E coli O157

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: Data suggested that the cluster patients with diarrhea and HUS might have been related to the pathogens from domestic animas and contaminated food or vegetables . The distribution of PFGE types also varied in different provinces of China.

Zhonghua Liu Xing Bing Xue Za Zhi, 2002 Apr, 23(2), 114 - 8
{Serological investigations on patients with hemolytic uremic syndromes due to enterohemorrhagic Escherichia coli O157:H7 infection}; Xu J et al.; OBJECTIVE: To investigate the etiological agent of patients with diarrhea followed by acute kidney failure symptoms in China, 1999 . METHODS: Western blot was used to detect serum specific antibodies of patients against entero-haemorrhagic Escherichia coli hemolysin (EHEC-Hly) and lipo-polysaccharide of E . coli O157 . RESULTS: Twenty-one and 16 of 42 patients showed positive reaction of specific IgG or IgM antibodies against EHEC-Hly respectively . Eleven of 42 serum samples were positive for having both IgG and IgM antibodies while 26 of 42 samples were positive for IgG or IgM . For E . coli O157 LPS test, 24 and 24 of 42 samples showed positive for IgG or IgM antibodies respectively . In 42 samples, 20 were positive for IgG and IgM while 29 were positive for IgG or IgM . CONCLUSIONS: Twenty-two of 42 samples were reacted with EHEC-Hly and E . coli O157 LPS, but 34 of 42 samples were positive for EHEC-Hly or E . coli O157 . In combination of western blot results, bacterial isolation clinical symptoms and epidemiological investigation findings, it was reasonable to conclude that this cluster of patients with distinguish clinical symptoms was caused by E . coli O157:H7, which had never been reported in China . Hence serological methods with EHEC-Hly and E . coli O157 LPS are valuable for diagnosis of infections of E . coli O157:H7, when bacterial isolation is failed.

Zhonghua Liu Xing Bing Xue Za Zhi, 2002 Apr, 23(2), 102 - 4
{Surveillance of Escherichia coli O157:H7 among animals in Jiangsu province in 1999}; Ni D et al.; OBJECTIVE: To understand the Escherichia coli O157:H7 carrier rate of host animals and the toxic gene of the strains in different areas in Jiangsu province . METHODS: Surveillance spots were set up in different areas, to collect feces of pigs, chickens, sheep, cattle to culture for O157:H7 with immunomagnetic separation as well as detection of toxic gene of the strain with MPCR were both carried out . RESULTS: One hundred and seventy strains of O157:H7 were separated from 1 767 feces of different animals in six spots, with a overall positive rate 9.62% . The positive rates of cattle and sheep were 19.05% and 12.01% respectively . Among 85 strains SLT1, SLT2, eaeA and hly toxic genes were detected . In which, 56.47% of the strains were positive curturely while 79.17% of them carried SLT2, eaeA and hly gene simultaneously . CONCLUSION: The positive rate of O157:H7 in animals and the positive rates of strains were correlated to the incidence of the area . The highest rates were seen in areas where there had been O157:H7 epidemic, followed by the areas where there were only scattered cases identified while the lowest was in areas with no patients . Data indicated that it was important to enforce the surveillance of O157:H7 in animals to better predict and control of the disease.

Infect Immun, 2002 Jun, 70(6), 3094 - 100
Characterization of a novel type IV pilus locus encoded on the large plasmid of locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli strains that are virulent for humans; Srimanote P et al.; The majority of Shiga-toxigenic Escherichia coli (STEC) strains isolated from humans with gastrointestinal disease carry large (approximately 90-kb) plasmids . We have been analyzing the megaplasmid (designated pO113) from an O113:H21 STEC strain (98NK2) . This strain lacks the locus for enterocyte effacement (LEE) and yet was responsible for an outbreak of hemolytic uremic syndrome . In the present study, we demonstrate that pO113 carries a novel type IV pilus biosynthesis locus (pil) related to those of the IncI plasmids R721, R64, and ColIb9 . The pO113 pil locus consists of 11 closely linked genes (pilL through pilV) with an additional separately transcribed upstream gene (pilI) . It directs the expression of long thin pili on the 98NK2 surface and the hemagglutination of guinea pig erythrocytes . We also demonstrate that pO113 can be transferred by conjugation . However, the type IV pilus encoded by pO113 does not appear to be involved in the adherence of 98NK2 to HEp-2 or Hct-8 cells in vitro . Homologues of the pO113 pil locus were present in several other LEE-negative STEC strains but not in LEE-positive STEC strains belonging to serogroup O26, O111, or O157.

J Appl Microbiol, 2002, 92(6), 1021 - 7
Comparison of ATP and in vivo bioluminescence for assessing the efficiency of immunomagnetic sorbents for live Escherichia coli O157:H7 cells; Sun W et al.; AIMS: To develop methods to assess the efficiency of immunomagnetic separation (IMS) . METHODS AND RESULTS: The capturing efficiency of biosorbents for Escherichia coli O157:H7, constructed using streptavidin-coated magnetic beads and biotinylated antibodies, was tested using both in vivo and ATP bioluminescence . Both methods were suitable for the enumeration of bacteria captured by the biosorbents . The level of both ATP and in vivo bioluminescence depended on the media used, but was unaffected by the magnetic beads . The capture efficiency depended on time and sample volume, but did not depend on the length of spacer arm of the biotinylation agent . For cell concentrations of <or= 105 cfu ml-1, in a 1-ml sample volume, nearly 80-85% recovery of the pathogen was observed after 0.5 h of incubation . For an 11-ml sample containing 104 cfu ml-1, maximum recovery (50% of cells) was achieved only after 2 h incubation . CONCLUSIONS: The detection limit of an ATP-based bioluminescent assay for E . coli O157:H7 was reduced by 1 log cycle after optimization of IMS . The bioluminescent methods could be used for screening and testing the affinity of antibodies or other affinity elements of biosorbents towards live bacterial cells . SIGNIFICANCE AND IMPACT OF THE STUDY: Bioluminescent assays provide an easy way to optimize conditions for the capture of bacteria by biosorbents in real time.

J Appl Microbiol, 2002, 92(6), 1015 - 20
Comparison of the sensitivity of manual and automated immunomagnetic separation methods for detection of Shiga toxin-producing Escherichia coli O157:H7 in milk; Reinders RD et al.; AIM: To determine the sensitivity of methods for detection of injured and uninjured Escherichia coli O157:H7 (E . coli O157) in raw and pasteurized milk . METHODS AND RESULTS: Raw milk, pasteurized milk with 1.5% fat content and pasteurized milk with 3.5% fat content were spiked with E . coli O157 at low levels . The samples were enriched in modified tryptone soya broth with novobiocin (mTSBn) at 37 degrees C . Aliquots of the enriched culture were analysed either by manual immunomagnetic separation (MIMS) and culturing on sorbitol MacConkey agar with or without cefixime and potassium tellurite (SMACct or SMAC), or by automated immunomagnetic separation and integrated ELISA (EiaFosstrade mark) . Uninjured E . coli O157 organisms were detected in milk by both methods at 1 cfu 10 ml-1 sample) . Injured organisms were detected at levels of about 4 cfu 10 ml-1 sample . Direct enrichment in mTSBn (22 h incubation) showed better sensitivity for injured cells than enrichment in buffered peptone water (BPW, 22 h incubation), or in a two-step enrichment consisting of BPW (6 h, 37 degrees C) and mTSBn (16 h, 37 degrees C), successively . CONCLUSIONS: The methods showed equal sensitivity in that they were both able to detect 1 cfu 10 ml-1 milk sample . Injured organisms can be detected and isolated at a level almost as low as this . A resuscitation step is not recommended for the detection and isolation of injured and non-injured E . coli O157 from milk . SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the dilution of contamination in the bulk tank, analysis of milk for the presence of E . coli O157 requires a very sensitive method . Both methods described here are useful for such analysis.

Luminescence, 2002 Mar-Apr, 17(2), 123 - 9
Simultaneous detection of two verotoxin genes using dual-label time-resolved fluorescence immunoassay with duplex PCR; Watanabe K et al.; Verotoxin (VT) produced by several Escherichia coli serotypes causes haemorrhagic colitis and has been associated with haemolytic uraemic syndrome in humans . Two types of verotoxin are known . Conventional diagnosis of verotoxin-producing Escherichia coli (VTEC) is conducted after isolation of bacteria from clinical specimens, followed by serological determination and identification of VTs . This method is complicated and time-consuming . Recently, rapid, direct immunological methods for identification of VTEC, i.e . immunochromatography and latex agglutination, have been developed . However, these techniques continue to suffer from limited sensitivity and a lack of specificity . These difficulties arise from the fact that the antibody used in these procedures reacts exclusively with the O157 antigen; moreover, VTEC strains with non-O157 antigens, such as O26, O103 and O111 antigens, exist . These VTEC groups did not react with anti-O157 antibody . Consequently, it is necessary to diagnose the VT gene in these bacteria . Therefore, we have designed a sensitive and specific method for the detection of two VT genes simultaneously, utilizing duplex PCR with time-resolved fluorescence immunoassay (TRFIA).

Emerg Infect Dis, 2002 May, 8(5), 525 - 7
Deer meat as the source for a sporadic case of Escherichia coli O157:H7 infection, Connecticut; Rabatsky-Ehr T et al.; We report a case of Escherichia coli O157:H7, which was acquired by eating wild White-Tailed deer (Odocoileus virginianus) . DNA fingerprint analysis verified venison as the source of infection . This pediatric case emphasizes the need for dissemination of information to hunters regarding the safe handling and processing of venison.

