Biotypes of enteropathogenic Escherichia coli strains from rabbits

The Isolator 1.5 Microbial tube (E. I. du Pont de Nemours & Co., Inc., Wilmington, Del.) is a commercially available blood culture system for use in pediatrics. The methodology is based on blood lysis followed by direct plating of the sample on culture media to detect bacteria and fungi. Comparative recovery rates of pathogens from blood collected in this and a conventional broth system were similar. The Isolator detected 104 of 120 clinically significant isolates, whereas 106 of 120 isolates were detected by the broth system. The major advantage of the Isolator methodology was early detection of septicemia. Initial detection of gram-negative bacteria occurred an average of 14.2 h earlier by the Isolator system than by the conventional broth method. The Isolator also permitted quantitation of bacteremia and fungemia. Probable contaminants were recovered from 10.0% of the cultures processed by the Isolator, but steps which could be taken to minimize this problem were identified. The Isolator is a useful method for pediatric blood cultures.     

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.     

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.     

Maternal milk contains antimicrobial factors that protect young rabbits from enteropathogenic Escherichia coli infection

Enteropathogenic Escherichia coli (EPEC) colibacillosis represents a major cause of lethal diarrhea in young children in developing countries. EPEC strains also infect numerous mammal species and represent a major economical problem in rabbit industry. Protection against this pathogen is a challenging goal both in humans and in other mammal species. Despite a good knowledge of the pathogenicity mechanisms of EPEC, the intrinsic and environmental factors that control the expression of EPEC virulence in mammals remain unknown. For instance, the exacerbated sensitivity of young mammals to EPEC infection is still unexplained. Our goal was to investigate if age or other factors, like milk consumption, could be determinants that trigger the disease. We used rabbits as an animal model to study the role of milk in the sensitivity to an EPEC infection. Weaned and suckling rabbits were orally inoculated with EPEC strain E22 (O103:H2:K-) at 28 days of age, and the evolution of the disease was investigated in the two groups. In addition, in order to better characterize the interactions between milk and EPEC, we determined in vitro bacterial growth and the abilities of EPEC cells to adhere to epithelial cells in the presence of milk. Our results demonstrate a protective role of milk in vivo in association with in vitro antibacterial activity. These effects are independent of the presence of specific anti-EPEC antibodies.     

Peyer's patch adherence of enteropathogenic Escherichia coli strains in rabbits.

RDEC-1 (serotype O15) is an attaching and effacing strain of rabbit enteropathogenic Escherichia coli (REPEC) that causes diarrhea in postweanling rabbits. It expresses AF/R1 pili that mediate Peyer's patch M-cell adherence. We investigated Peyer's patch adherence, the presence of virulence genes, ileal brush border aggregation, and pilus expression in 9 strains representing several serotypes of REPEC as well as in two commensal strains. Postweanling rabbits were inoculated with 10(6) organisms and sacrificed at 24 h, and tissues were prepared for examination by light microscopy. Strains B10 and RDEC-1 were also studied at 12 and 72 h postinoculation. All REPEC strains were eaeA positive, expressed pili, and adhered to ileal brush borders. Both commensal strains expressed pili, and one strain adhered to brush borders. All REPEC strains demonstrated some degree of Peyer's patch lymphoid follicle adherence, ranging from diffuse coverage to small patches covering two to three dome epithelial cells. Strains C102 and C110 had genes homologous with the structural subunit gene of the AF/R1 pilus (afrA) of RDEC-1, which correlated with greater degrees of lymphoid follicle adherence and lesser degrees of ileal villus adherence. The observation that all REPEC strains adhere to Peyer's patch epithelium suggests the possibility that human strains of enteropathogenic E. coli (EPEC) might do likewise. EPEC strains might thus serve as mucosal vaccine vectors in humans. Better understanding of the molecular mechanism of REPEC adherence should provide a model for the targeting of the Peyer's patch in humans.     

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.     

Identification of eae sequences in enteropathogenic Escherichia coli strains from rabbits.

DNA sequences coding for attachment and for verotoxin production were investigated in a collection of enteropathogenic Escherichia coli strains from rabbits. All of the strains produced diarrhea after experimental infection, attached to the brush borders of the intestinal lining, and possessed homology to the eae probe, whereas strains isolated from healthy rabbits did not. Sequences homologous to the AF/R1 fimbriae of strain RDEC-1 were not found. One strain reacted with the probe for the Shiga-like toxin type I gene.     

