H7 Shiga Toxin-producing Escherichia Coli Research Articles

Sequence-based typing of genetic targets encoded outside of the O-antigen gene cluster is indicative of Shiga toxin-producing Escherichia coli serogroup lineages

Serogroup classifications based upon the O-somatic antigen of Shiga toxin-producing Escherichia coli (STEC) provide significant epidemiological information on clinical isolates. Each O-antigen determinant is encoded by a unique cluster of genes present between the gnd and galF chromosomal genes. Alternatively, serogroup-specific polymorphisms might be encoded in loci that are encoded outside of the O-antigen gene cluster. Segments of the core bacterial loci mdh, gnd, gcl, ppk, metA, ftsZ, relA and metG for 30 O26 STEC strains have previously been sequenced, and comparative analyses to O157 distinguished these two serogroups. To screen these loci for serogroup-specific traits within a broader range of clinically significant serogroups, DNA sequences were obtained for 19 strains of 10 additional STEC serogroups. Unique alleles were observed at the gnd locus for each examined STEC serogroup, and this correlation persisted when comparative analyses were extended to 144 gnd sequences from 26 O-serogroups (comprising 42 O : H-serotypes). These included O157, O121, O103, O26, O5 : non-motile (NM), O145 : NM, O113 : H21, O111 : NM and O117 : H7 STEC; and furthermore, non-toxin encoding O157, O26, O55, O6 and O117 strains encoded distinct gnd alleles compared to STEC strains of the same serogroup. DNA sequencing of a 643 bp region of gnd was, therefore, sufficient to minimally determine the O-antigen of STEC through molecular means, and the location of gnd next to the O-antigen gene cluster offered additional support for the co-inheritance of these determinants. The gnd DNA sequence-based serogrouping method could improve the typing capabilities for STEC in clinical laboratories, and was used successfully to characterize O121 : H19, O26 : H11 and O177 : NM clinical isolates prior to serological confirmation during outbreak investigations.

L-Sorbose utilization by virulent Escherichia coli and Shigella: Different metabolic adaptation of pathotypes

The frequency of l-Sorbose utilization differs significantly between pathotypes of Escherichia coli and Shigella from 93% to 0%. Among 266 strains tested, this frequency increased in the order Shigella, enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), Shiga toxin-producing E. coli (STEC), enteroaggregative E. coli, enteropathogenic E. coli (EPEC), and neonatal bacterial meningitis (NBM) E. coli. This suggests an association of pathomechanism with the capability to degrade l-Sorbose. The use of a selective agar, containing l-Sorbose and antibiotics, facilitated the isolation of l-Sorbose-non-utilizing ETEC from stool specimens of patients. The sor operon, comprising seven genes in the order sorCDFBAME, confers l-Sorbose utilization. Surprisingly, l-Sorbose-non-degrading Shigella harbored all genes of the sor operon indicating l-Sorbose-utilizing E. coli as ancestor. Additionally, strains of several EIEC and STEC serotypes harbored an inactivated sor operon. These l-Sorbose-non-utilizing Shigella, EIEC, and STEC showed significantly reduced amounts of transcripts as examined for sorC and sorD. Common surface antigens, types of intimin gene, and hemolysin gene as well as use of l-Sorbose suggested the relatedness of attaching and effacing O26:H11 and O55:H7 EPEC and STEC, respectively. pepE and yibC genes flank the sor operon of E. coli and Shigella strains. Surprisingly, one O7:K1:H- NBM E. coli harbored an aroE-homologous gene between its sor operon and pepE as in Klebsiella pneumoniae suggesting a horizontal gene transfer. In conclusion, l-Sorbose utilization of virulent E. coli and Shigella is characterized by different adaptation that represents a valuable tool for evolutionary and diagnostic analysis of related patho- and serotypes.

