ORIGINAL ARTICLE Detection of genetic diversity by pulsed-field gel electrophoresis among Escherichia coli O157 isolated from bovine faecal samples by immunomagnetic separation technique L. Vali 1 , A. Hamouda 1 , M.C. Pearce 1 , H.I. Knight 2 , J. Evans 2 and S.G.B. Amyes 1 1 Molecular Chemotherapy, Centre for Infectious Diseases, University of Edinburgh, Edinburgh, UK 2 Scottish Agricultural College, Inverness, UK Introduction Escherichia coli O157 is a cause of nonbloody and bloody diarrhoea, haemorrhagic colitis, and haemolytic uraemic syndrome in humans (Riley et al. 1983; Akiba et al. 1999; Mead et al. 1999; Allison et al. 2000; Stampi et al. 2004). The presence and prolonged survival of E. coli O157 on pasture and in the farm environment is strongly related to the presence of cattle faeces (Fukushima et al. 1999). Diversity of E. coli O157 within herds of cattle has been demonstrated in cross-sectional (Faith et al. 1996; Akiba et al. 1999; Rice et al. 1999) and longitudinal (Rice et al. 1999; Renter et al. 2003; Vali et al. 2004) studies. During the isolation and detection process, it has become com- mon practice to pick one to a maximum of 12 colonies from isolation plates (Faith et al. 1996; Galland et al. 2001; Renter et al. 2003). These colonies probably repre- sent the dominant strain. As only a small dose of E. coli O157 is required, even minority strains may be important if they are pathogenic. Considering the importance of animal faeces in the transmission of E. coli O157 to humans and the serious clinical complications caused by this organism, it is important to select multiple isolates from a sample for genotypic typing and characterization of E. coli O157 isolates in epidemiological studies. Heterogeneity of E. coli O157 isolates between and within samples has been demonstrated with selective enrichment and isola- tion on sorbitol MacConkey agar (SMAC) plates fol- lowed by identification using a combination of bacterial Keywords bovine, CT-SMAC media, Escherichia coli O157, faecal samples, immunomagnetic separation, pulsed-field gel electrophoresis. Correspondence Sebastian G.B. Amyes, Molecular Chemotherapy, Centre for Infectious Diseases, The Chancellor’s Building, 49 Little France Crescent, University of Edinburgh, Edinburgh EH16 4SB, UK. E-mail: s.g.b.amyes@ed.ac.uk 2006/0461: received 4 April 2006, revised and accepted 10 August 2006 doi:10.1111/j.1472-765X.2006.02034.x Abstract Aims: Escherichia coli O157 is considered to be one of most important human pathogens of animal origin which causes serious clinical complications. One of the most common methods to isolate E. coli O157 is the immunomagnetic separation (IMS) technique which employs specific antibodies coupled to mag- netic beads to bind and extract cells from enrichment broths followed by pla- ting onto sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (CT-SMAC) plates. The aim of this study was to determine strain vari- ation by pulsed-field gel electrophoresis (PFGE) among E. coli O157 on IMS/ CT-SMAC plates. Methods and Results: Every suspect colony of E. coli O157 was tested following isolation by the IMS/CT-SMAC technique. From 124 colonies detected; six XbaI-PFGE profiles were identified. Conclusions: Our results demonstrate that mixed populations of E. coli O157 with distinguishable PFGE profiles that are simultaneously present in bovine faeces can be isolated with IMS/CT-SMAC technique. Significance and Impact of the Study: If the aim of the study was to analyse diversity of PFGE profiles of E. coli O157 in a faecal sample following isolation by the IMS/CT-SMAC technique, at least five colonies per sample should be analysed to detect different PFGE subtypes if present. Letters in Applied Microbiology ISSN 0266-8254 ª 2006 The Authors Journal compilation ª 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 44 (2007) 19–23 19