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Intramammary challenge with Escherichia coli following immunization with a curli-producing Escherichia coli.

Holstein and Jersey cattle were immunized with a curli-producing strain of Escherichia coli (pCRL65/A012) or a noncurli-producing strain (pUC18/HB101) to determine differences in resistance to establishment of experimental intramammary infection. Cows (n = 6 per group) were immunized at 14 d prior to drying off, 7 d of involution, and at calving with 3 x 10(10) E. coli in Freund's Incomplete Adjuvant. At 30 d of lactation, one mammary quarter of each cow was infused with a wild strain of E. coli (727). Escherichia coli 727 was isolated from a naturally occurring intramammary infection and produced curli. All challenged quarters became infected, and all cows developed acute clinical mastitis. Geometric mean duration of intramammary infections was 6 d for both immunization groups. All infections were spontaneously eliminated within 10 d. No differences occurred between immunization groups in blood selenium and glutathione peroxidase activity, plasma selenium, number of E. coli 727 isolated from secretion after challenge, rectal temperature and SCC response, clinical status of mammary quarters, or DMI. Reduction in milk production after challenge was greater for cows immunized with E. coli pCRL65/A012. Immunization of dairy cattle with a curli-producing strain of E. coli did not protect against experimental intramammary challenge during lactation.

Animals↗

Competitive inhibition of adherence of enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli and Clostridium difficile to intestinal epithelial cell line Lovo by purified adhesin of Bifidobacterium adolescentis 1027.

AIM: To observe competitive inhibition of adherence of enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC) and Clostridium difficile (C. difficile) to intestinal epithelial cell line Lovo by purified adhesin of Bifidobacterium adolescentis 1027 (B. ado 1027). METHODS: The binding of bacteria to intestinal epithelial cell line Lovo was counted by adhesion assay. The inhibition of adherence of ETEC, EPEC and C. difficile to intestinal epithelial cell line Lovo by purified adhesin of B. ado 1027 was evaluated quantitatively by flow cytometry. RESULTS: The purified adhesin at the concentration of 10 microg/mL, 20 microg/mL and 30 microg/mL except at 1 microg/mL and 5 microg/mL could inhibit significantly the adhesion of ETEC, EPEC and C. difficile to intestinal epithelial cell line Lovo. Moreover, we observed that a reduction in bacterial adhesion was occurred with increase in the concentration of adhesin, and MFI (Mean fluorescent intensity) was decreased with increase in the concentration of adhesin. CONCLUSION: The purified adhesin of B. ado 1027 can inhibit the adhesion of ETEC, EPEC and C. difficile to intestinal epithelial cell line Lovo in a dose-dependent manner.

Adhesins, Bacterial↗

Anticodon-anticodon interactions in solution. Studies of the self-association of yeast or Escherichia coli tRNAAsp and of their interactions with Escherichia coli tRNAVal.

The temperature-jump method was used to measure the thermodynamic and kinetic parameters of the yeast tRNAAsp (anticodon GUC) duplex, which involves a U/U mismatch in the middle position of the quasi self-complementary anticodon, and of the yeast tRNAAsp (GUC)-Escherichia coli tRNAVal (GAC) complex, in which the tRNAs have complementary anticodons. The existence of the tRNAAsp duplex involving GUC-GUC interactions as evidenced in the crystal structure has now been demonstrated in solution. However, the value of its association constant (Kass = 10(4)M-1 at 0 degrees C) is characteristic of a rather weak complex, when compared with that between tRNAAsp and tRNAVal (Kass = 4 X 10(6) M-1 at 0 degrees C), the effect being essentially linked to differences in the rate constant for dissociation. tRNAAsp split in the anticodon by T1 ribonuclease gives no relaxation signal, indicating that the effects observed with intact tRNA were entirely due to anticodon interactions. No duplex formation was observed with other tRNAs having quasi self-complementary GNC anticodons (where N is C, A or G), such as E. coli tRNAGly (GCC), E. coli tRNAVal (GAC) or E. coli tRNAAla (GGC). This is compatible with the idea that, probably as in the crystal structure, a short double helix is formed in solution between the two GUC anticodons. Because of steric effects, such a complex formation would be hindered if a cytosine, adenine or guanine residue were located in the middle position of the anticodon. Escherichia coli tRNAAsp possessing a modified G residue, the Q base, at the first position of the anticodon, showed a weaker self-association than yeast tRNAAsp but its complex with E. coli tRNAVal was found to be only 1.5 times less stable than that between yeast tRNAAsp and E. coli tRNAVal. Temperature-jump experiments conducted under conditions mimicking those used for the crystallization of yeast tRNAAsp (in the presence of 1.6 M-ammonium sulphate and 3mM-spermine) revealed an important stabilization of the yeast and E. coli tRNAAsp duplexes or of their complexes with E. coli tRNAVal. The effect is due exclusively to ammonium sulphate; it is entropy driven and its influence is reflected on the association rate constant; no influence on the dissociation rate constant was observed. For all tRNA-tRNA complexes, the melting temperature upon addition of ammonium sulphate was considerably increased. This study permits the definition of solution conditions in which tRNAs with appropriate anticodons exist mainly as anticodon-anticodon dimers.

