title: Detection and isolation of low levels of E. coli O157:H7 in cilantro by real-time PCR, immunomagnetic separation, and cultural methods with and without an acid treatment.
abstract: Leafy greens such as cilantro, contaminated with Escherichia coli O157:H7, have been implicated in cases of human illnesses. High levels of microflora in fresh cilantro make recovery of low numbers of E. coli O157:H7 difficult. To improve upon current methods, immunomagnetic separation (IMS) techniques in combination with real-time PCR (RTiPCR) and selective enrichment protocols were examined. Rinsates were prepared from cilantro samples inoculated with low (~0.02 CFU/g) and slightly higher (~0.05 CFU/g) levels of E. coli O157:H7. Rinsate portions were enriched in modified buffered peptone water with pyruvate (mBPWp) for 5 h at 37 °C. After 5 h, selective agents were added to samples and further incubated at 42 °C overnight. Detection and recovery were attempted at 5 and 24 h with and without IMS. IMS beads were screened by RTiPCR for simultaneous detection of stx1, stx2, and uidA SNP. Additionally, broth cultures and IMS beads were streaked onto selective agar plates (Rainbow(®) agar, R&F(®) E. coli O157 Chromogenic medium, TC-SMAC and CHROMagar™ 0157) for isolation of E. coli O157:H7. Both broth cultures and IMS beads were also acid treated in Trypticase Soy Broth pH 2 prior to plating to selective media to improve upon cultural recovery. Although E. coli O157 strains were detected in most samples by PCR after 5 h enrichment, cultural recovery was poor. However, after 24 h enrichment, both PCR and cultural recovery were improved. Acidification of the broths and the IMS beads prior to plating greatly improved recovery from 24 h enrichment broths by suppressing the growth of competing microorganisms.
  
  
  CONCLUSIONS
  Detection and recovery of Escherichia coli O157:H7 in fresh produce matrices (e.g., cilantro) can be complicated by high background microflora present in these foods. Rapid detection by molecular methods combined with effective enrichment and isolation procedures such as using immunomagnetic separation (IMS) techniques can quickly identify potential hazards to public health. Additional techniques such as acidification of enrichment broths can exploit acid resistance characteristics of pathogens such as E. coli O157:H7, facilitating their isolation in complex food matrices.
authors.affiliation: Food and Drug Administration, Pacific Regional Laboratory, Northwest, Bothell, WA 98021-4421, USA.
authors.forename: Ken J
authors.initials: KJ
authors.lastname: Yoshitomi
authors.forename: Karen C
authors.initials: KC
authors.lastname: Jinneman
authors.forename: Ruben
authors.initials: R
authors.lastname: Zapata
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Weagant
authors.forename: Willis M
authors.initials: WM
authors.lastname: Fedio
mesh_terms: D015169:Colony Count, Microbial; D028526:Coriandrum / Q000382:microbiology*; D003470:Culture Media / Q000737:chemistry; D019453:Escherichia coli O157 / Q000254:growth & development / Q000302:isolation & purification*; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D018189:Immunomagnetic Separation / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D003470:Culture Media