Can J Vet Res, 2002 Apr, 66(2), 65 - 72
Pathogenic Shiga toxin-producing Escherichia coli in the intestine of calves; Sandhu KS et al.; The purpose of this study was to compare the pathological effects of Shiga toxin-producing Escherichia coli (STEC) that vary in their association with bovine and human disease . Shiga toxin-producing E . coli of serotypes associated with both dysentery in calves and hemolytic uremic syndrome (HUS) in humans (O5:H-, O26:H11, O111:H-,O113:H21) were compared with O157:H7 STEC, which are associated with HUS in humans but not with disease in calves . The STEC were administered orally to 80 day-old chicks and into ligated loops in the ileum and colon of four 2- to 6-day-old calves . Examination of the ceca of the chickens 10 d postchallenge showed no adherence or tissue abnormality for any isolate . The calves were euthanized 8 to 10 h postinoculation, and sections of the intestinal loops were examined by light microscopy, transmission and scanning electron microscopy, and immunohistochemistry . All strains showed consistent focal adherence associated with mild lesions in the colon . Attaching and effacing lesions were observed with the eae-positive strains . Ileal lesions were similar to the colonic ones but were sometimes severe, with marked polymorphonuclear leukocyte proliferation in the lamina propria . It is concluded that chickens were unsuitable for studying interaction of STEC with the intestine and that there was no difference in the interaction of the ligated calf intestine with STEC of serotypes associated with disease in calves compared with O157:H7 STEC.

Immunology, 2002 Apr, 105(4), 509 - 14
Lack of Shiga-like toxin binding sites in germinal centres of mouse lymphoid tissues; Imai Y et al.; B cells in germinal centres are known to express carbohydrate antigen CD77 in human lymphoid tissues . The CD77 antigen is specifically recognized by Shiga-like toxins (SLTs) that are produced by enterohaemorrhagic Escherichia coli O157:H7 . To determine whether the binding subunits of Shiga-like toxin-1 (SLT-1B) could have adverse effects on the murine immune system when used as an immunogen, we investigated whether SLT-1B could bind to germinal centres of mouse lymphoid tissues . Frozen sections of peripheral lymph nodes and Peyer's patches from immunized mice were tested for the presence of SLT-1B-binding sites by immunohistological methods . Germinal centres were not stained with SLT-1B, while they were intensely stained with peanut agglutinin (PNA), another marker of germinal centres . On the other hand, SLT-1B specifically bound to renal tubules and collecting ducts in frozen sections of mouse kidney . This is consistent with results from human tissues . We also demonstrated that B220/PNA double-positive populations in lymph nodes from immunized mice exhibited only marginal staining with SLT-1B . The present results suggest that SLTs would not impede germinal centre functions of the murine immune system.

Kansenshogaku Zasshi, 2002 Mar, 76(3), 167 - 73
{The serotype and Shiga toxin type of Shiga toxin-producing Escherichia coli from humans and various animals}; Tsukamoto T et al.; To clear the route of STEC (Shiga toxin-producing Escherichia coli) infection to humans, we examined the serotype . Shiga toxin genotype and eae gene of STEC strains from humans and various animals . The most predominant serotype originated from humans was O157:H7, followed by O26:H11, and other serotypes were O91:H21, O103:H2, O111:NM, O121:H19, etc . The eae gene was found in 79 of 93 strains from human origin . The serotypes of STEC from cattle were significant by similar to that of STEC from humans . The eae gene was found in 44 of 87 strains from cattle . Shiga toxin genotypes possessed O157 strains from humans and cattle, were divided into six groups, stx1, stx2, stx2c, stx1 + stx2, stx1 + stx2c and stx2 + stx2c . Moreover, frequency rates of Shiga toxin genotypes of O157 were also similar to both human and cattle origins . The serotypes of STEC from sheep were also a little similar to that of STEC from humans . Seven of 8 strains from deer possessed stx2d gene that the strains from humans seldom possessed, and none of the strains possessed eae gene . In STEC originated from swine, 15 of 25 strains were O139:H1 that Shiga toxin genotype was stx2e . It was thought that the sources of STEC infection to human are cattle and sheep, and deer and swine had little possibility to human STEC infection.

Jpn J Infect Dis, 2002 Feb, 55(1), 19 - 22
High genomic diversity of enterohemorrhagic Escherichia coli isolates in Japan and its applicability for the detection of diffuse outbreak; Terajima J et al.; Genotyping of 1,102 enterohemorrhagic Escherichia coli isolates by the use of pulsed-field gel electrophoresis (PFGE) carried out from January to November 2000 has revealed the high genomic diversity of these isolates in Japan . By combining the results of genotyping of the isolates with the information from other epidemiological investigations of the cases, we identified a diffuse outbreak in Japan in the year 2000 that seemed to be sporadic but was actually linked . Isolates with only the Shiga toxin 2 gene derived from patient specimens and the contaminated food involved in this diffuse outbreak showed an indistinguishable PFGE profile and the same phage type . Based on the diversity of genotypes among the isolates of enterohemorrhagic E . coli O157:H7/- in Japan, we suggest the presence of a few other possible diffuse outbreaks due to the organisms, showing indistinguishable genotypes.

J Appl Microbiol, 2002, 92(4), 633 - 40
A reverse transcriptase-polymerase chain reaction assay for detection of viable Escherichia coli O157:H7: investigation of specific target genes; Yaron S et al.; AIMS: To determine suitable target genes for detection of the pathogen Escherichia coli O157:H7 by reverse transcriptase-polymerase chain reaction (RT-PCR) . METHODS AND RESULTs: Potential genes used as indicators for viability included rfbE, fliC, stx1, stx2, mobA, eaeA, hly and 16S rRNA . Under normal growth conditions, rfbE, stx1, hly and 16S rRNA amplicons were detected in association with all growth phases . The products of 16S rRNA, mobA, rfbE and stx1 were readily visualized in RNA isolated from viable but non-culturable cells . The 16S rRNA gene was not amplified following heat treatment of cells at 121 degrees C for 15 min and mRNA targets were not amplified after treatment at 60 degrees C for 20 min . In this instance, genes that are not amplified are good targets for determining viability . CONCLUSIONS: The results of RT-PCR amplification indicate that, under the conditions examined, the rfbE gene is the most appropriate target for detection of viable E . coli O157:H7 . SIGNIFICANCE AND IMPACT OF THE STUDY: Prior to detection or identification from an environmental or food sample E . coli O157:H7 may be exposed to many harsh conditions that influence nucleic acid (RNA and DNA) stability . This study gives an insight into the effects of temperature and nutrient deprivation on identification of viable cells using RT-PCR . It also suggests that, if RT-PCR is to be used for detection of live cells in a sample without enrichment, 10(7) cfu of the target organism are required.

Eur J Clin Microbiol Infect Dis, 2002 Mar, 21(3), 189 - 95 Epub 2002 Mar 19.
Use of phenotyping and genotyping to verify transmission of Escherichia coli O157:H7 from dairy farms; Lahti E et al.; A total of 80 human infections by Escherichia coli O157:H7 were documented in Finland in 1997 and 1998 . Most were sporadic and their sources undetermined . Five cases not associated with one another, one of which led to secondary transmission within a family, could be traced to five different dairy farms . These five case patients (age range 2-17 years, median age 3 years) were hospitalised with bloody diarrhoea; two of them developed haemolytic uraemic syndrome . All nine human isolates obtained were sorbitol negative, carried the verocytotoxin 2 and eae genes, and produced verocytotoxin and enterohaemolysin . The phage and pulsed-field gel electrophoresis types of the human and bovine isolates from the corresponding farms were indistinguishable . The cattle (20-70 animals per farm) were monitored for up to 2 years after the human cases . The proportion of cattle excreting the type that caused the human infections varied from 3.2 to 66.7% when sampled soon after the human cases, and from 0.0 to 5.3% about a year or so later . On most of the farms, the animals excreted the pathogen intermittently . On one farm, Escherichia coli O157 isolates with other characteristics were also occasionally isolated . Although the infections were traced back to the farms, it could not be established whether the source was unpasteurised milk or direct or indirect contact with cattle . The results of this study emphasise the need for special recommendations for children visiting or living on a farm to prevent these infections.

J Food Prot, 2002 Apr, 65(4), 596 - 602
Detection of Escherichia coli O157:H7 in 10- and 25-gram ground beef samples with an evanescent-wave biosensor with silica and polystyrene waveguides; Demarco DR et al.; A portable evanescent-wave fiber-optic biosensor was used to detect Escherichia coli O157:H7 in seeded 10- and 25-g ground beef samples . The biosensor works by launching light from a 635-nm laser diode into specially designed optical fiber probes, generating an evanescent field that extends approximately 1,000 nm from the fiber surface . Fluorescent molecules within the evanescent field are excited, and a portion of their emission recouples into the fiber probe . The return path emission is transported by an optical fiber to a photodiode within the biosensor that detects and quantifies the fluorescent signal . A sandwich immunoassay was performed on the fiber probes with cyanine 5 dye-labeled polyclonal anti-E . coli O157:H7 antibodies for generation of the specific fluorescent signal . Biotin-streptavidin interactions were used to attach polyclonal anti-E . coli O157:H7 antibodies to the surface of the fiber probe . A centrifugation method was developed to obtain samples suitable for biosensor analysis from 10- and 25-g ground beef samples . The assay was shown to be sensitive and repeatable . One hundred percent correct identification of positive samples was demonstrated at 9.0 x 10(3) CFU/g for 25-g ground beef samples with silica waveguides and at 5.2 x 10(2) CFU/g for 10-g ground beef samples with polystyrene waveguides . The reaction was highly specific . No false positives were observed for 10-g ground beef samples not spiked with the pathogen . In addition, when samples were spiked with high concentrations of a variety of non-E . coli O157:H7 organisms, no false positives were observed . The method was rapid, with results being obtained within 25 min of sample processing.

