Mouse model of enteropathogenic Escherichia coli infection

Enteropathogenic Escherichia coli (EPEC) is an important cause of diarrhea in humans. EPEC infection of cultured intestinal epithelial cells induces attaching and effacing (A/E) lesions, alters intestinal ion transport, increases paracellular permeability, and stimulates inflammation. The lack of a small-animal model has restricted in vivo studies examining EPEC-host interactions. The aim of this study was to characterize the C57BL/6J mouse as a model of EPEC infection. We have shown that EPEC can adhere to and colonize the intestinal epithelium of C57BL/6J mice. Animal weight and water intake were not altered during 10 days of EPEC infection. The proximal colon of infected mice contained semisolid stool, with stool pellets forming only in the distal colon. In contrast, the entire colon of control mice contained formed stool. Microvillous effacement and actin rearrangement, characteristic of A/E lesions, were seen in the intestine of infected mice but not control mice. Histological assessment revealed increased numbers of lamina propria neutrophils with occasional crypt abscesses, intraepithelial lymphocytes, and goblet cells in the intestine of EPEC-infected mice. Altogether, these data suggest that the C57BL/6J mouse is susceptible to infection by EPEC and will provide a suitable in vivo model for studying the consequences of EPEC infection.     

Effect of zinc in enteropathogenic Escherichia coli infection

Enteropathogenic Escherichia coli (EPEC) infection triggers the release of ATP from host intestinal cells, and the ATP is broken down to ADP, AMP, and adenosine in the lumen of the intestine. Ecto-5′-nucleotidase (CD73) is the main enzyme responsible for the conversion of 5′-AMP to adenosine, which triggers fluid secretion from host intestinal cells and also has growth-promoting effects on EPEC bacteria. In a recent study, we examined the role of the host enzyme CD73 in EPEC infection by testing the effect of ecto-5′-nucleotidase inhibitors. Zinc was a less potent inhibitor of ecto-5′-nucleotidase in vitro than the nucleotide analog α,β-methylene-ADP, but in vivo, zinc was much more efficacious in preventing EPEC-induced fluid secretion in rabbit ileal loops than α,β-methylene-ADP. This discrepancy between the in vitro and in vivo potencies of the two inhibitors prompted us to search for potential targets of zinc other than ecto-5′-nucleotidase. Zinc, at concentrations that produced little or no inhibition of EPEC growth, caused a decrease in the expression of EPEC protein virulence factors, such as bundle-forming pilus (BFP), EPEC secreted protein A, and other EPEC secreted proteins, and reduced EPEC adherence to cells in tissue culture. The effects of zinc were not mimicked by other transition metals, such as manganese, iron, copper, or nickel, and the effects were not reversed by an excess of iron. Quantitative real-time PCR showed that zinc reduced the abundance of the RNAs encoded by the bfp gene, by the plasmid-encoded regulator (per) gene, by the locus for the enterocyte effacement (LEE)-encoded regulator (ler) gene, and by several of the esp genes. In vivo, zinc reduced EPEC-induced fluid secretion into ligated rabbit ileal loops, decreased the adherence of EPEC to rabbit ileum, and reduced histopathological damage such as villus blunting. Some of the beneficial effects of zinc on EPEC infection appear to be due to the action of the metal on EPEC bacteria as well as on the host.     

Invasive potential of noncytotoxic enteropathogenic Escherichia coli in an in vitro Henle 407 cell model.

The invasive capacity of 13 enteropathogenic Escherichia coli strains was assessed in vitro in Henle 407 cell culture. Both fluorescent microscopy of infected monolayers stained with acridine orange and electron microscopy revealed the presence of intracellular bacteria. As shown by acridine orange-stained infected monolayers, the number of internalized bacteria increased with time. Monolayers infected for 3 h were treated with antibiotics and either [14C]glutamine or [3H]leucine and incubated for various time intervals, after which the amount of radioactivity present in the washed monolayers was measured. A significant (P less than 0.005) increase in uptake was evident for up to 4 h after the addition of radiolabeled amino acid. This finding was confirmed by an increase in bacterial number in cultured cells and in protein concentration of infected cells with time. None of the South African enteropathogenic E. coli isolates used in these studies produced Vero cytotoxin. These findings demonstrate that, in addition to adherence, cell penetration and intracellular multiplication take place in epithelial cell-derived tissue culture cells infected by enteropathogenic E. coli.     

