HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Mundy, R. Articles by Frankel, G. Search for Related Content PubMed PubMed Citation Articles by Mundy, R. Articles by Frankel, G. Agricola Articles by Mundy, R. Articles by Frankel, G.

Distribution of espI among clinical enterohaemorrhagic and enteropathogenic Escherichia coli isolates

¹Centre for Molecular Microbiology and Infection, Department of Biological Sciences, Imperial College London, London SW7 2AZ, UK ²Microbiology Department, Royal Free Hospital, Pond Street, London NW3 2QG, UK ³Laboratory of Enteric Pathogens, Specialist and Reference Microbiology Division, Health Protection Agency, 61 Colindale Avenue, London NW9 5HT, UK

Correspondence Gad Frankel g.frankel{at}imperial.ac.uk

Received March 25, 2004
Accepted July 22, 2004

Enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli are important diarrhoeagenic pathogens; infection is dependent on translocation of a number of type III effector proteins. Until recently all the known effectors were encoded on the LEE pathogenicity island, which also encodes the adhesin intimin and the type III secretion apparatus. Recently, a novel non-LEE effector protein, EspI/NleA, which is required for full virulence in vivo and is encoded on a prophage, was identified. The aim of this study was to determine the distribution of espI among clinical EHEC and EPEC isolates. espI was detected in 86 % and 53 % of LEE⁺ EHEC and EPEC strains, respectively. Moreover, the espI gene was more commonly found in patients suffering from a more severe disease.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Enteric bacteria have a conserved core genomic structure, common to both commensal and pathogenic strains, that provides micro-organisms with mechanisms required for survival in the competitive gut, as well as the ability to transmit between hosts and survive in the environment (Dougan et al., 2001). In pathogenic bacteria, the core genome framework is further decorated with novel genetic islands often associated with enhanced virulence (Wain et al., 2001). Striking examples of such pathogenic bacteria are the diarrhoeal agents enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC), also known as Verocytotoxin-producing E. coli (VTEC) or Shiga toxin-producing E. coli (STEC) (Nataro & Kaper, 1998).

EPEC is a frequent cause of infantile diarrhoea in the developing world while EHEC causes a wide spectrum of illnesses ranging from mild diarrhoea to severe diseases, such as haemorrhagic colitis and haemolytic uraemic syndrome (HUS). Strains of EHEC belonging to serogroup O157 are most commonly associated with severe human disease (Willshaw et al., 2001). However, infections with EHEC of other serogroups (i.e. O26, O103, O111 and O145) have also been documented (Caprioli et al., 1997).

By adhering to intestinal epithelial cells, EHEC and EPEC produce a histopathological feature known as the attaching and effacing (A/E) lesion (reviewed by Frankel et al., 1998), which is characterized by localized destruction of brush border microvilli and intimate attachment of the bacteria to the plasma membrane of the host epithelial cells. The capacity to form A/E lesions is encoded on the LEE pathogenicity island (McDaniel et al., 1995), which encodes the positive regulator Ler (Mellies et al., 1999), the outer-membrane adhesin intimin (Jerse et al., 1990), a type III secretion system (TTSS) and effector proteins (Tir, EspF, Map, EspG, EspH) (Elliott et al., 2001; Kenny et al., 1997; Kenny & Jepson, 2000; McNamara & Donnenberg, 1998; Tu et al., 2003) that are translocated into the epithelial cell for the benefit of the pathogen.

Recently, we and others identified a novel type III secreted protein, termed EspI (Mundy et al., 2004) or NleA (Gruenheid et al., 2004), that was found in the genome sequence of EHEC (Hayashi et al., 2001; Perna et al., 2001) and EPEC (http://www.sanger.ac.uk/projects/Escherichia_Shigella) to be encoded on a prophage outside of the LEE region. An espI/nleA homologue is found on Stx1-converting phage phi-4795 in EHEC strain O84 (AJ487680) but is missing from laboratory E. coli strains (Gruenheid et al., 2004). Importantly, despite not affecting A/E lesion formation in vitro (Gruenheid et al., 2004; Mundy et al., 2004), EspI was shown to be essential for virulence in vivo using the Citrobacter rodentium mouse model of infection (Mundy et al., 2004; Gruenheid et al., 2004), which became a popular surrogate model for in vivo studies of the mechanisms and processes of A/E E. coli pathogenesis. The aim of this study was to determine the prevalence and distribution of espI among clinical EPEC and EHEC isolates.

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Bacterial strains.

Ninety-three strains of EHEC, 29 from patients with HUS, 49 from patients with diarrhoea who did not develop HUS and 15 from a healthy control group, were obtained from the culture collection of the Laboratory of Enteric Pathogens (LEP), London. Strains from patients with HUS and diarrhoea were isolated between 1968 and 2000 either at the LEP from faecal samples sent in for tests for EHEC and EPEC, or at hospital laboratories in the UK, and sent to the LEP for confirmation and further tests (Jenkins et al., 2003a, b; Kleanthous et al., 1990; Willshaw et al., 1992, 2001). The strains from the healthy control group were isolated during a study on Infectious Intestinal Disease (IID) in England (Evans et al., 2002). Healthy controls were defined as individuals without loose stools or significant vomiting for 3 weeks before the faecal sample was taken. Twenty-five strains of EPEC were from patients with diarrhoea sent to the LEP for identification and further characterization between January and December 2000, and a further 207 strains were isolated during the IID study (Tompkins et al., 1999) from cases and control. The strains isolated during the IID study were from cases and controls in two study components: one was a community cohort and the other was a General Practice case-control component. Cases were individuals with loose stools and significant vomiting lasting less than 2 weeks, in the absence of a known non-infectious cause and proceeded by a symptom-free period of 3 weeks. Controls were individuals without loose stools or significant vomiting for 3 weeks.