Anticodon↗

Structural and immunochemical relationship between the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 396/C-1 and Escherichia coli O126.

The structure of the O-antigen polysaccharide (PS) from the enteroaggregative Escherichia coli strain 396/C-1 has been determined. Sugar and methylation analyses together with 1H and 13C NMR spectroscopy were the main methods used. Inter-residue correlations were determined by 1H,1H-NOESY, 1H,13C-heteronuclear multiple-bond correlation and dipole-dipole cross-correlated relaxation experiments. The PS is composed of pentasaccharide repeating units with the following structure: [structure: see text]. Analysis of NMR data reveals that on average the PS consists of approximately 13 repeating units and indicates that the biological repeating unit contains an N-acetylglucosamine residue at its reducing end. This structure is different to that reported for the O-antigen polysaccharide from E. coli O126. Monospecific anti-E. coli O126 rabbit serum from The International Escherichia and Klebsiella Centre did not distinguish between the E. coli strain 396/C-1 and the E. coli O126 reference strain, neither in slide agglutination nor in an indirect enzyme immunoassay. Subsequent successful serotyping of the E. coli strain 396/C-1 showed it to be E. coli O126:K+:H27.

Carbohydrate Sequence↗

Use of the RIM Escherichia coli Kit for rapid identification of Escherichia coli.

The Rapid Identification Method (RIM) Escherichia coli Kit (Austin Biologicals Laboratories Inc., Austin, TX) was evaluated as a means to rapidly differentiate at reduced cost clinical isolates of Escherichia coli from other members of the Enterobacteriaceae. A group of 408 gram-negative rods that were isolated on primary isolation plates were tested with the use of both the kit and complete bacterial identification. The RIM E. coli Kit identified 92.5% (272 of 294) of the E. coli isolates. Only 1 of 114 non-E. coli isolates was misidentified as E. coli (positive predictive value equal to 99.6). Although the RIM E. coli Kit was sufficiently rapid (less than one hour), any supply cost savings would depend upon the proportion of true E. coli in the group of organisms tested. Savings derived from the lower cost of using the RIM E. coli Kit would be reduced by the extra cost of screening non-E. coli isolates, which would then have to be completely identified.

Clinical Enzyme Tests↗

Physical characterisation of the Escherichia coli B gene encoding nitroreductase and its over-expression in Escherichia coli K12.

The Escherichia coli B gene (nfnB) encoding nitroreductase has been cloned in Escherichia coli K-12 and its nucleotide sequence determined. The translated amino acid sequence was found to share substantial identity (88.5%) with the equivalent proteins of Enterobacter cloacae and Salmonella typhimurium. When the structural gene was placed under the transcriptional control of either the trp or lac promoter, recombinant nitroreductase was accumulated to 33% and 25% of the cell's soluble protein, respectively. Substitution of the nfrB ribosome binding site with that of the E. coli lacZ gene reduced production levels of nitroreductase. The sequenced region also contained two incomplete open reading frames of unknown function.

Amino Acid Sequence↗

Atypical biogroups of Escherichia coli found in clinical specimens and description of Escherichia hermannii sp. nov.