Supplementation of enrichment broths by novobiocin for detecting Shiga toxin-producing Escherichia coli from food: a controversial use

To investigate the assumption that usage of novobiocin (20 mg l(-1)) in Shiga toxin-producing Escherichia coli (STEC) enrichment broths could achieve false-negative results. First, the minimum inhibitory concentration (MIC) of 74 E. coli O157:H7 and 55 non-O157:H7 STEC strains to novobiocin was determined. Second, to visualize the potential impact of novobiocin on the STEC growth during the enrichment step, the growth experiments were carried out in trypticase soy broth (TSB) with and without 20 mg l(-1) of novobiocin. The MIC values varied from 32 to > 64 mg l(-1) for the 74 E. coli O157:H7 strains, and from 16 to > 64 mg l(-1) for the 55 non-O157:H7 STEC strains. The E. coli O157:H7 strains were significantly (P < 0.001) more resistant to novobiocin than the non-O157:H7 STEC strains. The present study shows that the addition of novobiocin into enrichment broths inhibits the growth of some non-O157:H7 STEC strains, and slows down the growth of some STEC strains. Enrichment broths supplemented by novobiocin could lead to false-negative results for detecting STEC from food. We strongly suggest that novobiocin should not be systematically added into enrichment broths for detecting STEC from food.

Phenotypic characteristics, virulence profile and genetic relatedness of O157 Shiga toxin-producing Escherichia coli isolated in Brazil and other Latin American countries

Thirty-eight Shiga toxin-producing Escherichia coli (STEC) O157:H7/H(-) strains isolated from human infections, cattle and foods in Brazil and in some other Latin American countries were compared with regard to several phenotypic and genotypic characteristics. The genetic relatedness of the strains was also determined by pulsed-field gel electrophoresis (PFGE). Similar biochemical behaviour was identified, regardless of the origin and country of the strains. Most (89.5%) strains were sensitive to the antimicrobial agents tested, but resistance to at least one drug was observed among bovine strains. Although a diversity of stx genotypes was identified, most (77.8%) of the human strains harboured stx(2) or stx(2)stx(2c(2vha)), whereas stx(2c(2vha)) prevailed (64.2%) among strains isolated from cattle. stx(1) and stx(1)stx(2c(2vha)) were the genotypes identified less frequently, and occurred exclusively among strains isolated from food and cattle, respectively. Despite differences in the stx genotypes, all strains carried eae-gamma, efa1, ehx, iha, lpf(O157) and toxB sequences. Many closely related subgroups (more than 80% of similarity) were identified by PFGE, and the presence of a particular O157:H7 STEC clone more related to human infections in Brazil, as well as a common origin for some strains isolated from different sources and countries in Latin America can be suggested.

Bacterial Genetic Determinants of Non‐O157 STEC Outbreaks and Hemolytic‐Uremic Syndrome after Infection

Although O157:H7 Shiga toxin-producing Escherichia coli (STEC) are the predominant cause of hemolytic-uremic syndrome (HUS) in the world, non-O157:H7 serotypes are a medically important cause of HUS that are underdetected by current diagnostic approaches. Because Shiga toxin is necessary but not sufficient to cause HUS, identifying the virulence determinants that predict severe disease after non-O157 STEC infection is of paramount importance. Disease caused by O157:H7 STEC has been associated with a 26-gene pathogenicity island known as O island (OI) 122. To assess the public-health significance of this pathogenicity island, we examined the association between OI122 genes and outbreaks and HUS after non-O157 STEC infection. We found that a subset of OI122 genes is independently associated with outbreaks and HUS after infection with non-O157 STEC. The presence of multiple virulence genes in non-O157 serotypes strengthened this association, which suggests that the additive effects of a variable repertoire of virulence genes contribute to disease severity. In vivo, Citrobacter rodentium mutants lacking outbreak- and HUS-associated genes were deficient for virulence in mice; in particular, nleB mutant bacteria were unable to cause mortality in mice. The present study shows that virulence genes associated epidemiologically with outbreaks and HUS after non-O157 STEC infection are pivotal to the initiation, progression, and outcome of in vivo disease.