DNA hybridization.

The espI gene was amplified by PCR using Hotstar Taq (Invitrogen) from EHEC O157 : H7 (EDL933) using the forward primer 5'-ATGAACATTCAACCGACCATACAATCTG and the reverse primer 5'-TTAGACTCTTGTTTCTTGGATTATATCA. The amplified 1293 bp DNA fragment was purified using a PCR Clean-up kit (Qiagen) and labelled with fluorescein-deoxyuridine triphosphate (dUTP) using a random primer labelling kit (Amersham) according to the manufacturer's instructions. The eae gene and the EAF plasmid were detected using DNA probes described by Jerse et al. (1990) and Nataro et al. (1985). These probes were also labelled with fluorescein dUTP.

Probe tests were performed according to the method of Maniatis et al. (1982). Briefly, broth cultures were spotted on nylon membranes. The membranes were overlaid on nutrient agar, incubated for 6 h and, after alkaline lysis, the DNA was bound to the membrane. Filters were hybridized overnight at 68 °C with the fluorescein-labelled probes. Stringency washes were carried out at 68 °C. Colonies harbouring the target genes were detected using enhanced chemiluminescence, as described by the manufacturer (Amersham).

Subtyping of the eae gene.

The subtyping of the eae gene was performed using the methods described previously (Jenkins et al., 2003a). The eae gene subtypes of some of the strains have been reported (Jenkins et al., 2003a, c).

Statistics.

The chi-square (S²) test with the Yates’ correction was used to determine if there were any statistically significant differences in the distribution of the espI gene between groups of EPEC and EHEC strains. A S² test of association was carried out to assess the relationship between the presence of the espI gene and the presence of the eae gene. S² tests were also performed to determine the relationship between the espI gene and disease symptoms in patients with EHEC. In all cases, a P value of < 0.05 was taken to indicate significance.

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
We assembled a large collection of strains, isolated from patients with HUS and diarrhoeal disease and from a healthy control group, and tested the strains for the presence of espI using a specific DNA probe. Two hundred and thirty-two EPEC strains were tested for the presence of espI using colony hybridizations. espI was detected in 124 of the 232 (53.4 %) strains. Table 1 shows the distribution of espI in the 25 EPEC strains from the LEP culture collection. Nine of the 232 EPEC strains carried the EPEC adherence factor (EAF) plasmid and all of these isolates also had the espI gene. There was no difference in the prevalence of espI between strains isolated from either cases or controls in the IID study and there was no association between espI and defined EPEC serogroups. However, the espI gene was more commonly associated with strains harbouring the ß-, -, 2- or -intimin genes than with strains harbouring the - or -intimin genes (Table 2).

Ninety-three strains of EHEC were tested for the presence of the espI gene, 43 of which had the eae gene (LEE⁺). espI was detected in 37 of the 43 (86 %) EHEC intimin-positive isolates, which included 16 EHEC O157 and 8 EHEC O26 strains, and in none of the intimin-negative (LEE^–) strains (Table 3). Therefore, the espI gene appears to be highly associated with the presence of the eae gene in EHEC (S² = 67.9, with Yates’ correction; df = 1, P < 0.0001). However, there was no correlation between the presence of espI and a specific intimin type. Sixteen of 19 (84.2 %) intimin ß strains were positive for espI and 19 of 20 (95.0 %) intimin strains were positive for espI [S² = 0.34, with Yates’ correction (df = 1, P = 0.561), showing there is no statistically significant association]. As only two strains with the -intimin and two strains with the -intimin gene were isolated, no statistical analysis could be carried out to look for an association between these intimin subtypes and the presence of the espI gene.

The espI gene was also more commonly found in patients with more severe disease. Thirteen of 29 (44.8 %) strains isolated from patients with HUS and 24 of 49 (49 %) strains isolated from patients with diarrhoea contained espI, compared to none of the 15 strains isolated from asymptomatic carriers. Statistical analysis confirmed that espI is more frequently associated with strains isolated from symptomatic than from asymptomatic individuals, both from those with severe disease (HUS) (S² = 7.52, with Yates’ correction; df = 1, P < 0.006) and from those with diarrhoea (S² = 9.758, with Yates’ correction; df = 1, P < 0.002).

EspI is a member of a growing family of newly discovered type III secreted proteins of A/E E. coli (AEEC) that are not encoded on the LEE region (Marches et al., 2003). EspI is a type III secreted effector protein that is essential for full virulence in vivo (Mundy et al., 2004; Gruenheid et al., 2004) and is found more frequently among strains isolated from humans suffering from a symptomatic EHEC infection. The fact that espI is found more frequently in LEE⁺ EHEC (86 %) than EPEC (53.4 %) strains suggests that espI might also play a role during bacterial spread in the environment or in the animal reservoir. We intend to test this hypothesis experimentally by engineering an espI EHEC mutant that will be tested in a calf model of EHEC infection (Stevens et al., 2002).

	ACKNOWLEDGEMENTS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
This project was supported by the BBSRC and the Wellcome Trust (as part of the International Partnership Research Award in Veterinary Epidemiology project entitled Epidemiology and Evolution of Enterobacteriaceae Infections in Humans and Domestic Animals).

	REFERENCES

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES