / Q000302:isolation & purification / Q000502:physiology*
keywords: Antibodies; Detection; Immunomagnetic separation; Latex beads; Shiga toxin-producing E. coli O104; Sprouts