DNA relatedness was used to define the biochemical boundaries of Escherichia coli. A large number of biochemically atypical strains were shown to belong to biogroups of E. coli. These included strains negative in reactions for indole, all three decarboxylases, D-mannitol, lactose, or methyl red and strains positive in reactions for H2S, urea, citrate, KCN, adonitol, myo-inositol, or phenylalanine deaminase. Frequency and source data are presented for these atypical E. coli biogroups. One group of KCN-positive, cellobiose-positive, yellow-pigmented strains was 84 to 91% interrelated but only 35 to 45% related to E. coli. The name Escherichia hermannii sp. nov. is proposed for this group of organisms that was formerly called Enteric Group 11 by the Enteric Section, Centers for Disease Control, Atlanta, GA. Twenty-nine strains of E. hermannii have been isolated in the United States from a variety of clinical sources, principally wounds, sputum, and stools. Three additional strains were isolated from food. E. hermannii strains are gram-negative, oxidase-negative, fermentative, motile rods. In addition to yellow pigment and positive KCN and cellobiose tests, the biochemical reactions characteristic of 32 strains of E. hermannii were as follows: gas from D-glucose, acid from D-glucose, maltose, D-xylose, L-arabinose, L-rhamnose, and D-mannitol; no acid from adonitol or inositol; variable acid production from lactose and sucrose; positive tests for indole, methyl red, and mucate; negative tests for Voges-Proskauer. Simmons citrate, H2S, urea, phenylalanine deaminase, and gelatin hydrolysis; negative or delayed test for L-lysine decarboxylase and negative test for L-arginine dihydrolase; and positive test for ornithine decarboxylase. E. hermannii strains were resistant to penicillin, ampicillin, and carbenicillin and sensitive to other commonly used antibiotics. Wounds account for almost 50% of human isolates of E. hermannii, followed by sputum or lung isolates (ca. 25%) and stool isolates (20%).

Anti-Bacterial Agents↗

[Utilization of nylon membranes for the specific isolation of Escherichia coli O157 with DNA probes and checking for relationship to the shiga toxin Escherichia coli (STEC) group].

A method for specific isolation of Escherichia coli strains of serotype O157 is given. DNA-hybridization technique by using DIG-labeled specific O157 PCR-amplificates as probes is the basis. These investigations can be used for the detection and isolation of shigatoxin-producing Escherichia coli (STEC) of this serotype in combination with other tests for detection of shigatoxin genes (stx). No background is seen by using 'DIG Easy Hyb' solution and Nylon membranes for colony-and plaque-hybridization (Roche Diagnostics Boehringer Mannheim). Dark brown spots (E. coli colonies) are visible on the membranes after staining. After comparing the membrane with the masterplate it is possible to isolate the colonies from the masterplate, respectively. It is necessary to make proof that the isolates possess the rfb-genes (verification of serotype O157) and the stx-genes (test for belonging to the STEC-group). Both tests could be done by using PCR. In case of positive results other virulence factors of the isolates can be detected.

Bacterial Toxins↗

Screening the enteroaggregative Escherichia coli activity and detection of the aggA, aafA, and astA genes with novel PCR primers for the Escherichia coli isolates from diarrhea cases in Taiwan.

Enteroaggregative Escherichia coli (EAggEC) are emerging enteropathogens associated with human diarrhea diseases and food poisoning cases. They show distinctive aggregative pattern of adherence to cultured human epithelial cells. However, EAggEC strains are diverse and not all of them have the aggregative adherence fimbria I (AAF/I), AAF/II and heat-stable enterotoxin 1 (EAST1) genes. We attempted to determine the incidence of EAggEC in E. coli isolates from diarrhea patients in Taiwan and to characterize these EAggEC strains. We used three activity assays including HeLa cell adhesion, human blood hemagglutination and bacterial clumping tests and polymerase chain reaction (PCR) primers designed from an aggregative adherence pattern associated plasmid (pCVD432) to screen the EAggEC strains in 403 E. coli isolates including 63 laboratory isolates and 340 clinical isolates obtained from diarrheal disease cases. All these 403 E. coli strains were also assayed with novel PCR primers designed from AAF/I (aggA), AAF/II (aafA) and EAST1 (astA) genes. Results showed that except for the three EAggEC reference strains, only three clinical isolates were identified as EAggEC strains. Including the reference strains, all the E. coli strains with EAggEC activity generated positive PCR results to the aggA gene based primers, but not to the aafA and astA gene targeted primers.

Adhesins, Escherichia coli↗

In vitro inhibition of Salmonella enterica serovars choleraesuis and typhimurium, Escherichia coli F-18, and Escherichia coli O157:H7 by a porcine continuous-flow competitive exclusion culture.

