Serum antibodies to Escherichia coli serotype O157:H7 in patients with hemolytic uremic syndrome

Sera from 13 patients with hemolytic uremic syndrome (HUS) and 8 healthy control subjects were examined for antibodies specific for bacterial antigens of Eschericia coli serotype O157:H7. Bacterial components, including outer membrane proteins (OMPs), lipopolysaccharide (LPS), and flagella, were reacted with sera by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting and by enzyme-linked immunosorbent assay. All 13 serum samples from HUS patients contained high-titered antibodies of the immunoglobulin M class against O157 LPS and some OMPs. These same sera reacted weakly with some of the major OMPs, but not the LPS, of non-O157 strains of E. coli. Sera from patients did not contain antibodies to non-O157 LPS or H7 flagella. The possibility of using E. coli serotype O157 LPS in an enzyme-linked immunosorbent assay for the routine diagnostic testing of sera from HUS patients for evidence of O157:H7 infection is discussed.     

Inhibition of Water Absorption and Selective Damage to Human Colonic Mucosa Are Induced by Subtilase Cytotoxin Produced by Escherichia coli O113:H21

Shiga toxin-producing Escherichia coli O157:H7 (STEC) is by far the most prevalent serotype associated with hemolytic uremic syndrome (HUS) although many non-O157 STEC strains have been also isolated from patients with HUS. The main virulence factor of STEC is the Shiga toxin type 2 (Stx2) present in O157 and non-O157 strains. Recently, another toxin, named subtilase cytotoxin (SubAB), has been isolated from several non-O157 strains and may contribute to the pathogenesis of HUS. Here, we have demonstrated that an O113:H21 STEC strain expressing SubAB and Stx2 inhibits normal water absorption across human colon and causes damage to the surface epithelium, necrosis, mononuclear inflammatory infiltration, edema, and marked mucin depletion. This damage was less marked, but nevertheless significant, when purified SubAB or E. coli O113:H21 expressing only SubAB was assayed. This is the first study showing that SubAB may directly participate in the mechanisms of diarrhea in children infected with non-O157 STEC strains.     

Pathogenic potential of emergent sorbitol-fermenting Escherichia coli O157:NM

Non-sorbitol-fermenting (NSF) Escherichia coli O157:H7 is the primary Shiga toxin-producing E. coli (STEC) serotype associated with human infection. Since 1988, sorbitol-fermenting (SF) STEC O157:NM strains have emerged and have been associated with a higher incidence of progression to hemolytic-uremic syndrome (HUS) than NSF STEC O157:H7. This study investigated bacterial factors that may account for the increased pathogenic potential of SF STEC O157:NM. While no evidence of toxin or toxin expression differences between the two O157 groups was found, the SF STEC O157:NM strains adhered at significantly higher levels to a human colonic cell line. Under the conditions tested, curli were shown to be the main factor responsible for the increased adherence to Caco-2 cells. Notably, 52 of 66 (79%) European SF STEC O157:NM strains tested bound Congo red at 37^οC and this correlated with curli expression. In a subset of strains, curli expression was due to increased expression from the csgBAC promoter that was not always a consequence of increased csgD expression. The capacity of SF STEC O157:NM strains to express curli at 37^οC may have relevance to the epidemiology of human infections as curliated strains could promote higher levels of colonization and inflammation in the human intestine. In turn, this could lead to increased toxin exposure and an increased likelihood of progression to HUS.     

Serological response of Shiga toxin-producing Escherichia coli type III secreted proteins in sera from vaccinated rabbits, naturally infected cattle, and humans

