Kansenshogaku Zasshi, 2002 Nov, 76(11), 911 - 20 {Presence of the genes regarding adherence factors of Escherichia coli isolates and a consideration of the procedure for detection of diarrheagenic strain}; Kobayashi K et al.; Diarrheagenic Escherichia coli are differentiated from non-pathogenic members with enterotoxin production, enteroinvasiveness and serotyping . However, the serotypic members are rarely sufficient to reliably identify a strain as diarrheagenic on E . coli . Recently, there are many definite articles which the adhesive E . coli strain against intestinal epithelial cells is enterovirulent . In this study, 1,748 E . coli isolates of diarrheagenic and non-diarrheagenic categories which belonged to EHEC, ETEC, EIEC EPEC and non-EPEC were examinated by PCR method for the presence of eaeA, aggR and bfpA regarding adherence factor genes, and astA of EAST1 . The strains examined were recognized to variable carrying geno-patterns, and a large number of EHEC, EPEC and non-EPEC had carried either eaeA or aggR genes . In EHEC isolates, a carrying pattern with the most high frequency was only eaeA, and this type was recognized in the isolates of serotype O157, O26 and O111 . EPEC and non-EPEC isolates were recognized eaeA or aggR which harboring with astA or not . Of 508 EPEC isolates from human, a total of 137 isolates (27.0%) carried aggR, and a total of 74 isolates (14.6%) had eaeA, while of the 91 isolates from non-human were recognized aggR and eaeA with 2.2% (2 isolates) and 12.1% (11 isolates), respectively . Also, of 266 non-EPEC isolates from human, a total of 16 isolates (6.0%) carried aggR, and a total of 58 isolates (21.8%) had eaeA . On the other hand, 22 (7.0%) of 316 isolates examined from non-human had eaeA, however no isolate had aggR . Thirteen isolates of EIEC and 218 ETEC isolates were screened, and only 6 ETEC isolates had either eaeA or aggR . The astA gene was recognized in the isolates of all categories, and ETEC strains had more frequently . The bfpA gene was recognized with more frequently in a serotype O157: H45, which is obtained from human with diarrhea, however, this strain was not recognized a member of the EPEC serotype . There is no diagnostic system for the strain of E . coli that cause diarrheal diseases, therefore more laboratories are unable to identify them . The authors had confirmed which PCR technique is a useful simple and rapid method for the detection of adherence factor genes on E . coli strains . From the these results, we showed a differentiation method using PCR technique which have relation with adherence factor, enterotoxin-production and invasiveness, and we firmly believe that application of the procedure is a reasonable and useful method for the identification of diarrheagenic E . coli. J Food Prot, 2002 Dec, 65(12), 1970 - 5 Evidence for Escherichia coli O157:H7 attachment to water distribution pipe materials by scanning electron microscopy; Assanta MA et al.; Scanning electron microscopy observation was used to investigate the adhesion of Escherichia coli O157:H7 on water distribution pipe surfaces such as copper and polyethylene plastic at different contact times and storage temperatures . Our results indicated that E . coli cells could easily attach to both surface types after exposures as short as 1 or 4 h at ambient (20 degrees C) and refrigeration temperatures (4 degrees C) . Also, we found that copper surfaces have a higher number of attached E . coli cells than plastic surfaces . The number of cells attached to each type of material depended on the nature of the water distribution pipe surfaces and the length of contact time . In addition, the surface energy value of each surface estimated by contact angle measurements using water, alpha-bromonaphthalene, and dimethyl sulfoxide as wetting agents showed that both copper (41.2 megajoules {MJ} m(-2)) and plastic (45.8 MJ x m(-2)) have a low energy surface . In no cases could evidence of extracellular material be observed on surfaces with either exposure condition. Pediatr Nephrol, 2002 Dec, 17(12), 1047 - 52 Epub 2002 Nov 05. Platelet-activating factor and Escherichia coliO157:H7 infections; Smith JM et al.; The role of platelet-activating factor (PAF), a phospholipid inflammatory mediator, in Escherichia coli O157:H7-associated hemolytic uremic syndrome (HUS) is unknown . PAF is synthesized by diverse cells and is degraded by PAF-acetylhydrolase (PAF-AH) . Deficient PAF-AH activity results from a G-->T transversion at position 994 of exon 9 . We examined children infected with E . coliO157:H7 to determine if PAF levels or the PAF-AH ( G994T) mutation reflects the risk of developing HUS . Plasma PAF concentrations were determined using chloroform/methanol extraction, thin layer chromatography purification, and scintillation proximity assay in 10 patients with uncomplicated infection (UI), 10 infected patients who subsequently developed HUS (pre-HUS), 5 HUS patients, and 8 healthy controls . The PAF-AH ( G994T) allele frequency was determined in 52 UI children, 15 with HUS, and 11 controls . Wilcoxon rank sum tests were performed to test differences in location (median) of pairs of groups . PAF levels were higher in the UI ( P=0.04) and pre-HUS ( P=0.01) groups than in healthy controls . No subject had the PAF-AH ( G994T) allele . Thus, elevated plasma PAF levels occur in E . coliO157:H7-infected children, even without HUS, but diminish when HUS develops . The PAF-AH ( G994T) allele does not contribute to the risk of developing HUS. Public Health Rep, 2002 Jul-Aug, 117(4), 380 - 5 A cluster of Escherichia coli O157: nonmotile infections associated with recreational exposure to lake water; Feldman KA et al.; OBJECTIVES: To identify cases and determine risk factors for an outbreak of Escherichia coli (E . coli) O157: nonmotile (NM) infections in children attending a summer day care program in California . METHODS: The authors conducted a retrospective cohort study; the cohort comprised first and second graders who attended the day care program during the last week in August 1999 . Shiga toxin testing and molecular subtyping using pulsed-field gel electrophoresis were performed on isolates . Lake water, lake bottom sediment samples, and waterfowl feces from the lake environs were cultured for E . coli O157 . RESULTS: Three cases of Shiga toxin-producing E . coli O157: NM infections with matching pulsed-field gel electrophoresis patterns and four probable cases were found . Children who swallowed more than a mouthful of water had a higher attack rate than those who swallowed less than a mouthful or none at all (43% vs . 10%, relative risk = 4.43, 95% confidence interval 1.12, 17.50) . CONCLUSIONS: E . coli O157: NM infections were associated with swallowing water from a freshwater lake . Potential sources of contamination include feces from humans, cattle, or deer . This outbreak illustrates the value in screening patients with diarrhea for E . coli O157, submitting isolates to public health laboratories, and using molecular techniques to identify related cases . Outbreaks associated with contaminated freshwater could be averted by prevention and early detection of contamination. Mol Cell Probes, 2002 Oct, 16(5), 335 - 9 Two methods for construction of internal amplification controls for the detection of Escherichia coli O157 by polymerase chain reaction; Abdulmawjood A et al.; For the detection of food born bacteria by polymerase chain reaction (PCR) in food products, an internal amplification control (IAC) is required in order to prevent false negative results that might be caused by PCR inhibitors . In the present study, two IACs were constructed using two different methods . These IACs were designed in a way that the same primer pair can be used to amplify the target DNA and coamplify the IAC . The first IAC with a size of approximately 200 bp was constructed by deleting a part of the amplicon of the original target DNA (500 bp) between the two primer sites to produce an IAC smaller than the target DNA . The second IAC with a size of approximately 600 bp was synthesized in a one step PCR reaction . The primers used in this reaction possessed 5' over-hanging ends, which were identical to the primers used in the diagnostic reaction, whereas their 3' ends were complementary to the (pUC19) predetermined DNA sequence of defined length and sequence . The concentration of IACs appeared to be critical . Too much IAC DNA template would out-compete the target DNA template, thus giving a false negative result . However the use of an optimal IAC concentration increased the reliability of the PCR assays and appeared to be useful for food diagnostics. Proc Natl Acad Sci U S A, 2002 Dec 24, 99(26), 17020 - 4 Epub 2002 Dec 05. Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli; Welch RA et al.; We present the complete genome sequence of uropathogenic Escherichia coli, strain CFT073 . A three-way genome comparison of the CFT073, enterohemorrhagic E . coli EDL933, and laboratory strain MG1655 reveals that, amazingly, only 39.2% of their combined (nonredundant) set of proteins actually are common to all three strains . The pathogen genomes are as different from each other as each pathogen is from the benign strain . The difference in disease potential between O157:H7 and CFT073 is reflected in the absence of genes for type III secretion system or phage- and plasmid-encoded toxins found in some classes of diarrheagenic E . coli . The CFT073 genome is particularly rich in genes that encode potential fimbrial adhesins, autotransporters, iron-sequestration systems, and phase-switch recombinases . Striking differences exist between the large pathogenicity islands of CFT073 and two other well-studied uropathogenic E . coli strains, J96 and 536 . Comparisons indicate that extraintestinal pathogenic E . coli arose independently from multiple clonal lineages . The different E . coli pathotypes have maintained a remarkable synteny of common, vertically evolved genes, whereas many islands interrupting this common backbone have been acquired by different horizontal transfer events in each strain. J Immunol, 2002 Dec 15, 169(12), 6822 - 30 Antibody repertoire development in fetal and neonatal piglets . VIII . Colonization is required for newborn piglets to make serum antibodies to T-dependent and type 2 T-independent antigens; Butler JE et al.; Cesarean-derived piglets were reared for 5 wk under germfree conditions or monoassociated with a benign Escherichia coli (G58-1) or a enterohemorrhagic strain (933D) derived from O157:H7, and immunized i.p . with the T-dependent (TD) Ags fluorescein-labeled (FL) keyhole limpet hemocyanin or trinitrophenylated (TNP) keyhole limpet hemocyanin and the type 2 T-independent Ags TNP-Ficoll or FL-Ficoll . Only colonized piglets showed an increase in serum IgG, IgA, and IgM and had serum Abs to FL, TNP, and colonizing bacteria . While serum Abs to FL or TNP appeared following colonization alone, secondary responses were restricted to piglets immunized using TD carriers . While animals colonized with 933D had significantly higher total serum IgG and IgM levels and specific IgG Abs than those colonized with G58-1, no differences were seen in serum IgA levels, B cell diversification in the ileal Peyer's patches, and specific activity (ELISA activity per micrograms of Ig) of pre-boost serum IgG and IgM anti-TNP and anti-FL Abs . Serum IgA Abs to TNP, FL, or bacteria were not detected . Ag-driven responses, as measured by an increase in specific Ab activity, were only observed in secondary responses to TD Ags and to colonizing, pathogenic E . coli . We propose that germline-encoded, isotype-switched B cells in newborn piglets differentiate to Ab-secreting cells 1) after stimulation by bacteria-activated APCs or 2) through direct stimulation by bacterial products . We further propose that Ag-driven systemic responses require both bacterial colonization and TD Ags translocated to the peritoneum. Clin Pediatr (Phila), 2002 Nov-Dec, 41(9), 705 - 9 Recurrent hemolytic uremic syndrome; Siegler RL et al.; Hemolytic uremic syndrome (HUS) in children follows a diarrheal prodrome (D+) approximately 90% of the time, and recurrence due to enteric reinfection with Shiga toxin producing E . coli (e.g., O157:H7) can occur but is rare . It is not well recognized that nondiarrheal (D-) recurrences can also follow an episode of D+ HUS; we report 2 unrelated females who experienced multiple D- episodes following an initial episode of D+ HUS . We also present an HUS classification system that includes recurrence risk . It illustrates that recurrence is seen most frequently with familial HUS but can also occur in cases that are secondary to drugs, cancer, and pregnancy. Eur J Clin Microbiol Infect Dis, 2002 Nov, 21(11), 810 - 3 Epub 2002 Nov 07. Verocytotoxin-producing Escherichia coli in patients with diarrhoea in Switzerland; Schmid H et al.; The present study was conducted between October 1996 and October 1998 to estimate the frequency of verocytotoxin-producing Escherichia coli among outpatients with diarrhoea in Switzerland . Among 3,041 subjects studied, 16 (0.5%) verocytotoxin-producing Escherichia coli infections were identified . Eleven cases were in infants and children </= 6 years of age . In 15 cases, a verocytotoxin-producing Escherichia coli strain was isolated . These