Adherence of Escherichia coli O157:H7 to epithelial cells in vitro and in pig gut loops is affected by bacterial culture conditions.
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The objectives of this study were to determine the effect of bacterial culture conditions on adherence of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain 86-24 in vivo to pig enterocytes and to compare the results with adherence in vitro to cultured HEp-2 and IPEC-J2 cells. Growth of O157:H7 in MacConkey broth (MB) resulted in almost no adherence to both HEp-2 and IPEC-J2 cells; prior exposure of the bacteria to pH 2.5 reduced adherence. There was greater adherence by bacteria from static cultures than by those from shaken cultures and by bacteria cultured in brain-heart infusion (BHI) plus NaHCO₃ (BHIN) than by bacteria cultured in BHI. In contrast, in pig ileal loops, bacteria cultured in MB adhered well to enterocytes, and prior exposure to pH 2.5 had no effect on adherence. Among several media tested for their effect on bacterial adherence in the pig intestine, MB and BHIN proved to be the best. Bacterial adherence was dose-dependent and was more extensive in the ileum than in the colon. This study demonstrated that there are remarkable differences between culture conditions that promote adherence of an EHEC O157:H7 strain in vitro and in vivo, that culture conditions profoundly affect adherence to epithelial cells in vitro and in vivo, and that pig ileal loops are better suited to adherence studies than are colon loops.Keywords:
Microbiological culture
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Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli O157:H7 are intestinal pathogens that profoundly damage the microvilli and subapical cytoskeleton of epithelial cells. Here we report finding in EPEC a 35-kbp locus containing several regions implicated in formation of these lesions. DNA probes throughout this locus hybridize to E. coli O157:H7 and other pathogens of three genera that cause similar lesions but do not hybridize to avirulent members of the same species. The EPEC locus and a different virulence locus of uropathogenic E. coli insert into the E. coli chromosome at the identical site and share highly similar sequences near the point of insertion.
Enteropathogenic Escherichia coli
Pathogenicity island
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Pathogenic Escherichia coli
Cloning (programming)
Escherichia
Human pathogen
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ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a diarrheal pathogen that causes attaching and effacing (A/E) lesions on intestinal epithelial cells. Strains of the O157 serogroup carry the large virulence plasmid pO157, which encodes the etp type II secretion system that secretes the genetically linked zinc metalloprotease StcE. The Ler regulator controls expression of many genes involved in A/E lesion formation, as well as StcE, suggesting StcE may be important at a similar time during colonization. Our laboratory has previously demonstrated that StcE cleaves C1-esterase inhibitor, a regulator of multiple inflammation pathways. Here we report two new substrates for StcE, mucin 7 and glycoprotein 340, and that purified StcE reduces the viscosity of human saliva. We tested the hypothesis that StcE contributes to intimate adherence of EHEC to host cells by cleavage of glycoproteins from the cell surface. The fluorescent actin stain (FAS) test was used to observe the intimate adherence represented by fluorescently stained bacteria colocalized with regions of bundled actin formed on HEp-2 cells. An E. coli O157:H7 strain with a stcE gene deletion was not affected in its ability to generally adhere to HEp-2 cells, but it did score threefold lower on the FAS test than wild-type or complemented strains. Addition of exogenous recombinant StcE increased intimate adherence of the mutant to wild-type levels. Thus, StcE may help block host clearance of E. coli O157:H7 by destruction of some classes of glycoproteins, and it contributes to intimate adherence of E. coli O157:H7 to the HEp-2 cell surface.
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The adherence of radiolabeled Salmonella typhimurium to freshly isolated enterocytes of rats was studied. The results established that type 1 fimbriated strains adhered in significantly higher numbers than did related nonfimbriated strains. Adherence was inhibited by D-mannose and methyl alpha-D-mannoside. Results of kinetic studies indicated that adherence was biphasic; the number of bacteria that adhered per enterocyte remained constant for approximately 20 min and then increased rapidly under the assay conditions. The second phase was associated with structural damage to the enterocytes. The addition of chloramphenicol did not prevent the initial attachment of bacteria to enterocytes but did prevent the second phase. Viable and nonviable bacterial cells adhered to enterocytes, but only viable bacteria were destructive. Freshly isolated enterocytes (trypan blue impermeable) and enterocytes stored overnight (trypan blue permeable) were infected by viable S. typhimurium in a similar manner, suggesting that metabolic activity of the host cell was of less consequence than metabolic activity of the bacterial cells. A model for the role of mannose-sensitive fimbriae as a virulence factor is proposed.
Enterocyte
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The effects of culture conditions on the extent of Escherichia coli O157:H7 attaching-effacing (A/E) adherence in an adult bovine large intestinal mucosal explant model were assessed by three different morphometric methods. Measurement of the percent of tissue sections with A/E adherence and the number of foci of A/E adherence mm(-1) of surface epithelium was more sensitive than measurement of the percent of surface epithelium with A/E adherent bacteria for detection of treatment effects. Culture of bacterial inoculum in tryptic soy broth, incubation of explants in 5% CO(2), and rocking of explants on a platform rocker at 18 cycles min(-1) provided optimal conditions for A/E adherence. In future studies, the model may be used for preliminary testing of intervention strategies aimed at reduction of E. coli O157:H7 intestinal colonization of cattle.