Growth and survival of non-O157:H7 Shiga-toxin-producing Escherichia coli in cow manure

The main objective of this study was to evaluate the behaviour of non-O157:H7 Shiga-toxin-producing Escherichia coli (STEC) strains in cow manure. A mixture of eight green-fluorescent-protein-labelled STEC strains was inoculated around 10(6)-10(7) CFU g(-1) into four manure heaps. Two heaps were regularly turned and the two others remained unturned. STEC counts and physical parameters (temperature, pH, moisture content and oxido-reduction potential) were monitored for 1000 manure samples. The highest mean pH values were obtained near the surface at the base of all manure heaps. At the surface, the moisture content decreased from 76.5% to 42% in turned heaps. Temperatures reached 65 degrees C near the main body of all manure heaps, and only 35 degrees C near the superficial parts located at the base of them. These two sites (the centre and the base) were associated with D values for the STEC counts of 0.48 and 2.39 days, respectively. We were able to detect STEC strains during 42 days in turned manure heaps and during at least 90 days in unturned ones. These results emphasize the long-term survival of non-O157:H7 STEC in cow manure. Good management practices (e.g. turning) should be respected in order to minimize the risk of environmental contamination by STEC.

Genetic Heterogeneity of Shiga Toxin-Producing Escherichia coli Strains Isolated in São Paulo, Brazil, from 1976 through 2003, as Revealed by Pulsed-Field Gel Electrophoresis

The pulsed-field gel electrophoresis (PFGE) patterns of 46 Shiga toxin-producing Escherichia coli (STEC) strains isolated in São Paulo, Brazil, during the period from 1976 to 2003 were compared with those found among 30 non-STEC strains that carried eae and that belonged to the same serogroups as the STEC strains. All except two of the STEC and non-STEC strains of human origin were from sporadic and unrelated cases of infection; two O111 strains originated from the same patient. Multiple PFGE patterns were found among STEC strains of distinct serotypes. Moreover, the PFGE restriction patterns of STEC strains differed substantially from those observed among non-STEC strains of the same serogroup except serotype O26 strains. Based on the indistinguishable PFGE pattern for two O157:H7 STEC strains isolated in the same geographic area at an interval of approximately 15 days and toxin profile data, the first occurrence of an O157:H7 outbreak in Brazil during that period can be suggested. In general, a close relationship between types of intimin, serotypes, and diarrheagenic groups of E. coli was observed. This is the first time that a large collection of STEC strains from Brazil has been analyzed, and a great genetic diversity was shown among O157:H7 and non-O157:H7 STEC strains isolated in São Paulo, Brazil.

Isolation of E. coli O157:H7 and non-O157 STEC in different matrices: review of the most commonly used enrichment protocols

To review and characterize the enrichment protocols used for detecting all Shiga-Toxin producing Escherichia coli (STEC) from different matrices. Firstly, the frequency distribution of the factors characterizing the enrichment protocols is described; secondly, a multiple correspondence analysis is performed to display profiles of association of these factors, and thirdly, published results concerning the relative performances of the protocols are summarized. Trypticase Soy Broth (TSB) is reported as the most frequently used enrichment broth. More often, one antibiotic is added in enrichment broths and these broths are incubated for a duration of 16-24 h at 35-37 degrees C. It also appears that the incubation temperature does not seem to be related to the type of serogroup looked for and that antibiotics are used regardless of the matrix analysed. Finally, results relating to the enrichment protocol efficacy are rare and differ from one study to another. Statistical studies must be conducted so as to assess the efficacy of the main enrichment protocols investigated in this study. This study reviews the most commonly used enrichment protocols and highlights the lack of results as to their relative efficacy.

Serotypes, intimin variants and other virulence factors of eae positive Escherichia coli strains isolated from healthy cattle in Switzerland. Identification of a new intimin variant gene (eae-eta2).