strains belonged to 11 different serotypes, and only two were serogroup O157 . In five cases, the infection was probably acquired outside Switzerland . Verocytotoxin-producing Escherichia coli apparently play a minor role in the aetiology of diarrhoeal disease in adult outpatients in Switzerland, but they are important pathogens in preschool children in whom the most severe symptoms are observed. J Clin Microbiol, 2002 Dec, 40(12), 4685 - 90 Novel single-tube agar-based test system for motility enhancement and immunocapture of Escherichia coli O157:H7 by H7 flagellar antigen-specific antibodies; Murinda SE et al.; This paper describes a novel single-tube agar-based technique for motility enhancement and immunoimmobilization of Escherichia coli O157:H7 . Motility indole ornithine medium and agar (0.4%, wt/vol) media containing either nutrient broth, tryptone broth, or tryptic soy broth (TSBA) were evaluated for their abilities to enhance bacterial motility . Twenty-six E . coli strains, including 19 O157:H7 strains, 1 O157:H(-) strain, and 6 generic E . coli strains, were evaluated . Test bacteria were stab inoculated in the center of the agar column, and tubes were incubated at 37 degrees C for 18 to 96 h . Nineteen to 24 of the 26 test strains (73.1 to 92.3%) were motile in the different media . TSBA medium performed best and was employed in subsequent studies of motility enhancement and H7 flagellar immunocapture . H7 flagellar antiserum (30 and 60 micro l) mixed with TSBA was placed as a band (1 ml) in the middle of an agar column separating the top (3-ml) and bottom (3-ml) agar layers . The top agar layer was inoculated with the test bacterial strains . The tubes were incubated at 37 degrees C for 12 to 18 h and for 18 to 96 h . The specificity and sensitivity of the H7 flagellar immunocapture tests were 75 and 100%, respectively . The procedure described is simple and sensitive and could be adapted easily for routine use in laboratories that do not have sophisticated equipment and resources for confirming the presence of H7 flagellar antigens . Accurate and rapid identification of H7 flagellar antigen is critical for the complete characterization of E . coli O157:H7, owing to the immense clinical, public health, and economic significance of this food-borne pathogen. J Clin Microbiol, 2002 Dec, 40(12), 4585 - 93 Clinical Escherichia coli strains carrying stx genes: stx variants and stx-positive virulence profiles; Eklund M et al.; Altogether, 173 Shiga toxin-producing Escherichia coli (STEC) serotype O157 (n = 111) and non-O157 (n = 62) isolates from 170 subjects were screened by PCR-restriction fragment length polymorphism for eight different stx genes . The results were compiled according to serotypes, phage types of O157, production of Stx toxin and enterohemolysin, and the presence of eae . The stx genes occurred in 11 combinations; the most common were stx(2) with stx(2c) (42%), stx(2) alone (21%), and stx(1) alone (16%) . Of the O157 strains, 64% carried stx(2) with stx(2c) versus 2% of the non-O157 strains (P < 0.001) . In the non-O157 strains, the prevailing gene was stx(1) (99% versus 1% in O157 strains; P < 0.001) . In addition, one strain (O Rough:H4:stx(2c)) which has not previously been described as associated with hemolytic-uremic syndrome (HUS) was found . Ten stx-positive virulence profiles were responsible for 71% of all STEC infections . Of these profiles, five accounted for 71% of the 21 strains isolated from 20 patients with HUS or thrombotic thrombocytopenic purpura (TTP) . The strains having the virulence profile that caused mainly HUS or TTP or bloody diarrhea produced Stx with titers of >/=1:128 (90%) more commonly than did other strains (51%; P < 0.001) . These strains were also more commonly enterohemolytic (98% versus 68% for other strains; P < 0.001) and possessed the eae gene (100%) more commonly than did other strains (74%; P < 0.001) . A particular virulence profile, O157:H7:PT2:stx(2):stx(2c):eae:Ehly, was significantly more frequently associated with HUS and bloody diarrhea than were other profiles (P = 0.02) and also caused the deaths of two children . In this study, the risk factors for severe symptoms were an age of <5 years and infection by the strain of O157:H7:PT2 mentioned above. J Appl Microbiol, 1997 Feb, 82(2), 197 - 203 Invasion of bovine epithelial cells by verocytotoxin-producing Escherichia coli O157:H7; Matthews KR et al.; The ability of verocytotoxin-producing Escherichia coli (VTEC) O157:H7 to enter selected human (RPMI-4788 and HeLa) and bovine (MAC-T, mammary secretory; MDBK, kidney) epithelial cell lines was evaluated . All VTEC evaluated efficiently entered RPMI-4788 and MAC-T cell lines . VTEC entered MDBK cells at approximately 4% of MAC-T cells . VTEC were not able to invade HeLa cells . Presence of plasmid had no influence on efficiency of entry, nor did production of shiga-like toxin (SLT I or SLT II) . Internalization required microfilaments, but not microtubules . Two types of adherence, localized and diffuse, were exhibited depending on isolate and cell line evaluated . Ability of VTEC to invade bovine mammary epithelial cells may be important in pathogenesis in the bovine, may indicate a route by which raw milk may potentially become contaminated, and may provide a reservoir of bacteria for the contamination of workers, equipment and carcass at time of slaughter. Brain Res, 2002 Nov 29, 956(2), 246 - 53 Shiga-like toxin II modifies brain distribution of a P-glycoprotein substrate, doxorubicin, and P-glycoprotein expression in mice; Zhao YL et al.; The effect of Shiga-like toxin II (SLT-II), which was derived from Escherichia coli O157:H7, on doxorubicin transport across the blood-brain barrier (BBB) and P-glycoprotein function, was investigated in ddY mice . Doxorubicin (30 mg kg(-1)) was administered intravenously or fluorescein isothiocyanate labeled dextran (FD-4) was infused (20 microg min(-1)) to the mice, who had received an intravenous injection of SLT-II (0.2 microg/animal) 6 or 24 h earlier . Blood and brain were removed 4 h after injection of doxorubicin or 60 min after infusion of FD-4 . SLT-II significantly elevated the brain concentration and brain-to-plasma concentration ratio (K(p)) of doxorubicin and FD-4 24 h after injection, but did not alter 6 h after . Cyclosporin A (200 mg kg(-1)) significantly increased the K(p) value of doxorubicin in the control mice, but did not alter it in mice treated 24 h earlier with SLT-II . Pentoxifylline (100 mg kg(-1)) a TNF-alpha production inhibitor, ameliorated SLT-II-induced increases in the brain concentrations of both drugs and the K(p) value of FD-4, suggesting that TNF-alpha, at least in part, causes damage to the brain capillaries . Western blot analysis revealed that SLT-II increased the protein level of P-glycoprotein in the brain of mice 6 h after injection and the increased level remained unchanged for 24 h . SLT-II did not change ATP content in the brain of mice . These results suggest that the increased P-glycoprotein level cannot explain SLT-II-induced increase in the doxorubicin accumulation in brain . The present findings indicate that SLT-II impairs the BBB function and doxorubicin transport across the BBB, while it overexpresses P-glycoprotein . FEMS Immunol Med Microbiol, 2002 Dec 13, 34(4), 289 - 97 Subtyping of Shiga toxin 2 variants in human-derived Shiga toxin-producing Escherichia coli strains isolated in Japan; Nakao H et al.; Shiga toxin 2 (Stx2) variants have been found to exhibit not only antigenic divergence, but also differences in toxicity for tissue culture cells and animals . To clarify whether all or just a subset of Stx2 variants are important for the virulence of Shiga toxin-producing Escherichia coli, we designed PCR primers to detect and type all reported variants . We classified them into four groups according to the nucleotide sequences of the Stx2 family; for example, group 1 (G1) contains VT2vha and group 2 (G2) contains VT2d-Ount . The 120 strains of Shiga toxin-producing E . coli used in this study were isolated from humans in Japan between 1986 and 1999 . Among the four variant groups, the G1 gene only was detected in 23 of the 120 clinical strains (19.2%) and all belonged to the O157 serotype . G1 is considered the most important Stx2 variant group in terms of human pathogenicity . A multiplex PCR that can detect the stx1, stx2, and G1 genes was developed as a means of rapid and easy typing to better understand the roles of the different types of Stx. Pediatr Res, 2002 Dec, 52(6), 928 - 34 Circulating granulocyte colony-stimulating factor, C-X-C, and C-C chemokines in children with Escherichia coli O157:H7 associated hemolytic uremic syndrome; Proulx F et al.; Leukocytes are implicated in the pathogenesis of diarrhea-associated hemolytic uremic syndrome (D(+) HUS) . We hypothesized that increased circulating levels of granulocyte colony-stimulating factor (G-CSF), and the chemokines epithelial cell-derived neutrophil-activating protein-78 (ENA-78), growth related oncogen-alpha (GRO-alpha), macrophage inflammatory protein-1beta (MIP-1beta), and monocyte chemotactic protein-1 (MCP-1) are related to the severity of illness in Escherichia coli O157:H7 infections . We compared the circulating concentrations of these mediators in the course of E . coli O157:H7 enteritis, hemorrhagic colitis, and HUS . Our data show that, on admission, children with HUS presented 10-fold abnormally increased levels of G-CSF (p < 0.007), 3-fold increased MIP-1beta concentrations (p < 0.001), and 2-fold lower values of ENA-78 (p < 0.0001) . One week later, a further 4-fold decrease in ENA-78 concentration was noted (p < 0.0001) whereas MIP-1beta levels returned to normal . HUS patients requiring peritoneal dialysis showed 6-fold increased G-CSF (p < 0.001) and 5-fold decreased ENA-78 (p < 0.001) levels . On admission, children with uncomplicated O157:H7 hemorrhagic colitis (HC) presented 3-fold abnormally increased concentrations of G-CSF (p < 0.001) and MIP-1beta (p < 0.0001) . Those with O157:H7 enteritis but no bloody stools showed higher rates of abnormal GRO-alpha, MIP-1beta, and MCP-1 measurements than children with O157:H7 HC or HUS: GRO-alpha (50% enteritis, 36% HC, 17% HUS; p < 0.06), MIP-1beta (40% enteritis, 22% HC, 11% HUS; p < 0.02), MCP-1 (77% enteritis, 20% HC, 18% HUS; p < 0.0001) . The data indicates that GRO-alpha, MIP-1beta, and MCP-1 are produced during E . coli O157:H7 enteritis, whether or not HC or HUS develops . Our data suggest that children with O157:H7 associated HUS may present abnormally increased circulating levels of G-CSF and decreased ENA-78 concentrations . The mechanisms responsible for leukocytes recruitment in O157:H7 infections are unclear and await further studies. Infect Immun, 2002 Dec, 70(12), 6853 - 9 Molecular characterization of a serotype O121:H19 clone, a distinct Shiga toxin-producing clone of pathogenic Escherichia coli; Tarr CL et al.; Most illnesses caused by Shiga toxin-producing Escherichia coli (STEC) have been attributed to E . coli serotype O157:H7, but non-O157 STEC infections are now increasingly recognized as public health problems worldwide . The O121:H19 serotype is being isolated more frequently from clinical specimens and has been implicated in one waterborne outbreak . We used multilocus virulence gene profiling, a PCR-based assay, to characterize the virulence gene content of 24 isolates of serotype O121:H19 and nonmotile variants . We also performed multilocus enzyme electrophoresis and multilocus sequencing to establish the clonal relatedness of O121 isolates and to elucidate the relationship of O121 to common STEC clones . The 24 isolates were found to represent a single bacterial clone, as there was no allelic variation across 18 enzyme loci among the isolates . The complete nucleotide sequence of the intimin gene differed by four substitutions from that of the epsilon (Int- epsilon ) allele of O103:H2 strain PMK5 . The typical O121 virulence gene profile was similar to the profiles of enterohemorrhagic E . coli (EHEC) clones of E . coli: it included a Shiga toxin 2 gene (stx(2)), two genes on the EHEC plasmid (toxB and ehxA), and the gene encoding intimin (eae) . Despite the similarities, putative virulence genes distributed on O islands-large chromosomal DNA segments present in the O157:H7 genome-were useful for discriminating among STEC serotypes and the O121:H19 clone had a composite profile that was distinct from the profiles of the other major EHEC clones of pathogenic E . coli . On the basis of sequencing analysis with 13 housekeeping genes, the O121:H19 clone did not fall into any of the four classical EHEC and enteropathogenic E . coli groups but instead was closely related to two eae-negative STEC strains. Infect Immun, 2002 Dec, 70(12), 6761 - 9 Identification of a novel fimbrial gene cluster related to long polar fimbriae in locus of enterocyte effacement-negative strains of enterohemorrhagic Escherichia coli; Doughty S et al.; Enterohemorrhagic Escherichia coli (EHEC) is a food-borne cause of bloody diarrhea and the hemolytic-uremic syndrome (HUS) in humans . Most strains of EHEC belong to a group of bacterial pathogens that cause distinctive lesions on the host intestine termed attaching-and-effacing (A/E) lesions . A/E strains of EHEC, including the