Escherichia coli O157:H7 is an important zoonotic pathogen, causing hemolytic uremic syndrome (HUS). The colonization of cattle and human hosts is mediated through the action of effectors secreted via a type III secretion system (T3SS). The structural genes for the T3SS and many of the secreted effectors are located on a pathogenicity island called the locus of enterocyte effacement (LEE). We cloned and expressed the genes coding for 66 effectors and purified each to measure the cross-reactivity of type III secreted proteins from Shiga toxin-producing Escherichia coli (STEC) serotypes. These included 37 LEE-encoded proteins and 29 non-LEE effectors. The serological response against each protein was measured by Western blot analysis and enzyme-linked immunosorbent assay (ELISA) using sera from rabbits immunized with type III secreted proteins (T3SPs) from four STEC serotypes, experimentally infected cattle, and human sera from six HUS patients. Twenty proteins were recognized by at least one of the STEC T3SP-vaccinated rabbits by Western blotting. Several structural proteins (EspA, EspB, and EspD) and a number of effectors (Tir, NleA, and TccP) were recognized by O26-, O103-, O111-, and O157-specific sera. Sera from experimentally infected cattle and HUS patients were tested using an ELISA against each of the proteins. Tir, EspB, EspD, EspA, and NleA were recognized by the majority of the samples tested. A number of other proteins also were recognized by individual serum samples. Overall, proteins such as Tir, EspB, EspD, NleA, and EspA were highly immunogenic in vaccinated and naturally infected subjects and could be candidates for a cross-protective STEC vaccine.     

Petting zoos as sources of Shiga toxin-producing Escherichia coli (STEC) infections

Despite their general low incidence, Shiga toxin-producing Escherichia (E.) coli (STEC) infections are considered an important public health issue due to the severity of illness that can develop, particularly in young children. We report on two Austrian petting zoos, one in Tyrol (2015) and one in Vorarlberg (2016), which were identified as highly likely infection sources of STEC infections. The petting zoo related cases involved a case of hemolytic uremic syndrome (HUS) due to STEC O157:HNM in 2015 and an outbreak of STEC O157:H7 infections affecting five young children and two adults in 2016. The HUS case accounted for 2.8% of the 36 STEC O157:HNM/H7 infections notified in Austria in 2015 (5,9% of 17 HUS cases). The seven cases described for 2016 accounted for 4.0% of the 177 human STEC infections documented for Austria in 2016, and for 19.4% of the 36 STEC O157:HNM/H7 infections notified that year. The evaluation of the STEC infections described here clearly underlines the potential of sequence-based typing methods to offer suitable resolutions for public health applications. Furthermore, we give a state-of-the-art mini-review on the risks of petting zoos concerning exposure to the zoonotic hazard STEC and on proper measures of risk-prevention.     

Antibodies to lipopolysaccharide block adherence of Shiga toxin-producingEscherichia colito human intestinal epithelial (Henle 407) cells

Shiga toxin-producingEscherichia coli(STEC) are a diverse group of organisms known to cause diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome (HUS) in humans. During the early stage of infection, numbers of STEC in the gut may be very high (of the order of 10⁹/g faeces), but as disease progresses, the numbers may drop rapidly such that STEC are undetectable within a week. Convalescent sera from patients recovering from HUS frequently contain high levels of antibody toE. colilipopolysaccharide (LPS) of the infecting serotype, and it is possible that a local immune response to LPS contributes to elimination of the organism from the gut. We have recently demonstrated that STEC strains isolated from HUS cases have enhanced adherence to a human intestinal epithelial cell line (Henle 407) compared with STEC strains from non-human sources. In this study, we examined the capacity of STEC strains belonging to O-antigen types O111 and O157 to adhere to human intestinal epithelial (Henle 407) cells in the presence or absence of anti-LPS. Adherence was inhibited by up to 95% by anti-LPS of the homologous, but not heterologous serotype. This effect was not an artefact of serum bactericidal or agglutinating activity. Preincubation with purified homologous or heterologous LPS did not prevent adherence, suggesting that LPS was not acting as an adhesin per se. Nevertheless, these findings raise the possibility that oral administration of preparations containing anti-LPS may interfere with colonization of the human gut by STEC, and therefore could be of potential therapeutic value if administered early in the course of infection.     