BMC Microbiol . 2002 Apr 08;2(1):6.
First isolation of the enterohaemorrhagic Escherichia coli O145:H- from cattle in feedlot in Argentina; Padola NL et al.; BACKGROUND: Enterohaemorrhagic Escherichia coli (EHEC) is considered to be common cause of haemorrhagic colitis (HC), thrombotic thrombocytopenic purpura and haemolytic uraemic syndrome (HUS) in humans . In a previous paper, we have demonstrated that EHEC are commonly found in the intestines of livestock . Infections in humans are, in part, a consequence of consumption of undercooked meat or raw milk . Argentina has one of the highest records of HUS (300-400 cases/year; 22/100,000 children under 4 years of age) . The aim of this work is to communicate the first isolation of O145:H-from cattle in this country and characterize the virulence cassette, providing useful information to evaluate the risk of foodborne transmission of this emergent non-O157:H7 serotype . RESULTS: EHEC O145:H- was isolated from cattle in an Argentinian feedlot . Pheno- and genotype of nine strains were characterized, corresponding to several virulence cassettes: VT2+eaeA+ Mp+ (n = 5), VT2+eaeA+ (n = 1), VT1+eaeA+ Mp+ (n = 2), and VT1+eaeA+ (n = 1) . Strains isolated from the same animal were considered only when they showed a different virulence pattern . The clonal relationship was studied by RAPD . Strains were distributed in two RAPD profiles, which corresponded to the presence of either, VT1+ or VT2+ genotype . No difference was detected by RAPD analysis between Mp+ or Mp- strains . CONCLUSIONS: This was the first isolation of EHEC O145:H- serotype in Argentina enlarging the list of non-O157:H7 serotypes isolated from cattle in this country by us . All O145:H-strains carried several virulence factors which allow us to predict their potential ability to develop haemolytic uraemic syndrome in humans.

Endoscopy, 2002 Apr, 34(4), 311 - 4
The clinical significance of colonoscopy in hemorrhagic colitis due to enterohemorrhagic Escherichia coli O157:H7 infection; Shigeno T et al.; BACKGROUND AND STUDY AIMS: Although hemorrhagic colitis due to enterohemorrhagic Escherichia coli O157:H7 (O157) infection has recently attracted increasing attention as an important enteric infection, the colonoscopic findings associated with this disease have not been sufficiently characterized . The aim of this study is to characterize the colonoscopic features of hemorrhagic colitis due to O157 infection . PATIENTS AND METHODS: The colonoscopic findings in ten patients with hemorrhagic colitis due to O157 infection were retrospectively reviewed . To assess the severity of inflammation in each part of the large intestine, colonoscopic findings were categorized into four grades: grade 1, intact mucosa; grade 2, sporadic erythema and erosion; grade 3, mostly diffuse inflammation; and grade 4, diffuse, severe inflammation . RESULTS: Eight out of ten patients had grade 4 findings in the cecum and ascending colon, grade 3 in the transverse colon and descending colon, and grade 2 in the sigmoid colon . Two of these eight patients also had grade 4 inflammation in the proximal transverse colon . Five of these eight patients revealed longitudinal ulcer-like lesions in the transverse colon and/or descending colon . The remaining two patients had grade 3 findings in the cecum to the descending colon and grade 2 findings in the sigmoid colon . All patients exhibited grade 1 finding in the terminal ileum and the rectum . Based on these colonoscopic findings, the ten patients were divided into the typical group (eight patients) and the mild-type group (two patients) . CONCLUSIONS: The characteristic colonoscopic findings in most patients with hemorrhagic colitis due to O157 infection were as follows: 1) severe inflammation, including primarily marked edema and facile hemorrhage, and 2) inflammation predominating at the right-side colon; and 3) frequent appearance of longitudinal ulcer-like lesions.

Int J Food Microbiol, 2002 Mar 25, 74(1-2), 37 - 45
Comparison of methods for fluorescent detection of viable, dead, and total Escherichia coli O157:H7 cells in suspensions and on apples using confocal scanning laser microscopy following treatment with sanitizers; Burnett SL et al.; The influence of treating Escherichia coli O157:H7 cells labeled with an enhanced green fluorescent protein (EGFP) plasmid with 20 microg/ml active chlorine, 100 mg/ml hydrogen peroxide, and 80 mg/ml acetic acid on fluorescence intensity was determined . In addition, fluorescent staining methods to differentiate viable and dead E . coli O157:H7 cells on the cuticle of Red Delicious cv . apples following treatment with water or 200 microg/ml active chlorine were evaluated . Suspensions of E . coli O157:H7 EGFP+ cells were exposed to chemical treatment solutions for 0, 30, 60, 120, or 300 s before populations (log10 cfu/ml) were determined by surface plating, and fluorescence intensities of suspensions and individual cells were measured using spectrofluorometry and confocal scanning laser microscopy (CSLM), respectively . The relative fluorescence intensity of suspensions and individual cells changed upon exposure to various treatments . Results indicate that the use of EGFP to tag E . coli O157:H7 may not be appropriate for investigations seeking to microscopically differentiate viable and dead cells on produce following surface treatment with sanitizers . SYTOX Orange and SYTOX Green nucleic acid stains fluorescently labeled dead E . coli O157:H7 cells attached to apple cuticles more intensely than did propidium iodide . A cross-signal occurred between CSLM photomultipliers when examining tissues treated with SYTOX Orange to detect dead cells and antibody labeled with Alexa Fluor 488 to detect total (dead and viable) cells . Because of the possibility of cross-signal resulting in an overestimation of the number of dead cells on apples and, perhaps, other produce treated with these stains, SYTOX Green is preferred to detect dead cells and antibody labeled with Alexa Fluor 594 is preferred to detect the total number of cells on apple surfaces following treatment with sanitizers . The performance of SYTOX Green in combination with Alexa Fluor 594 to detect dead and total cells of E . coli O157:H7 on other produce remains to be determined.

Int J Food Microbiol, 2002 Mar 25, 74(1-2), 161 - 3
Evaluation of sorbitol-salicin MacConkey medium containing cefixime and tellurite (CT-SSMAC medium) for isolation of Escherichia coli O157:H7 from raw vegetables; Fujisawa T et al.; The utility of CT-SSMAC medium (sorbitol-salicin MacConkey medium containing cefixime and tellurite) for the isolation of Escherichia coli O157:H7 from raw vegetables was investigated . The colonies of all E . coli O157:H7 and O157:NM strains tested were colorless and beta-galactosidase-positive on CT-SSMAC medium . Furthermore, the number of colorless colonies on the CT-SSMAC medium was less than that on the sorbitol MacConkey medium containing cefixime and tellurite (CT-SMAC medium) from several raw vegetable samples . All colorless colonies grown on CT-SSMAC medium from raw vegetable samples were beta-galactosidase-negative . These findings suggest that the CT-SSMAC medium is useful for the isolation of E . coli O157:H7 from raw vegetable samples.

J Med Microbiol, 2002 Apr, 51(4), 336 - 43
Experimental infection of germ-free mice with hyper-toxigenic enterohaemorrhagic Escherichia coli O157:H7, strain 6; Taguchi H et al.; A mouse enterohaemorrhagic Escherichia coli (EHEC) infection model was developed with a combination of germ-free (GF) mice and hyper-toxigenic EHEC (HT-EHEC) O157:H7 strain 6 . The HT-EHEC strain 6 produced both Shiga-like toxin (SLT)-1 1.0 microg/mI and SLT-2 8.2 microg/ml in vitro . Eight-week-old germ-free mice were inoculated intragastrically with 5.0 x 10(7) cfu of HT-EHEC strain 6 . All GF mice challenged with the HT-EHEC developed ruffled fur and convulsion of limbs or hindleg weakness within 3 days after the challenge, culminating in death within 5 days . The HT-EHEC colonised well at a level of 10(8) - 10(9) cfu/g of faeces 5 days after the challenge . Both SLT-1 and SLT-2 were demonstrated in the faeces of the mice for 5 days after challenge . Histological examination of the colons of the infected mice showed congestion of the lamina propria mucosa, mild inflammatory cell infiltration and goblet cell depletion . In proximal tubules of the renal cortex, epithelial swelling with scattered necrotic cells was recognised . Endothelial swelling and mononuclear cell infiltration were also observed in the glomeruli . The brain showed acute neuronal necrosis in the cerebrum and slight loss of Purkinje cells in the cerebellum . Passive immunisation with anti-SLT antisera prolonged the life of the mice without anyneural symptoms . Microscopically, all tissue specimens from the passively immunised mice were not remarkable . These results indicate that the infection of GF mice with HT-EHEC is a useful animal model to study the pathogenicity of SLT-producing E . coliand the toxins.

J Med Microbiol, 2002 Apr, 51(4), 332 - 5
Isolation and characterisation of Shiga toxigenic Escherichia coli strains from northern Palestine; Adwan K et al.; Shiga toxigenic Escherichia coli (STEC) isolates from symptomatic and asymptomatic patients in northern Palestine in 1999 were screened for serotype O157 and characterised for virulence genes by multiplex PCR assay . Of the 176 STEC isolates, 124 (70.5%) were of serotype O157 . All these isolates carried the gene for Shiga toxin type 1 (stx,) and 112 (90.3%) carried stx2 . The intimin encoding gene locus eae was detected in 16 isolates (12.9%) and the enterohaemolysin encoding gene, hlyA, in 18 (14.5%) . Statistical analysis showed a significant association between the presence of eaeA and hlyA, either alone or combined with stx1 and stx2 genes in O157 isolates from symptomatic infection . ERIC-PCR analysis of DNA from 80 serotype O157 isolates revealed three major clonal populations.