predominant serotype, O157:H7, are responsible for the majority of HUS outbreaks worldwide . However, several serotypes of EHEC are not A/E pathogens because they lack the locus of enterocyte effacement (LEE) pathogenicity island . Nevertheless, such strains have been associated with sporadic cases and small outbreaks of hemorrhagic colitis and HUS . Of these LEE-negative organisms, O113:H21 is one of the most commonly isolated EHEC serotypes in many regions . Clinical isolates of LEE-negative EHEC typically express Shiga toxin 2 and carry an approximately 90-kb plasmid that encodes EHEC hemolysin, but in the absence of LEE, little is known about the way in which these pathogens colonize the host intestine . In this study we describe the identification of a novel fimbrial gene cluster related to long polar fimbriae in EHEC O113:H21 . This chromosomal region comprises four open reading frames, lpfA to lfpD, and has the same location in the EHEC O113:H21 genome as O island 154 in the prototype EHEC O157:H7 strain, EDL933 . In a survey of EHEC of other serotypes, homologues of lpfA(O113) were found in 26 of 28 LEE-negative and 8 of 11 non-O157:H7 LEE-positive EHEC strains . Deletion of the putative major fimbrial subunit gene, lpfA, from EHEC O113:H21 resulted in decreased adherence of this strain to epithelial cells, suggesting that lpf(O113) may function as an adhesin in LEE-negative isolates of EHEC. FEMS Microbiol Lett, 2002 Nov 5, 216(2), 243 - 8 Amino acid alterations in Gp38 of host range mutants of PP01 and evidence for their infection of an ompC null mutant of Escherichia coli O157:H7; Morita M et al.; The previously isolated T-even type coliphage PP01, specifically infective to Escherichia coli O157:H7, uses the outer membrane protein OmpC as a receptor . The characterization of a spontaneous PP01-resistant strain indicated that it had lost ompC due to the deletion of a 14-kbp region upstream of and partially including ompC . Two host range mutants, able to infect an ompC null mutant, were isolated . Sequencing of gene 38, which codes for the receptor recognition protein Gp38, indicated three mutations in one mutant and two in the other . Both mutant proteins had a Gly208Arg, a Gly161Arg or Gly101His replacement, respectively, and the one mutant phage in addition a Trp189Arg replacement . These alterations suggest that the host range was mediated by a more positively charged Gp38. FEMS Microbiol Lett, 2002 Oct 29, 216(1), 117 - 22 Survival of Escherichia coli O157:H7 in private drinking water wells: influences of protozoan grazing and elevated copper concentrations; Artz RR et al.; The survival characteristics of Escherichia coli O157:H7 in private drinking water wells were investigated to assess the potential for human exposure . A non-toxigenic, chromosomally lux-marked strain of E . coli O157:H7 was inoculated into well water from four different sites in the North East of Scotland . These waters differed significantly in their heavy metal contents as well as nutrient and bacterial grazer concentrations . Grazing and other biological factors were studied using filtered (3 and 0.2 microm) and autoclaved water . The survival of E . coli O157:H7 was primarily decreased by elevated copper concentrations . This hypothesis was supported by acute toxicity assay data . In addition, significant protozoan predation effects were observed in untreated water when compared with survival rates in filtered water . The combination of these two factors in particular determines the survival time of the pathogen in a private water well . It therefore appears that wells with higher water quality as assessed using the European Union Drinking Water Directive standards will also allow survival of E . coli O157:H7 for much longer periods. FEMS Microbiol Lett, 2002 Oct 8, 215(2), 229 - 36 Genetic variation in the flanking regions of Shiga toxin 2 gene in Shiga toxin-producing Escherichia coli O157:H7 isolated in Japan; Hamabata T et al.; We found frequent IS1 integration nearby the stx(2) gene during in vitro mutagenesis of an stx(2) variant, stx(2vhd) . To examine the possibility that such insertions have been contributing to generate new stx(2) variants, we screened 86 strains of Escherichia coli O157:H7 isolated in Japan for variations in the ca . 4-kb region flanking the stx(2) locus using PCR methods . Two major classes were identified based on the PCR amplicon size . DNA sequence analysis revealed that the stx(2) subtype of the two classes were stx(2) (referred to as stx(2-EDL933)) and stx(2vhd) . IS1203v insertions were found in three stx(2vhd)-positive strains and two stx(2-EDL933)-positive strains, and no other insertions were found . These results suggest that the DNA sequences surrounding the stx(2) genes are preferably integrated by IS1203v in wild-type Shiga toxin-producing E . coli strains. Clin Infect Dis, 2002 Nov 1, 35(9), e103 - 5 Epub 2002 Oct 10. Colonic necrosis and perforation secondary to Escherichia coli O157:H7 gastroenteritis in an adult patient without hemolytic uremic syndrome; Kravitz GR et al.; During a multistate outbreak of Escherichia coli O157:H7 diarrhea, we encountered a woman who had hemorrhagic colitis complicated by ischemic colitis with perforation . To our knowledge, this has not previously been described in adult patients . Because of the insensitivity of the commonly used diagnostic methods, this condition may be underrecognized. Ren Fail, 2002 Sep, 24(5), 567 - 75 Cytokine expression in the renal tubular epithelial cells stimulated by Shiga toxin 2 of Escherichia coli O157:H7; Lee JE et al.; Hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in children worldwide . Shiga toxin (Stx) associated HUS, the most common type, is now known to be caused by Escherichia coli O157:7, which produces Stxl or the more potent, Stx2 . Since the renal tubule is the major tissue affected in the course of HUS and Stx2 is known to be toxic to the renal tubular cells (RTC), we attempted to elucidate the mechanism of renal injury in HUS by studying the alteration of cytokines in the RTC evoked by Stx2 . cDNA-array is a powerful tool for evaluating changes in the expression of a group of critical genes and also gives insights on the overview of the gene activation . In this study, we purified Stx2 from the E . coli O157:7, which was isolated from a typical diarrhea-associated HUS patient and then tried to compare the cytokine gene expression between the stimulated RTC and un-stimulated RTC using cDNA-array . Our results showed that one third of the examined cytokine genes were up regulated at least twice by the addition of Vtx2 . These up-regulated genes represented the chemokines (macrophage related cytokines), fibrosis-related cytokine (TNF, PDGF) and leukemia inhibitory factors . However, the expression of IL-6, one of the pleiotropic cytokines, was significantly decreased and this finding was confirmed by northern analysis . Our results suggest that VT2 up-regulates the pro-inflammatory cytokines and fibrosis prone growth factors in RTC and that the inhibition of the activation of these cytokines may ameliorate the renal tubular injury in the HUS caused by E . coli O157:7. J Food Prot, 2002 Oct, 65(10), 1641 - 5 Growth and survival of Escherichia coli O157:H7 on fresh-cut apples in modified atmospheres at abusive temperatures; Gunes GG et al.; The effects of reduced-O2 and elevated-CO2 modified atmospheres (MAs) and abusive temperatures on the growth and survival of E . coli O157:H7, yeast, and molds and on changes in the visual quality of fresh-cut apples were evaluated . High-CO1 and low-O2 (> or = 15% and < 1%, respectively) atmospheres inhibited the growth of the pathogen on apple slices at 15 and 20 degrees C . However, the population of the pathogen increased by 1 log cycle after 2 weeks of storage in air . The high-CO2 MA resulted in the inhibition of yeast and mold growth, less browning, and better visual quality than did air and ambient-CO2 atmospheres . The results of this study confirm that E . coli O157:H7 can grow on apple slices in air . These results also show that these organisms survive but are inhibited in MAs with high CO2 levels at abusive temperatures . An MA can increase the shelf life of fresh-cut apples by improving retention of visual quality and inhibiting yeast and molds . Thus, contamination of minimally processed apples with E . coli O157:H7 can be a safety issue for both air- and MA-packaged cut apples. J Food Prot, 2002 Oct, 65(10), 1637 - 40 Frequency of Escherichia coli O157:H7 in Turkish cattle; Yilmaz A et al.; In this study, five abattoirs in Istanbul were visited between January 2000 and April 2001 . During these visits, 330 cattle were selected by a systematic sampling method . Cattle were examined clinically and breed, age, and sex were recorded . Rectal swabs were taken immediately after slaughter . Immunomagnetic separation was performed, and sorbitol-negative colonies were selected on sorbitol MacConkey agar with cefixime and tellurite (CT-SMAC agar) . These colonies were checked for 4-methylenebelliferyl-beta-D-glucuronide, indol, rhamnose, and urease activity and motility . Serotypes of bacteria were determined by using antisera specific for Escherichia coli O157 and H7 . All cattle selected were clinically healthy . Of 88 sorbitol-negative colonies selected on CT-SMAC agar, isolates from only 14 (4.2%) cattle reacted with anti-O157, and 13 of these isolates also reacted with anti-H7 . E . coli O157:H7 was isolated from all breeds, but the numbers of isolates were largest for Holstein and Swiss Brown cows . E . coli O157:H7 was most frequently isolated from 2-year-old cattle . Similarly, it was most frequently isolated from male cattle . E . coli O157:H7 was isolated from cattle slaughtered in four of the five abattoirs studied. Int J Food Microbiol, 2002 Dec 15, 79(3), 183 - 92 The effect of commercial production and product formulation stresses on the heat resistance of Escherichia coli O157:H7 (NCTC 12900) in beef burgers; Byrne CM et al.; The effects of commercial beef burger production and product formulation on the heat resistance of Escherichia coli O157:H7 (NCTC 12900) in beef burgers were investigated . Fresh beef trimmings were inoculated with E . coli O157:H7 to approximately log10 7.0 cfu g(-1) and subjected to standard beef burger production processes, including freezing, frozen storage and tempering . The tempered trimmings were processed in line with commercial practice to produce burgers of two formulations, a 'Quality' burger containing 100% beef and an 'Economy' burger containing 70% beef and 30% other ingredients (salt, seasoning, soya, onion and water) . The burgers were then frozen and stored . Control 'unprocessed' burgers were produced to each of the above formulations using fresh beef trimmings . All burger types were heat-treated at 55, 60 or 65 degrees C . Samples were examined by plating on Tryptone Soya Agar (TSA), incubated at 37 degrees C for 2 h, before overlaying with SMAC (TSA/SMAC) and incubation at 37 degrees C . The resultant counts were used to derive D-values for E . coli O1 57:H7 . At each treatment temperature, the D-values from each burger formulation using frozen tempered trimmings were significantly lower (P < 0.001) than the D-values from that formulation using fresh trimmings . At each treatment temperature, the D-values from Economy burgers using processed trimmings were significantly higher (P < 0.001) than the D-values from Quality burgers using processed trimmings . A similar trend of significantly higher (P<0.001) D-values for Economy burgers was observed using fresh trimmings . This study found that commercial processing and product formulation have profound effects on the heat resistance of E . coli O157:H7 in beef burgers. Cell Microbiol, 2002 Oct, 4(10), 635 - 48 Role of EHEC O157:H7 virulence factors in the activation of intestinal epithelial cell NF-kappaB and MAP kinase pathways and the upregulated expression of interleukin 8; Berin MC et al.; Enterohaemorrhagic Escherichia coli O157:H7 (EHEC) is a gastrointestinal pathogen that is generally non-invasive for intestinal epithelial cells, yet causes acute intestinal inflammation, diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome . To study signal transduction pathways activated in human intestinal epithelial cells by EHEC, we took advantage of EHEC O157:H7 and isogenic mutants deficient in the major EHEC virulence factors, intimin (eae-) and Shiga toxin (stx-) . Infection with wild-type EHEC activated p38 and ERK MAP kinases and the nuclear translocation of the transcription factor NF-kappaB . Downstream, this was accompanied by increased expression of mRNA and protein for the neutrophil chemoattractant IL-8 . Isogenic eae- and stx- mutants also activated p38 and ERK MAP kinases, and NF-kappaB and stimulated increases in IL-8 protein secretion similar to those of wild-type EHEC . Further, inhibition of either p38, ERK or NF-kappaB activation abrogated the IL-8 response induced by wild-type EHEC and the mutants . Epithelial cell MAP kinase and NF-kappaB pathways leading to IL-8 secretion were also activated by isolated EHEC H7 flagellin, which was active when added to either the apical or basolateral surface of polarized human intestinal epithelial cells . We conclude that EHEC interacting with intestinal epithelial