A competitive exclusion (CE) culture of porcine cecal bacteria was developed as a continuous-flow culture in chemostats, was designated RPCF, and was used as a model to determine its usefulness against in vitro colonization by Salmonella enterica serovars Typhimurium and Choleraesuis, Escherichia coli strain F-18, and E. coli serotype O157:H7 (933). Chemostats with or without RPCF were inoculated with 10(6) colony-forming units (CFU)/ml of Typhimurium, Choleraesuis, F-18, or O157:H7. Chemostats were sampled for salmonellae and E. coli at 15 min, 7 h, and every 24 h thereafter. In control chemostats without RPCF, Typhimurium, Choleraesuis, F-18, and O157:H7 rapidly established colonization and had concentrations of 10(6) CFU/ml for 96-120 h post-inoculation. In the chemostats that contained RPCF, reductions (P < 0.05) of Choleraesuis, F-18, and O157:H7 were observed at 24 h post-inoculation. Typhimurium was decreased (P < 0.05) at 48 h post-inoculation, and by 120 h post-inoculation, all chemostats were negative for the four challenge microorganisms. These results demonstrate that RPCF cultures were able to inhibit the growth of Typhimurium, Choleraesuis, and E. coli strains F-18 and O157:H7 in vitro and suggest the potential for the use of CE in swine to prevent disease induced by these microorganisms.

Animals↗

Efficacy of an Escherichia coli J-5 mutant strain bacterin in the protection of calves from endotoxin disease caused by subcutaneous challenge with endotoxins from Escherichia coli.

The purpose of this trial was to examine the potential of a new Escherichia (E) coli J-5 mutant strain bacterin to reduce the severity of clinical disease caused by subcutaneous challenge with endotoxins of Gram-negative bacteria in calves. Day-old to 3-day old calves (n = 40 per study phase) were randomly assigned to either of two treatment groups, i.e. a vaccinated or a placebo group. Calves in the vaccinated group received an inactivated bacterin containing a J-5 mutant strain of E. coli via subcutaneous route at 2-4 days of age and at 14 days thereafter. The placebo contained only adjuvant and saline in lieu of the antigen. Lipopolysaccharides (LPS) originating from E. coli were administered subcutaneously 3 weeks after the booster dose. The LPS challenge dosages were 1 and 8 microg/kg in study phases I and II, respectively. Various clinical, physiological, hematological, and serological parameters were measured at specific time intervals after challenge. The data were mostly analysed using peak changes from baseline recorded during the observation period. By the time of challenge the titers in vaccinated calves had increased significantly more than in the unvaccinated controls. Disease severity following subcutaneous challenge was dose dependent. In phase I, placebo calves were only mildly challenged whereas in phase II placebo calves showed a moderate challenge. After a mild challenge, there was little evidence of protection due to vaccination as only attitude was significantly improved in the vaccinates. In contrast, after a moderate challenge rectal temperature, hematocrit, blood glucose concentrations, and leukocyte changes were significantly better in the vaccinated group. In conclusion, the results of this study show that following a subcutaneous endotoxin challenge that induces a moderate clinical response, calves that were previously vaccinated with the E. coli J-5 bacterin were better protected than those in the placebo group.

Animals↗

Different kinetic of antibody responses following infection of newly weaned pigs with an F4 enterotoxigenic Escherichia coli strain or an F18 verotoxigenic Escherichia coli strain.

To develop a vaccine against Escherichia coli-induced post-weaning diarrhea and edema disease, insights in the induction of the protective immune response following infection with these pathogenic E. coli is needed. Therefore, the fimbriae-specific antibody response of newly weaned pigs following infection with the Shiga-like toxin type II variant (SLT-IIv) producing F18(+) verotoxigenic E. coli (VTEC) (strain 107/86) was compared with the response following an infection with LT producing F4(+) enterotoxigenic E. coli (ETEC) (strain GIS 26). F4(+) ETEC were able to colonize the gut very soon after infection, as peak excretion of F4(+) E. coli bacteria was seen 2 days post-infection (dpi), but had already disappeared 7dpi. On the other hand, F18(+) VTEC infection resulted in a slower colonization of the gut as the peak excretion of F18(+) E. coli was observed between 3 and 5dpi, but this colonization remained longer as F18(+) E. coli were detected till 9dpi in feces. Furthermore, this fast colonization pattern of F4(+) ETEC is accompanied with the presence of F4-specific antibodies in mucosal tissues and serum from 4dpi onward, with maximal amounts of F4-specific IgA in the jejunal lamina propria and serum 7dpi. In contrast, F18-specific IgA was only readily detected in the jejunal lamina propria 15dpi and showed a maximum serum titer 21dpi. Besides this faster induction and higher antibody response, the switch from IgM to IgA and IgG was also earlier following the F4(+) ETEC infection.