PCR-Based Detection and Molecular Characterization of Shiga Toxin-Producing Escherichia coli Strains in a Routine Microbiology Laboratory over 16 years

Shiga toxin-producing Escherichia coli (STEC) is a heterogeneous group of bacteria causing disease ranging from asymptomatic carriage and mild infection to hemolytic uremic syndrome (HUS). Here, we describe patients with STEC infection and characterize the STEC strains detected in our laboratory by use of PCR for stx₁, stx₂, and eae from 1996 through 2011. Patient information was collected from referral forms and from the Norwegian Surveillance System for Communicable Diseases. STEC isolates were characterized with respect to serogroup or serotype, selected potential virulence genes, and multilocus variable-number tandem-repeat analysis (MLVA) genotype. STEC strains were isolated from 138 (1.09%) of 12,651 patients tested. STEC strains of serogroups O26, O103, O121, O145, and O157 were the most frequent. These serogroups, except non-sorbitol-fermenting O157, were also the most frequent among the 11 patients (all ≤5 years old) who developed HUS. Twenty-four STEC strains were classified as being HUS associated based on an epidemiological link to a HUS case, including an MLVA genotype identical to that of the STEC strain. The age of the patient (≤5 years) and the genes eae and stx_2a were significantly associated with HUS-associated STEC (P < 0.05 for each parameter), while stx₁ was associated with non-HUS-associated STEC (P < 0.05). All of the potential virulence genes analyzed, except ehxA, were significantly more frequent among HUS-associated than non-HUS-associated strains (P < 0.05 for each gene). However, these genes were also present in some non-HUS-associated STEC strains and could therefore not reliably differentiate between HUS-associated and non-HUS-associated STEC strains.     

Shiga Toxin-Producing Escherichia coli in Finland from 1990 through 1997: Prevalence and Characteristics of Isolates

During the past 10 years Shiga toxin-producing Escherichia coli (STEC) has emerged as one of the most important causes of food-borne infections in industrialized countries. In Finland, with a population of 5.1 million, however, only four STEC O157:H7 infections were identified from 1990 through 1995; the occurrence of non-O157 STEC infections was unknown. In 1996, we established a national prospective study to determine the prevalence of STEC serotypes in feces of Finns with bloody diarrhea. During this enhanced 1-year study period eight sporadic cases of STEC infection were found; of them, only two were indigenously acquired O157:H7 infections. In 1997, O157 infections increased dramatically, with O157 strains causing 51 of all 61 STEC infections. Altogether 14 non-O157:H7 STEC strains were found in Finland in the 1990s: O26:H11 (four strains), O26:HNM (HNM indicates nonmotile), O2:H29, O91:H21, O91:H40, O101:HNM, O107:H27, O157:HNM, O165:H25, OX3:H21, and Rough:H49. All O157:H7 and O26:H11 isolates produced enterohemolysin, but seven of the other STEC strains did not. Most (n = 63) of the 71 STEC strains isolated carried the stx₂ gene only, five carried the stx₁ gene only, and three carried both genes. The eaeA gene was detected in all other isolates except five non-O157 strains. There were seven distinct pulsed-field gel electrophoresis (PFGE) genotypes among 57 O157 strains and three distinct PFGE types among four O26:H11 strains. The main PFGE type was found among 65% of all O157 isolates.     

Divergent Signal Transduction Responses to Infection with Attaching and Effacing Escherichia coli

Shiga toxin-producing Escherichia coli (STEC) O157:H7 is an attaching and effacing pathogen that causes hemorrhagic colitis and the hemolytic-uremic syndrome. Although this organism causes adhesion pedestals, the cellular signals responsible for the formation of these lesions have not been clearly defined. We have shown previously that STEC O157:H7 does not induce detectable tyrosine phosphorylation of host cell proteins upon binding to eukaryotic cells and is not internalized into nonphagocytic epithelial cells. In the present study, tyrosine-phosphorylated proteins were detected under adherent STEC O157:H7 when coincubated with the non-intimately adhering, intimin-deficient, enteropathogenic E. coli (EPEC) strain CVD206. The ability to be internalized into epithelial cells was also conferred on STEC O157:H7 when coincubated with CVD206 ([158 ± 21] % of control). Neither the ability to rearrange phosphotyrosine proteins nor that to be internalized into epithelial cells was evident following coincubation with another STEC O157:H7 strain or with the nonsignaling espB mutant of EPEC. E. coli JM101(pMH34/pSSS1C), which overproduces surface-localized O157 intimin, also rearranged tyrosine-phosphorylated and cytoskeletal proteins when coincubated with CVD206. In contrast, JM101(pMH34/pSSS1C) demonstrated rearrangement of cytoskeletal proteins, but not tyrosine-phosphorylated proteins, when coincubated with intimin-deficient STEC (strains CL8KO1 and CL15). These findings indicate that STEC O157:H7 forms adhesion pedestals by mechanisms that are distinct from those in attaching and effacing EPEC. Taken together, these findings point to diverging signal transduction responses to infection with attaching and effacing bacterial enteropathogens.     