cells activates intracellular signalling pathways and an epithelial cell proinflammatory response independent of either Shiga toxin or intimin, two of the major known virulence factors of EHEC . The activation of proinflammatory signals in human colon epithelial cells in response to this non-invasive pathogen appears to depend to a significant extent on H7 flagellin. J Med Microbiol, 2002 Sep, 51(9), 755 - 63 Production of attaching-effacing lesions in ligated large intestine loops of 6-month-old sheep by Escherichia coli O157:1H7; Wales AD et al.; Shiga-toxigenic Escherichia coli O157:H7 (STEC O157:H7) is associated with potentially fatal human disease, and a persistent reservoir of the organism is present in some farm animal species, especially cattle and sheep . The mechanisms of persistent colonisation of the ruminant intestine by STEC O157:H7 are poorly understood but may be associated with intimate adherence to eukaryotic cells . Intimate adherence, as evidenced by induction of attaching-effacing (AE) lesions by STEC O157, has been observed in 6-day-old conventional lambs after deliberate oral infection but not in older animals . Thus, the present study used a ligated intestinal loop technique to investigate whether STEC O157:H7 and other attaching-effacing E . coli may adhere intimately to the sheep large intestinal mucosa . To do this, four STEC O157:H7 strains, one STEC O26:K60:H11 and one Shiga toxin-negative E . coli O157:H7 strain, suspended in either phosphate-buffered saline or Dulbecco's modified Eagle's medium, were inoculated into ligated spiral colon loops of each of two lambs . The loops were removed 6 h after inoculation, fixed and examined by light and electron microscopy . AE lesions on the intestinal mucosa were produced by all the inoculated strains . However, the lesions were sparse and small, typically comprising bacterial cells intimately adhered to a single enterocyte, or a few adjacent enterocytes . There was little correlation between the extent of intimate adherence in this model and the bacterial cell density, pre-inoculation growth conditions of the bacteria or the strain tested. J Clin Microbiol, 2002 Oct, 40(10), 3613 - 9 Detection in Escherichia coli of the genes encoding the major virulence factors, the genes defining the O157:H7 serotype, and components of the type 2 Shiga toxin family by multiplex PCR; Wang G et al.; Strains of Shiga toxin-producing Escherichia coli (STEC) have been associated with outbreaks of diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans . Most clinical signs of disease arise as a consequence of the production of Shiga toxin 1 (Stx1), Stx2 or combinations of these toxins . Other major virulence factors include enterohemorrhagic E . coli hemolysin (EHEC hlyA), and intimin, the product of the eaeA gene that is involved in the attaching and effacing adherence phenotype . In this study, a series of multiplex-PCR assays were developed to detect the eight most-important E . coli genes associated with virulence, two that define the serotype and therefore the identity of the organism, and a built-in gene detection control . Those genes detected were stx(1), stx(2), stx(2c), stx(2d), stx(2e), stx(2f), EHEC hlyA, and eaeA, as well as rfbE, which encodes the E . coli O157 serotype; fliC, which encodes the E . coli flagellum H7 serotype; and the E . coli 16S rRNA, which was included as an internal control . A total of 129 E . coli strains, including 81 that were O157:H7, 10 that were O157:non-H7, and 38 that were non-O157 isolates, were investigated . Among the 129 samples, 101 (78.3%) were stx positive, while 28 (21.7%) were lacked stx . Of these 129 isolates, 92 (71.3%) were EHEC hlyA positive and 96 (74.4%) were eaeA positive . All STEC strains were identified by this procedure . In addition, all Stx2 subtypes, which had been initially identified by PCR-restriction fragment length polymorphism, were identified by this method . A particular strength of the assay was the identification of these 11 genes without the need to use restriction enzyme digestion . The proposed method is a simple, reliable, and rapid procedure that can detect the major virulence factors of E . coli while differentiating O157:H7 from non-O157 isolates. Anal Chem, 2002 Sep 15, 74(18), 4814 - 20 Immunobiosensor chips for detection of Escherichia coil O157:H7 using electrochemical impedance spectroscopy; Ruan C et al.; Impedance biosensor chips were developed for detection of Escherichia coli O157:H7 based on the surface immobilization of affinity-purified antibodies onto indium tin oxide (ITO) electrode chips . The immobilization of antibodies onto ITO chips was carried out using an epoxysilane monolayer to serve as a template for chemical anchoring of antibodies . The surface characteristics of chips before and after the binding reaction between the antibodies and antigens were characterized by atomic force microscopy (AFM) . The patterns of the epoxysilanes monolayer, antibodies, and E . coli cells were clearly observed from the AFM images . Alkaline phosphatase as the labeled enzyme to anti-E . coli O157:H7 antibody was used to amplify the binding reaction of antibody-antigen on the chips . The biocatalyzed precipitation of 5-bromo-4-chloro-3-indolyl phosphate by alkaline phosphatase on the chips in pH 10 PBS buffer containing 0.1 M MgCl2 increased the electron-transfer resistance for a redox probe of Fe(CN)6(3-/4-) at the electrode-solution interface or the electrode resistance itself . Electrochemical impedance spectroscopy and cyclic voltammetric method were employed to follow the stepwise assembly of the systems and the electronic transduction for the detection of E . coli . The biosensor could detect the target bacteria with a detection limit of 6 x 10(3) cells/mL . A linear response in the electron-transfer resistance for the concentration of E . coli cells was found between 6 x 10(4) and 6 x 10(7) cells/mL. Arch Dis Child, 2002 Oct, 87(4), 335 - 6 Daily bowel movements and Escherichia coli O157 infection; Kitajima H et al.; The usual bowel habits of 259 school age children before the large outbreak of E coli O157 infection in Sakai City in the summer of 1996 were investigated . A daily morning movement and/or frequent bowel movements may be a protective factor against infection, while being constipated contributed to a worsening of symptoms. Gene, 2002 Jul 10, 294(1-2), 25 - 33 A systematic investigation identifies a significant number of probable pseudogenes in the Escherichia coli genome; Homma K et al.; Pseudogenes are open reading frames (ORFs) encoding dysfunctional proteins with high homology to known protein-coding genes . Although pseudogenes were reported to exist in the genomes of many eukaryotes and bacteria, no systematic search for pseudogenes in the Escherichia coli genome has been carried out . Genome comparisons of E . coli strains K-12 and O157 revealed that many protein-coding sequences have prematurely terminated orthologs encoding unstable proteins . To systematically screen for pseudogenes, we selected ORFs generated by premature termination of the orthologous protein-coding genes and subsequently excluded those possibly arising from sequence errors . Lastly we eliminated those with close homologs in this and other species, as these shortened ORFs may actually have functions . The process produced 95 and 101 pseudogene candidates in K-12 and O157, respectively . The assigned three-dimensional structures suggest that most of the encoded proteins cannot fold properly and thus are dysfunctional, indicating that they are probably pseudogenes . Therefore, the existence of a significant number of probable pseudogenes in E . coli is predicted, awaiting experimental verification . Most of them were found to be genes with paralogs or horizontally transferred genes or both . We suggest that pseudogenes constitute a small fraction of the genomes of free-living bacteria in general, reflecting the faster elimination than production of pseudogenes. J Food Prot, 2002 Sep, 65(9), 1371 - 80 Detection and quantitation of enterohemorrhagic Escherichia coli O157, O111, and O26 in beef and bovine feces by real-time polymerase chain reaction; Sharma VK; Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and certain non-O157 EHEC serotypes (such as O26:H11, O26: NM, O11:H8, and O111:NM) have emerged as significant causes of human disease throughout the world . Important virulence attributes of EHEC are the intimin protein (encoded by the eae gene) and Shiga toxins 1 and 2 (encoded by the stx1 and stx2 genes, respectively) . Two sets of real-time polymerase chain reaction (R-PCR) assays were developed for the simultaneous detection and quantitation of EHEC through the monitoring of the presence of the eae and stx genes, and these assays were evaluated . In the eaeR-PCR assay, three sets of primers and TaqMan probes were designed for the amplification and real-time detection of a portion of the eae gene specific to the EHEC O26, O111, and O157 serotypes . In the stxR-PCR assay, two sets of primers and TaqMan probes were used to amplify and detect the stx1 and stx2 genes . DNA prepared from 67 bacterial strains carrying known virulence markers was tested to determine the specificities of the two assays . In the eaeR-PCR assay, eaeO157- and eaeO111-specific primer-probe sets identified only EHEC O157 and O111 strains, respectively . The eaeO26-specific primer-probe set identified all EHEC 026 isolates and some Shiga toxin-negative serotypes of enteropathogenic E . coli and rabbit diarrheagenic E . coli . The stxR-PCR assay was able to identify only those strains carrying either or both of the Shiga toxin-encoding genes . The detection range of both R-PCR assays was linear over DNA concentrations corresponding to 10(3) to 10(8) CFU/ml of an EHEC strain . Both assays were able to detect and quantify very low levels (1 to 10 CFU/g of food or feces) of EHEC in feces and ground beef enriched for 16 h in a modified Trypticase soy broth . In conclusion, eae- and stx-based R-PCR assays are reliable and sensitive methods for the rapid screening and specific and quantitative detection of important serotypes of EHEC in cattle and in foods of bovine origin. Infect Immun, 2002 Oct, 70(10), 5370 - 80 Essential role for verotoxin in sustained stress-activated protein kinase and nuclear factor kappa B signaling, stimulated by Escherichia coli O157:H7 in Vero cells; Cameron P et al.; The effects of Escherichia coli O157:H7 (strains E30480 and PM601) and the associated verotoxins (VTs), VT1 and VT2, on stress-activated protein kinase and nuclear factor kappa B (NF-kappaB) signaling were investigated with Vero cells, which are extremely sensitive to the cytotoxic effects of E . coli O157:H7 in vitro . Cell-free supernatants prepared from E30480 and PM601 cultures and purified VT1 and VT2 stimulated a strong and prolonged (>4-h) activation of both c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase . However, JNK activity stimulated in response to E30480 supernatants was substantially reduced following pretreatment with anti-VT1 and anti-VT2 antibodies, while a VT1 and VT2 gene knockout mutant of PM601 was unable to stimulate JNK activity . E30480 supernatants also caused a sustained activation of NF-kappaB DNA binding, degradation of inhibitory kappa B alpha (IkappaBalpha), and an increase in inhibitory kappa B kinase alpha activity, although PM601 supernatants and VT1 and VT2 had no effect . However, preincubation with VTs prolonged the transient activation of NF-kappaB and IkappaBalpha degradation stimulated by either tumor necrosis factor alpha or interleukin 1beta, while preincubation with anti-VT antibodies prevented the prolonged loss of IkappaBalpha and partially reduced DNA binding in response to E30480 supernatants . These results strongly suggest that in Vero cells, VT plays an essential role in sustained JNK and NF-kappaB signaling in response to E . coli O157:H7 and that this action may underpin their cell-selective cytotoxic effects . These studies also suggest that another component released by strain E30480 contributes to the early activation of JNK and NF-kappaB. Vet Microbiol, 2002 Oct 2, 89(1), 69 - 81 Persistence of Escherichia coli O157:H7 in experimentally infected swine; Booher SL et al.; These experiments determined the ability of Escherichia coli O157:H7 to colonize and persist in pigs simultaneously inoculated with other pathogenic E . coli strains . Three-months-old pigs were inoculated with a mixture of five E . coli strains . The mixture included two Shiga toxigenic E . coli (STEC) O157:H7 strains, two enterotoxigenic E . coli (ETEC) strains and one enteropathogenic E . coli (EPEC) strain . A high dose mixture with all five strains at 10(10)CFU/animal (CFU: colony forming units) and a low dose mixture with the STEC strains at 10(7)CFU and the EPEC and ETEC strains remaining at 10(10)CFU were used . The STEC strains persisted in the alimentary tracts of some pigs at 2 months post-inoculation, following inoculation with both the high and low dose mixtures . When all strains were given at 10(10)CFU (high dose) the STEC strains persisted in greater numbers and in more pigs than did the other E . coli strains . The results demonstrated that persistent colonization (> or =2 months) by E . coli O157:H7 can occur in pigs . These