J Clin Microbiol, 2002 Apr, 40(4), 1530 - 3
Four strains of Escherichia coli O157:H7 isolated from patients during an outbreak of disease associated with ground beef: importance of evaluating multiple colonies from an outbreak-associated product; Proctor ME et al.; This report describes the investigation of a ground-beef-associated outbreak that involved five genetically distinct patient strains of Escherichia coli O157:H7 . Human and product isolates were evaluated by pulsed-field gel electrophoresis (PFGE) with two endonucleases . The multiple-strain etiology of this outbreak underscores the importance of isolating and evaluating multiple colonies from outbreak-related products and comparing two endonuclease PFGE patterns of all product and human isolates identified during outbreak periods . This investigation emphasizes the importance of interviewing all confirmed and suspected case patients during the outbreak period, regardless of the PFGE pattern of their isolate, to confirm or rule out an epidemiologic link to the outbreak.

J Clin Microbiol, 2002 Apr, 40(4), 1152 - 9
Polymorphic amplified typing sequences provide a novel approach to Escherichia coli O157:H7 strain typing; Kudva IT et al.; Escherichia coli O157:H7 (O157) strains are commonly typed by pulsed-field gel electrophoresis (PFGE) following digestion of genomic DNA with the restriction enzyme XbaI . We have shown that O157 strains differ from each other by a series of discrete insertions or deletions, some of which contain recognition sites for XbaI, suggesting that these insertions and deletions are responsible for the differences in PFGE patterns . We have devised a new O157 strain typing protocol, polymorphic amplified typing sequences (PATS), based on this information . We designed PCR primer pairs to amplify genomic DNA flanking each of 40 individual XbaI sites in the genomes of two O157 reference strains . These primer pairs were tested with 44 O157 isolates, 2 each from 22 different outbreaks of infection . Thirty-two primer pairs amplified identical fragments from all 44 isolates, while eight primer pairs amplified regions that were polymorphic between isolates . The isolates could be differentiated solely on the basis of which of the eight polymorphic amplicons was detected . PATS correctly identified 21 of 22 outbreak pairs as being identical or highly related, whereas PFGE correctly identified 14 of the 22 outbreak pairs as being identical or highly related; PATS was also able to type isolates from three outbreaks that were untypeable by PFGE . However, PATS was less sensitive than PFGE in discriminating between outbreaks . These data suggest that typing by PATS may provide a simple procedure for strain typing of O157 and other bacteria and that further evaluation of the utility of this method for epidemiologic investigations is warranted.

Kansenshogaku Zasshi, 2002 Feb, 76(2), 96 - 101
{Molecular epidemiological investigation of vero toxin-producing Escherichia coli (VTEC) isolated from diarrhea patients and dairy cattle}; Sakata S et al.; To elucidate the source and route of VTEC infection, we performed pulse field gel electrophoresis (PFGE) an 50 isolates from human diarrhea typed as serotypes O157, O111, and O26, which were very frequently isolated from patients with VTEC infection between 1986 and 1997, and 32 isolates from dairy cattle, a total of 82 isolates . The isolates were genetically analyzed based on the electrophoresis patterns of DNA, and a phylogenetic tree was prepared . The isolates were classified based on similarity > or = 89 . The following results of the molecular epidemiological investigation were obtained . 1) Based on the electrophoresis patterns of DNA obtained by PFGE, 34 of the 49 O157 isolates (69.4%) were divided into groups 1-9, 15 of the 18 O111 isolates (83.3%) were divided into groups 1-3, and 12 of the 15 O26 isolates (80%) were divided into groups 1-3 . Of the grouped isolates, group 8 of O157, groups 2 and 3 of O111, and group 3 of O26 included isolates from human diarrhea and dairy cattle, but the other groups included isolates from only one of the two sources . 2) With regard to regional investigation, groups 6 and 9 of O157 included human diarrhea-derived isolates from Yokohama and Ehime, and group 8 included a human diarrhea-derived isolate from Yokohama and a dairy cattle-derived isolate from Tokushima . Group 3 of O111 included a human diarrhea-derived isolate from Ehime and a dairy cattle-derived isolate from Hokkaido . Group 3 of O26 included human diarrhea-derived isolate from Ehime and dairy cattle-derived isolate from Sagamihara and Hokkaido . Since the above findings showed that although the frequency was low, isolates from human diarrhea and dairy cattle were included in the same groups, it was demonstrated that dairy cattle are closely related to the human infectious disease of the intestinal tract as a source of infection . However, classification using the PFGE method is difficult due to diversity of the electrophoresis pattern of DNA . It is necessary to investigate the classification by a combination of the PFGE method with phage typing, ribotyping, and RAPD-PCR, and to investigate more numbers of patient-derived and animal-derived isolates.

FEBS Lett, 2002 Mar 6, 514(1), 84 - 9
Tandem termination signals: myth or reality?
Major LL, Edgar TD, Yee Yip P, Isaksson LA, Tate WP.
In two Escherichia coli genomes, laboratory strain K-12 and pathological strain O157:H7, tandem termination codons as a group are slightly over-represented as termination signals . Individually however, they span the range of representations, over, as expected, or under, in one or both of the strains . In vivo, tandem termination codons do not make more efficient signals . The second codon can act as a backstop where readthrough of the first has occurred, but not at the expected efficiency . UGAUGA remains an enigma, highly over-represented, but with the second UGA a relatively inefficient back up stop codon.

J Food Prot, 2002 Mar, 65(3), 465 - 70
Cross-contamination of lettuce with Escherichia coli O157:H7; Wachtel MR et al.; Contamination of produce by bacterial pathogens is an increasingly recognized problem . In March 1999, 72 patrons of a Nebraska restaurant were infected with enterohemorrhagic Escherichia coli (EHEC) O157:H7, and shredded iceberg lettuce was implicated as the food source . We simulated the restaurant's lettuce preparation procedure to determine the extent of possible EHEC cross-contamination and growth during handling . EHEC inoculation experiments were conducted to simulate the restaurant's cutting procedure and the subsequent storage of shredded lettuce in water in the refrigerator . All lettuce pieces were contaminated after 24 h of storage in inoculated water (2 x 10(9) CFU of EHEC per 3 liters of water) at room temperature or at 4 degrees C; EHEC levels associated with lettuce increased by > 1.5 logs on the second day of storage at 4 degrees C . All lettuce pieces were contaminated after 24 h of storage in water containing one inoculated lettuce piece (approximately 10(5) CFU of EHEC per lettuce piece) at both temperatures . The mixing of one inoculated dry lettuce piece with a large volume of dry lettuce, followed by storage at 4 degrees C or 25 degrees C for 20 h resulted in 100% contamination of the leaves tested . Microcolonies were observed on lettuce stored at 25 degrees C, while only single cells were seen on leaves stored at 4 degrees C, suggesting that bacterial growth had occurred at room temperature . Three water washes did not significantly decrease the number of contaminated leaves . Washing with 2,000 mg of calcium hypochlorite per liter significantly reduced the number of contaminated pieces but did not eliminate contamination on large numbers of leaves . Temperature abuse during storage at 25 degrees C for 20 h decreased the effectiveness of the calcium hypochlorite treatment, most likely because of bacterial growth during the storage period . These data indicate that storage of cut lettuce in water is not advisable and that strict attention must be paid to temperature control during the storage of cut lettuce.

J Food Prot, 2002 Mar, 65(3), 447 - 51
Inactivation of Escherichia coli O157:H7 on inoculated alfalfa seeds with ozonated water and heat treatment; Sharma RR et al.; Alfalfa seeds inoculated with a five-strain mixture of Escherichia coli O157:H7 were immersed in water containing 4, 8, 10, and 21 ppm of ozone for 2, 4, 8, 16, 32, and 64 min at 4 degrees C . Direct ozone sparging of seeds in water was used as an alternative mode of ozone treatment . Ozone-sparged seeds were also subsequently exposed to heat treatment at 40, 50, and 60 degrees C for 3 h . Populations of E . coli O157:H7 on untreated and treated seeds were determined by spread plating diluted samples on tryptic soy agar supplemented with 50 microg/ml of nalidixic acid . Since E . coli O157:H7 was released from inoculated seeds during treatment with ozone, the rate of release of cells from inoculated seeds soaked in 0.1% peptone water for up to 64 min was also determined . The overall reduction of E . coli O157:H7 on seeds treated with ozonated water without continuous sparging ranged from 0.40 to 1.75 log10 CFU/g (59.6 to 98.2%), whereas reductions for control seeds were 0.32 to 1.03 log10 CFU/g (51.7 to 90.5%) . Treatment with higher ozone concentrations enhanced inactivation, but contact time longer than 8 min did not result in significantly higher reductions (P > 0.05) . For seeds treated by ozone sparging, a 1.12-log10 CFU/g (92.1%) reduction was achieved using a 2-min contact time, and a 2.21-log10 CFU/g (99.4%) reduction was achieved with a 64-min contact time . The corresponding reductions for control seeds were 0.71 log10 CFU/g (79.5%) and 2.21 log10 CFU/g (99.4%), respectively . Treatment of ozone-sparged seeds at 60 degrees C for 3 h reduced the population to an undetectable level by direct plating (4 to 4.8 log10 CFU/g), although survivors were detected by enrichment . Ozone did not have a detrimental effect on seed germination percentage.

