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1 Centre for Paediatric Gastroenterology, Royal Free Hospital, Imperial College, London, UK
2 Division of Cell and Molecular Biology, Imperial College, London, UK
Correspondence
Alan D. Phillips
a.phillips{at}medsch.ucl.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These authors contributed equally to this work.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Tarr, 1995). Systemic diseases associated with EHEC infections are linked to the expression of the bacteriophage-associated Shiga toxin (Stx) (Karmali et al., 1983, 1985); however, an O157 : H7 strain depleted of the bacteriophage-encoded Stx is as pathogenic, in terms of diarrhoeal disease, as the parent EHEC strain in animal models (Li et al., 1993).
The most prominent EHEC serotype is O157 : H7 (Slutsker et al., 1997). Although attaching/effacing (A/E) lesion formation has been shown for EHEC in animal models (Dean-Nystrom et al., 1997; Tzipori et al., 1986) and on cell culture (Knutton et al., 1989), in vivo examples in man have not been demonstrated. We demonstrated in vitro A/E lesion formation on human intestine in organ culture, and found it was restricted to the follicle-associated epithelium (FAE) from the Peyer's patch (PP) region of the distal ileum (Phillips et al., 2000). This tropism has also been demonstrated for an O103 : H2 EHEC strain (Fitzhenry et al., 2003), for the initial stages of infection in rabbit enteropathogenic E. coli (REPEC) strains (Cantey & Inman, 1981; Heczko et al., 2000), and for the mouse pathogen Citrobacter rodentium (Wiles et al., 2004). Enteropathogenic E. coli (EPEC) strains show a variable phenotype, with the prototype strain E2348/69 exhibiting adhesion to proximal and distal small intestine, as well as to FAE (Phillips et al., 2000), whereas O55 strains show an FAE-restricted adhesion (Fitzhenry et al., 2002b). One bacterial factor that influences tropism is the outer-membrane protein intimin (Fitzhenry et al., 2002a; Phillips & Frankel, 2000; Tzipori et al., 1995), encoded by the eae gene (Jerse et al., 1990) and common to A/E organisms (Frankel et al., 1998), including EPEC, EHEC and C. rodentium.
Distinct intimin types (
, 
, 
, 
, 
, etc.) have been identified (Adu-Bobie et al., 1998; Jenkins et al., 2003; Oswald et al., 2000) and results suggesting that intimin type plays a role in the pattern of colonization include intimin exchange studies in piglets (Tzipori et al., 1995) and our human ex vivo investigations. The latter showed that while EPEC expressing intimin colonized PP as well as proximal and distal small intestine (Phillips et al., 2000), a restricted tropism towards PP followed expression of intimin from EHEC in the EPEC background (Phillips & Frankel, 2000). These results were based on one prototype strain, 85/170, and, although we have shown that an EHEC intimin -expressing O103 : H2 strain also shows PP-restricted tropism (Fitzhenry et al., 2003), it is unclear if other intimin -expressing O157 : H7 strains are similarly restricted. In particular, although O157 : H7 is associated with colonic pathology (Griffin et al., 1990; Kelly et al., 1987) and is considered a colonic pathogen (Nataro & Kaper, 1998), strain 85/170 does not show adhesion to large bowel (Phillips et al., 2000). We have shown colonic colonization by both enteroaggregative E. coli (EAEC) (Hicks et al., 1996) and EPEC (Phillips et al., 2000), indicating that the lack of colonic adhesion is not a general result of the organ culture conditions or host related. The aim of this study was to determine if FAE-restricted tropism was typical for EHEC strains and to see if modulation of the in vitro organ culture (IVOC) system could change this phenotype.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. We included an O26 : H11 strain for comparison and used O103 : H2 strain PMK5 (Fitzhenry et al., 2003) for further colonic studies. In consideration of health and safety (Nataro & Kaper, 1998), only Stx-negative strains were used. EAEC strain O42, which colonizes colonic samples in IVOC (Hicks et al., 1996), was used as a positive control.
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). Briefly, 25 µl (2.5x107 c.f.u.) of an overnight bacterial culture was inoculated onto the biopsy samples. IVOC medium was changed every 2 h and the assay terminated at 8 h. Each bacterial strain was examined on at least three occasions using tissue from different children. An uninoculated specimen was included with each experiment to exclude in vivo bacterial colonization. After incubation with bacteria or appropriate control solutions, IVOC specimens were washed thoroughly three times to remove non-adherent bacteria and prepared for scanning electron microscopy (SEM). Samples were fixed with glutaraldehyde and post-fixed in osmium tetroxide. Specimens were critical-point dried using liquid carbon dioxide in an Emitech K850 apparatus, sputter coated with gold–palladium using a Polaron E5100 sputter coater, and viewed in a JEOL 5300 SEM at an accelerating voltage of 25 kV.