Reactivity of convalescent-phase hemolytic-uremic syndrome patient sera with the megaplasmid-encoded TagA protein of Shiga toxigenic Escherichia coli O157

A cosmid library of Shiga toxigenic Escherichia coli (STEC) O157:H7 strain EDL933 DNA was screened for clones capable of reacting with convalescent-phase serum from a patient with hemolytic-uremic syndrome (HUS), in an attempt to identify candidate virulence genes. One of the immunoreactive clones contained a portion of the large plasmid pO157, and the immunoreactive gene product was identified as TagA. The function of this 898-amino-acid protein is unknown, but it exhibits 42% amino acid sequence identity and 63% similarity to a 312-amino-acid region of a ToxR-regulated lipoprotein of Vibrio cholerae. Antibodies to E. coli O157 TagA were detected in sera from other HUS patients with O157 STEC infection but not in those from patients whose illnesses were caused by other STEC types or in healthy controls. These data demonstrate that TagA is expressed in vivo and provide circumstantial evidence for a role in the pathogenesis of the disease. The tagA gene is present only in STEC strains belonging to serogroup O157, and so antibodies to TagA are a potentially useful serological marker for infections due to such strains.     

Molecular Analysis of Shiga Toxigenic Escherichia coli O111:H− Proteins Which React with Sera from Patients with Hemolytic-Uremic Syndrome

Western blot analysis was used to assess the reactivity of convalescent-phase sera from patients who were associated with an outbreak of hemolytic-uremic syndrome (HUS) caused by fermented sausage contaminated with Shiga toxin-producing Escherichia coli (STEC). The predominant STEC isolated from HUS patients belonged to serotype O111:H⁻, and reactivity to O111:H⁻ whole-cell lysates, treated or untreated with proteinase K, was examined. As expected, all five serum samples demonstrated a marked anti-lipopolysaccharide response, but several protein bands were also immunoreactive, particularly one with an apparent size of 94 kDa. One convalescent-phase serum sample was subsequently used to screen an O111:H⁻ cosmid bank and 2 of 900 cosmid clones were found to be positive, both of which contained a similar DNA insert. Western blot analysis of one of these clones identified three major immunoreactive protein bands of approximately 94, 70, and 50 kDa. An immune response to the three proteins was detectable with all five convalescent-phase serum samples but not with healthy human serum. Immunoreactive 94- and 50-kDa species were produced by a deletion derivative of the cosmid containing a 7-kb STEC DNA insert. Sequence analysis of this region indicated that it is part of the locus for enterocyte effacement, including the eaeA gene which encodes intimin. The deduced amino acid sequence of the O111:H⁻ intimin was 88.6% identical to intimin from O157:H7 STEC, and the most divergent region was the 200 residues at the carboxyl terminus, which were only 75% identical. Such variation may be antigenically significant as serum from a HUS patient infected only with the O111:H⁻ STEC reacted with intimin from an enteropathogenic E. coli O111 strain, as well as several other eaeA-positive STEC isolates, but not with an eaeA-positive STEC belonging to serotype O157:H⁻. Sera from two of the other HUS patients also failed to react with intimin from this latter strain. However, intimin from O157:H⁻ STEC did react with serum from a patient infected with both O111:H⁻ and O157:H⁻ STEC.     