J Bacteriol, 2002 Apr, 184(7), 1873 - 9
Strains of Escherichia coli O157:H7 differ primarily by insertions or deletions, not single-nucleotide polymorphisms; Kudva IT et al.; Escherichia coli O157:H7 (O157) strains demonstrate varied pulsed-field gel electrophoresis patterns following XbaI digestion, which enable epidemiological surveillance of this important human pathogen . The genetic events underlying PFGE differences between strains, however, are not defined . We investigated the mechanisms for strain variation in O157 by recovering and examining nucleotide sequences flanking each of the XbaI restriction enzyme sites in the genome . Our analysis demonstrated that differences between O157 strains were due to discrete insertions or deletions that contained the XbaI sites polymorphic between strains rather than single-nucleotide polymorphisms in the XbaI sites themselves . These insertions and deletions were found to be uniquely localized within the regions of the genome that are specific to O157 compared to E . coli K-12 (O islands), suggesting that strain-to-strain variation occurs in these O islands . These results may be utilized to devise novel strain-typing tools for this pathogen.

J Clin Microbiol, 2002 Mar, 40(3), 959 - 64
Genetic profiling of enterohemorrhagic Escherichia coli strains in relation to clonality and clinical signs of infection; Welinder-Olsson C et al.; Sixty-seven human strains of enterohemorrhagic Escherichia coli (EHEC) (from patients with more or less severe symptoms) were serogrouped and arranged according to pulsed-field gel electrophoresis (PFGE) patterns . We used PCR to investigate the strains according to known or putative virulence factors, and associations with disease were studied . All EHEC strains with the same PFGE pattern belonged to the same serogroup . On the contrary, two serogroups (O157 and O8) included strains with different PFGE patterns . We found several different combinations of chromosomal and plasmid-borne determinants, encoding the putative virulence factors, among the strains . As judged from clinical symptoms, there was no marked difference in pathogenicity among the strains and their combinations of virulence traits . All strains of O157 had the genes coding for verocytotoxin (VT) 2, intimin (eaeA), E . coli hemolysin (E-hly), and secreted serine protease (espP) . Among EHEC non-O157 strains, the genes coding for VT1 and VT2 were equally dispersed . EaeA positivity was just as common among VT1- as VT2-positive strains . Among the plasmid-borne determinants, E-hly and espP were the most common and E-hly might be a pathogenicity marker among EHEC non-O157 strains . The conclusion is that PFGE is a very useful tool in epidemiological studies . The EHEC plasmids are heterogeneous in their gene composition, with the four plasmid-borne determinants found in many combinations . There was no reliable correlation between chromosomal and plasmid-borne virulence factors and human disease.

Clin Microbiol Infect, 1996 Mar, 1(3), 175 - 178
Screening for Escherichia coli O157:H7 in Illinois; Fischer SA et al.; OBJECTIVE: To determine the incidence of infection with Escherichia coli O157:H7 in a tertiary referral center in Chicago, where a similar study had been performed in 1984, to evaluate cases of disease reported to the Illinois Department of Public Health (IDPH) in 1993, and to determine laboratory practices used to detect this infection throughout the state . METHODS: During a 6-month period in 1993, all stool specimens at Rush-Presbyterian-St Luke's Medical Center (RPSLMC) were tested for E . coli O157:H7 . Reports of diagnosed E . coli O157:H7 cases investigated by IDPH were also reviewed . A survey of 73 hospitals in the Chicago area was performed to determine routine culturing practices, specifically, the selection of stool specimens for evaluation for this pathogen . RESULTS: In the RPSLMC survey, two cases were identified among 1985 samples (incidence 0.1%), similar to the 0.08% incidence detected in a similar study conducted at the same institution in 1984 . Through passive surveillance, the IDPH received 44 reports of E . coli O157:H7 in 1993 . The hospital survey revealed that, in the seven labs testing all stool specimens for E . coli O157:H7, an incidence of 16/8137 specimens (0.2%) was determined . CONCLUSIONS: These data suggest that sporadic E . coli O157:H7 remains uncommon in Illinois and that the incidence may not have changed over a 9-year period . The low yield and substantial cost of culturing all stools suggest that only specimens from patients with bloody diarrhea should be evaluated routinely in areas of low endemicity.

J Med Microbiol, 2002 Feb, 51(2), 143 - 9
Expression of receptors for verotoxin 1 from Escherichia coli O157 on bovine intestinal epithelium; Hoey DE et al.; Human enterohaemorrhagic Escherichia coli (EHEC) infection most commonly arises, either directly or indirectly, from cattle, which act as a reservoir host for these bacteria . In man, EHEC disease can be severe, whereas EHEC do not normally cause disease in cattle . Verotoxins (VTs) are the main virulence factors in human disease but no role for VT has been ascribed in cattle; however, this study shows for the first time that VT receptor is expressed by the bovine intestinal tract . VT bound to crypt epithelial cells of the small (ileum and jejunum) and large (caecum and colon) intestine independently of the animals' age . VT also bound to discrete cell subsets in the bovine kidney and to submucosal lymphoid cells but not to vasculature . Analysis of tissues for isoforms of the VT receptor, Gb3, confirmed the presence of the receptor in the bovine intestinal epithelium and kidney . A distinct pattern of Gb3 receptor isoform mixtures was observed in the bovine kidney . This, together with the general absence of receptors on vasculature, could contribute to the apparent resistance of cattle to systemic effects of VT . Expression of Gb3 on the bovine intestinal epithelium, together with previously described effects, may affect EHEC colonisation in its reservoir hosts and hence the potential for distribution to man.

Clin Microbiol Infect, 1997 Feb, 3(1), 117 - 119
Direct detection of verotoxin genes in stool samples by polymerase chain reaction in hemolytic uremic syndrome patients in France; Mariani-Kurkdjian P et al.; OBJECTIVE: To reassess the occurrence of verocytotoxin-producing Escherichia coli (VTEC) in French hemolytic uremic syndrome (HUS) patients . METHODS: From March 1991 to January 1995, direct detection of verotoxin genes (VT) by the polymerase chain reaction (PCR) was performed on stool samples from 169 patients suffering from HUS . RESULTS: Fifty-one were PCR positive (30.1%); one was positive for the VT1 gene and the others for the VT2 gene . VTEC was isolated from only 32 of the 51 PCR-positive samples . E . coli O157:H7 was isolated from five patients . E . coli O111 was isolated from seven patients during an outbreak of HUS . Among the other VT2 E . coli strains, only four were serotypable . Of the 51 PCR-positive stools, 19 were culture negative for VTEC . CONCLUSIONS: This study provides evidence that in France E . coli O157 and other VTEC serotypes are involved in HUS.

Zhonghua Liu Xing Bing Xue Za Zhi, 2000 Dec, 21(6), 410 - 2
{Using multiplex PCR for the detection of virulence genes in Escherichia coli O157:H7}; Guo X et al.; OBJECTIVE: To detect and characterize the virulence genes in E . coli O157:H7 isolated from various reservoir in six areas of Jiangsu province . METHOD: The virulence genes of Shiga-like toxin (SLT(1) and SLT(2)), intimin (eaeA) and hemolysin (hlyA) were chosen as the target genes and amplified in multiplex PCR assays . RESULTS: Of the eighty-five E . coli O157:H7 strains, the overall virulence gene prevalence was found to be 56.5% (48/85) . The prevalence rates virulence genes of isolates from various areas were different from 0% up to 90.5% . It seemed to exist a relationship between the virulence gene prevalence and the level of incidence . In the areas where rates of incidence were divided into high, low, sporadic or zero, the prevalence rates were 85.7% (36/42), 52.6% (10/19) and 8.3% (2/24), respectively . The prevalence rates of isolates were also different from various reservoirs, decreasing by sheep, cattle, pig and poultry . One isolate from a rabbit was positive for SLT(2), eaeA and hly genes . Of forty-eight isolates carrying virulence genes, 38 (79.2%) had SLT(2), eaeA and hly genes, taking the dominate virulence gene pattern, 8 (16.6%) had all of the four virulence genes 2 (4.2%) had both SLT(2) and hly genes respectively . In addition, SLT(1) gene showed a lower prevalence, which was different from some findings abroad . CONCLUSION: Since virulence gene pattern of E . coli O157:H7 is an important molecular epidemiological marker, it can provide an useful information for epidemiologic studies, and helpful to the design of prevention and control strategies . For virulence gene detection, multiplex PCR seems to be a simple, rapid, specific and sensitive method.

J Appl Microbiol, 2001 Dec, 91(6), 1004 - 10
Detection of Escherichia coli O157:H7 in soil and water using multiplex PCR; Campbell GR et al.; AIMS: To evaluate the suitability of a multiplex PCR-based assay for sensitive and rapid detection of Escherichia coli O157:H7 in soil and water . METHODS AND RESULTS: Soil and water samples were spiked with E . coli O157:H7 and subjected to two stages of enrichment prior to multiplex PCR . Detection sensitivities were as high as 1 cfu ml(-1) drinking water and 2 cfu g(-1) soil . Starvation of E . coli O157:H7 for 35 d prior to addition to soil did not affect the ability of the assay to detect initial cell numbers as low as 10 cfu g(-1) soil . Use of an 8-h primary enrichment enabled detection of as few as 6 cfu g(-1) soil, and 10(4) cfu g(-1) soil with a 6-h primary enrichment . When soil was inoculated with 10(5) cfu g(-1), the PCR assay indicated persistence of E . coli O157:H7 during a 35 d incubation . However, when soil was inoculated with lower numbers of pathogen, PCR amplification signals indicated survival to be dependent on cell concentration . CONCLUSIONS: A multiplex PCR-based assay, in combination with an enrichment strategy enabled sensitive and rapid detection of E . coli O157:H7 in soil and water . SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to sensitively detect E.coli O157:H7 in environmental material within one working day represents a considerable advancement over alternative more time-consuming methods for detection of this pathogen.

Lett Appl Microbiol, 2002, 34(2), 139 - 43
The ineffectiveness of organic acids, freezing and pulsed electric fields to control Escherichia coli O157:H7 in beef burgers; Bolton DJ et al.; AIMS: The objective of this study was to investigate the potential value of individual and combined applications of some GRAS (generally regarded as safe) additives with freezing and pulsed electric field (PEF) application, in reducing the risks associated with the presence of E . coli O157:H7 in beef burgers . METHODS AND RESULTS: Beef burgers, trimmings and filter paper were inoculated with E . coli O157:H7 and subjected to a range of chemical and physical treatments . Sequential application of 2% (v/v) lactic acid and freezing (at - 20 degrees C for 2 h) resulted in a decrease of approximately 6 log10 cfu cm(-1) in E . coli O157:H7, but only on filter paper . All other treatments were ineffective . CONCLUSIONS: Currently available methods for controlling E.coli O157:H7 in beef burgers during production are ineffective . SIGNIFICANCE AND IMPACT OF THE STUDY: Further research is needed to develop controls for E . coli O157:H7 during beef burger production.

J Food Prot, 2002 Feb, 65(2), 408 - 10
Synergistic effect of nisin and heat treatment on the growth of Escherichia coli O157:H7; Lee JI et al.; A combination of nisin and heat treatment was found to inhibit Escherichia coli O157:H7 effectively . After organisms were heated at 50, 52.5, and 55 degrees C for 5, 10, and 15 min, respectively, nisin was incorporated into the plates of E . coli O157:H7 at 0, 25, 50, and 100 IU/ml . The concentration of 100 IU/ml nisin significantly inhibited the growth of E . coli O157:H7 heated at 50 and 52.5 degrees C for 15 min . Nisin treatment at 100 IU/ml for 6 h resulted in the elimination of E . coli O157:H7 heated at 55 degrees C for 10 and 15 min.

J Food Prot, 2002 Feb, 65(2), 260 - 5
Influence of acid adaptation on the tolerance of Escherichia coli O157:H7 to some subsequent stresses; Cheng HY et al.; Three stains of Escherichia coli O157:H7, including ATCC 43889, ATCC 43895, and 933, were first subjected to acid adaptation at a pH of 5.0 for 4 h . Thermal tolerance at 52 degrees C and survival of the acid-adapted as well as the nonadapted cells of E . coli O157:H7 in the presence of 10% sodium chloride, 0.85% bile salt, or 15.0% ethanol were investigated . Results showed that the effect of acid adaptation on the survival of E . coli O157:H7 varied with the strains and types of subsequent stress . Acid adaptation caused an increase in the thermal tolerance of E . coli O157:H7 ATCC 43889 and ATCC 43895, but no significant difference in the thermal tolerance was noted between acid-adapted and nonadapted cells of E . coli O157:H7 933 . Although the magnitude of increase varied with strains of test organisms, acid adaptation generally led to an increase in the tolerance of E . coli O157:H7 to sodium chloride . On the other hand, the susceptibility of acid-adapted cells of the three strains of E . coli O157:H7 tested did not show a significant difference from that of their nonadapted counterparts when stressed with bile salt . The acid-adapted cells of E . coli O157:H7 ATCC 43889 and ATCC 43895 were less tolerant than the nonadapted cells to ethanol, whereas the tolerance of adapted and nonadapted cells of E . coli O157:H7 933 showed no significant differences.

J Food Prot, 2002 Feb, 65(2), 251 - 9
Differentiation of viable and dead Escherichia coli O157:H7 cells on and in apple structures and tissues following chlorine treatment; Burnett SL et al.; Confocal scanning laser microscopy (CSLM) was used to differentiate viable and nonviable cells of Escherichia coli O157:H7 on and in raw apple tissues following treatment with water and 200 or 2,000 ppm active chlorine solution . Whole unwaxed Red Delicious cultivar apples at 25 degrees C were inoculated by dipping in a suspension of E . coli O157:H7 (8.48 log10 CFU/ml) at 4 degrees C, followed by treatment in water or chlorine solution at 21 degrees C for 2 min . The dead cells on and in apples were distinguished from live cells by treating tissue samples with SYTOX green nucleic acid stain . Viable and dead cells were then labeled with an antibody conjugated with a fluorescent dye (Alexa Fluor 594) . The percentage of viable cells on the apple surface, as well as at various depths in surface and internal structures, was determined . The mean percentages of viable cells located at the sites after treatment with water or chlorinated water were in the following order, which also reflects the order of protection against inactivation: floral tube wall (20.5%) > lenticels (15.0%) > damaged cuticle surrounding puncture wounds (13.0%) > intact cuticle (8.1%) . The location of viable cells within tissues was dependent on the structure . Except for lenticels, the percentage of viable cells increased as depth into the CSLM stacks increased, indicating that cells attached to subsurface structures were better protected against inactivation with chlorine than were cells located on exposed surfaces . Further research is warranted to investigate the efficacy of other chemical sanitizers . as well as that of surfactants and solvents in combination with sanitizers, in removing or killing E . coli O157:H7 lodgedin protective structures on the surface and within tissues of apples.

Comp Immunol Microbiol Infect Dis, 2002 Mar, 25(2), 77 - 84
Serotypes of non-O157 verocytotoxigenic Escherichia coli isolated from meat in New Zealand; Bennett J et al.; Verocytotoxigenic Escherichia coli (VTEC) were isolated from meat in New Zealand . They were tested for the presence of virulence factors associated with VTEC and serotyped . Some of the serotypes found were identical to ones reported from other parts of the world, but some serotypes were also found which had not been reported elsewhere . This study confirms the world-wide distribution of these emerging food-borne pathogens.

Biomed Chromatogr, 2002 Feb, 16(1), 41 - 6
Detection of two variant Vero toxin genes in Escherichia coli by capillary electrophoresis; Arakawa H et al.; Three methods {capillary electrophoresis (CE)-allele-specific PCR, CE-single-strand conformation polymorphism (SSCP) and CE-cleavase fragment length polymorphism (CFLP)} were developed in order to effect rapid and specific analysis of the vero toxin (VT)1 and VT2 genes of O157 . The allele-specific polymerase chain reaction (PCR) method, which utilized specific duplex PCR with specific primers for VT1 and VT2, showed that VT1 and VT2 consisted of 174 and 128 bp, respectively . Subsequent CE analysis was carried out . Separation time was 4 min . SSCP, which utilized one primer set which reacted with both VT1 and VT2 in the PCR method, was followed by CE analysis of secondary structure of single-strand DNA . Two genes could be analyzed in approximately 18 min . CFLP, like SSCP, is a method for detecting mutation-induced changes in secondary structure of single-stranded DNA . The endonuclease cleavase I recognizes and cleaves the 5' side of hairpin loops in self-annealed single-strand DNA of PCR product 169 bp obtained from VT1 and VT2 . The produced DNA fragments are analyzed by CE and the electrophelogram reveals a sequence-specific CFLP . Separation time was 6 min . These techniques are suitable for the detection and the identification of O157 .

Epidemiol Infect, 2001 Dec, 127(3), 413 - 20
Monitoring patients in the community with suspected Escherichia coli O157 infection during a large outbreak in Scotland in 1996; Wood R et al.; During outbreaks of Escherichia coli O157 a minority of patients with suspected infection develop haemolytic uraemic syndrome (HUS) . The ability to identify this subgroup at an early stage is beneficial as mortality from HUS is high and may be influenced by intervention . During the 1996 Central Scotland E . coli O157 outbreak, of 886 patients from the community with suspected infection monitored at an outbreak clinic, nine developed HUS . We assessed factors associated with the development of HUS in this group . Children and the elderly were at increased risk of HUS . However, high white cell count was as least as good a predictor of HUS as age . High white cell counts predicted development of HUS with a sensitivity of 89%, specificity of 87%, positive predictive value of 7% and a negative predictive value of over 99% . We have used the results from this study along with other currently available evidence to propose a monitoring protocol for patients from the community with suspected E . coli O157 infection.

J Food Prot, 2002 Jan, 65(1), 66 - 72
Economic impact of an Escherichia coli O157:H7 outbreak in Japan; Abe K et al.; We estimated the economic impact of an outbreak of foodborne diseases occurring from elementary school lunches in 1996 in which 268 persons in Iwate prefecture, Japan were infected with Escherichia coli O157:H7 . This study assessed the impact of direct economic losses and indirect economic consequences due to this outbreak . The economic impact of the outbreak was estimated to be about 82,686,000 yen . The laboratory costs, about 21,204,000 yen, showed the highest ratio of the total cost of this outbreak (about 26%) . Also, the cost of foodstuffs that were not purchased during the suspension of the lunch service (about 19%), personnel expenses paid to lunch service employees (about 17%), human illness costs (about 15%), and the repair costs of facilities (about 15%) showed up as a high ratio in the total cost, respectively . Because all patients were children, the productivity losses estimated were low as children were considered as dependants with no income . Instead, we estimated the lost income of the mothers of the children . The source of the contamination could not be identified . Therefore, no food industries suffered any setbacks where certain food items could not be used for daily consumption due to the outbreak.

J Food Prot, 2002 Jan, 65(1), 5 - 11
Polymerase chain reaction assay for detection of Escherichia coli O157: H7 and Escherichia coli O157: H-; Miyamoto T et al.; The DNA band patterns generated by polymerase chain reaction (PCR) using the du2 primer and template DNAs from various strains of Escherichia coli and non-E . coli bacteria were compared . Among three to five prominent bands produced, the three bands at about 1.8, 2.7, and 5.0 kb were detected in all of the E . coli O157 strains tested . Some nonpathogenic E . coli and all pathogenic E . coli except E . coli O157 showed bands at 1.8 and 5.0 kb . It seems that the band at 2.7 kb is specific to E . coli O157 . Sequence analysis of the 2.7-kb PCR product revealed the presence of a DNA sequence specific to E . coli O157:H- and E . coli O157:H7 . Since the DNA sequence from base 15 to base 1,008 of the PCR product seems to be specific to E . coli O157, a PCR assay was carried out with various bacterial genomic DNAs and O157-FHC1 and O157-FHC2 primers that amplified the region between base 23 and base 994 of the 2.7-kb PCR product . A single band at 970 bp was clearly detected in all of the strains of E . coli O157:H- and E . coli O157:H7 tested . However, no band was amplified from template DNAs from other bacteria, including both nonpathogenic and pathogenic E . coli except E . coli O157 . All raw meats inoculated with E . coli O157:H7 at 3 x 10(0) to 3.5 x 10(2) CFU/25 g were positive both for our PCR assay after cultivation in mEC-N broth at 42 degrees C for 18 h and for the conventional cultural method.

J Food Prot, 2002 Jan, 65(1), 26 - 32
Effectiveness of spraying with tween 20 and lactic acid in decontaminating inoculated Escherichia coli O157:H7 and indigenous Escherichia coli biotype I on beef; Calicioglu M et al.; Beef carcass quarters and fat-covered subprimal cuts were suspended vertically and inoculated with a bovine manure slurry containing a five-strain mixture of Escherichia coli O157:H7 to deliver about 4 to 5 log10 CFU/cm2 . To identify treatments that would improve the effectiveness of spraying with lactic acid (LA), the inoculated quarters and cuts were treated as follows: experiment A, (i) not treated (control), (ii) sprayed with 2% (vol/vol) LA, (iii) tempered at 21 degrees C for 4 h, and (iv) tempered and then sprayed with LA; experiment B, (v) sprayed with water, (vi) sprayed with LA, (vii) sprayed with LA containing 0.5% (vol/vol) sodium benzoate (SB), and (viii) sprayed with LA containing SB and 5% (vol/vol) Tween 20 (TW20); and experiment C, (ix) sprayed with water (no prespray), (x) presprayed with TW20 and then sprayed with LA, and (xi) presprayed with TW20 and then sprayed with LA containing SB . In experiment A, spraying carcasses with LA significantly (P < 0.05) reduced the numbers of E . coli Biotype I and serotype O157:H7 after 1 and 3 days of storage, respectively . The tempering process employed did not affect the effectiveness of the LA spray on either type of E . coli . In experiment B, there was no significant difference in the reduction of E . coli O157:H7 on subprimal cuts sprayed with water and that on cuts sprayed with LA alone or with LA in combination with SB and TW20 after 1 or 3 days of storage (total reductions ranged from about 1.6 to 2.8 log10 CFU/cm2) . In experiment C, prespraying subprimal cuts with TW20 significantly (P < 0.05) increased the effectiveness of LA (reductions of 2.8 and 3.2 log10 CFU/cm2, respectively) and that of LA with SB (reductions of 2.6 and 3.3 log10 CFU/cm2, respectively) compared with spraying with water alone (reductions of ca . 1.0 and 2.0 log10 CFU/cm2, respectively) after I and 3 days of storage, respectively . In a separate experiment, the incorporation of TW20 (0.1 or 0.25%) into buffered peptone water prior to the maceration of excised carcass surface samples resulted in the recovery of significantly larger numbers (ca . 5.1 to 5.2 log10 CFU/cm2) of E . coli O157:H7 cells than did the control treatment without added TW20 (ca . 3.8 to 4.6 log10 CFU/cm2) . These results demonstrate that the treatment of beef carcasses with LA reduces the number of viable E . coli O157:H7 cells and that this inactivation or removal by LA is enhanced by prespraying of the carcass with a 5% solution of TW20.

J Food Prot, 2002 Jan, 65(1), 18 - 25
Association of Escherichia coli O157:H7 with preharvest leaf lettuce upon exposure to contaminated irrigation water; Wachtel MR et al.; Recent foodborne outbreaks have linked infection by enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 to the consumption of contaminated lettuce . Contamination via food handler error and on-the-farm contamination are thought to be responsible for several outbreaks . Though recent studies have examined the application of EHEC to store-bought lettuce, little is known about the attachment of EHEC to growing plants . We investigated the association of lettuce seedlings with EHEC O157:H7 strains implicated in lettuce or fruit outbreaks using hydroponic and soil model systems . EHEC strains that express the green fluorescent protein were observed by stereomicroscopy and confocal laser scanning microscopy to determine adherence patterns on growing lettuce seedlings . Bacteria adhered preferentially to plant roots in both model systems and to seed coats in the hydroponic system . Two of five nonpathogenic E . coli strains showed decreased adherence to seedling roots in the hydroponic system . EHEC was associated with plants in as few as 3 days in soil, and contamination levels were dose-dependent . EHEC levels associated with young plants inoculated with a low dose suggested that the bacteria had multiplied . These data suggest that preharvest crop contamination via contaminated irrigation water can occur through plant roots.

J Infect Dis, 2002 Feb 1, 185(3), 393 - 6 Epub 2002 Jan 08.
Blood group and susceptibility to disease caused by Escherichia coli O157; Blackwell CC et al.; Patients (n=186) infected during the Escherichia coli O157 outbreak in Scotland in 1996 were assessed for blood group markers (ABO, Lewis, and P) associated with other gastrointestinal infections . Binding of bacteria to epithelial cells was assessed by flow cytometry . Buffy coats from blood donors were examined for inflammatory responses to culture filtrates of the outbreak strain . Individuals of blood group O comprised 63.4% of patients, compared with 53.4% (P <.05) and 53.9% (P <.01) of neighboring populations in Airdrie and Glasgow, respectively; group O also comprised 64.3% of patients with hemolytic uremic syndrome (HUS) and 87.5% of patients who died (P <.05) . No or weak agglutination by anti-P antiserum was observed for 40.7% of control persons (n=122), 61.5% of all patients (P =.0027), and 83.3% of patients with HUS (P =.013) . The susceptibility of group O to E . coli was not associated with increased binding of bacteria to epithelial cells or with higher production of tumor necrosis factor (TNF)-alpha or interleukin-6 . Leukocytes of P-negative blood donors produced higher levels of TNF-alpha than those of P-positive donors.

J Infect Dis, 2002 Jan 15, 185(2), 214 - 9 Epub 2002 Jan 03.
ABO and P1 blood group antigen expression and stx genotype and outcome of childhood Escherichia coli O157:H7 infections; Jelacic S et al.; P1 and ABO antigens and bacterial stx genotypes might influence the risk of developing hemolytic uremic syndrome (HUS) after Escherichia coli O157:H7 infections . We determined ABO status and P1 antigen expression in 130 infected and 17 uninfected children, and we determined the stx genotype of the infecting isolate . P1 expression was weakly and directly related to HUS risk (P=.04), but this risk did not extend to the group with the greatest P1 expression . P1 expression remained constant as HUS evolved . The ABO frequency was similar in all groups . These associations were not affected by the stx genotype . stx1(-)/stx2(+) E . coli O157:H7 strains were more commonly associated with HUS than were stx1(+)/stx2(+) strains (P=.21), and 1 child with HUS was infected with a rare stx1(+)/stx2(-) isolate . In the present study, ABO antigens and stx genotypes were not major determinants of the outcome of E . coli O157:H7 infections, and P1 expression did not protect against the development of HUS.

Infect Immun, 2002 Feb, 70(2), 945 - 52
Intimin, tir, and shiga toxin 1 do not influence enteropathogenic responses to shiga toxin-producing Escherichia coli in bovine ligated intestinal loops; Stevens MP et al.; Shiga toxin-producing Escherchia coli (STEC) comprises a group of attaching and effacing (A/E) enteric pathogens of animals and humans . Natural and experimental infection of calves with STEC may result in acute enteritis or subclinical infection, depending on serotype- and host-specific factors . To quantify intestinal secretory and inflammatory responses to STEC in the bovine intestine, serotypes that are associated with human disease (O103:H2 and O157:H7) were introduced into ligated mid-ileal loops in gnotobiotic and conventional calves, and fluid accumulation and recruitment of radiolabeled neutrophils were measured after 12 h . STEC serotype O103:H2, but not serotype O157:H7, elicited strong enteropathogenic responses . To determine if the inflammatory response to STEC O103:H2 in calves requires Shiga toxin 1 or intimate bacterial attachment to the intestinal epithelium, defined mutations were made in the stx1, eae, and tir genes . Our data indicate that some STEC induce intestinal inflammatory responses in calves by a mechanism that is independent of A/E-lesion formation, intimin, or Shiga toxin 1 . This may have implications for strategies to reduce STEC carriage in cattle.