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.     

Host specificity of enteropathogenic Escherichia coli from rabbits: lack of correlation between adherence in vitro and pathogenicity for laboratory animals.

The pathogenicity of four attaching and effacing strains of enteropathogenic Escherichia coli originally isolated from diarrheic rabbits was investigated by inoculating them perorally into rabbits, guinea pigs, and mice. The ability of the four strains to adhere to cultured epithelial cells, erythrocytes, and intestinal brush borders from various animal species, including rabbits, guinea pigs, and mice, varied considerably. Only one strain carried AF/R1 fimbriae, which are believed to determine the host specificity of these bacteria. Despite these differences, the pattern of behavior of the four strains in experimentally infected animals was similar. Each strain caused fatal diarrhea in rabbits (although the virulence of individual strains for rabbits differed significantly), and none was virulent for guinea pigs or mice. None of the strains colonized the intestinal tract of guinea pigs, but all were able to cause attaching-effacing lesions in ligated loops of guinea pig small intestine. By contrast, all four strains colonized mice, in particular the distal intestine, but none induced attaching-effacing lesions in mouse intestinal loops. These findings suggest that there may be previously unrecognized host-restricted adhesins in enteropathogenic E. coli and indicate that adherence to erythrocytes or intestinal brush borders in vitro does not necessarily reflect colonizing ability or pathogenicity in vivo.     

Pathogenicity of attaching effacing enteropathogenic Escherichia coli isolated from diarrheic suckling and weanling rabbits for newborn rabbits

The pathogenicity of six strains of Escherichia coli originating from different commercial rabbitries was tested in neonatal rabbits. Two strains isolated from healthy weaned rabbits (O7:H6 and O9:H?) did not induce any clinical sign or lesion. Two strains (O109:H2) isolated from diarrheic suckling rabbits caused yellow diarrhea 36 to 60 h after inoculation and high mortality between 60 and 72 h after infection. At 12 h after infection, light and electron microscopy showed attachment to epithelial cells and effacement of microvilli from proximal small intestine to colon. Bacteria were often present in the apical cytoplasm of epithelial cells. The two strains isolated from diarrheic weanling rabbits (O109:H2 and O15:H-) did not induce any clinical sign. Attachment to epithelial cells and effacement of microvilli was observed 48 h after inoculation in distal small intestine, cecum, and colon. These data are further evidence for the existence of two groups of attaching effacing enteropathogenic E. coli in rabbits, showing different preferences for age group and intestinal compartment.     

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.     

Thymidine-dependent Escherichia coli infection and some associated laboratory problems

The rapid emergence of a thymidine-dependent strain of Escherichia coli in a patient treated with co-trimoxazole is described. The organism was recovered from blood cultures during a septicaemia which developed after only a few days of co-trimoxazole treatment. The cultural characteristics of three thymidine-dependent strains were studied and a remarkable variation was found in their ability to grow on different batches of blood agar, depending upon the age of the plates and the type of agar base used.     

Diffuse adherence of enteropathogenic Escherichia coli strains

For the identification and characterization of the factor(s) responsible for the diffuse adherence (DA) pattern of enteropathogenic Escherichia coli strains, E. coli strain 2787 isolated from a case of infantile diarrhoea was employed. A plasmid-derived 11-kb fragment was cloned into pBR322. The recombinant plasmid pIB6 was shown to confer the diffuse adherence phenotype on different E. coli K12 strains as well as pIB4, a plasmid with a 9.2-kb insert. The DNA fragment necessary for the expression of the DA phenotype could be reduced to 6.0 kb. Antiserum obtained against pIB4-encoded proteins recognized a surface-associated protein of about 100 kDa in Western blotting. The isolated 100-kDa protein was found to bind to HeLa cells. The antiserum against C600(pIB4) inhibits adherence of E. coli 2787 and C600(pIB6) to HeLa cells. For this reason, the protein is called adhesin involved in diffuse adherence (AIDA-I).     