Comparative Genomics and Immunoinformatics Approach for the Identification of Vaccine Candidates for Enterohemorrhagic Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains are major human food-borne pathogens, responsible for bloody diarrhea and hemolytic-uremic syndrome worldwide. Thus far, there is no vaccine for humans against EHEC infections. In this study, a comparative genomics analysis was performed to identify EHEC-specific antigens useful as potential vaccines. The genes present in both EHEC EDL933 and Sakai strains but absent in nonpathogenic E. coli K-12 and HS strains were subjected to an in silico analysis to identify secreted or surface-expressed proteins. We obtained a total of 65 gene-encoding protein candidates, which were subjected to immunoinformatics analysis. Our criteria of selection aided in categorizing the candidates as high, medium, and low priority. Three members of each group were randomly selected and cloned into pVAX-1. Candidates were pooled accordingly to their priority group and tested for immunogenicity against EHEC O157:H7 using a murine model of gastrointestinal infection. The high-priority (HP) pool, containing genes encoding a Lom-like protein (pVAX-31), a putative pilin subunit (pVAX-12), and a fragment of the type III secretion structural protein EscC (pVAX-56.2), was able to induce the production of EHEC IgG and sIgA in sera and feces. HP candidate-immunized mice displayed elevated levels of Th2 cytokines and diminished cecum colonization after wild-type challenge. Individually tested HP vaccine candidates showed that pVAX-12 and pVAX-56.2 significantly induced Th2 cytokines and production of fecal EHEC sIgA, with pVAX-56.2 reducing EHEC cecum colonization. We describe here a bioinformatics approach able to identify novel vaccine candidates potentially useful for preventing EHEC O157:H7 infections.     

Identification of Potential Diagnostic and Vaccine Candidates of Helicobacter pylori by Two-Dimensional Gel Electrophoresis, Sequence Analysis, and Serum Profiling

There is great interest in characterizing the proteins of the gastric pathogen Helicobacter pylori, especially those to which humans respond immunologically, because of the potential importance of such proteins in diagnosis and vaccine development. Two-dimensional gel electrophoresis was used to separate and identify potential antigens of H. pylori ATCC 43504. Over 30 proteins were reactive in Western blots with pooled sera from 14 infected patients. These proteins were analyzed by N-terminal sequence analysis. Fourteen proteins were determined to be distinct from any proteins previously described from H. pylori; the others were previously isolated and characterized proteins. Analysis of eight distinct H. pylori strains showed that most of these antigens were produced by all of the strains. We propose that collection of new antigens such as those recognized here will be useful in serologic tests for detecting and monitoring H. pylori infection and may also serve as potential targets for antimicrobial agent or vaccine development.     

Evaluation of CHROMagar STEC and STEC O104 Chromogenic Agar Media for Detection of Shiga Toxin-Producing Escherichia coli in Stool Specimens

The performance of CHROMagar STEC and CHROMagar STEC O104 (CHROMagar Microbiology, Paris, France) media for the detection of Shiga toxin-producing Escherichia coli (STEC) was assessed with 329 stool specimens collected over 14 months from patients with suspected STEC infections (June 2011 to August 2012). The CHROMagar STEC medium, after an enrichment broth step, allowed the recovery of the STEC strain from 32 of the 39 (82.1%) Shiga toxin-positive stool specimens, whereas the standard procedure involving Drigalski agar allowed the recovery of only three additional STEC strains. The isolates that grew on CHROMagar STEC medium belonged to 15 serotypes, including the prevalent non-sorbitol-fermenting (NSF) O157:H7, O26:H11, and O104:H4 serotypes. The sensitivity, specificity, and positive and negative predictive values for the CHROMagar STEC medium were between 89.1% and 91.4%, 83.7% and 86.7%, 40% and 51.3%, and 98% and 98.8%, respectively, depending on whether or not stx-negative eae-positive E. coli was considered atypical enteropathogenic E. coli (EPEC) or STEC that had lost Shiga toxin genes during infection. In conclusion, the good performance of CHROMagar STEC agar medium, in particular, the high negative predictive value, and its capacity to identify NSF O157:H7 as well as common non-O157 STEC may be useful for clinical bacteriology, public health, and reference laboratories; it could be used in addition to a method targeting Shiga toxins (detection of stx genes by PCR, immunodetection of Shiga toxins in stool specimens, or Vero cell cytotoxicity assay) as an alternative to O157 culture medium. This combined approach should allow rapid visualization of both putative O157 and non-O157 STEC colonies for subsequent characterization, essential for real-time surveillance of STEC infections and investigations of outbreaks.     