findings were similar to those reported from sheep inoculated with the same mixture of E . coli strains . The results are consistent with reports suggesting that pigs have the potential to be reservoir hosts for STEC O157:H7. Epidemiol Infect, 2002 Aug, 129(1), 173 - 85 A long-term study on the prevalence of shiga toxin-producing Escherichia coli (STEC) on four German cattle farms; Geue L et al.; The occurrence of Shiga toxin-producing Escherichia coli (STEC) was studied on four cattle farms . STEC were detected in 29-82% of the cattle . STEC with additional EHEC markers were detected on all farms . The occurrence of the complete virulence marker pattern (stx1 and/or stx2, eae, EHEC(hlyA), katP, espP) was correlated with the presence of known STEC serotypes . STEC O26:H11 and O165:H25 with the complete pattern of virulence markers were the most prevalent . STEC O157 (H7/H-) STEC O103:H2 and STEC O145:H- were found sporadically . Five clonal subgroups of the STEC O26:H11 isolates were identified by pulsed-field gel electrophoresis . STEC O26:H11 were present in three groups of cattle . This serotype was detected in a single group over the entire fattening period . Most STEC O26:H11 with the complete pattern of potential virulence markers were found in clinically healthy cattle . These animals may represent a risk factor for farmers and consumers. Vet Res, 2002 Jul-Aug, 33(4), 405 - 12 Effect of diet on Shiga toxin-producing Escherichia coli (STEC) growth and survival in rumen and abomasum fluids; Boukhors K et al.; 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. J Infect Dis, 2002 Aug 15, 186(4), 566 - 9 Epub 2002 Aug 02. Escherichia coli O157 fails to induce a long-lasting lipopolysaccharide-specific, measurable humoral immune response in children with hemolytic-uremic syndrome; Ludwig K et al.; Escherichia coli O157 lipopolysaccharide (LPS)-specific antibodies were measured in sequential serum samples from 131 children with serologically defined E . coli O157-associated hemolytic-uremic syndrome (HUS), using an enzyme immunoassay . On the basis of evaluation of 66 children with culture-proven E . coli O157 infection and serum samples from 132 age-matched control subjects, the assay showed a sensitivity of 95%, 88%, and 74% and a specificity of 99%, 99%, and 98% for IgM, IgA, and IgG, respectively . Anti-O157 LPS antibodies decreased below the cut-off levels in >50% of the children at 11 (IgM), 5 (IgA), and 11 weeks (IgG) after onset of diarrhea and 10, 4, and 10 weeks, respectively, after the onset of HUS . Children with enteropathic HUS fail to develop a long-lasting humoral immune response to the O157 antigen . Incomplete immunity to E . coli O157 may signal a risk for recurrent infections and has implications for serodiagnostic studies. N Engl J Med, 2002 Aug 22, 347(8), 555 - 60 An outbreak of Escherichia coli O157:H7 infections among visitors to a dairy farm; Crump JA et al.; BACKGROUND: Outbreaks of Escherichia coli O157:H7 infections have involved direct transmission from animals and their environment to humans . We describe an outbreak among visitors to a Pennsylvania dairy and petting farm that provides public access to animals . METHODS: We conducted both a case-control study among visitors to a farm to identify risk factors for infection and a household survey to determine the rates of diarrheal illness among these visitors . We performed an extensive environmental study to identify sources of E . coli O157:H7 on the farm . RESULTS: Fifty-one patients with confirmed or suspected E . coli O157:H7 infection were enrolled in the case-control study . The median age of the patients was four years, and the hemolytic-uremic syndrome developed in eight . Contact with calves and their environment was associated with an increased risk of infection, whereas hand washing was protective . The household survey indicated that visitors to the farm during the outbreak had higher than expected rates of diarrhea . Environmental studies showed that 28 of the 216 cattle on the farm (13 percent) were colonized with E . coli O157:H7 that had the same distinct pattern on pulsed-field gel electrophoresis that was found in isolates from the patients . This organism was also recovered from surfaces that were accessible to the public . CONCLUSIONS: In a large outbreak of E . coli O157:H7 infections among visitors to a dairy farm, predominantly children, high rates of carriage of E . coli O157:H7 among calves and young cattle most likely resulted in contamination of both the animals' hides and the environment . MMWR Morb Mortal Wkly Rep, 2002 Jul 26, 51(29), 637 - 9 Multistate outbreak of Escherichia coli O157:H7 infections associated with eating ground beef--United States, June-July 2002; Efa1 influences colonization of the bovine intestine by shiga toxin-producing Escherichia coli serotypes O5 and O111; Division of Environmental Microbiology, Institute for Animal Health, Compton Laboratory, Berkshire RG20 7NN, United KingdomShiga toxin-producing Escherichia coli (STEC) comprises a broad group of bacteria, some of which cause attaching and effacing (AE) lesions and enteritis in animals and humans . Non-O157 STEC serotypes contain a gene (efa1) that mediates attachment to cultured epithelial cells . An almost-identical gene in enteropathogenic E . coli (lifA) encodes lymphostatin, which inhibits the proliferation of mitogen-activated lymphocytes and the synthesis of proinflammatory cytokines . We have investigated the role of the efa1 gene in colonization of 4- and 11-day-old conventional calves by STEC serotypes O5 and O111 . Our findings show that Efa1 is required for efficient colonization of the bovine intestinal tract by STEC, since efa1 deletion and insertion mutants were shed in the feces in significantly lower numbers . In addition, efa1 mutations dramatically reduced the number of bacteria associated with the intestinal epithelium . Expression and secretion of locus for enterocyte effacement-encoded type III secreted proteins that are required for adhesion and AE-lesion formation were impaired by mutation of efa1 in STEC but not by mutation of lifA in enteropathogenic E . coli . However, STEC efa1 mutants retain the ability to nucleate filamentous actin under sites of bacterial attachment to cultured eukaryotic cells . Efa1 is only the second STEC factor shown to influence carriage of the bacteria in the bovine intestine . Our data may have implications for strategies to reduce the prevalence of STEC in cattle. Lett Appl Microbiol, 2002, 35(3), 233 - 6 Survival of Escherichia coli O157 in abattoir waste products; Hepburn NF et al.; AIMS: This study monitored survival and growth of Escherichia coli O157 in ovine and bovine abattoir waste . METHODS: Blood and gut contents were inoculated separately with cocktails of E . coli O157 . Samples were stored aerophilically and microaerophilically at 5 degrees C, 15 degrees C and 30 degrees C to represent storage at different container depths and at extremes of UK ambient temperature . CONCLUSIONS: Results showed survival of E . coli O157 was irrespective of oxygen content with no significant differences observed between aerophilic and microaerophilic environments . Numbers of E . coli O157 in ovine and bovine gut contents showed no change when stored at 5 degrees C and increased 1-2 log(10) at 15 degrees C and 30 degrees C in 28 h . In ovine and bovine blood, irrespective of storage temperature, there was a 0.5-2 log(10) reduction or no change in numbers except in ovine blood stored at 30 degrees C where the fall in numbers was followed by a 3 log(10) increase . In aged (stored at 4 degrees C for 18 h before spiking) bovine blood there was no significant change in numbers at 5 degrees C while at 15 degrees C there was 2 log(10) rise after 48 h . At 30 degrees C there was an initial 1 log(10) decrease in numbers followed by a 1 log(10) rise over the following 40 h . SIGNIFICANCE AND IMPACT OF STUDY: Abattoir wastes may become contaminated from animals infected with Verocytotoxigenic E . coli O157 and in certain storage conditions these pathogens could significantly increase in numbers . There is need for care in abattoir waste disposal, not only for personnel subject to direct contact, but also in the prevention of cross contamination to adjacent land and water courses which could indirectly infect humans. Hybrid Hybridomics, 2002 Jun, 21(3), 161 - 8 Development of humanized monoclonal antibody TMA-15 which neutralizes Shiga toxin 2; Kimura T et al.; A murine monoclonal antibody (MAb), VTm1.1, specifically recognizing and neutralizing Shiga toxin 2 (Stx2), was obtained . To prevent a humoral response against murine antibody when used clinically, a humanized antibody was constructed by combining the complementarity-determining regions of VTm1.1 with human framework and constant regions . In addition, several amino acids in the framework were changed to improve the binding affinity of the antibody and further reduce its potential immunogenicity . The humanized antibody, TMA-15, recognized the B-subunit of Stx2 and had affinity for Stx2 of 3.3 x 10(-9) M, within two-fold of that of the original murine antibody . TMA-15 neutralized the cytotoxicity of Stx2 and several different Stx2 variants in vitro, and it completely protected mice from death in a Stx2-challenged mice model . These results suggest that TMA-15 will have clinical potency in Stx-producing Escherichia coli infections, including E . coli O157 infections. Int J Food Microbiol, 2002 Aug 25, 77(3), 213 - 21 Applicability of the draft standard method for the detection of Escherichia coli O157 in dairy products; Voitoux E et al.; According to the draft standard method, EN ISO 16654 for the detection of Escherichia coli O157 in foods, samples of milk products inoculated with E . coli O157 were cultured in modified Tryptone Soya Broth supplemented with novobiocin . After immuno-magnetic separation (IMS) of the micro-organisms with magnetic beads coated with an antibody against E . coli O157 (Dynabeads anti E . coli O157, Dynal), the enrichment broth was subcultured onto both Cefixim Tellurite Sorbitol MacConkey agar and CHROMagar O157 . IMS alone appeared not to be very specific to E . coli O157; however, IMS combined with CT-SMAC plating enabled a greater exclusivity . The method displayed a low limit of detection (1-2 cfu/25 g) of E . coli O157 strains in milk products after only 6 h of incubation of the enrichment broth . However, the detection was affected by storage of the inoculum at 4 degrees C, and another 12 to 18 h of incubation was necessary to recover potentially stressed cells. Pediatr Res, 2002 Aug, 52(2), 307 - 13 Saliva IgM and IgA are a sensitive indicator of the humoral immune response to Escherichia coli O157 lipopolysaccharide in children with enteropathic hemolytic uremic syndrome; Ludwig K et al.; Saliva antibodies to Escherichia coli O157 were investigated as markers of the immune response in children with enteropathic hemolytic uremic syndrome (HUS) . Paired serum and saliva samples were collected from 22 children with HUS during acute disease and convalescence and were tested for E . coli O157 lipopolysaccharide (LPS)-specific IgM and IgA antibodies by ELISA . Serum and saliva samples from 44 age-matched controls were used to establish the cut-off values . Elevated levels of IgM and/or IgA antibodies to O157 LPS were detected in saliva of 13/13 HUS patients with Shiga toxin-producing E . coli (STEC) O157 in stool culture and from 4 of 5 HUS patients in whom STEC were not detected . These results closely mirrored the results obtained with paired serum samples . In contrast, saliva and serum samples from four children with STEC isolates belonging to O-groups O26, O145 (n = 2), and O165 lacked detectable O157 LPS-specific antibodies . The specificity of the ELISA was confirmed by western blotting . In STEC O157 culture-confirmed cases, the sensitivity of the ELISA was 92% for saliva IgM and IgA, based on the first available sample, and 100% and 92%, respectively, when subsequent samples were included . The specificity was 98% for IgM and 100% for IgA . Children with E . coli O157 HUS demonstrate a brisk, easily detectable immune response as reflected by the presence of specific antibodies in their saliva . Saliva-based immunoassays offer a reliable, noninvasive method for the diagnosis of E . coli O157 infection in patients with enteropathic HUS. J Clin Microbiol, 2002 Aug, 40(8), 3079 - 81 Effects of repeated subculturing and prolonged storage at room temperature of enterohemorrhagic Escherichia coli O157:H7 on pulsed-field gel electrophoresis profiles; Iguchi A et al.; Three clinical strains of enterohemorrhagic Escherichia coli O157:H7 which were subcultured repeatedly or stored at room temperature over a 25-week period showed appreciable variations in their pulsed-field gel electrophoresis fragment patterns . The variations could be explained by a couple of spontaneous genetic events at most and thus did not invalidate the genetic lineage of the strains. J Clin Microbiol, 2002 Aug, 40(8), 2806 - 12 Combined use of two genetic fingerprinting methods, pulsed-field gel electrophoresis and ribotyping, for characterization of Escherichia coli O157 isolates from food animals, retail meats, and cases of human disease; Avery SM et al.; Two genetic fingerprinting