Effect of vitamin E nutritional supplementation on the pathological changes induced in the ileum of rabbits by experimental infection with enteropathogenic Escherichia coli

A well-established rabbit model of enteropathogenic E. coli (EPEC) disease was used to examine whether vitamin E (VE) nutritional supplementation had an effect on the pathological changes induced in the bowel by EPEC. Quantitative methods were used to evaluate the influence of VE on bacterial colonization, intestinal mucosal architecture and inflammation, and intestinal epithelial proliferation and apoptosis. VE did not affect EPEC colonization and did not give significant protection against EPEC-induced changes and diarrhoea. Although VE had no effect on the EPEC-related increase of enterocyte apoptosis, it clearly contributed to an acceleration of epithelial cell proliferation in the ileal crypts. This finding may explain why ileal morphometry undertaken in this study showed that VE ameliorated somewhat the effects of EPEC on intestinal mucosal architecture. Quantitative studies on inflammatory cells in the intestinal mucosa revealed that VE nutritional supplementation resulted in an increased neutrophilic and mononuclear inflammatory cell response to EPEC infection, which did not contribute, however, to the clearance of infection.     

Implication of mitogen-activated protein kinases in T84 cell responses to enteropathogenic Escherichia coli infection

Enteropathogenic Escherichia coli (EPEC) infection of T84 cells induces a decrease in transepithelial resistance, the formation of attaching and effacing (A/E) lesions, and cytokine production. The purpose of this study was to investigate the ability of EPEC to activate mitogen-activated protein (MAP) kinases in T84 cells and to correlate these signaling pathways with EPEC-induced cell responses. T84 cells were infected with either the wild-type (WT) EPEC strain E2348/69 or two mutants, intimin deletion strain CVD206 (deltaeaeA) and type III secretion apparatus mutant strain CVD452 (deltaescN::aphA). Infection of T84 cells with WT but not mutant EPEC strains induced tyrosine phosphorylation of several proteins in T84 cells, including the p46 and p52 Shc isoforms. Kinetics studies revealed that ERK1/2, p38, and c-Jun N-terminal kinase (JNK) MAP kinases were activated in cells infected with strain E2348/69 but not with the mutant strains. Inhibition of MAP kinases with PD98059 or SB203580 did not affect the EPEC-induced decrease in transepithelial resistance or actin accumulation beneath the WT bacteria, but these two inhibitors significantly decreased interleukin-8 (IL-8) synthesis. We demonstrate that EPEC induces activation of ERK1/2, p38, and JNK cascades, which all depend on bacterial adhesion and expression of the bacterial type III secretion system. ERK1/2 and p38 MAP kinases were equally implicated in IL-8 expression but did not participate in A/E lesion formation or transepithelial resistance modification, indicating that the signaling pathways involved in these events are distinct.     

Experimental infection of infant rabbits with verotoxin-producing Escherichia coli.

To study the pathogenesis of diarrheal disease due to verotoxin (VT)-producing Escherichia coli, 3-day-old rabbits were inoculated intragastrically with live E. coli O157:H7 (high VT producer), E. coli O113:K75:H21 (low VT producer), or O157:H45 (VT negative) and were examined for clinical symptoms, bacterial colonization, presence of detectable free VT in the intestines, and histological changes. Diarrhea developed consistently with 10(8) bacteria of E. coli O157:H7 but was observed only infrequently with even a higher dose of E. coli O113:K75:H21. VT-negative strains failed to cause diarrhea under the same experimental conditions. E. coli O157:H7 was recovered from the colon of infected animals in a significantly higher concentration than from the small intestine, and the clinical symptoms correlated with the presence of detectable free VT in the colon. Histological changes were seen mainly in the mid- and distal colon; these changes were characterized by a vast increase in apoptosis in the surface epithelium, increased mitotic activity in the crypts, mucin depletion, and a mild to moderate infiltration of neutrophils in the lamina propria and epithelium. Multiple foci of attached bacteria were seen on the surface epithelium of the gut-associated lymphoid tissue, cecum, and colon. Bacteria were never seen in epithelial cells or the lamina propria. These mucosal abnormalities as well as clinical symptoms were reproduced in infant rabbits by the intragastric administration of VT alone. These results are consistent with the hypothesis that VT plays a major role in the pathogenesis of diarrhea caused by E. coli O157:H7 and other VT-producing E. coli.