BackgroundEnteropathogenic Escherichia coli (EPEC) and Shigatoxin-producing Escherichia coli (STEC) share the ability to introduce attaching-and-effacing (A/E) lesions on intestinal cells. The genetic determinants for the production of A/E lesions are located on the locus of enterocyte effacement (LEE), a pathogenicity island that also contains the genes encoding intimin (eae). This study reports information on the occurrence of eae positive E. coli carried by healthy cattle at the point of slaughter, and on serotypes, intimin variants, and further virulence factors of isolated EPEC and STEC strains.ResultsOf 51 eae positive bovine E. coli strains, 59% were classified as EPEC and 41% as STEC. EPEC strains belonged to 18 O:H serotypes, six strains to typical EPEC serogroups. EPEC strains harbored a variety of intimin variants with eae-β1 being most frequently found. Moreover, nine EPEC strains harbored astA (EAST1), seven bfpA (bundlin), and only one strain was positive for the EAF plasmid. We have identified a new intimin gene (η2) in three bovine bfpA and astA-positive EPEC strains of serotype ONT:H45. STEC strains belonged to seven O:H serotypes with one serotype (O103:H2) accounting for 48% of the strains. The majority of bovine STEC strains (90%) belonged to five serotypes previously reported in association with hemolytic uremic syndrom (HUS), including one O157:H7 STEC strain. STEC strains harbored four intimin variants with eae-ε1 and eae-γ1 being most frequently found. Moreover, the majority of STEC strains carried only stx1 genes (13 strains), and was positive for ehxA (18 strains) encoding for Enterohemolysin. Four STEC strains showed a virulence pattern characteristic of highly virulent human strains (stx2 and eae positive).ConclusionOur data confirm that ruminants are an important source of serologically and genetically diverse intimin-harboring E. coli strains. Moreover, cattle have not only to be considered as important asymptomatic carriers of O157 STEC but can also be a reservoir of EPEC and eae positive non-O157 STEC, which are described in association with human diseases.

Sorbitol-Negative Shiga Toxin-Producing Escherichia coli in Beef Heifers Grazing Rangeland Forages

Escherichia coli O157:H7 has been a major cause of human illness outbreaks in the U.S., mainly attributable to the consumption of contaminated beef. Worldwide, however, other shiga toxin-producing E. coli (STEC) have been responsible for major outbreaks of human illnesses. Because of the lack of sorbitol fermentation and β-glucuronidase activity, E. coli O157:H7 is relatively easier to detect than other STEC. The objective of this experiment was to examine prevalence of sorbitol-negative STEC in beef heifers grazing rangeland forages over the four seasons of 1999. However, because of the loss of heifers and vegetation to wild fires during the summer, the study was terminated, and the results of two seasons (winter and spring) were reported. A random number of heifers (22 in winter; 23 in spring) from a herd of 70 were fecal-sampled; nine were sampled in both seasons. A random sample (n = 115) of potential sorbitol-negative STEC isolates was screened for the presence and expression of toxin genes. Fifty-six STEC isolates were detected in feces of five heifers in the winter and two heifers in the spring with occurrence rates of 22.7 and 8.7%, respectively. Of these isolates, 37 had and expressed Shiga Toxin 1(Stx1), 14 had and expressed Shiga Toxin 2 (Stx2), and five had but did not express stx2. Only non-O157:H7 STEC (O118:H–[non-motile] and O138:H–) were detected. Because E. coli O118:H–is pathogenic to humans, it is important to determine its incidence in cattle and to determine the risk of beef contamination with such a pathogen.

Isolation and Characterization of Escherichia coli Recovered from Maryland Apple Cider and the Cider Production Environment

Contaminated apple cider has been implicated in several Escherichia coli O157:H7 outbreaks. In an attempt to investigate sources and modes of entry of E. coli into apple cider, samples of fresh apple, pomace, and cider and equipment and mill floor swabs were analyzed for standard plate counts (SPC), total coliforms (TC), fecal coliforms (FC), and E. coli. E. coli was isolated from 14 (33%) of 42 samples of bottled fresh cider, from food equipment in 6 (67%) of 9 mills, and from apples, pomace, or cider in 7 (78%) of 9 mills. Seventy-five E. coli isolates were further characterized for Shiga toxin–producing E. coli (STEC)–associated virulence factors, antimicrobial susceptibility, and pulsed-field gel electrophoresis (PFGE) type. No E. coli O157:H7 or other STEC was identified. Serotyping and PFGE revealed 64 distinct profiles, suggesting that recovered E. coli arose from multiple independent sources. However, on one occasion, E. coli isolated from the source apple sample was closely related to the E. coli identified in the finished cider sample. E. coli isolates were further tested for antimicrobial susceptibility to 17 antimicrobial agents of human and veterinary importance. Fourteen (19%) of the 75 isolates were resistant to at least one of the antimicrobial agents tested, and 9 (12%) were resistant to at least two of these agents. Of the resistant isolates recovered, 64% were resistant to tetracycline and 57% were resistant to streptomycin. Overall, the level of E. coli contamination in source apple samples did not differ significantly from those in samples of pomace, cider at the press, and cider entering the bottling tank; therefore, source apples cannot be dismissed as a potential contributor of E. coli to the cider-making process.