techniques, pulsed-field gel electrophoresis (PFGE) and ribotyping, were used to characterize 207 Escherichia coli O157 isolates from food animals, foods of animal origin, and cases of human disease (206 of the isolates were from the United Kingdom) . In addition, 164 of these isolates were also phage typed . The isolates were divided into two general groups: (i) unrelated isolates not known to be epidemiologically linked (n = 154) and originating from food animals, foods and the environment, or humans and (ii) epidemiologically related isolates (n = 53) comprised of four related groups (RGs) originating either from one farm plus the abattoir where cattle from that farm were slaughtered or from one of three different English abattoirs . PFGE was conducted with the restriction endonuclease XbaI, while for ribotyping, two restriction endonucleases (PstI and SphI) were combined to digest genomic DNAs simultaneously . The 207 E . coli O157 isolates produced 97 PFGE profiles and 51 ribotypes . The two genetic fingerprinting methods had similar powers to discriminate the 154 epidemiologically unrelated E . coli O157 isolates in the study (Simpson's index of diversity {D} = 0.98 and 0.94 for PFGE typing and ribotyping, respectively) . There was no correlation between the source of an isolate (healthy meat or milk animals, retail meats, or cases of human infection) and either particular PFGE or ribotype profiles or clusters . Combination of the results of both genetic fingerprinting methods produced 146 types, significantly more than when either of the two methods was used individually . Consequently, the superior discriminatory performance of the PFGE-ribotyping combination was proven in two ways: (i) by demonstrating that the majority of the E . coli O157 isolates with unrelated histories were indeed distinguishable types and (ii) by identifying some clonal groups among two of the four RGs of E . coli O157 isolates (comprising PFGE types different by just one or two bands), the relatedness of which would have remained unconfirmed otherwise. J Appl Microbiol, 2002, 93(2), 250 - 60 A PCR test for detecting Escherichia coli O157:H7 based on the identification of the small inserted locus (SILO157); Perelle S et al.; AIMS: This paper provides identification of a DNA sequence derived from Shiga toxin-producing Escherichia coli (STEC) O157:H7 and information on its utilization for detecting STEC O157 by PCR . METHODS AND RESULTS: Random Amplified Polymorphic DNA and DNA library were used to identify in STEC O157:H7 (strain EDL 933) a 2634-bp Small Inserted Locus, designated SILO157 . Analysis of 211 bacterial strains showed that the PCR assays amplifying the SILO157 region could be used to detect STEC O157 with a good specificity . CONCLUSIONS: Characterization of a novel locus in STEC O157 is attractive since the serotype O157:H7 of STEC is still by far the most important serotype associated with more serious diseases . This island encodes putative proteins and especially one that is predicted to be an outer membrane protein designated IHP1 . SIGNIFICANCE AND IMPACT OF THE STUDY: Further investigations could now be developed to appreciate the role of the SILO157 in pathogenicity. Nephrol Nurs J, 2001 Oct, 28(5), 547 - 50, 553-5; quiz 556-7 Escherichia coli O157:H7: etiology, clinical features, complications, and treatment; Peacock E et al.; In recent years, Escherichia coli (E . coli) O157:H7 has developed into an emerging cause of foodborne illness . It has been identified as the leading cause of post-diarrheal hemolytic-uremic syndrome (HUS) and acute renal failure in infancy and childhood . This article examines, the etiology, clinical features, complications, and treatment of this illness . Prevention strategies are also presented as well as a disaster management case study. Comp Immunol Microbiol Infect Dis, 2002 Jul, 25(4), 249 - 68 Escherichia coli 'O' group serological responses and clinical correlations in epidemic HUS patients; Kulkarni H et al.; This first comprehensive serological analysis of an haemolytic uraemic syndrome (HUS) outbreak in which a wide range of 'O' group Escherichia coli antibody responses in patients and controls provided a unique insight into the epidemiology of such epidemics . Possible answers to clinical aspects related to severity of disease and complications were revealed . A microagglutination assay was used to examine E . coli 'O' group serological responses in 49 serum samples of 21 children hospitalised with HUS and 14 single samples from contemporaneous age-matched controls . A total of 51 O serogroup strains were used, including those reported to be associated with cases of HUS, with six isolates from patients associated with the Adelaide outbreak, environmental verocytotoxi-genic/shiga-toxin producing E . coli (VTEC/STEC) strains and common human commensal strains . Amongst the 21 patients, there were 226 instances of seroreactivity (titre > or = 100) against 34 E . coli serogroups while six instances of seroreactivity against four serogroups occurred in controls . There were 128 instances in patients and one instance in controls in which titres > or = 400 were observed . All 21 patients were seroreactive (titre > or = 100 and <400) to one or more of the 17 HUS-associated serogroups included in the study . Titres ranged from 100 to 6,400, some of the highest in three patients were against O157, whose faeces yielded only EHEC O111, and only one developed O111 antibody . Mixed infection was demonstrated serologically by microagglutination (confirmed by western blot) and was consistent with the multiple serogroups of VTEC found in the mettwurst incriminated as the source, and suggests further strains (not found in the source or in patients' faeces) were probably involved . In HUS-associated EHEC infection, multiple strain infection may be the rule rather than the exception . Analysis of 34 of the 51 serogroup antibody responses in the HUS patients revealed clues to possible relationships with clinical severity and complications . Patients with severe renal failure tended to develop antibodies to a larger number of serogroups than those with moderate or mild impairment . The same was true for central nervous system complications . Other associations were observed . While VTEC O157 remains an important causal serogroup in HUS, non-O157 serogroups also appear to play a significant role and therefore the latter should always be sought in all future HUS cases as new insights into pathogenicity may be discovered . This study indicates that co-infection with different VTEC serogroups may affect clinical outcome. J Infect Dis, 2002 Aug 1, 186(3), 419 - 22 Epub 2002 Jul 11. A multistate outbreak of Shiga toxin-producing Escherichia coli O26:H11 infections in Germany, detected by molecular subtyping surveillance; Werber D et al.; In the spring of 2000, a cluster of indistinguishable Shiga toxin-producing Escherichia coli (STEC) O26:H11 was identified in Germany by molecular subtyping surveillance . An investigation was prompted to identify a common source of exposure . A case subject was defined as a person having a polymerase chain reaction-confirmed STEC O26 infection between March and April 2000, irrespective of clinical signs, and whose isolate was indistinguishable from the index strain by use of pulsed-field gel electrophoresis . Eleven case subjects were found in 5 institutions that were supplied by 4 kitchens located in 3 states . The median age was 2 years (range, 2-31 years) . No bloody diarrhea was reported, and 5 persons remained asymptomatic . Comparison of invoices revealed a certain type of beef ("Seemerrolle") as possible source of infection . This is, to our knowledge, the first multistate outbreak associated with a non-O157 STEC detected by laboratory-based surveillance . Molecular subtyping was pivotal, as disease occurrence was sporadic or family-related. Chin Med J (Engl), 2002 Jun, 115(6), 900 - 3 Prolongation of functional life-span of neutrophils by recombinant verotoxin 2; Liu J et al.; OBJECTIVE: Verotoxin-producing Escherichia coli (VTEC) strains of serotype O157 : H7 have been implicated in a wide spectrum of diseases, including blood diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS) . To further explore the pathological role of verotoxin (VT) in HUS and other VTEC associated diseases, we investigated the effects of recombinant verotoxin 2 (rVT2) on the biological activity of neutrophils . METHODS: The technique of flow cytometry, a fluorescent probe 2,7-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF/AM), and the assay of reduced cytochrome c to detect superoxide production were used in this study . RESULTS: gammaVT2 significantly inhibited spontaneous apoptosis in neutrophils . Neutrophils with prolonged survival due to gammaVT2 maintained various biological functions, such as the expression of adhesion molecules (shading CD62L and raising CD11b/CD18), adherence to human umbilical vein endothelial cells (HUVECs), and generation of superoxide (O(2)(-)) . CONCLUSION: Prolongation of the functional life-span of neutrophils by gammaVT2 may accelerate inflammatory responses at sites of inflammation . This may play a crucial role in neutrophil-mediated tissue injury in HUS and other VTEC-associated diseases. Mol Microbiol, 2002 Jul, 45(2), 277 - 88 StcE, a metalloprotease secreted by Escherichia coli O157:H7, specifically cleaves C1 esterase inhibitor; Lathem WW et al.; Escherichia coli O157:H7 causes diarrhoea, haemorrhagic colitis, and the haemolytic uraemic syndrome . We have identified a protein of previously unknown function encoded on the pO157 virulence plasmid of E . coli O157:H7, which is the first described protease that specifically cleaves C1 esterase inhibitor (C1-INH), a member of the serine protease inhibitor family . The protein, named StcE for secreted protease of C1 esterase inhibitor from EHEC (formerly Tagn), cleaves C1-INH to produce (unique) approximately 60-65 kDa fragments . StcE does not digest other serine protease inhibitors, extracellular matrix proteins or universal protease targets . We also observed that StcE causes the aggregation of cultured human T cells but not macrophage-like cells or B cells . Substitution of aspartic acid for glutamic acid at StcE position 435 within the consensus metalloprotease active site ablates its abilities to digest C1-INH and to aggregate T cells . StcE is secreted by the etp type II secretion pathway encoded on pO157, and extracellular StcE levels are positively regulated by the LEE-encoded regulator, Ler . StcE antigen and activity were detected in the faeces of a child with an E . coli O157:H7 infection, demonstrating the expression of StcE during human disease . Cleavage of C1-INH by StcE could plausibly cause localized pro-inflammatory and coagulation responses resulting in tissue damage, intestinal oedema and thrombotic abnormalities. Infect Immun, 2002 Aug, 70(8), 4362 - 8 Tissue tropism of enteropathogenic Escherichia coli strains belonging to the O55 serogroup; Fitzhenry RJ et al.; Four enteropathogenic Escherichia coli (EPEC) strains belonging to the O55 serogroup (G21 and G30 {both O55:H6}, G35 {O55:H-}, and G58 {O55:H7}) were tested for their tissue tropism by using human intestinal in vitro organ culture . Strains showed restricted adhesion with attaching-and-effacing activity to follicle-associated epithelium of Peyer's patches, with no apparent adhesion to duodenum or colon . G35 and G58 express intimin gamma and show a similar tropism to intimin gamma-expressing enterohemorrhagic E . coli (EHEC) O157:H7 . However, strains G21 and G30 were unusual because they expressed intimin alpha and had a restricted tissue tropism of intimin gamma phenotype . The amino acid sequence of the carboxy-terminal 280 amino acids of intimin from G21 was determined . Comparison with the prototype intimin alpha from strain E2348/69 (O127:H6) showed a single amino acid difference (corresponding to Val907 and Ala907 in the whole intimins) . This mutation was reproduced by site-directed mutagenesis in an intimin alpha plasmid template, pCVD438, with the hypothesis that it may induce a change in tropism . However, when the mutated plasmid was placed in both EPEC and EHEC backgrounds, duodenal adhesion in a manner similar to strain E2348/69 was evident upon in vitro organ culture . Thus, additional factor(s) unrelated to intimin exist in the O55:H6 genome that influence human intestinal tissue tropism. J Food Prot, 2002 Jul, 65(7), 1172 - 6 Pulsed-field gel electrophoresis characterization of Shiga toxin-producing Escherichia coli O157 from hides of cattle at slaughter; Avery SM et al.; Contamination of the brisket areas of the hides of healthy adult cattle with Shiga toxin-producing Escherichia coli O157 at slaughter in England was studied . In total, 73 cattle consignments comprising 584 animals delivered to one abattoir over 3 days during 1 week in July 2001 were studied: 26 cattle consignments arriving on Monday, 32 consignments arriving on Wednesday, and 15 consignments arriving on Friday . Consignment sizes ranged from 1 to 23 animals, with a mean consignment size of 8 . The hide of the first animal to be slaughtered in each consignment was sampled by using a sponge swab moistened with 0.85% saline to rub an unmeasured brisket (ventral) area (ca . 