Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Fri, 08 Feb 2019 21:53:09 Adherence and associated virulence gene expression in acid-treated Escherichia coli O157 : H7 in vitro and in ligated pig intestine Xianhua Yin, 1,2 Yanni Feng, 3 Yang Lu, 4 James R. Chambers, 1 Joshua Gong 1 and Carlton L. Gyles 2 Correspondence Carlton L. Gyles cgyles@ovc.uoguelph.ca Joshua Gong gongj@agr.gc.ca Received 4 November 2011 Revised 17 January 2012 Accepted 27 January 2012 1 Guelph Food Research Center, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, ON N1G 5C9, Canada 2 Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada 3 College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109, PR China 4 Institute of Animal Husbandry and Veterinary Science, Shanghai Academy of Agricultural Sciences, 2901 Beidi Road, Shanghai 201106, PR China Escherichia coli O157 : H7 cells that interact with intestinal epithelial cells in animals and humans do so after passage through the low pH of the stomach. This study compared adherence and its associated virulence gene expression in acid-treated (AT) and non-acid treated (NAT) E. coli O157 : H7 strain 86-24 in vitro and in ligated pig intestine. It was found that in vitro, AT O157 : H7 had significantly decreased adherence accompanied by decreased expression of stcE and toxB but not of the locus of enterocyte effacement (LEE) genes. Expression of gadE, genes involved in quorum sensing, and the global regulators cyaA, hfq, lrp, fis and himA was significantly increased; notably, ureD expression was increased 29-fold compared with NAT O157 : H7. AT O157 : H7 colonized the pig intestine as effectively as NAT O157 : H7 bacteria. Expression of 70 of 72 virulence genes from bacteria recovered from the intestine was similar between AT and NAT O157 : H7, except ureD, pagC and bax, whose level of expression was reduced in the AT bacteria. Genes involved in acid response, regulators gadE, cyaA and hfq, and toxin synthesis genes (stx2A and stx2B) were expressed at significantly reduced levels in the intestine by both AT and NAT strains. Expression in the intestine of the LEE and putative adhesion factors cahA, iha and lpf2 was at levels similar to those in vitro, while ehaA and ureD in NAT O157 : H7 were expressed significantly more highly in vivo than in vitro. These data indicate that AT and NAT O157 : H7 behave differently, and that expression of their virulence genes is regulated differently in vitro from in vivo. INTRODUCTION Enterohaemorrhagic Escherichia coli (EHEC), represented by the serotype O157 : H7, have emerged as important pathogens over the last three decades (Karmali et al., 2010). EHEC O157 : H7 can colonize the intestine of humans and cause severe clinical manifestations, such as haemorrhagic colitis (HC) and haemolytic–uraemic syndrome (HUS) (Manning et al., 2008). The bovine gastrointestinal tract (GIT) is the principal reservoir of EHEC O157 : H7 (Grauke et al., 2002), and outbreaks of EHEC disease are due to the consumption of contaminated water and foods, such as beef, lettuce, spinach, sprouts and juice, and by direct animal contact (Ferens & Hovde, 2011; Kulasekara et al., 2009). EHEC O157 : H7 is characterized by its ability to produce attaching and effacing (AE) lesions, resulting in localized destruction of microvilli, cytoskeleton rearrangement, and formation of pedestal-like structures underneath the bacteria, leading to intimate adherence to and colonization of the host intestine (Kaper et al., 2004). Formation of the AE lesion requires genes on a pathogenicity island named Abbreviations: AE, attaching and effacing; AR, acid resistance; AT, acid- treated; EHEC, enterohaemorrhagic E. coli; GDAR system, glutamate- dependent acid resistance system; GIT, gastrointestinal tract; HC, haemorrhagic colitis; HUS, haemolytic–uraemic syndrome; LEE, locus of enterocyte effacement; NAT, non-acid-treated; qPCR, quantitative PCR; QS, quorum sensing; Stx, Shiga toxin. A supplementary table, showing primers used for quantitative PCR and their target genes, is available with the online version of this paper. Microbiology (2012), 158, 1084–1093 DOI 10.1099/mic.0.056101-0 1084 056101 G 2012 SGM Printed in Great Britain