Shiga toxin-producing Escherichia coli in Montana: bacterial genotypes and clinical profiles.

The diseases and virulence genes associated with Shiga toxin-producing Escherichia coli (STEC) are characterized incompletely. We analyzed, by polymerase chain reaction, 82 STEC isolates collected prospectively in Montana and profiled associated illnesses by patient chart review. All E. coli O157:H7 contained stx2-group genes, as well as eae, iha, espA, and ehxA; 84% contained stx1. Non-O157:H7 STEC less frequently contained stx1 (P=.046), stx2 (P<.001), iha (P<.001), eae, and espA (P=.039 for both), were isolated less often from patients treated in emergency departments (P=.022), and tended to be associated less frequently with bloody diarrhea (P=.061). There were no significant associations between stx genotype and bloody diarrhea, but isolates containing stx2c or stx(2d-activatable) were recovered more often from patients who underwent diagnostic or therapeutic procedures (P=.033). Non-O157:H7 STEC are more heterogeneous and cause bloody diarrhea less frequently than do E. coli O157:H7. Bloody diarrhea cannot be attributed simply to the stx genotype of the infecting organism.

Clinical course and the role of shiga toxin-producing Escherichia coli infection in the hemolytic-uremic syndrome in pediatric patients, 1997-2000, in Germany and Austria: a prospective study.

Hemolytic-uremic syndrome (HUS) is mainly associated with foodborne infections by Shiga toxin-producing Escherichia coli (STEC). From January 1997 through December 2000, 394 children with HUS were evaluated in a prospective multicenter surveillance study in Germany and Austria (incidences, 0.7/100,000 and 0.4/100,000 children <15 years old, respectively). Blood leukocytosis was associated with increased detection of STEC in stool cultures (P<.01) and a more severe disease course. Risk of death was associated with cerebral involvement (P<.01). Most strikingly, non-O157:H7 STEC were detected in 43% of stool cultures of patients with HUS: O26 was detected in 15%, sorbitol-fermenting O157:H(-) in 10%, O145 in 9%, O103 in 3%, and O111 in 43%. Patients with O157:H7 serotypes required dialysis for a longer time and had bloody diarrhea detected more frequently, compared with patients with non-O157:H7 serotypes (P<.05). This large study in children with HUS underlines the rising importance of non-O157:H7 serotypes, and, despite increased public awareness, the number of patients remained unchanged.

Effect of diet on Shiga toxin-producing Escherichia coli (STEC) growth and survival in rumen and abomasum fluids.

The gastrointestinal tract of ruminants is the main reservoir for Shiga toxin-producing Escherichia coli (STEC) strains, potentially pathogenic for humans. We used for the first timerumen fluid in which no exogenous carbon source or other supplement was added to compare acid resistance and growth of STEC in physiological physico-chemical conditions. We showed that acidic conditions resulting from the combination of high volatile fatty acid concentration and moderately acidic pH did not alter the survival of STEC, and that human non-O157:H7 STEC isolates were able to persist in the rumen contents in spite of acid stress, low oxygen availability and nutrient deprivation, in the same manner as bovine STEC isolates do. Furthermore, our results support the hypothesis that a grain-rich diet may induce mechanisms of STEC acid resistance in the rumen that allow STEC survival in the abomasum.

Shiga Toxin–Producing Escherichia coli in Beef Heifers Grazing an Irrigated Pasture

Shiga toxin–producing Escherichia coli (STEC) produce toxins that have been associated with several human illnesses. E. coli O157:H7 is the most well-studied STEC and was first associated with consumption of improperly cooked ground beef in 1982. E. coli O157:H7 is not the only foodborne STEC because other STEC serotypes are also associated with human illnesses. The objective of this study was to assess prevalence of STEC in 23 yearling beef (Angus) heifers grazing an irrigated grass pasture in spring (April), summer (July), fall (October), and winter (December) of 1999. A total of 86 fecal samples were rectally collected and were subjected to microbiological testing for the presence of STEC. Nine E. coli isolates from five heifers (one in spring and fall and three in winter) were toxic to Vero cells. Of these isolates, four were E. coli O157:H7, two belonged to the serogroup O6, one O39:NM, one O113:H−, and the final isolate was untypable. The STEC prevalence rate in our herd ranged from 4% (spring) to 15% (winter). Based on detecting both O157:H7 and non-O157:H7 STEC in our heifers, it is clear that screening fecal samples should not be limited to E. coli O157:H7. Identification of STEC-positive cattle prior to slaughter should help in reducing the risk of beef contamination with such foodborne pathogens if pre- and/or postharvest control measures are applied to such animals.

