title: An adapted ImmunoMagnetic cell separation method for use in quantification of Escherichia coli O157:H7 from bovine faeces.
abstract: Detection of Escherichia coli O157:H7 organisms in food, clinical or environmental samples is necessary for diagnosis of infection and epidemiological investigations. However, this pathogen may be present in low numbers and difficult to identify among high numbers of other background bacteria. In order to increase the sensitivity of culture- and PCR detection, pre-enrichment of E. coli O157:H7 in broth culture combined with ImmunoMagnetic cell Separation (IMS) is routinely employed. These methods, although able to detect levels as low as 2 cfu/g (from 10 to 25 g samples), are qualitative detection strategies only. If the actual numbers of E. coli O157:H7 are to be quantified, growth enrichment must be excluded and the organisms isolated directly from the sample of interest. Such quantification is necessary, for example, to determinate contamination levels on beef carcasses and for determination of bacterial numbers in in vivo gene expression studies. In the present study, it was not possible to recover organisms from bovine faecal suspensions using the customary IMS system and so a range of alternative buffers and other paramagnetic beads was tested. Combination of a 6.2-microm diameter bead with a detergent-based buffer gave optimal recovery of E. coli O157:H7 organisms from faecal suspensions. This system was validated for recovery of E. coli O157:H7 by comparing it with that obtained with the standard Dynabeads IMS protocol, using both the traditional broth enrichment method and a quantitative detection approach. We conclude that a 6.2-microm diameter Aureon bead can be used for quantitative isolation of E. coli O157:H7 directly from bovine faeces and, for this purpose, is preferred to the 2.8-microm diameter Dynal bead.
authors.affiliation: Department of Veterinary Pathology, Institute of Comparative Medicine, Glasgow University Veterinary School, Bearsden Road, Glasgow G61 1QH, UK.
authors.forename: Nick
authors.initials: N
authors.lastname: Parham
authors.forename: Janice
authors.initials: J
authors.lastname: Spencer
authors.forename: David
authors.initials: D
authors.lastname: Taylor
authors.forename: Helen
authors.initials: H
authors.lastname: Ternent
authors.forename: Giles
authors.initials: G
authors.lastname: Innocent
authors.forename: Dominic
authors.initials: D
authors.lastname: Mellor
authors.forename: Mark
authors.initials: M
authors.lastname: Roberts
authors.forename: Alun
authors.initials: A
authors.lastname: Williams
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000382:microbiology*; D003470:Culture Media; D004927:Escherichia coli Infections / Q000175:diagnosis / Q000662:veterinary*; D019453:Escherichia coli O157 / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D005243:Feces / Q000382:microbiology*; D018189:Immunomagnetic Separation / Q000145:classification / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D003470:Culture Media