title: Optimization and evaluation of a modified enrichment procedure combined with immunomagnetic separation for detection of E. coli O157:H7 from artificially contaminated alfalfa sprouts.
abstract: Escherichia coli O157:H7 has been linked to foodborne disease outbreaks with alfalfa sprouts. Detection of the organism in sprouts by standard cultural methods can be difficult due to the high background microflora. The objective of this study was to develop and optimize an enrichment protocol with and without post-enrichment immunomagnetic separation (IMS) for the rapid detection by real-time PCR (RTiPCR) and cultural recovery of E. coli O157:H7 from artificially contaminated alfalfa sprouts. Initially we found that the FDA BAM procedure, enriching samples in modified buffered peptone water with pyruvate and at 37°C for 5h, followed by the addition of acriflavin, cefsulodin and vancomycin (mBPWp+ACV) and static incubation at 42°C gave poor results for both PCR detection and isolation for alfalfa sprouts artificially contaminated at 0.2cfu/g. The addition of post-enrichment IMS improved detection but not isolation. This procedure was modified and optimized by changing to mBPWp with cefsulodin and vancomycin at 42°C and shaking for 24h with and without IMS prior to PCR detection and cultural isolation. Using the resulting protocol we were able to detect E. coli O157:H7 in 100% of samples of alfalfa sprouts contaminated at 0.2cfu/g. This was validated for five strains of E. coli O157:H7. Isolation was 84% without added post-enrichment IMS and 100% with IMS. The optimized procedure was effective for detection and isolation of E. coli O157:H7 from this difficult food matrix.
authors.affiliation: Food and Drug Administration, Retired, Poulsbo, WA, USA.
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Weagant
authors.forename: Karen C
authors.initials: KC
authors.lastname: Jinneman
authors.forename: Ken J
authors.initials: KJ
authors.lastname: Yoshitomi
authors.forename: Ruben
authors.initials: R
authors.lastname: Zapata
authors.forename: Willis M
authors.initials: WM
authors.lastname: Fedio
mesh_terms: D004926:Escherichia coli; D004927:Escherichia coli Infections / Q000517:prevention & control; D019453:Escherichia coli O157 / Q000302:isolation & purification*; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D000455:Medicago sativa / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D014675:Vegetables / Q000382:microbiology