Nationwide study of haemolytic uraemic syndrome: clinical, microbiological, and epidemiological features

AIMSTo establish the incidence and aetiology of haemolytic uraemic syndrome (HUS) in Australia and compare clinical and microbial characteristics of sporadic and outbreak cases.METHODSNational active surveillance through the Australian Paediatric...

Heterogeneity of Shiga toxin-producing Escherichia coli strains isolated from hemolytic-uremic syndrome patients, cattle, and food samples in central France.

A detailed analysis of the molecular epidemiology of non-O157:H7 Shiga toxin-producing Escherichia coli (STEC) was performed by using isolates from sporadic cases of hemolytic-uremic syndrome (HUS), animal reservoirs, and food products. The isolates belonged to the O91 and OX3 serogroups and were collected in the same geographical area over a short period of time. Five typing methods were used; some of these were used to explore potentially mobile elements like the stx genes or the plasmids (stx(2)-restriction fragment length polymorphism [RFLP], stx(2) gene variant, and plasmid analyses), and others were used to study the whole genome (ribotyping and pulsed-field gel electrophoresis [PFGE]). The techniques revealed that there was great diversity among the O91 and OX3 STEC strains isolated in central France. A close relationship between strains of the same serotype having the same virulence factor pattern was first suggested by ribotyping. However, stx(2)-RFLP and stx(2) variant analyses differentiated all but 5 of 21 isolates, and plasmid analysis revealed further heterogeneity; a unique combination of characteristics was obtained for all strains except two O91:H21 isolates from beef. The latter strains were shown by PFGE to be the most closely related isolates, with >96% homology, and hence may be subtypes of the same strain. Overall, our results indicate that the combination of stx(2)-RFLP, stx(2) variant, and plasmid profile analyses is as powerful as PFGE for molecular investigation of STEC diversity. Finally, the non-O157:H7 STEC strains isolated from HUS patients were related to but not identical to those isolated from cattle and food samples in the same geographical area. The possibility that there are distinct lineages of non-O157:H7 STEC, some of which are more virulent for humans, should be investigated further.

Semi-automated fluorogenic PCR assays (TaqMan) forrapid detection of Escherichia coli O157:H7 and other Shiga toxigenic E. coli

Semi-automated detection of Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 and non-O157:H7 Shiga toxin-producing E. coli (STEC) was achieved using fluorogenic polymerase chain reaction (PCR). These PCR assays were designed to amplify 80, 120 and 150 bp regions of virulence genes stx1 , stx2 and eaeA , respectively, using specific primers. The fluorogenic probes were used for specific detection of amplified products of the stx1 andstx2 genes of STEC, and the eaeA gene of EHEC O157:H7. For multiplex PCR assay, the three sets of primers and fluorogenic probes were included in one reaction to simultaneously amplify and detect any of the three targeted virulence genes. In non-multiplex PCR assay, each of the three virulence genes was amplified and detected in independent reactions. The specificity of these assays was evaluated using suspensions of STEC and other bacterial species lacking stx1 , stx2 andeaeA . The multiplex assay detected all STEC harbouring any combination of three virulence genes. Three non-multiplex PCR reactions identified types of Shiga toxin genes carried by a STEC and identified STEC as either EHEC O157:H7 or non-O157:H7 STEC. Sensitivity limits of these assays in beef and faeces inoculated with EHEC O157:H7 were 5.8 to 580 cfu and 1.2 to 1200 cfu, respectively. These assays can be completed within 8–10 h when performed simultaneously or within 13 h if the multiplex assay is used as an initial screen for detecting STEC and the non-multiplex assay is used for subsequent detection of stx1 andstx2 of STEC and eaeA of EHEC O157:H7

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.

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.