Long Polar Fimbriae Contribute to Colonization by \u3ci\u3eEscherichia coli\u3c/i\u3e O157:H7 In Vivo

The contribution of long polar fimbriae to intestinal colonization by Escherichia coli O157:H7 was evaluated in sheep, conventional pigs, and gnotobiotic piglets. E. coli O157:H7 strains with lpfA1 and lpfA2 mutated were recovered in significantly lower numbers and caused fewer attachment and effacement lesions than the parent strain

Detection of enterohemorrhagic \u3ci\u3eEscherichia coli\u3c/i\u3e O157:H7 by using a multiplex real-time PCR assay for genes encoding intimin and Shiga toxins

A multiplex real-time PCR (R-PCR) assay was designed and evaluated on the ABI 7700 sequence detection system (TaqMan) to detect enterohemorrhagic Escherichia coli (EHEC) O157:H7 in pure cultures, feces, and tissues. Three sets of primers and fluorogenic probes were used for amplification and real-time detection of a 106-bp region of the eae gene encoding EHEC O157:H7-specific intimin, and 150-bp and 200-bp segments of genes stx1 and stx2 encoding Shiga toxins 1 and 2, respectively. Analysis of 67 bacterial strains demonstrated that the R-PCR assay successfully distinguished EHEC O157:H7 serotype from non-O157 serotypes and provided accurate profiling of genes encoding intimin and Shiga toxins. Bacterial strains lacking these genes were not detected with this assay. The detection range of the R-PCR assay for the three genes was linear over DNA concentrations corresponding from 103 to 108 CFU/ml of EHEC O157:H7. The R-PCR allowed construction of standard curves that facilitated quantification of EHEC O157:H7 in feces and intestinal tissues. Detection sensitivity of the R-PCR assay ranged from 104 to 108 CFU/g of feces or tissues without enrichment. Enrichment of feces in a non-selective broth for 4 and 16 h resulted in the detection of levels (from 100 to 103 CFU/g of feces) considered sufficient for infection in humans. The R-PCR assay for eaeO157:H7, stx1, and stx2 proved to be a rapid test for detection of EHEC O157:H7 in complex biological matrices and could also potentially be used for quantification of EHEC O157:H7 in foods or fecal samples

Identification of Plasmid and Chromosomal Copies of 987P Pilus Genes in Enterotoxigenic \u3ci\u3eEscherichia coli\u3c/i\u3e 987

A DNA probe derived from the subunit gene of a cloned 987P determinant was used to characterize the locations of the 987P genes in several Escherichia coli strains. We examined E. coli 987, a nonpiliated mutant of strain 987 (136) that does not express 987P in vitro, and a variant of I36 that expressed 987P following growth in vivo. We found that plasmid and chromosomal copies of the 987P subunit gene could be differentiated in strain 987 by restriction endonuclease analysis and Southern blot hybridization. The nonpiliated mutant 136 had lost the plasmid copy but retained the chromosomal copy of the 987P gene. A 987P-piliated variant of 136, which did not contain the 987P plasmid, colonized and caused diarrhea in neonatal pigs similarly to wild-type 987. The plasmid that hybridized with the 987P probe was transferred from strain 987 to an E. coli K-12 strain by conjugation. We were unable to demonstrate expression of 987P by these transconjugants. The data suggest that the chromosomal and plasmid copies of the 987P genes may interact to influence 987P expression

Evaluation of \u3ci\u3ehha\u3c/i\u3e and \u3ci\u3ehha sepB\u3c/i\u3e mutant strains of \u3ci\u3eEscherichia coli\u3c/i\u3e O157:H7 as bacterins for reducing E. coli O157:H7 shedding in cattle