Technical factors and colonic adhesion.
Routine IVOC incorporates a medium change every 2 h (Hicks et al., 1998). This could remove secreted bacterial factors that may influence colonization of target host epithelium via quorum-sensing-mediated regulation of virulence mechanisms, such as locus of enterocyte effacement (LEE) gene expression and flagella production (Sperandio et al., 1999). The impact of reducing the frequency of medium change to one change at 4 h and to no change at all was tested on adhesion of strains Sakai 813 and EAEC O42 to transverse colon.
A modified version of the IVOC system (Cleary et al., 2004) used DMEM-activated bacterial cultures to inoculate explants immersed in organ culture medium for 1.5 h on a rotary mixer at 37 °C. Using this protocol, the authors demonstrated adhesion of the 
eae mutant of strain E2348/69 (CVD206) to duodenal explants whereas CVD206 is non-adherent in routine IVOC (Hicks et al., 1998). This immersion system was tested to see if it influenced colonic adhesion. Explants from the transverse colon were fully immersed in organ culture medium at 37 °C on a rotator with Sakai 813 or O42 either for 1.5 h followed by 6.5 h of the standard IVOC system, or for 8 h. Samples were then processed for SEM as above.
Environmental factors and colonic adhesion.
Various compounds were either added to (40 mM sodium bicarbonate, 30 mM sodium acetate and 20 mg taurocholic acid ml–1 as a representative bile acid), or removed from (D-mannose and newborn calf serum), the organ culture medium to determine if they influenced the adhesion of O157 : H7 to colonic mucosa.
HEp-2 : HEp-2 and HEp-2 : IVOC relay experiments.
HEp-2 cell monolayers were grown on 13 mm glass coverslips in 24-well tissue culture plates. Cells were seeded at 5x105 cells (ml culture medium)–1 and incubated at 37 °C in 5 % CO2 for at least 24 h to achieve 70–80 % confluency. For infection assays, the wells were inoculated with 10 µl of an overnight culture of bacteria (1x107 c.f.u.) grown in BHI broth and incubated for 3 h (strain E2348/69) or 6 h (strain TUV 93-0) at 37 °C in 5 % CO2. After incubation, the HEp-2 cell monolayers were washed with sterile PBS to remove non-adherent bacteria and lysed in 1 ml of 0.1 % Triton X-100. The lysed contents were transferred to a 1.5 ml Eppendorf tube and washed twice with sterile PBS to remove traces of Triton X-100. The cell debris and bacterial pellet were resuspended in 100 µl LB [(1–5)x108 c.f.u. ml–1] and 10 µl [(1–5)x106 c.f.u.] was added to fresh uninfected HEp-2 cell monolayers for the relay assay. The relay assay continued for 1 h (strain E2348/69) or 3 h (strain TUV 93-0) when the cells were washed with sterile PBS to remove non-adherent bacteria and processed for fluorescent actin staining (FAS) (Knutton et al., 1989). The appearances at these times were compared to those at the same incubation times in initial assays. In addition, 25 µl of the inoculum recovered from the initial assay was added onto intestinal explants for the HEp-2 : IVOC relay assay, which was performed for 8 h (Hicks et al., 1998).
Distal ileal IVOC : colonic IVOC relay experiments.
In order to establish the procedure, initial experiments were performed with duodenal explants in duplicate. Paired duodenal samples were taken from a single patient and underwent 8 h IVOC, one being inoculated with strain E2348/69. After 8 h, the infected sample was placed next to the uninfected sample and IVOC was continued for a further 12 h. An uninfected D4 sample was incubated for 20 h as a negative control.
For O157 : H7 strains, distal ileal (PP containing) and transverse colon biopsy samples were obtained from a single patient. The colonic sample was placed in organ culture without bacterial inoculation, while the ileal sample was incubated with bacterial strain 12900 (n=6) or TUV 93-0 (n=5) for 8 h routine IVOC. At 8 h the sample was washed in PBS, placed in close proximity to the colonic sample in fresh organ culture medium and IVOC was continued for a further 12 h. The medium was changed every 4 h. Each of the 11 experiments was performed with different patients providing paired ileal and colonic biopsy samples. Control IVOC included uninfected colonic samples incubated for 20 h and colonic samples incubated with O157 : H7 strains 12900 and TUV 93-0 for 20 h.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). For this study, five further O157 : H7 strains and an O26 : H11 Stx-negative strain (Table 1
) were tested for tropism to duodenum, distal ileum, PP and transverse colon using IVOC.
Table 2 shows the results. Prototype strain 85/170 again showed FAE-restricted tropism, as did AGT300 (Fig. 1a). Other strains showed FAE adherence (Table 2); however, four strains (12900, Sakai 813, TUV 93-0 and 3801) also adhered to ileal villi around PP, sometimes in large colonies (Fig. 1b, c), and strains 12900, TT12B, Sakai 813 and TUV 93-0 showed some adhesion to proximal small intestine (Fig. 1d). On two occasions isolated follicles were present in D4 biopsies, to which both TT12B (Fig. 1e) and 85/170 (Table 2) adhered. In contrast to the accepted dogma that EHEC strains adhere to colon, only 1 of 40 transverse colon IVOC showed A/E lesion formation (strain TT12B, Fig. 1f).
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), one O26 : H11 (intimin ) and one O103 : H2 (intimin ). We performed 113 colonic IVOCs to determine if there was selective adhesion to a particular colonic region (Table 3), but only 4 (3.5 %) IVOCs from distal colonic sites (transverse colon to rectum) showed A/E lesion formation in a similar appearance to that shown in Fig. 1(f); there was no adhesion in proximal colonic samples (caecum and ascending colon). EAEC strain O42 demonstrated colonic adhesion on all occasions. This suggested that either IVOC conditions prevented Stx-negative EHEC colonic adhesion, or that initial adhesion to FAE is followed at an undetermined time by colonization of other intestinal regions. This prompted an investigation of environmental factors.
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) but its tropism did not change in mannose-free medium. Due to health and safety considerations we were not able to use Stx-positive strains and it is possible that Stx itself may influence tropism through the modulation of receptor expression (Robinson et al., 2006).
HEp-2 cell : HEp-2 cell relay infection assay of EPEC and O157 : H7
HEp-2 cells incubated for 1 h with strain E2348/69 showed scanty adherence (Fig. 2a), whereas the 1 h relay infection assay following Triton extraction from a standard 3 h HEp-2 cell assay produced large bacterial colonies on the majority of the cells (Fig. 2b). Strain TUV 93-0 adhered extremely poorly at both 2 h and 3 h infection of HEp-2 cells (Fig. 2c), whereas both 2 and 3 h relay assays showed large colonies (Fig. 2d). Hence, adherence in the HEp-2 relay infection assay occurred more rapidly and was enhanced in comparison to the standard assay for strains E2348/69 and TUV 93-0.
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IVOC : IVOC relay infection
These experiments involved generating O157 : H7-mediated A/E lesions on FAE, which were then incubated with transverse colonic explants for a further 12 h, to allow time for the development of colonic adhesion via bacterial spread from FAE. The ability of bacteria to spread from explant to explant was confirmed in D4 : D4 relay infections carried out using strain E2348/69 (Fig. 3a). No adhesion was noted on uninfected D4 controls after 20 h incubation (data not shown).
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) and bacterial-like imprints were seen on the epithelial surface of 12900-infected FAE among A/E adhering bacteria (Fig. 3c). These probably represent sites of A/E lesion formation where bacteria have become detached.
IVOC incubations of strains TUV 93-0 and 12900 with transverse colon explants for a period of 20 h generated adhesion on 0/3 and 1/3 occasions respectively without A/E lesion formation (Fig. 3d), indicating that prolonged incubation per se did not promote O157 : H7 colonic adhesion. The FAE : transverse colon relay assay with TUV 93-0 and 12900 produced small foci of adhering bacteria on 2/5 and 2/6 transverse colon explants respectively, again without A/E lesion formation (Fig. 3e, f). No bacterial adhesion occurred on uninfected IVOC incubated for 20 h (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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O26 : H11 strain and by an intimin -expressing O103 : H2 (Fitzhenry et al., 2003) strain. More strains should be tested before this can be assumed to be representative for these serotypes. However, we have now shown that Stx-negative EHEC adhesion is not limited to FAE, but includes normal absorptive epithelium, albeit in a region where lymphoid follicles are commonly found.
We were unable to demonstrate reproducible colonic adhesion of any strain using direct inoculation of IVOC samples, although four strains did adhere on single occasions. The positive control strain, an EAEC, adhered on each occasion, and EPEC strain E2348/69 shows colonic adhesion (Hicks et al., 1998), indicating that this is not an absolute problem of IVOC. It is possible that technical reasons precluded colonic adhesion but simple environmental factors were investigated and no positive results were seen.