Characterization of Shiga toxin-producing Escherichia coli strains isolated from human patients in Germany over a 3-year period

We have investigated 677 Shiga toxin-producing Escherichia coli (STEC) strains from humans to determine their serotypes, virulence genes, and clinical signs in patients. Six different Shiga toxin types (1, 1c, 2, 2c, 2d, and 2e) were distributed in the STEC strains. Intimin (eae) genes were present in 62.6% of the strains and subtyped into intimins alpha1, beta1, gamma1, epsilon, theta, and eta. Shiga toxin types 1c and 2d were present only in eae-negative STEC strains, and type 2 was significantly (P < 0.001) more frequent in eae-positive STEC strains. Enterohemorrhagic E. coli hemolysin was associated with 96.2% of the eae-positive strains and with 65.2% of the eae-negative strains. Clinical signs in the patients were abdominal pain (8.7%), nonbloody diarrhea (59.2%), bloody diarrhea (14.3%), and hemolytic-uremic syndrome (HUS) (3.5%), and 14.3% of the patients had no signs of gastrointestinal disease or HUS. Infections with eae-positive STEC were significantly (P < 0.001) more frequent in children under 6 years of age than in other age groups, whereas eae-negative STEC infections dominated in adults. The STEC strains were grouped into 74 O:H types by serotyping and by PCR typing of the flagellar (fliC) genes in 221 nonmotile STEC strains. Eleven serotypes (O157:[H7], O26:[H11], O103:H2, O91:[H14], O111:[H8], O145:[H28], O128:H2, O113:[H4], O146:H21, O118:H16, and O76:[H19]) accounted for 69% of all STEC strains. We identified 41 STEC strains belonging to 31 serotypes which had not previously been described as human STEC. Twenty-six of these were positive for intimins alpha1 (one serotype), beta1 (eight serotypes), epsilon (two serotypes), and eta (three serotypes). Our study indicates that different types of STEC strains predominate in infant and adult patients and that new types of STEC strains are present among human isolates.     

Discrimination of Enterohemorrhagic Escherichia coli (EHEC) from Non-EHEC Strains Based on Detection of Various Combinations of Type III Effector Genes

Enterohemorrhagic Escherichia coli (EHEC) strains comprise a subgroup of Shiga-toxin (Stx)-producing E. coli (STEC) and are characterized by a few serotypes. Among these, seven priority STEC serotypes (O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7) are most frequently implicated in severe clinical illness worldwide. Currently, standard methods using stx, eae, and O-serogroup-specific gene sequences for detecting the top 7 EHEC serotypes bear the disadvantage that these genes can be found in non-EHEC strains as well. Here, we explored the suitability of ureD, espV, espK, espN, Z2098, and espM1 genes and combinations thereof as candidates for a more targeted EHEC screening assay. For a very large panel of E. coli strains (n = 1,100), which comprised EHEC (n = 340), enteropathogenic E. coli (EPEC) (n = 392), STEC (n = 193), and apathogenic strains (n = 175), we showed that these genetic markers were more prevalent in EHEC (67.1% to 92.4%) than in EPEC (13.3% to 45.2%), STEC (0.5% to 3.6%), and apathogenic E. coli strains (0 to 2.9%). It is noteworthy that 38.5% of the EPEC strains that tested positive for at least one of these genetic markers belonged to the top 7 EHEC serotypes, suggesting that such isolates might be Stx-negative derivatives of EHEC. The associations of espK with either espV, ureD, or Z2098 were the best combinations for more specific and sensitive detection of the top 7 EHEC strains, allowing detection of 99.3% to 100% of these strains. In addition, detection of 93.7% of the EHEC strains belonging to other serotypes than the top 7 offers a possibility for identifying new emerging EHEC strains.     