Escherichia coli O157:H7 colonizes cattle intestines by using the locus of enterocyte effacement (LEE)- encoded proteins. The induction of systemic immune response against LEE-encoded proteins, therefore, will prove effective in reducing E. coli O157:H7 colonization in cattle. The previous studies have demonstrated that a hha (encodes for a hemolysin expression modulating protein) deletion enhances expression of LEE-encoded proteins and a sepB (encodes an ATPase required for the secretion of LEE-encoded proteins) deletion results in intracellular accumulation of LEE proteins. In this study, we demonstrate the efficacy of the hha and hha sepB deletion mutants as bacterins for reducing fecal shedding of E. coli O157:H7 in experimentally inoculated weaned calves. The weaned calves were injected intramuscularly with the bacterins containing 109 heat-killed cells of the hha+ wild-type or hha or hha sepB isogenic mutants, and boosted with the same doses 2- and 4-weeks later. The evaluation of the immune response two weeks after the last booster immunization revealed that the calves vaccinated with the hha mutant bacterin had higher antibody titers against LEE proteins compared to the titers for these antibodies in the calves vaccinated with the hha sepB mutant or hha+ wild-type bacterins. Following oral inoculations with 1010 CFU of the wild-type E. coli O157:H7, the greater numbers of calves in the group vaccinated with the hha or hha sepB mutant bacterins stopped shedding the inoculum strain within a few days after the inoculations compared to the group of calves vaccinated with the hha+ wild-type bacterin or PBS sham vaccine. Thus, the use of bacterins prepared from the hha and hha sepB mutants for reducing colonization of E. coli O157:H7 in cattle could represent a potentially important pre-harvest strategy to enhance post-harvest safety of bovine food products, water and produce

The Established Intimin Receptor Tir and the Putative Eucaryotic Intimin Receptors Nucleolin and B1 Integrin Localize at or near the Site of Enterohemorrhagic \u3ci\u3eEscherichia coli\u3c/i\u3e O157:H7 Adherence to Enterocytes In Vivo

For enterohemorrhagic Escherichia coli (EHEC) O157:H7 to adhere tightly to the intestinal epithelium and produce attach and efface (A/E) lesions, the organism must express the adhesin intimin and insert the bacterially encoded translocated intimin receptor Tir into the plasma membrane of the host enterocyte. Additionally, some reports based on tissue culture experiments indicate that intimin has affinity for the eucaryotic proteins nucleolin and β1 integrin. To address the potential biological relevance of these eucaryotic proteins in the infection process in vivo, we sought to compare the proximity of Tir, nucleolin, and β1 integrin to regions of EHEC O157:H7 attachment in intestinal sections from three different inoculated animals: piglets, neonatal calves, and mice. Piglets and neonatal calves were chosen because intimin-mediated adherence of EHEC O157:H7 and subsequent A/E lesion formation occur at high levels in these animals. Mice were selected because of their ease of manipulation but only after we first demonstrated that in competition with the normal mouse gut flora, an EHEC O157:H7 strain with a nonpolar deletion in the intimin gene was cleared faster than strains that produced wild-type or hybrid intimin. In all three animal species, we noted immunostained Tir beneath and stained nucleolin closely associated with adherent bacteria in intestinal sections. We also observed immunostained β1 integrin clustered at locations of bacterial adherence in porcine and bovine tissue. These findings indicate that nucleolin and β1 integrin are present on the luminal surface of intestinal epithelia and are potentially accessible as receptors for intimin during EHEC O157:H7 infection

Shiga Toxin Binding to Isolated Porcine Tissues and Peripheral Blood Leukocytes

Shiga toxin (Stx) binding sites in porcine tissues and leukocytes were identified by the use of Stx overlay and anti-CD77/Gb3 immunoassays. Stx1 and Stx2 bound to similar tissue locations and leukocytes, although some differences were noted. Previously unreported Stx binding sites were identified in kidney tubules, intestinal lymphoid aggregates, sinusoidal liver cells, alveolar macrophages, and peripheral blood leukocytes

\u3ci\u3eEscherichia coli\u3c/i\u3e O157:H7 in the gallbladders of experimentally infected calves