Tryptic soy broth
Microbiological culture
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Two in vitro adherence assays involving isolated mucosal cells or mucosal explants were used to study the adherence of five Candida albicans strains to murine gastrointestinal mucosal surfaces. Adherence was found to be dependent on the strain used, and on the cellular arrangement, as well as the site of origin of the mucosal surface. Adherence of strains NCPF 3436 and 3310 to stomach and jejunal surfaces was affected by the pH of the medium. Binding between the C. albicans strains and stomach mucosal cells fluctuated as the pH was raised from pH 1·2 to pH 3·4. However, adherence increased with a rise in pH when the strains were incubated with stomach mucosal explants. Optimal adherence by both strains to jejunal mucosal surfaces occurred at neutral pH.
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The correlation between affinity of adherence in vitro to mice intestinal segments and infectivity in vivo was examined in an enterotoxigenic Escherichia coli (ETEC) strain. Two unstable phenotypes of the same strain were obtained by growing bacteria in agar or broth. Affinity of adherence in vitro calculated by Scatchard plot analysis of agar-grown bacteria was significantly higher than that of broth-grown bacteria. The effective dose of infection of agar-grown bacteria at 3, 24 and 72 h after infection, was one-tenth, one-half and the same, respectively, of that of broth-grown bacteria. The results suggest that the differences in the adhering ability of the inoculum influenced the number of bacteria retained in the intestine during the early phases of infection.
Enterotoxigenic Escherichia coli
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Guinea pig colonic epithelial cells released by treating sections of the colon with solutions containing EDTA, dithiothreitol, and citrate avidly adhered Shigella flexneri bacteria. Separation of the intestinal cells from nonbound bacteria was achieved by differential sedimentation on a Percoll gradient. Adherence of S. flexneri to the colonic cells was Ca2+ (1 mM) and time dependent. The pH optimum was pH 6.2, and almost no attachment (less than 5%) was observed at low temperature (4 degrees C). The average number of bacteria which bound to colonic cells was 70 bacteria per cell, whereas attachment to cells isolated from the ileum region was 6 bacteria per cell. Colonic cells obtained from the intestine of rabbits or rats did not adhere Shigella. Adherence to guinea pig colonic cells was inhibited (50%) by several carbohydrates, such as 0.1% fucose or 0.5% glucose, as well as by a lipopolysaccharide preparation (10 micrograms /ml) isolated from S. flexneri. Fixation of the bacteria with glutaraldehyde or preincubation of the bacteria with lectins or proteolytic enzymes did not affect their adherence. Proteolytic digestions or fixation of the epithelial cells, as well as pretreatments with lipopolysaccharide or fucose solutions, abolished their ability to adhere bacteria. These results indicate that a carbohydrate-binding substance on the surface of guinea pig colonic epithelial cells is responsible for the attachment of the Shigella bacilli.
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Background Escherichia coli O157:H7 strain 86–24 grown in MacConkey broth (MB) shows almost no adherence to cultured epithelial cells but adheres well in pig ligated intestines. This study investigated the mechanisms associated with the difference between in-vitro and in-vivo adherence of the MB culture. Methodology/Principal Findings It was found that decreased adherence in vitro by bacteria grown in MB was mainly due to lactose, possibly implicating the involvement of carbon catabolite repression (CCR). Expression of selected virulence-related genes associated with adherence and CCR was then examined by quantitative PCR. When bacteria were grown in MB and Brain Heart Infusion with NaHCO3 (BHIN) plus lactose, pH was reduced to 5.5–5.9 and there was a significant decrease in expression of the locus of enterocyte effacement (LEE) genes eae, tir, espD, grlA/R and ler, and an increase in cya (cAMP), and two negative regulators of the LEE, gadE and hfq. Putative virulence genes stcE, hlyA, ent and nleA were also decreased in vitro. Reversal of these changes was noted for bacteria recovered from the intestine, where transcripts for qseF and fis and putative virulence factors AidA15, TerC and Ent/EspL2 were significantly increased, and transcripts for AIDA48, Iha, UreC, Efa1A, Efa1B, ToxB, EhxA, StcE, NleA and NleB were expressed at high levels. Conclusions/Significance Presence of lactose resulted in decreased expression of LEE genes and the failure of EHEC O157:H7 to adhere to epithelial cells in vitro but this repression was overcome in vivo. CCR and/or acidic pH may have played a role in repression of the LEE genes. Bacterial pathogens need to integrate their nutritional metabolism with expression of virulence genes but little is known of how this is done in E. coli O157:H7. This study indicates one aspect of the subject that should be investigated further.
Enterocyte
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