30 by 30 cm) . The process of isolating E . coli O157 from the swabs consisted of enrichment, screening with immunoprecipitation assay kits, and immunomagnetic separation . E . coli O157 was found on 24 of 73 (32.9%) cattle hides examined, and 21 of these 24 isolates produced Shiga toxins . The 24 E . coli O157 isolates produced six different pulsed-field gel electrophoresis profiles, and 18 (75%) of the isolates were of one prevalent clone . The high prevalence of one E . coli O157 clone on the hides of cattle at slaughter could be due to a high prevalence of that clone on the 18 farms involved (not investigated in the current study), in the postfarm transport or lairage environments, or both . Since the lairage environment, but not the farm of origin or the postfarm transport vehicle, was a factor common to all 18 cattle consignments, it could have played an important role in spreading the prevalent E . coli O157 clone to the cattle hides . Lairage pen floors and the stunning box floor were identified as the most probable sites along the unloading-to-slaughter route at which the brisket areas of cattle hides could become contaminated. J Food Prot, 2002 Jul, 65(7), 1106 - 9 Fate of field-isolated Escherichia coli O157 in ground beef at different storage temperatures; Barkocy-Gallagher GA et al.; The survival of six Escherichia coli O157 strains, including five strains recently isolated from beef carcasses and strain ATCC 43895, was evaluated at 0, 1, 7, and 14 days in ground beef held at -20, 1, 4, and 7 degrees C . Only small losses in cell numbers occurred at -20 and 1 degree C; in general, cell numbers decreased during the first day of storage and then remained unchanged through day 14 . At -20 degrees C, statistically significant reductions in cell numbers were observed only for strains 55AC1 and 299AB3 due to greater losses in the first day . At 1 degree C, strain 131AC1 did not decrease in cell numbers during the first day of storage, but both this strain and strain 55AC1 experienced statistically significant reductions in viable cell numbers by day 14, primarily due to losses after day 7 . At 4 degrees C, after an initial loss of cell numbers for four strains, minor increases were observed for all six strains by day 14 . The differences were statistically significant for strains 114AC1, 299AB3, and ATCC 43895, but were small enough to question whether they refect actual growth . When the inoculated ground beef was stored at 7 degrees C for 14 days, growth of all six strains was statistically significant, with populations increasing between 0.9 and 1.5 log10 CFU/g . This study demonstrates that there are small differences in the abilities of various E . coli O157 strains to survive and sometimes grow in fresh ground beef at cold storage temperatures, but overall these differences do not appear to be meaningful . The differences cannot be attributed to recency of isolation, since strain ATCC 43895 behaved similarly to recently isolated strains . Storage temperatures of 4 degrees C or below limited growth of E . coli O157 isolates, but did not have a noteworthy effect on survival. J Food Prot, 2002 Jul, 65(7), 1075 - 80 Survival differences of Escherichia coli O157:H7 strains in apples of three varieties stored at various temperatures; Janes ME et al.; Differences in survival and growth among five different Escherichia coli O157:H7 strains in three apple varieties were determined at various temperatures . Jonathan, Golden Delicious, and Red Delicious apples were wounded and inoculated with E coli O157:H7 strains C7929 (apple cider isolate), 301C (chicken isolate), 204P (pork isolate), 933 (beef isolate), and 43890 (human isolate) at an initial level of 6 to 7 log CFU/g . The inoculated apples were stored at a constant temperature of 37, 25, 8, or 4 degrees C or at 37 degrees C for 24 h and then at 4 degrees C, and bacterial counts were determined every week for 28 days . By day 28, for Jonathan apples at 25 degrees C, the apple isolate counts were significantly higher than the chicken and human isolate counts . At 4 degrees C for 28 days, the human isolate inoculated into Jonathan, Golden Delicious, and Red Delicious apples was present in significantly smaller numbers than the other strains . The apple isolate survived significantly better at 4 degrees C, yielding the highest number of viable cells . By days 21 and 28, for apples stored at 37 degrees C for the first 24 h and then at 4 degrees C, the counts of viable E . coli O157:H7 apple and human isolates were 6.8 and 5.8 log CFU/g at the site of the wound, whereas for apples kept at 4 degrees C for the duration of storage, the respective counts were 5.6 and 1.5 log CFU/g . Our study shows that E . coli O157:H7 strains responded differentially to their ability to survive in these three apple varieties at 25 or 4 degrees C and produced higher viable counts when apples were temperature abused at 37 degrees C for 24 h and then stored at 4 degrees C for 27 days. Epidemiol Infect, 2002 Jun, 128(3), 357 - 62 Comparison between human and animal isolates of Shiga toxin-producing Escherichia coli O157 from Australia; Fegan N et al.; There is very little human disease associated with enterohaemorrhagic Escherichia coli O157 in Australia even though these organisms are present in the animal population . A group of Australian isolates of E . coli O157:H7 and O157:H- from human and animal sources were tested for the presence of virulence markers and compared by XbaI DNA macrorestriction analysis using pulsed-field gel electrophoresis (PFGE) . Each of 102 isolates tested contained the gene eae which encodes the E . coli attaching and effacing factor and all but one carried the enterohaemolysin gene, ehxA, found on the EHEC plasmid . The most common Shiga toxin gene carried was stx2c, either alone (16%) or in combination with stx1 (74%) or stx2 (3%) . PFGE grouped the isolates based on H serotype and some clusters were source specific . Australian E . coli O157:H7 and H- isolates from human, animal and meat sources carry all the virulence markers associated with EHEC disease in humans therefore other factors must be responsible for the low rates of human infection in Australia. BMC Microbiol . 2002 Jul 09;2(1):18. Genomic comparisons among Escherichia coli strains B, K-12, and O157:H7 using IS elements as molecular markers; Schneider D et al.; BACKGROUND: Insertion Sequence (IS) elements are mobile genetic elements widely distributed among bacteria . Their activities cause mutations, promoting genetic diversity and sometimes adaptation . Previous studies have examined their copy number and distribution in Escherichia coli K-12 and natural isolates . Here, we map most of the IS elements in E . coli B and compare their locations with the published genomes of K-12 and O157:H7 . RESULTS: The genomic locations of IS elements reveal numerous differences between B, K-12, and O157:H7 . IS elements occur in hok-sok loci (homologous to plasmid stabilization systems) in both B and K-12, whereas these same loci lack IS elements in O157:H7 . IS elements in B and K-12 are often found in locations corresponding to O157:H7-specific sequences, which suggests IS involvement in chromosomal rearrangements including the incorporation of foreign DNA . Some sequences specific to B are identified, as reported previously for O157:H7 . The extent of nucleotide sequence divergence between B and K-12 is < 2% for most sequences adjacent to IS elements . By contrast, B and K-12 share only a few IS locations besides those in hok-sok loci . Several phenotypic features of B are explained by IS elements, including differential porin expression from K-12 . CONCLUSIONS: These data reveal a high level of IS activity since E . coli B, K-12, and O157:H7 diverged from a common ancestor, including IS association with deletions and incorporation of horizontally acquired genes as well as transpositions . These findings indicate the important role of IS elements in genome plasticity and divergence. Curr Opin Nephrol Hypertens, 2002 Jul, 11(4), 431 - 5 The genetics and pathogenesis of haemolytic uraemic syndrome and thrombotic thrombocytopenic purpura; Richards A et al.; PURPOSE OF REVIEW: In recent years there has been a substantial increase in the understanding of the genetics and pathogenesis of haemolytic uraemic syndrome and thrombotic thrombocytopenic purpura . RECENT FINDINGS: In diarrhoeal associated haemolytic uraemic syndrome it has been established that the virulence of Escherichia coli O157 is related to intimin adhesion and the transport of verocytotoxin on polymorphonuclear cells . It has been shown that early changes in the coagulation pathway predate the onset of diarrhoeal haemolytic uraemic syndrome . Mutations in factor H, a fluid-phase regulator of the alternative complement pathway, have been identified in 10-20% of patients with both familial and sporadic (non-diarrhoeal-associated) haemolytic uraemic syndrome . The mutations mainly cluster in the C terminal part of factor H, a region that is important for both binding to C3b and also polyanionic structures on cell surfaces . The identification of antibodies against a plasma metalloproteinase responsible for cleaving ultralarge von Willebrand factor multimers in thrombotic thrombocytopenic purpura has been followed by the elucidation of the identity of the proteinase . It has been shown to be a member of the ADAMTS family, and mutations have been identified in the gene in families with inherited thrombotic thrombocytopenic purpura . SUMMARY: The molecular pathogenesis of haemolytic uraemic syndrome and thrombotic thrombocytopenic purpura is an exciting and rapidly evolving field . These recent advances will lead to logical, targetted changes in the management of these conditions. Vet J, 2002 Mar, 163(2), 128 - 46 Wild animals as reservoirs of infectious diseases in the UK; Simpson VR; This review aims to illustrate the extent to which wildlife act as reservoirs of infectious agents that cause disease in domestic stock, pet and captive animals and humans . More than 40 agents are described . In the case of some of these, e.g . Cryptosporidium spp., Escherichia coli O157 and malignant catarrhal fever, the current evidence is that wildlife either does not act as a reservoir or is of limited importance . However, in the case of many important diseases, including bovine tuberculosis, Weil's disease, Lyme disease, avian influenza, duck virus enteritis and louping ill, wild animals are considered to be the principal source of infection . Wildlife may be involved in the epidemiology of other major diseases, such as neosporosis, Johne's disease, mucosal disease and foot and mouth disease, but further studies are needed . The UK would benefit from a more positive approach to the study of wildlife and the infections they harbour . Lett Appl Microbiol, 2002, 35(1), 7 - 11 Prevalence of Escherichia coli O157:H7 in industrial minced beef; Vernozy-Rozand C et al.; AIMS: The lack of baseline data on the prevalence of Escherichia coli O157:H7 in retail minced beef in France prompted this survey of industrial minced beef production . METHODS AND RESULTS: An automated enzyme-linked fluorescence immunoassay (ELFA), the VIDAS E . coli O157 method, was used to detect E . coli O157 in industrial minced beef samples . Confirmation of samples positive according to the ELFA was performed using an automated immunoconcentration (ICE) system, VIDAS ICE, which allows the selective capture and release of target organisms . The ICE was followed by culture on cefixime tellurite sorbitol MacConkey agar and a chromogenic medium, O157:H7 ID . Of the 3450 minced beef samples tested, 175 samples were positive with the ELFA method and, of these, four were confirmed by the ICE method . They were identified as sorbitol-negative, O157-positive, H7-positive, mobile, verotoxin-producing E . coli . CONCLUSIONS: The prevalence of E . coli O157:H7 in industrial French minced beef was 0.12%, consistent with many other reports . SIGNIFICANCE AND IMPACT OF THE STUDY: The low infective dose of E . coli O157:H7 presents a major threat . The main means of combating this organism are thermal destruction and good food hygiene covering activities on-farm, in the abattoir and in minced beef industries. Nippon Rinsho, 2002 Jun, 60(6), 1108 - 13 {Indicators for early diagnosis of enterohemorrhagic Escherichia coli infection and methods for final diagnosis}; Shiomi M; Bloody diarrhea, abdominal cramp, history of barbecued beef for dinner at a restaurant and evidence of outbreak are important signs of Enterohemorrhagic Escherichia coli (EHEC) infections . Although early antibiotics therapy, such as fosfomycin and fluoroquinolones, is considered to be effective in Japan, stool cultures before antibiotics are essential for final diagnosis . In cases of hemorrhagic colitis or hemolytic uremic syndrome, when cultures are negative, detection of anti O-type specific lipopolysaccharides IgM antibodies is useful for estimate of causative EHEC . In our hospital, non-O157 EHECs increase in number as the causes of pediatric HUS since 1999. Nippon Rinsho, 2002 Jun, 60(6), 1101 - 7 {Mechanism of A/E lesion formation produced by enterohemorrhagic Escherichia coli: O157--role of EspB, Tir and cortactin}; Kodama T; Enterohemorrhagic Escherichia coli(EHEC) belongs to a family of pathogens which cause