Does EHEC colonize the colon in man? Although EPEC colonic colonization has been reported in vivo (Lewis et al., 1987; Rothbaum et al., 1982), there are no such reports from studies of in vivo EHEC infection in man, despite colonic pathology being clearly described (Griffin et al., 1990). Stx, as toxin levels can reach high levels in the intestinal lumen (Gamage et al., 2003), may induce marked mucosal damage via endothelial (Jacewicz et al., 1999) or epithelial (Schuller et al., 2004) interaction, without bacteria being present at that site. One study specifically looked for adhering organisms in acute O157 : H7 infection and concluded, as no bacteria were seen, that the pathology resulted from toxin-mediated ischaemia (Kelly et al., 1987). Other reports have described a mixture of acute infective and ischaemic changes (Griffin et al., 1990), leaving the debate open. If human tissue samples are taken at late stages in the illness or post-mortem, then bacterial adhesion may have diminished and/or be difficult to identify.
It seems likely that the PP-rich distal ileum represents the initial site of EHEC adhesion, and colonization spreads from there to other regions of the gut. The extent of the spread and the regions that are targeted are unknown. This pattern of colonization is shown by other bacteria, including REPEC (Cantey & Inman, 1981; Heczko et al., 2000) and C. rodentium (Wiles et al., 2004), but is not a universal phenotype as EPEC strain E2348/69 appears able to colonize the small intestine directly (Hicks et al., 1998; Knutton et al., 1987). The infective dose for EHEC is low (102 c.f.u. ml–1) whereas that for EPEC is much higher (Nataro & Kaper, 1998). It is possible that FAE colonization affords a ‘toe hold’ without inducing a strong host response, allowing multiplication and spread. This could be termed a ‘stealth’ approach. Down-regulation of antibacterial peptides of the innate immune response has been shown in the initial stages of Shigella infection in man (Islam et al., 2001), where the infective dose is also low (DuPont et al., 1989), and such interaction may be a factor that allows colonization to spread from follicular sites of infection. In comparison, direct infection of mucosal surfaces, as a ‘frontal assault’ approach may require higher numbers of organisms to overcome the innate responses.
In mice, C. rodentium spreads from the caecal follicular region to the large intestine within 4 days of infection (Wiles et al., 2004). E. coli strain RDEC-1 takes a similar time in the rabbit (Cantey & Inman, 1981). This time is outside the possibilities of the IVOC system as tissue viability is limited to 24–48 h. However, a recent study demonstrated rapid colonization when C. rodentium from infected animals was used to infect mice within 24 h of excretion. They showed a temporary hyperinfective state that facilitated colonic colonization without transient infection of the caecal patch area (Wiles et al., 2005). Here we tested if colonization of the FAE surface could mediate subsequent colonic colonization and found enhanced colonic adhesion but no A/E lesion formation in the time permitted. A similar enhancement, but with A/E lesion formation, was found for strain E2348/69 when transferring infection from duodenum to duodenum. There may be phase variation within the EHEC population so the initial colonization of FAE may act as a selection process for adherent bacteria, increasing the chances of colonic colonization. Alternatively, the process of FAE colonization may induce activation of genes which mediate colonization of other gut regions.
Mediators of FAE colonization have been identified in some species, i.e. long polar fimbriae (Lpf) in Salmonella (Baumler et al., 1996) and AF/R1 fimbriae in REPEC (Von Moll & Cantey, 1997). Homologous genes to Lpf have been identified in EHEC strains, and the operons show a high degree of similarity (Fitzhenry et al., 2006). However, deletion of one or both of the Lpf operons in O157 : H7 did not prevent FAE adhesion but resulted in additional colonization of the small intestine (Fitzhenry et al., 2006), questioning their role in targeting PP in O157 : H7.
It appears possible that O157 : H7 can colonize FAE beyond the distal ileum, as we demonstrated adhesion to FAE from the duodenum. Mucosal lymphoid follicles are found along the entire length of the small and large intestine (Cornes, 1965), giving a widespread potential for sites of adhesion. Whether host factors come into play in vivo to limit this remains to be determined. Lymphoid follicles in the rectal region have been implicated in O157 : H7 carriage in cattle (Gally et al., 2003), indicating that FAE adhesion may be important in continuing colonization as well as in the initial stages of infection.
In summary, we have shown an initial ex vivo tropism of the distal ileal region and FAE for Stx-negative EHEC strains, and that adherent O157 : H7 from distal ileal FAE can colonize the colon in a novel, non-A/E manner. Direct colonization of the colon is not seen ex vivo, despite the finding of extensive colonic pathology in vivo, and the relative contributions of direct bacterial infection and Stx to colonic pathology in EHEC infections remain to be established in man.
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ACKNOWLEDGEMENTS |
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Edited by: P. H. Everest
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Received 5 October 2006;
revised 26 November 2006;
accepted 6 December 2006.
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