Enzootic enteropathogenic Escherichia coli infection in laboratory rabbits

Enteropathogenic Escherichia coli (EPEC) is the most important cause of persistent diarrhea in children, particularly in developing countries. Animals serve as pathogenic E. coli reservoirs, and compelling evidence for cross-species EPEC transmission exists. In this report, enzootic EPEC infection associated with up to 10.5% diarrhea-associated morbidity in a large laboratory Dutch Belted rabbit colony was investigated. These rabbits were obtained from a commercial vendor and had acute diarrhea following shipment. Fecal culture of 20 rabbits yielded 48 E. coli isolates, 83% of which were eae positive. Repetitive sequence-based PCR (REP-PCR) and serologic analysis identified a single disease-associated EPEC O145:H2 strain. In sampled rabbits, EPEC-positive culture and the presence of diarrhea were significantly associated. This strain displayed a localized adherence-like HEp-2 cell adherence pattern, as seen in diarrheic human infant EPEC isolates. Treatment was instituted with the fluoroquinolone antibiotic enrofloxacin, to which all isolates were susceptible. Preshipment parenteral enrofloxacin administration reduced diarrhea-associated morbidity 22-fold and mortality 12-fold in subsequent deliveries. This report emphasizes the zoonotic potential of animal EPEC strains and the need for virulence determinant-based screening of E. coli isolates from diarrheic animals.     

Regulation of virulence and motility by acetate in enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli colonizes the human small intestine and causes severe diarrhea. Short-chain fatty acids are abundant in the intestine owing to the metabolic activity of the microflora and are important for intestinal health. Here, we found that acetate promotes the adherence of enteropathogenic E. coli O127:H6 to Caco-2 intestinal epithelial cells and its motility on semi-solid Luria–Bertani agar by activating the expression of locus of enterocyte effacement genes and flagellar genes, respectively. The effect of acetate on locus of enterocyte effacement gene expression is mediated by Ler, the master regulator of locus of enterocyte effacement genes, whereas the regulation of flagellar genes by acetate is dependent on the RNA polymerase sigma factor FliA. Conversely, formate, propionate, and butyrate had little or no effect on enteropathogenic E. coli O127:H6 adherence and motility. Finally, the acetate-mediated regulatory pathway was found to be a widespread mechanism used by a range of enteropathogenic E. coli to mediate bacterial virulence and motility. Therefore, upon entering the human small intestine, enteropathogenic E. coli may respond to the higher acetate level to enhance its virulence and motility, leading to efficient colonization of the target niche.     

Mutual enhancement of virulence by enterotoxigenic and enteropathogenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) and enteropathogenic E. coli (EPEC) are common causes of diarrhea in children in developing countries. Dual infections with both pathogens have been noted fairly frequently in studies of diarrhea around the world. In previous laboratory work, we noted that cholera toxin and forskolin markedly potentiated EPEC-induced ATP release from the host cell, and this potentiated release was found to be mediated by the cystic fibrosis transmembrane conductance regulator. In this study, we examined whether the ETEC heat-labile toxin (LT) or the heat-stable toxin (STa, also known as ST) potentiated EPEC-induced ATP release. We found that crude ETEC culture filtrates, as well as purified ETEC toxins, did potentiate EPEC-induced ATP release in cultured T84 cells. Coinfection of T84 cells with live ETEC plus EPEC bacteria also resulted in enhanced ATP release compared to EPEC alone. In Ussing chamber studies of chloride secretion, adenine nucleotides released from the host by EPEC also significantly enhanced the chloride secretory responses that were triggered by crude ETEC filtrates, purified STa, and the peptide hormone guanylin. In addition, adenosine and LT had additive or synergistic effects in inducing vacuole formation in T84 cells. Therefore, ETEC toxins and EPEC-induced damage to the host cell both enhance the virulence of the other type of E. coli. Our in vitro data demonstrate a molecular basis for a microbial interaction, which could result in increased severity of disease in vivo in individuals who are coinfected with ETEC and EPEC.     

Endogenous CO2 may inhibit bacterial growth and induce virulence gene expression in enteropathogenic Escherichia coli

Analysis of the growth kinetics of enteropathogenic Escherichia coli (EPEC) revealed that growth was directly proportional to the ratio between the exposed surface area and the liquid culture volume (SA/V). It was hypothesized that this bacterial behavior was caused by the accumulation of an endogenous volatile growth inhibitor metabolite whose escape from the medium directly depended on the SA/V. The results of this work support the theory that an inhibitor is produced and indicate that it is CO(2). We also report that concomitant to the accumulation of CO(2), there is secretion of the virulence-related EspB and EspC proteins from EPEC. We therefore postulate that endogenous CO(2) may have an effect on both bacterial growth and virulence.     