attaching and effacing(A/E) lesion on target cells . The following event is the alteration of the host intestinal cell cytoskeleton to form a pedestal-like structure . As a result of some recent breakthrough discoveries, EHEC injects effector proteins, EspB and Tir, into the host cells by type III secretion machinery and they modify cellular function and lead the signaling events by its direct binding with cytoskeletal proteins, alpha-catenin and talin . Cortactin is also accumulated to adherent site and involved in EHEC-induced A/E lesion . As a result of those interaction between bacterial effector proteins and host cytoskeletal proteins, EHEC can trigger the rearrangement of the host cell's actin cytoskeleton and induce the A/E lesion. Nippon Rinsho, 2002 Jun, 60(6), 1070 - 6 {PulseNet Japan--network system for the utilization of epidemiological information and the results of pulsed-field gel electrophoresis}; Terajima J et al.; Since 1996, we have been analyzing DNA pattern of enterohemorrhagic Escherichia coli(EHEC) O157: H7 isolates in Japan by the use of pulsed-field gel electrophoresis . The method, capable of discriminating genotypical difference of the isolates, enabled us to find the contaminated food such as salmon roe which was the causative agent for the multiprefectual outbreaks in Japan . These outbreaks which we are referring as diffuse outbreaks seem to be increasing in number, because it reflects that some of widely distributed or mass-produced food products are being contaminated by pathogens such as EHEC . In order to find a diffuse outbreak promptly and prevent it becoming large, we are constructing a network, called PulseNet Japan, for sharing the results of pulsed-field gel electrophoresis and epidemiological information among municipal public health institutes and National Institute of Infectious Diseases. FEMS Microbiol Lett, 2002 Jun 18, 212(1), 107 - 10 Effect of culture conditions on Escherichia coli O157:H7-mediated attaching-effacing lesions in a bovine large intestinal mucosal explant model; Baehler AA et al.; The effects of culture conditions on the extent of Escherichia coli O157:H7 attaching-effacing (A/E) adherence in an adult bovine large intestinal mucosal explant model were assessed by three different morphometric methods . Measurement of the percent of tissue sections with A/E adherence and the number of foci of A/E adherence mm(-1) of surface epithelium was more sensitive than measurement of the percent of surface epithelium with A/E adherent bacteria for detection of treatment effects . Culture of bacterial inoculum in tryptic soy broth, incubation of explants in 5% CO(2), and rocking of explants on a platform rocker at 18 cycles min(-1) provided optimal conditions for A/E adherence . In future studies, the model may be used for preliminary testing of intervention strategies aimed at reduction of E . coli O157:H7 intestinal colonization of cattle. Vet Rec, 2002 Jun 1, 150(22), 689 - 92 Identification of Escherichia coli O157:H7-strains from pigs with postweaning diarrhoea and amplification of their virulence marker genes by PCR; Osek J; Escherichia coli isolated from pigs with postweaning diarrhoea were examined by PCR for the presence of the O157 rfb gene responsible for the biosynthesis of E coli O157 lipopolysaccharide . Among the 372 isolates tested, 38 (10.2 per cent) were of the O157 serogroup, but none of these possessed the H7 determinant . Further analysis of the E coli O157 isolates revealed that seven of them had the genes responsible for the production of Shiga toxin 1 and eaeA intimin, four other strains had genes responsible for the production of Shiga toxin 2, and four other strains were positive for the enterohaemolysin gene. Infect Immun, 2002 Jul, 70(7), 3500 - 9 Bicarbonate ion stimulates the expression of locus of enterocyte effacement-encoded genes in enterohemorrhagic Escherichia coli O157:H7; Abe H et al.; Enterohemorrhagic Escherichia coli (EHEC) strains adhere to the intestinal mucosa and produce an attaching and effacing (A/E) lesion . Most of the genes required to produce A/E lesions are thought to be encoded by the 36-kb pathogenicity island termed the locus for enterocyte effacement (LEE) . Although the mechanisms underlying the bacterial adherence, including the genes involved, are still poorly understood, the preferential adherence phenotype of EHEC is thought to depend on the nature of the genes and/or the response of these genes to changes in environmental conditions . To explore the environmental factors affecting EHEC adherence, we used an O157:H7 strain and investigated the optimal growth conditions for its adherence to Caco-2 cells . We observed that EHEC grown in Dulbecco's modified Eagle's medium (DMEM) adhered more efficiently to Caco-2 cells than EHEC grown in Luria-Bertani (LB) broth . Among the components of DMEM, only NaHCO(3) was found to remarkably stimulate bacterial adherence . When bacteria were grown in LB broth containing NaHCO(3), the production of intimin, Tir, EspA, and EspB was greatly enhanced compared with the production in LB broth . Indeed, the transcription of ler required for LEE-encoded gene expression was promoted in response to the concentration of NaHCO(3) in LB broth . Since the concentration of NaHCO(3) in the lower intestinal tract has been shown to be relatively high compared with that in the upper small intestine, our results may imply that NaHCO(3) is an important signaling factor for promoting colonization of EHEC in the lower intestinal tract in humans. Appl Environ Microbiol, 2002 Jun, 68(6), 3169 - 71 Rapid detection of Escherichia coli O157:H7 with multiplex real-time PCR assays; Jothikumar N et al.; A SYBR Green LightCycler PCR assay using a single primer pair allowed simultaneous detection of stx1 and/or stx2 of Escherichia coli O157:H7 . A distinct sequence of the Shiga-like toxin genes was amplified to yield products of 227 and/or 224 bp, respectively . The two products were distinguished by melting point curve analysis. Proc Natl Acad Sci U S A, 2002 May 28, 99(11), 7669 - 74 A therapeutic agent with oriented carbohydrates for treatment of infections by Shiga toxin-producing Escherichia coli O157:H7; Nishikawa K et al.; Infection with Shiga toxin (Stx)-producing Escherichia coli O157:H7, which causes diarrhea and hemorrhagic colitis in humans, often results in fatal systemic complications, such as neurological damage and hemolytic-uremic syndrome . Because Stx circulating in the blood is a major causative factor of these complications, the development of a Stx neutralizer that functions in the circulation holds promise as a viable therapy . Here we developed a series of carbosilane dendrimers, in which trisaccharides of globotriaosyl ceramide, a receptor for Stx, were variously oriented at their termini (referred to as SUPER TWIG), and identified a SUPER TWIG with six trisaccharides as a Stx neutralizer functioning in the circulation . This SUPER TWIG specifically bound to Stx with high affinity (K(d) = 1.1 x 10(-6) M) and inhibited the incorporation of the toxin into target cells . Intravenous administration of the SUPER TWIG along with Stx to mice substantially reduced the fatal brain damage and completely suppressed the lethal effect of Stx . Moreover, the SUPER TWIG protected mice from challenge with a fatal dose of E . coli O157:H7, even when administered after the establishment of the infection . The SUPER TWIG neutralized Stx in vivo by a mechanism in which the accumulation and immediate degradation of Stx by phagocytic macrophages present in the reticuloendothelial system were induced . Taken together, our findings indicate that this SUPER TWIG is therapeutic agent against infections by Stx-producing E . coli. J Food Prot, 2002 May, 65(5), 845 - 7 Fate of Escherichia coli O157:H7 in coleslaw during storage; Wu FM et al.; An outbreak of Escherichia coli O157:H7 infection associated with the consumption of coleslaw in several units of a restaurant chain prompted a study to determine the fate of the pathogen in two commercial coleslaw preparations (pH 4.3 and 4.5) held at 4, 11, and 21 degrees C for 3 days . At an initial population of 5.3 log10 CFU/g of coleslaw, E . coli O157:H7 did not grow in either coleslaw stored at the three temperatures . Rather, the population of E . coli O157:H7 decreased by 0.1 to 0.5 log10 CFU/g within 3 days . The greatest reduction (0.4 and 0.5 log10 CFU/g) in population occurred at 21 degrees C, whereas only slight decreases (0.1 to 0.2 log10 CFU/g) occurred at 4 and 11 degrees C . A pH of 4.3 to 4.5 of coleslaw had little effect on reducing E . coli O157:H7 populations . Results suggest that the tolerance of E . coli O157:H7 to acid pH, not temperature abuse, is a major factor influencing the pathogen's fate in restaurant-prepared coleslaw. J Med Microbiol, 2002 Jun, 51(6), 522 - 5 The kinetics of antibody production to antigens of Escherichia coli O157 in a pregnant woman with haemolytic uraemic syndrome; Chart H et al.; Sequential blood samples taken from a pregnant woman with haemolytic uraemic syndrome caused by verocytotoxin (VT)-producing Escherichia coli O157 were used to examine the kinetics of serum antibody production to E . coli O157 lipopolysaccharide (LPS), intimin and the conserved region of the translocated intimin receptor (Tir-M) . Umbilical cord blood and two samples of blood from the newborn baby were also examined for antibodies to these antigens . In the mother, antibodies of the IgM class, specific for E . coli O157 LPS, were produced in the initial stages of the infection, reaching a peak at 9 days after onset of diarrhoea and subsiding 3 days later . High levels of IgG class antibodies, specific for E . coli O157 LPS, were detected 8 days after the onset of diarrhoea and were present at high titres on day 18 . Serum antibodies of the IgA class to E . coli O157 LPS were not detected . Antibodies binding to Tir-M were detected 8 days after the onset of diarrhoea and high titres of these antibodies were still present on day 18 . Serum antibodies to intimin were not detected in the mother and no antibodies to any of the antigens tested were detected in either the baby's blood or cord blood . This study describes for the first time the kinetics of serum antibody production during pregnancy, to selected antigens expressed by E . coli O157. Int Arch Allergy Immunol, 2002 Apr, 127(4), 294 - 8 Protection of monkeys against Shiga toxin induced by Shiga toxin-liposome conjugates; Suzaki Y et al.; BACKGROUND: We previously reported that the purified Shiga toxins (Stx) Stx1 and Stx2, when coupled with liposomes, induced substantial production of anti-Stx1 and anti-Stx2 IgG antibody, respectively, in mice . The levels of anti-Stx antibody in the sera of mice immune to Stx-liposome correlated well with the protection against subsequent challenge with Stx . Furthermore, mice immunized with a mixture of Stx1-liposome and Stx2-liposome were successfully protected against oral infection with cytotoxin-producing Escherichia coli O157:H7 . METHODS: In this study, the induction of protection against Stx2 by Stx2-liposomes was evaluated in monkeys . RESULTS: Stx2-liposomes induced a substantial amount of anti-Stx2 IgG antibodies as well as Stx2 neutralizing antibodies in monkeys . Test monkeys were successfully protected against challenge with lethal doses of Stx2 . Moreover, these monkeys showed no apparent symptoms, while nonimmunized control monkeys died within 4 days with hemorrhagic gastroenteritis and renal disorder . In addition, as shown by other cases involving antigen-liposome conjugates, Stx2-liposome did not induce anti-Stx2 IgE antibody production, though it stimulated the production of a substantial amount of anti-Stx2 IgG antibodies . CONCLUSION: These results suggest that Stx-liposome conjugates may serve as candidate vaccines to induce protection against death caused by cytotoxin-producing E . coli infection . Zhonghua Liu Xing Bing Xue Za Zhi, 2002 Apr, 23(2), 123 - 6 {Molecular typing of Shiga-toxin producing Escherichia coli O157:H7 isolated in China with pulsed field gel electrophresis}; Pang B et al.; OBJECTIVE: To type and group the Shiga-toxin producing Escherichia coli O157:H7 strains isolated recent years in China to understand the epidemiological features caused by the pathogen . METHODS: Pulsed field gel electrophoresis of large restriction fragments of bacterial chromosomal DNA was used . RESULTS: The 51 isolates of E . coli O157:H7 collected in recent years in China could be divided into 8 Pulsed Field Gel Electrophoresis (PFGE) types based on the size and number of restriction fragments and patterns, that were digested by XbaI . Strains isolated from Ningxia province showed only two types- PFGE1 and PFGE2 . Strains isolated in Xuzhou of Jiangsu province had 6 PFGE types . Isolates identified between 1986 - 1988 belonged to PFGE7 . Strains isolated from patients in 1999 - 2000 were PFGE5 and PFGE3 . Strains isolated from stool samples of domestic animal, food and vegetable were PFGE3 - 6, of which the predominant type was PFGE5 . All of the 5 strains isolated from patients with diarrhea and hemolytic uremic syndrome (HUS) belonged to PFGE type 5, which was the dominant type of the isolates from stool samples of domestic animal and samples of food and vegetable contaminated . CONCLUSION: