Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Sun, 02 Dec 2018 13:58:13 Haemolysin E- and enterohaemolysin-derived haemolytic activity of O55/O157 strains and other Escherichia coli lineages Kazunori Murase, 1 Tadasuke Ooka, 1 Atsushi Iguchi, 2 3 Yoshitoshi Ogura, 1,2 Keisuke Nakayama, 1 Md Asadulghani, 2 4 Md Rakibul Islam, 1 Hirotaka Hiyoshi, 3 Toshio Kodama, 4 Lothar Beutin 5 and Tetsuya Hayashi 1,2 Correspondence Tetsuya Hayashi thayash@med.miyazaki-u.ac.jp Received 17 September 2011 Revised 14 December 2011 Accepted 16 December 2011 1 Division of Microbiology, Department of Infectious Diseases, Faculty of Medicine, University of Miyazaki, 5200 Kiyotake, Miyazaki 889-1692, Japan 2 Division of Bioenvironmental Science, Frontier Science Research Center, University of Miyazaki, 5200 Kiyotake, Miyazaki 889-1692, Japan 3 Laboratory of Genomic Research on Pathogenic Bacteria, International Research Center for Infectious Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan 4 Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan 5 National Reference Laboratory for Escherichia coli, Department of Biological Safety, Federal Institute for Risk Assessment (BfR), Diedersdorfer Weg 1, D-12277 Berlin, Germany Among three haemolysins identified thus far in Escherichia coli, alpha-haemolysin (HlyA) is encoded on the pathogenicity islands of extraintestinal pathogenic strains, while enterohaemolysin (EhxA) is encoded on the virulence plasmids of enterohaemorrhagic E. coli (EHEC) strains. In contrast, the gene for haemolysin E (HlyE) is located on the E. coli chromosome backbone and is therefore widely distributed among E. coli strains. However, because hlyE gene expression is repressed by the H-NS protein and because the gene has been disrupted in many strains, its haemolytic activity cannot be detected in wild-type strains by routine screening on blood agar plates. In this study, we found that the HlyE-derived haemolytic activity of enteropathogenic E. coli (EPEC) O55 : H7 can be detected after anaerobic cultivation on a washed blood agar plate (EHX plate) that is used to detect the production of EhxA. We also found that the haemolytic activity of EHEC O157 : H7 observed on EHX plates under aerobic and anaerobic growth conditions is derived from EhxA and HlyE, respectively; this differential expression of the two haemolysins occurs at the transcriptional level. Our analysis of 60 E. coli strains of various pathotypes and phylogenies for their repertoires of haemolysin genes, haemolytic phenotypes and hlyE gene sequences revealed that HlyE activity can generally be detected on EHX plates under anaerobic growth conditions if the gene is intact. Furthermore, our results indicate that hlyE gene inactivation occurred in three of the five E. coli lineages (phylogroups A, B1 and B2), which demonstrates phylogroup-specific gene disruption patterns. INTRODUCTION Haemolysins are often regarded as one of the major virulence factors in various pathogenic bacteria, and a variety of haemolysins have been identified in a wide range of bacterial species; in Escherichia coli, three types of haemolysins have been identified thus far. Among these, the most extensively studied is alpha-haemolysin (HlyA), which is produced mainly by extraintestinal pathogenic E. coli (ExPEC) strains (Welch et al., 1981), and occasionally by enterotoxigenic E. coli (ETEC), Shiga toxin (Stx)-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) (Burgos & Beutin, 2010). HlyA belongs to the RTX (repeat in toxin) 3Present address: Interdisciplinary Research Organization, University of Miyazaki, 5200 Kihara, Miyazaki 889-1692, Japan. 4Present address: International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B), Mahakhali, Dhaka, Bangladesh. Abbreviations: EHEC, enterohaemorrhagic E. coli; EIEC, enteroinvasive E. coli, EPEC, enteropathogenic E. coli; ETEC, enterotoxigenic E. coli; RTX family, repeat in toxin family; Stx, Shiga toxin; UPEC, uropathogenic E. coli. The GenBank/EMBL/DDBJ accession numbers for the sequences of the hlyE gene and its 59 and 39 non-coding regions of various E. coli strains are AB646137–AB646188. A supplementary figure, showing PCR primers for hlyE gene detection, and a supplementary table, showing PCR primers used in this study, are available with the online version of this paper. Microbiology (2012), 158, 746–758 DOI 10.1099/mic.0.054775-0 746 054775 G 2012 SGM Printed in Great Britain