Adhesion and its role in the virulence of enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) organisms are an important cause of diarrheal disease in young children. The virulence of EPEC is a multifactorial process and involves a number of distinct stages. Initial adherence to intestinal mucosa is mediated by fimbriae which bring about a distinct form of adhesion, localized adhesion. Intimate adhesion of the bacterium to the eukaryotic membrane occurs, resulting in the activation of signal transduction pathways. Microvilli are disrupted and effaced from the apical membrane which then cups around the organism to form pedestal structures, the attaching and effacing lesion. Diarrhea may be produced by alteration of the permeability of the apical membrane and also through a malabsorption mechanism. The pathways involved in the production of the attaching and effacing lesion are described. EPEC organisms were originally thought to belong to a number of distinct serogroups; it is now apparent that many isolates belonging to these serogroups are not pathogenic or belong to other pathogenic groups of E. coli. In addition, isolates falling outside of these serogroups are considered to be true EPEC. The definition of EPEC based on serotyping is inaccurate and should be replaced by methods that specifically detect the virulence properties of EPEC.     

Cross-reactive protection against enterohemorrhagic Escherichia coli infection by enteropathogenic E. coli in a mouse model

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are related attaching and effacing (A/E) pathogens. The genes responsible for the A/E pathology are carried on a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). Both pathogens share a high degree of homology in the LEE and additional O islands. EHEC prevalence is much lower in areas where EPEC is endemic. This may be due to the development of antibodies against common EPEC and EHEC antigens. This study investigated the hypothesis that EPEC infections may protect against EHEC infections. We used a mouse model to inoculate BALB/c mice intragastrically, first with EPEC and then with EHEC (E. coli O157:H7). Four control groups received either a nonpathogenic E. coli (NPEC) strain followed by EHEC (NPEC/EHEC), phosphate-buffered saline (PBS) followed by EHEC (PBS/EHEC), EPEC/PBS, or PBS/PBS. Mice were monitored for weight loss and symptoms. EPEC colonized the intestine after challenge, and mice developed serum antibodies to intimin and E. coli secreted protein B (encoded in the LEE). Prechallenge with an EPEC strain had a protective effect after EHEC infection, as only a few mice developed mild symptoms, from which they recovered. These mice had an increase in body weight similar to that in control animals, and tissue morphology exhibited mild intestinal changes and normal renal histology. All mice that were not prechallenged with the EPEC strain developed mild to severe symptoms after EHEC infection, with weight loss as well as intestinal and renal histopathological changes. These data suggest that EPEC may protect against EHEC infection in this mouse model.     

Role of EspB in experimental human enteropathogenic Escherichia coli infection

Enteropathogenic Escherichia coli (EPEC), a leading cause of diarrhea among infants in developing countries, induces dramatic alterations in host cell architecture that depend on a type III secretion system. EspB, one of the proteins secreted and translocated to the host cytoplasm via this system, is required for numerous alterations in host cell structure and function. To determine the role of EspB in virulence, we conducted a randomized, double-blind trial comparing the ability of wild-type EPEC and an isogenic DeltaespB mutant strain to cause diarrhea in adult volunteers. Diarrhea developed in 9 of 10 volunteers who ingested the wild-type strain but in only 1 of 10 volunteers who ingested the DeltaespB mutant strain. Marked destruction of the microvillous brush border adjacent to adherent organisms was observed in a jejunal biopsy from a volunteer who ingested the wild-type strain but not from two volunteers who ingested the DeltaespB mutant strain. Humoral and cell-mediated immune responses to EPEC antigens were stronger among recipients of the wild-type strain. In addition, four of the volunteers who ingested the wild-type strain had lymphoproliferative responses to EspB. These results demonstrate that EspB is a critical virulence determinant of EPEC infections and suggest that EspB contributes to an immune response.     

An ultrastructural study of enteropathogenic Escherichia coli infection in human infants

Over the past 2 years, we have studied and treated 18 infants with protracted diarrhea due to an enteropathogenic Escherichia coli serogroup 0119. All patients had persistent stool escretion and jejunal overgrowth with this pathogenic E. coli. Jejunal biopsy revealed atrophy of villi with a chronic inflammatory cell infiltrate in the lamina propria. E. coli 0119 adhered to the luminal surface of enterocytes. Electron microscopy showed disappearance of glycocalyx and microvilli at the areas of bacterial adherence. Intracellular damage was indicated by dilatation of rough endoplasmic reticulum, mitochondrial changes, and cytoplasmic pallor. Similar changes in histology and ultrastructure occurred in ileal epithelial cells. Glandular crypt epithelium showed prominent subnuclear vacuolation and separation of lateral intercellular junctions throughout the small intestine. Rectal mucosal biopsy showed mucus depletion and irregular atrophy of the epithelium, with E. coli 0119 adherent to the luminal surface. Ultrasuctural damage paralleled that in the small intestine. E. coli 0119 causes damage to epithelial cells throughout the infant intestinal tract. This damage leads to atrophy of villi and a marked reduction in absorptive surface area, resulting in protracted diarrhea.     

Contribution of long polar fimbriae to the virulence of rabbit-specific enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) is a major of cause of diarrhea among children in developing countries. Although EPEC is a human specific pathogen, some related strains are natural pathogens of animals, including laboratory-bred rabbits. We have identified two chromosomal loci in rabbit-specific EPEC (REPEC) O15:H- strain 83/39, which are predicted to encode long polar fimbriae (LPF). lpf(R154) was identical to a fimbrial gene cluster, lpf(O113), identified previously in enterohemorrhagic E. coli (EHEC) O113:H21. The second locus, lpf(R141), comprised a novel sequence with five predicted open reading frames, lpfA to lpfE, that encoded long fine fimbriae in nonfimbriated E. coli ORN103. The predicted products of lpf(R141) shared identity with components of the lpfABCC'DE gene cluster from EHEC O157:H7, and the fimbriae were similar in morphology and length to LPF from EHEC O157:H7. Interruption of lpf(R141) resulted in significant attenuation of REPEC 83/39 for rabbits with respect to the early stages of colonization and severity of diarrhea. However, there was no significant difference in the number of bacteria shed at later time points or in overall body weight and mortality rate of rabbits infected with lpf(R141) mutant strains or wild-type REPEC 83/39. Although rabbits infected with the lpf(R141) mutants did not develop severe diarrhea, there was evidence of attaching and effacing histopathology, which was indistinguishable in morphology, location, and extent compared to rabbits infected with wild-type REPEC 83/39. The results suggested that lpf(R141) contributes to the early stages of REPEC-mediated disease and that this is important for the development of severe diarrhea in susceptible animals.     

Recognition of enteropathogenic Escherichia coli virulence determinants by human colostrum and serum antibodies

Human colostra and sera collected from Mexican mothers and their children at birth and 6 months thereafter were studied for the presence of antibodies against the bundle-forming pilus and several chromosomal virulence gene products (intimin and secreted proteins EspA and EspB) of enteropathogenic Escherichia coli (EPEC). Among 21 colostrum samples studied, 76, 71.5, 57, and 47% of them contained immunoglobulin A (IgA) antibodies against EspA, intimin, EspB, and BfpA, respectively. Interestingly, there was a difference in IgG response to EPEC antigens between the sera from neonates and sera from the same children 6 months later. While the number of neonates reacting to Esps and intimin diminished when they reached 6 months of age, those reacting with BfpA increased from 9 to 71%. Intimin from an enterohemorrhagic E. coli strain was also recognized by most of the samples reacting with EPEC intimin. These data suggest that Bfp and Esps elicit an antibody response during the early days of life of neonates and support the value of breast-feeding in areas of the world where bacterial diarrheal infections are endemic.     

Identification of virulence genes linked with diarrhea due to atypical enteropathogenic Escherichia coli by DNA microarray analysis and PCR

The role of atypical enteropathogenic Escherichia coli (EPEC) in childhood diarrhea is controversial. The aim of the present study was to search for genes linked with diarrhea in atypical EPEC strains from a case-control study among Norwegian children. Using DNA microarray analysis, genomic DNAs from strains isolated from children with (n = 37) and without (n = 20) diarrhea were hybridized against 242 different oligonucleotide probes specific for 182 virulence genes or markers from all known E. coli pathotypes. PCR was performed to test the strains for seven putative virulence genes not included in the microarray panel. The OI-122 gene efa1/lifA was the gene with the strongest statistical association with diarrhea (P = 0.0008). Other OI-122 genes (set/ent, nleB, and nleE) and genes with other locations (lpfA, paa, ehxA, and ureD) were also associated with diarrheal disease. The phylogenetic marker gene yjaA was negatively associated with diarrhea (P = 0.0004). Atypical EPEC strains could be classified in two main virulence groups based on their content of OI-122, lpfA, and yjaA genes. Among children with diarrhea, atypical EPEC isolates belonging to virulence group I (OI-122 and lpfA positive, yjaA negative) were the most common, while the majority of isolates from healthy children were classified as virulence group II strains (OI-122 negative, lpfA and yjaA positive; P < 0.001). In conclusion, using DNA microarray analysis to determine the virulence gene profile of atypical EPEC isolates, several genes were found to be significantly associated with diarrhea. Based on their composition of virulence genes, the majority of strains could be classified in two virulence groups, of which one was seen mainly in children with diarrhea.     