Animals↗

SdiA, an Escherichia coli homologue of quorum-sensing regulators, controls the expression of virulence factors in enterohaemorrhagic Escherichia coli O157:H7.

The quorum-sensing system in bacteria is a well-known regulatory system that controls gene expression in a cell density-dependent manner. A transcriptional regulator (LuxR homologue), signal synthase (LuxI homologue) and autoinducer (acyl homoserine lactone) are indispensable for this system in most Gram-negative bacteria. In this study, we found that SdiA, an Escherichia coli LuxR homologue, is a negative regulator of the expression of virulence factors EspD and intimin in enterohaemorrhagic E. coli (EHEC) O157:H7. The expression of EspD and intimin was inhibited at the RNA level upon SdiA overexpression. SdiA has a DNA-binding motif in its C-terminal part and can bind to the promoter regions of the esp and eae genes in vitro. Extracellular factors, which accumulate in culture supernatants of O157:H7 at the stationary phase of growth and inhibit EspD and intimin synthesis, bind to the N-terminal part of SdiA in vivo and in vitro. O157:H7 overproducing the N-terminal part of SdiA exhibited hypertranscription of EspD and intimin, suggesting that the overproduced N-terminal part had inhibited the activity of intact SdiA through titration of the extracellular factors. These results indicate that a quorum-sensing system including the SdiA protein controls colonization by O157:H7.

Bacterial Proteins↗

Studies of Escherichia coli cultured on Rainbow Agar O157 with particular reference to enterohaemorrhagic Escherichia coli (EHEC).

Rainbow Agar O157 is designed for the rapid isolation and identification of enterohaemorrhagic Escherichia coli (EHEC), particularly O157, characterised by black colonies. Five-hundred-eighty-five E. coli strains, including O157, O111 and O113 serogroups from many sources were examined on Rainbow Agar O157. EHEC O157 could readily be isolated and recognized uniquely by typical black colonies. Some other EHEC also stand out as blue-black, whereas O113 and some other EHEC strains were mauve, red or pink and indistinguishable from SLT-negative strains of E. coli.

Agar↗

A genome rearrangement has orphaned the Escherichia coli K-12 AcpT phosphopantetheinyl transferase from its cognate Escherichia coli O157:H7 substrates.

Phosphopantetheinyl transferases (PPTases) are enzymes that catalyse the transfer of a 4'-phosphopantetheine moiety from CoA to a conserved serine residue of a carrier protein. These carrier proteins use the 4'-phosphopantetheine thiol to shuttle intermediates between the active sites of biosynthetic enzymes involved in fatty acid, non-ribosomal peptide and polyketide synthesis. Three PPTases have been previously been identified in Escherichia coli K-12 and other E. coli strains by homology searches and are encoded by the genes acpS, entD and acpT. Both AcpS and EntD have been well studied whereas the function of AcpT has been an enigma because no carrier protein substrate could be found. We report genetic and biochemical evidence that AcpT modifies two carrier proteins encoded in O-island 138, a cluster of fatty acid biosynthesis-like genes located adjacent to acpT in the genome of the pathogenic E. coli strain O157:H7 (E. coli K-12 and several other sequenced E. coli and Shigella strains lack O-island 138). The two carrier proteins of O-island 138 of strain O157:H7 are not modified (or only very poorly modified) by AcpS, the PPTase responsible for 4'-phosphopantetheine attachment to the acyl carrier protein (AcpP) of fatty acid synthesis. We demonstrate that AcpT cannot functionally replace AcpS in E. coli K-12 either in its native chromosomal location or upon insertion of acpT into the acpS chromosomal location. However, in the absence of AcpS activity AcpT does allow very slow growth thus providing a rationale for its retention in the absence of its cognate substrates. These results together with phylogenetic analyses and comparisons of the E. coli and Shigella strains of known genome sequence strongly argue that AcpT has been orphaned from its cognate substrates by a deletion event that occurred in a common ancestor of these organisms. This seems one of the few cases where a chromosomal rearrangement has been functionally demonstrated to be a deletion event rather than an insertion event in the reference organism. We also show that the previously reported suppression of an acpS mutation by the deletion of Lon protease is an artifact of the increased capsular polysaccharide production of lon strains.