Fifteen weaned calves (age 89–141 days) were treated with dexamethasone (0.25 mg/kg, IV) for 3 days before, the day of, and the day after inoculation with 10 colony-forming units of either Escherichia coli O157:H7 (strain 86-24, which produces Shiga toxin 2 and intimin; n=13) or nonpathogenic E. coli (strain 123, which does not produce Shiga toxin or intimin; n=2). All calves were necropsied 4 days after inoculation. Histologic lesions of attaching and effacing bacteria were observed in the large intestine (12/13) and in the gallbladder mucosa (5/13) of calves inoculated with E. coli 86-24. Cholecystitis was present in 12 of 13 calves that received E. coli 86-24. Inoculum bacteria were recovered from the distal colons or feces (13/13) and gallbladders (3/4) of calves inoculated with 86-24

Differentiation of F18ab+ from F18ac+ \u3ci\u3eEscherichia coli\u3c/i\u3e by Single-Strand Conformational Polymorphism Analysis of the Major Fimbrial Subunit Gene (\u3ci\u3efedA\u3c/i\u3e)

Toxin-producing Escherichia coli expressing F18 fimbriae colonizes the small intestines of weaned pigs and causes diarrhea, edema disease, or both. The F18 family is composed of two antigenic variants, F18ab and F18ac. Because many strains do not express F18 fimbriae in vitro, identification and differentiation of these two variants are difficult. Single-strand conformational polymorphism (SSCP) analysis is a rapid method for identifying genetic mutations and polymorphisms. The F18 major fimbrial subunit genes (fedA) of 138 strains were amplified by PCR, and genetic differences were detected by SSCP analysis. The SSCP analysis of the fedA gene differentiated F18ab+ strains from F18ac+ strains. Most strains classified as F18ab+ by SSCP analysis contained Shiga toxin 2e and enterotoxin genes. Most strains classified as F18ac+ by SSCP analysis contained only enterotoxin genes. The SSCP analysis was a useful method for predicting the antigenicity of F18+ E. coli and could also be used for analysis of other virulence genes in E. coli and other pathogenic bacteria

The Structural Gene Encoding Human Enterotoxigenic \u3ci\u3eEscherichia coli\u3c/i\u3e PCFO20 Is Homologous to That for Porcine 987P

Putative colonization factor PCFO20 was recently identified in an enterotoxigenic Escherichia coli (ETEC) strain of serogroup O20 isolated from a child with diarrhea in Argentina. The gene encoding the structural subunit of PCFO20 fimbriae, fotA, was cloned from strain ARG-2 in the expression phage vector lambda ZAP Express. One positive clone, pGV29, that carried a 3.3-kb fragment was identified on the basis of fimbrillin production by using a monospecific rabbit anti-PCFO20 serum. Nucleotide sequencing of a 1.3-kb Sau3A-ClaI fragment of the subclone pGV292 containing the region coding for PCFO20 fimbrillin revealed two open reading frames of which one was complete. A Western blot (immunoblot) showed that the cloned protein, FotA, migrated like the PCFO20 fimbrial subunit protein did. Fimbriae were not detected on the surface of E. coli host bacteria containing pGV292 or pGV29, suggesting that the genes needed for assembly of PCFO20 fimbriae are lacking in both clones. The fotA gene encodes a 20,574-Da prefimbrillin protein which contains a 21-aminoacid signal sequence; the mature protein has a size of 18.1 kDa. The subunit protein FotA was found to be more homologous to the subunit of porcine 987P than to any fimbrial subunit produced by human ETEC. Alignments of the amino acid sequences of the two proteins indicate that they are partly identical, with an overall similarity of 82%. FotA fimbrillin was shown to be transported and assembled by the fimbria assembly machinery in porcine ETEC strain 987. PCFO20 and 987P may have evolved from a common ancestral gene. They are immunologically related but have affinity for different host cell receptors, since PCFO20-producing bacteria do not bind to neonatal piglet enterocytes

\u3ci\u3eEscherichia coli\u3c/i\u3e O157:H7 Requires Intimin for Enteropathogenicity in Calves

Enterohemorrhagic Escherichia coli (EHEC) strains require intimin to induce attaching and effacing (A/E) lesions in newborn piglets. Infection of newborn calves with intimin-positive or intimin-negative EHEC O157: H7 demonstrated that intimin is needed for colonization, A/E lesions, and disease in cattle. These results suggest that experiments to determine if intimin-based vaccines reduce O157:H7 levels in cattle are warranted