Plasmid-encoded expression of lipopolysaccharide O-antigenic polysaccharide in enteropathogenic Escherichia coli.

The role of a plasmid in the virulence activity of an enteropathogenic Escherichia coli (EPEC) strain belonging to serotype 0111:NM was examined. EPEC strain B171, which is resistant to chloramphenicol, streptomycin, sulfathiazole, and tetracycline, harbors a 54-megadalton plasmid, pYR111, and exhibits localized adherence (LA) with HeLa cells. Curing the plasmid yielded strain B171-4, which had lost the ability to exhibit LA, resistance to the antibiotics, and the lipopolysaccharide (LPS) O-antigenic polysaccharide. To confirm that these phenotypic characteristics were specified by pYR111, the plasmid was transferred by conjugation into a nalidixic acid-resistant strain of E. coli HB101. LA and antimicrobial resistance were expressed in most of the transconjugants examined. The O-polysaccharide side chains, antigenically reactive with O111-specific antiserum, were also expressed by the transconjugants. Although EPEC plasmids coding for both drug resistance and LA have been described, an EPEC plasmid encoding the expression of an LPS O antigen has not been previously reported. Similar findings described for some Shigella and Salmonella strains suggest that plasmid-encoded modification of the LPS in some enteric bacterial species may be more common than previously recognized and may contribute to the characteristic virulence activity of the organism.     

Differentiation in virulence patterns of Escherichia coli possessing eae genes

In this study 98 Escherichia coli strains which belonged to traditional enteropathogenic (EPEC) serotypes and 82 enterohemorrhagic E. coli (EHEC) strains were screened by polymerase chain reaction (PCR) for the presence of E. coli -attaching and -effacing (eae) genes. These strains were also hybridized with the enteropathogenic adherence factor (EAF) probe and examined in the fluorescence actin staining (FAS) test. The results obtained from the individual strains demonstrated that all 26 class I EPEC with localized adherence to HEp-2 cells carried EAF and eae genes. In contrast, of 72 EPEC strains with no or diffuse adherence only 1 strain was EAF positive and 6 strains had eae. Of 82 EHEC strains a total of 75 carried eae sequences. Of considerable interest, 15 of 21 E. coli strains that lost their slt genes during subcultivation were found to be eae positive. As controls a total of 53 enterotoxigenic and enteroinvasive E. coli, and 125 E. coli strains from the normal flora were investigated and all displayed negative results in the eae-PCR. From the 201 strains comprising classical EPEC serotypes, EHEC and E. coli with lost slt genes, a total of 126 displayed a positive FAS test and 122 reacted in the eae-PCR. Only 4 strains were FAS test positive but eae-PCR negative. Our data indicate that E. coli strains possessing the eae genes are heterogenous with respect to their virulence determinants. Loss of virulence plasmids and phage-encoded slt genes either in the host or during storage may contribute to this heterogeneity. The eae-PCR detected all class I EPEC and 91.5% of the EHEC. For diagnostic purposes we, therefore, recommend the combination of eae- and slt-specific gene probes which allowed a 100% detection of the class I EPEC and EHEC strains investigated here.     

Multiplex PCRs for identification of Escherichia coli virulence genes

PCRs were developed to detect 11 Escherichia coli virulence genes. Primers amplified the respective genes without cross-reaction with other genes. Specificity was maintained in multiplex reactions; excellent amplification of target genes was possible with a minimum of four multiplex reactions. These reactions successfully identified genes in E. coli from the feces of four dogs.     

espC pathogenicity island of enteropathogenic Escherichia coli encodes an enterotoxin

At least five proteins are secreted extracellularly by enteropathogenic Escherichia coli (EPEC), a leading cause of infant diarrhea in developing countries. However only one, EspC, is known to be secreted independently of the type III secretion apparatus encoded by genes located within the 35.6-kb locus of enterocyte effacement pathogenicity island. EspC is a member of the autotransporter family of proteins, and the secreted portion of the molecule is 110 kDa. Here we determine that the espC gene is located within a second EPEC pathogenicity island at 60 min on the chromosome of E. coli. We also show that EspC is an enterotoxin, indicated by rises in short-circuit current and potential difference in rat jejunal tissue mounted in Ussing chambers. In addition, preincubation with antiserum against the homologous Pet enterotoxin of enteroaggregative E. coli eliminated EspC enterotoxin activity. Like the EAF plasmid, the espC pathogenicity island was found only in a subset of EPEC, suggesting that EspC may play a role as an accessory virulence factor in some but not all EPEC strains.