Acyl Carrier Protein↗

Transduction of enteric Escherichia coli isolates with a derivative of Shiga toxin 2-encoding bacteriophage phi3538 isolated from Escherichia coli O157:H7.

We investigated the ability of a detoxified derivative of a Shiga toxin 2 (Stx2)-encoding bacteriophage to infect and lysogenize enteric Escherichia coli strains and to develop infectious progeny from such lysogenized strains. The stx(2) gene of the patient E. coli O157:H7 isolate 3538/95 was replaced by the chloramphenicol acetyltransferase (cat) gene from plasmid pACYC184. Phage phi3538(Deltastx(2)::cat) was isolated after induction of E. coli O157:H7 strain 3538/95 with mitomycin. A variety of strains of enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), Stx-producing E. coli (STEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and E. coli from the physiological stool microflora were infected with phi3538(Deltastx(2)::cat), and plaque formation and lysogenic conversion of wild-type E. coli strains were investigated. With the exception of one EIEC strain, none of the E. coli strains supported the formation of plaques when used as indicators for phi3538(Deltastx(2)::cat). However, 2 of 11 EPEC, 11 of 25 STEC, 2 of 7 EAEC, 1 of 3 EIEC, and 1 of 6 E. coli isolates from the stool microflora of healthy individuals integrated the phage in their chromosomes and expressed resistance to chloramphenicol. Following induction with mitomycin, these lysogenic strains released infectious particles of phi3538(Deltastx(2)::cat) that formed plaques on a lawn of E. coli laboratory strain C600. The results of our study demonstrate that phi3538(Deltastx(2)::cat) was able to infect and lysogenize particular enteric strains of pathogenic and nonpathogenic E. coli and that the lysogens produced infectious phage progeny. Stx-encoding bacteriophages are able to spread stx genes among enteric E. coli strains.

Bacterial Toxins↗

Association of genomic O island 122 of Escherichia coli EDL 933 with verocytotoxin-producing Escherichia coli seropathotypes that are linked to epidemic and/or serious disease.

The distribution of EDL 933 O island 122 (OI-122) was investigated in 70 strains of Verocytotoxin-producing Escherichia coli (VTEC) of multiple serotypes that were classified into five "seropathotypes" (A through E) based on the reported occurrence of serotypes in human disease, in outbreaks, and/or in the hemolytic-uremic syndrome (HUS). Seropathotype A comprised 10 serotype O157:H7 and 3 serotype O157:NM strains. Seropathotype B (associated with outbreaks and HUS but less commonly than serotype O157:H7) comprised three strains each of serotypes O26:H11, O103:H2, O111:NM, O121:H19, and O145:NM. Seropathotype C comprised four strains each of serotypes O91:H21 and O113:H21 and eight strains of other serotypes that have been associated with sporadic HUS but not typically with outbreaks. Seropathotype D comprised 14 strains of serotypes that have been associated with diarrhea but not with outbreaks or HUS, and seropathotype E comprised animal VTEC strains of serotypes not implicated in human disease. All strains were tested for four EDL 933 OI-122 virulence genes (Z4321, Z4326, Z4332, and Z4333) by PCR. Negative PCRs were confirmed by Southern hybridization. Overall, 28 (40%) strains contained OI-122 (positive for all four virulence genes), 27 (38.6%) contained an "incomplete" OI-122 (positive for one to three genes), and 15 (21.4%) strains did not contain OI-122. The seropathotype distribution of complete OI-122 was as follows: 100% for seropathotype A, 60% for B, 36% for C, 15% for D, and 0% for E. The differences in the frequency of OI-122 between seropathotypes A, B, and C (associated with HUS) and seropathotypes D and E (not associated with HUS) and between seropathotypes A and B (associated with epidemic disease) and seropathotypes C, D, and E (not associated with epidemic disease) were highly significant (P < 0.0001).

Animals↗