Enhanced Virulence of the Escherichia coli O157:H7 Spinach-Associated Outbreak Strain in Two Animal Models Is Associated with Higher Levels of Stx2 Production after Induction with Ciprofloxacin

Shiga toxin (Stx)-producing Escherichia coli (STEC) causes hemorrhagic colitis and the hemolytic-uremic syndrome (HUS). STEC strains may produce Stx1a and/or Stx2a or variants of either toxin. A 2006 spinach-associated outbreak of STEC O157:H7 resulted in higher hospitalization and HUS rates than previous STEC outbreaks. The spinach isolate, strain K3995, contains both stx_2a and stx_2c. We hypothesized that the enhanced virulence of K3995 reflects the combination of stx₂ alleles (carried on lysogenic phages) and/or the amount of Stx2 made by that strain. We compared the virulence of K3995 to those of other O157:H7 isolates and an isogenic Stx2 mutant in rabbits and mice. We also measured the relative levels of Stx2 produced from those strains with or without induction of the stx-carrying phage. Some rabbits infected with K3995 exhibited intestinal pathology and succumbed to infection, while none of those infected with O157:H7 strain 2812 (Stx1a⁺ Stx2a⁺) died or showed pathological signs. Rabbits infected with the isogenic Stx2a mutant K3995 stx_2a::cat were not colonized as well as those infected with K3995 and exhibited no signs of disease. In the streptomycin-treated mouse model, more animals infected with K3995 died than did those infected with O157:H7 strain 86-24 (Stx2a⁺). Additionally, K3995 produced higher levels of total Stx2 and toxin phage DNA in cultures after phage induction than did 86-24. Our results demonstrate the greater virulence of K3995 compared to other O157:H7 strains in rabbits and mice. We conclude that this enhanced virulence is linked to higher levels of Stx2 expression as a consequence of increased phage induction.     

Specific Detection of Enteroaggregative Hemorrhagic Escherichia coli O104:H4 Strains by Use of the CRISPR Locus as a Target for a Diagnostic Real-Time PCR

In 2011, a large outbreak of an unusual bacterial strain occurred in Europe. This strain was characterized as a hybrid of an enteroaggregative Escherichia coli (EAEC) and a Shiga toxin-producing E. coli (STEC) strain of the serotype O104:H4. Here, we present a single PCR targeting the clustered regularly interspaced short palindromic repeats locus of E. coli O104:H4 (CRISPR_O104:H4) for specific detection of EAEC STEC O104:H4 strains from different geographical locations and time periods. The specificity of the CRISPR_O104:H4 PCR was investigated using 1,321 E. coli strains, including reference strains for E. coli O serogroups O1 to O186 and flagellar (H) types H1 to H56. The assay was compared for specificity using PCR assays targeting different O104 antigen-encoding genes (wbwC_O104, wzx_O104, and wzy_O104). The PCR assays reacted with all types of E. coli O104 strains (O104:H2, O104:H4, O104:H7, and O104:H21) and with E. coli O8 and O9 strains carrying the K9 capsular antigen and were therefore not specific for detection of the EAEC STEC O104:H4 type. A single PCR developed for the CRISPR_O104:H4 target was sufficient for specific identification and detection of the 48 tested EAEC STEC O104:H4 strains. The 35 E. coli O104 strains expressing H types other than H4 as well as 8 E. coli strains carrying a K9 capsular antigen tested all negative for the CRISPR_O104:H4 locus. Only 12 (0.94%) of the 1,273 non-O104:H4 E. coli strains (serotypes Ont:H2, O43:H2, O141:H2, and O174:H2) reacted positive in the CRISPR_O104:H4 PCR (99.06% specificity).