Infect Immun, 2002 Feb, 70(2), 612 - 9
Human Stx2-specific monoclonal antibodies prevent systemic complications of Escherichia coli O157:H7 infection; Mukherjee J et al.; Hemolytic-uremic syndrome (HUS) is a serious complication predominantly associated with infection by enterohemorrhagic Escherichia coli (EHEC), such as E . coli O157:H7 . EHEC can produce Shiga toxin 1 (Stx1) and/or Shiga toxin 2 (Stx2), both of which are exotoxins comprised of active (A) and binding (B) subunits . In piglets and mice, Stx can induce fatal neurological symptoms . Polyclonal Stx2 antiserum can prevent these effects in piglets infected with the Stx2-producing E . coli O157:H7 strain 86-24 . Human monoclonal antibodies (HuMAbs) against Stx2 were developed as potential passive immunotherapeutic reagents for the prevention and/or treatment of HUS . Transgenic mice bearing unrearranged human immunoglobulin (Ig) heavy and kappa light chain loci (HuMAb___Mouse) were immunized with formalin-inactivated Stx2 . Thirty-seven stable hybridomas secreting Stx2-specific HuMAbs were isolated: 33 IgG1kappa A-subunit-specific and 3 IgG1kappa and 1 IgG3kappa B-subunit-specific antibodies . Six IgG1kappa A-subunit-specific (1G3, 2F10, 3E9, 4H9, 5A4, and 5C12) and two IgG1kappa B-subunit-specific (5H8 and 6G3) HuMAbs demonstrated neutralization of > 95% activity of 1 ng of Stx2 in the presence of 0.04 microg of HuMAb in vitro and significant prolongation of survival of mice given 50 microg of HuMAb intraperitoneally (i.p.) and 25 ng of Stx2 intravenously . When administered i.p . to gnotobiotic piglets 6 or 12 h after infection with E . coli O157:H7 strain 86-24, HuMAbs 2F10, 3E9, 5H8, and 5C12 prolonged survival and prevented development of fatal neurological signs and cerebral lesions . The Stx2-neutralizing ability of these HuMAbs could potentially be used clinically to passively protect against HUS development in individuals infected with Stx-producing bacteria, including E . coli O157:H7.

Int J Food Microbiol, 2001 Dec 30, 71(2-3), 125 - 30
Effect of oral sodium chlorate administration on Escherichia coli O157:H7 in the gut of experimentally infected pigs; Anderson RC et al.; Strategies are sought to reduce pathogenic Escherichia coli concentrations in food animals . Because E . coli possess respiratory nitrate reductase activity, which also reduces chlorate to cytotoxic chlorite, we tested and found that oral sodium chlorate administration reduced gut concentrations of E . coli O157:H7 in experimentally infected pigs and wildtype E . coli concentrations in nonchallenged pigs . Mean +/- S.E . concentrations (log10 CFU/g) of E . coli O157:H7 in ileal, cecal, colonic and rectal contents from placebo-treated pigs were 4.03 +/- 0.66, 3.82 +/- 0.24, 4.42 +/- 0.25 and 4.03 +/- 0.16, respectively . In contrast, E . coli O157:H7 concentrations were reduced (P < 0.05) in ileal (1.56 +/- 0.22) cecal (2.65 +/- 0.38), colonic (3.05 +/- 0.38) and rectal (3.00 +/- 0.29) contents from pigs orally administered three successive (8 h apart) 10-ml doses of 100 mM chlorate . Wildtype E . coli concentrations in gut contents of non-E . coli O157:H7-challenged pigs likewise treated with chlorate were reduced by 1.1 to 4.5 log10 units compared to concentrations in placebo-treated pigs, which exceeded 6.0 log10 CFU/g . As before, the reductions were greater in anterior regions of the gut than regions more caudal . Similar treatment of E . coli O157:H7-challenged pigs with 200 mM chlorate caused reductions in gut concentrations of E . coli O157:H7; however, the reductions were not necessarily greater than those achieved with the 100 mM chlorate treatment.

Gut, 2002 Feb, 50(2), 180 - 5
Intimin type influences the site of human intestinal mucosal colonisation by enterohaemorrhagic Escherichia coli O157:H7; Fitzhenry RJ et al.; BACKGROUND: Enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli epithelial cell adhesion is characterised by intimate attachment, and attaching and effacing (A/E) lesion formation . This event is mediated in part by intimin binding to another bacterial protein, Tir (translocated intimin receptor), which is exported by the bacteria and integrated into the host cell plasma membrane . Importantly, EPEC (O127:H6) and EHEC (O157:H7) express antigenically distinct intimin types known as intimin alpha and gamma, respectively . EHEC (O157:H7) colonises human intestinal explants although adhesion is restricted to the follicle associated epithelium of Peyer's patches . This phenotype is also observed with EPEC O127:H6 engineered to express EHEC intimin gamma . AIMS: To investigate the influence of intimin on colonisation of human intestine by E coli O157:H7, and intimin types on tissue tropism in humans . METHODS: Human intestinal in vitro organ culture with wild type and mutant strains of O157:H7 were employed . RESULTS: Introducing a deletion mutation in the eae gene encoding intimin gamma in EHEC (O157:H7) caused the strain (ICC170) to fail to colonise human intestinal explants . However, colonisation of Peyer's patches and A/E lesion formation were restored with intimin gamma expression from a plasmid (ICC170 (pICC55)) . In contrast, complementing the mutation with intimin alpha resulted in a strain (ICC170 (pCVD438)) capable of colonising and producing A/E lesions on both Peyer's patch and other small intestinal explants . CONCLUSION: Intimin is necessary for human intestinal mucosal colonisation by E coli O157:H7 . Intimin type influences the site of colonisation in a Tir type independent mechanism; intimin gamma appears to restrict colonisation to human follicle associated epithelium.

J Biol Chem, 2002 Mar 29, 277(13), 11247 - 54 Epub 2002 Jan 08.
Expression of the Gb3/CD77 synthase gene in megakaryoblastic leukemia cells: implication in the sensitivity to verotoxins; Furukawa K et al.; Expression levels of Gb3/CD77 synthase together with Gb3/CD77 antigen were analyzed using human hematopoietic tumor cell lines and normal cells . Among about 40 kinds of cells, Burkitt lymphoma cells showed the highest gene expression concomitant with the expression levels of Gb3/CD77 . Unexpectedly, megakaryoblastic leukemia lines also expressed fairly high levels of mRNA of Gb3/CD77 synthase and its product . A megakaryoblastic leukemia line, MEG-01 was sensitive to verotoxins from Escherichia coli O157 and apoptosis was induced via the caspase pathway . We also demonstrated that the cell surface Gb3/CD77 expression was reduced on differentiated MEG-01 although the mRNA level of the alpha1,4Gal-T gene increased . In this case, the localization of Gb3/CD77 was changed from the cell surface to the cytoplasm as stained with a granular pattern, co-localizing with platelet GPIIb-IIIa, indicating that some of them were platelet precursors . Small particles outside of cells also showed similar staining patterns . These results agreed with the previous report that platelets produced in mature megakaryoblasts abundantly contained Gb3/CD77 antigen . Here, we propose the possibility that verotoxins bind immature megakaryoblasts and induce their apoptosis, leading to the arrest of platelet generation in the bone marrow . This may be one of the causes of thrombocytopenia in patients with hemolytic uremic syndrome.

Clin Diagn Lab Immunol, 2002 Jan, 9(1), 46 - 53
Immunological characterization of Escherichia coli O157:H7 intimin gamma1; Son WG et al.; Portions of the intimin genes of Escherichia coli O157:H7 strain E319 and of the enteropathogenic E . coli O127:H6 strain E2348/69 were amplified by PCR and cloned into pET-28a+ expression vectors . The entire 934 amino acids (aa) of E . coli O157:H7 intimin, the C-terminal 306 aa of E . coli O157:H7 intimin, and the C-terminal 311 aa of E . coli O127:H6 intimin were expressed as proteins fused with a six-histidine residue tag (six-His tag) in pET-28a+ . Rabbit antisera raised against the six-His tag-full-length E . coli O157:H7 intimin protein fusion cross-reacted in slot and Western blots with outer membrane protein preparations from the majority of enterohemorrhagic and enteropathogenic E . coli serotypes which have the intimin gene . The E . coli strains tested included isolates from humans and animals which produce intimin types alpha (O serogroups 86, 127, and 142), beta1 (O serogroups 5, 26, 46, 69, 111, 126, and 128), gamma 1 (O serogroups 55, 145, and 157), gamma 2 (O serogroups 111 and 103), and epsilon (O serogroup 103) and a nontypeable intimin (O serogroup 80), results based on intimin type-specific PCR assays . Rabbit antisera raised against the E . coli O157:H7 C-terminal fusion protein were much more intimin type-specific than those raised against the full-length intimin fusion protein, but some cross-reaction with other intimin types was also observed for these antisera . In contrast, the monoclonal antibody Intgamma1.C11, raised against the C-terminal E . coli O157 intimin, reacted only with preparations from intimin gamma 1-producing E . coli strains such as E . coli O157:H7.

Appl Environ Microbiol, 2002 Jan, 68(1), 397 - 400
Transmission of Escherichia coli O157:H7 from contaminated manure and irrigation water to lettuce plant tissue and its subsequent internalization; Solomon EB et al.; The transmission of Escherichia coli O157:H7 from manure-contaminated soil and irrigation water to lettuce plants was demonstrated using laser scanning confocal microscopy, epifluorescence microscopy, and recovery of viable cells from the inner tissues of plants . E . coli O157:H7 migrated to internal locations in plant tissue and was thus protected from the action of sanitizing agents by virtue of its inaccessibility . Experiments demonstrate that E . coli O157:H7 can enter the lettuce plant through the root system and migrate throughout the edible portion of the plant.

J Food Prot, 2001 Dec, 64(12), 1922 - 8
A probability of growth model for Escherichia coli O157:H7 as a function of temperature, pH, acetic acid, and salt; McKellar RC et al.; Data accumulated on the growth of Escherichia coli O157:H7 in tryptic soy broth (TSB) were used to develop a logistic regression model describing the growth-no growth interface as a function of temperature, pH, salt, sucrose, and acetic acid . A fractional factorial design with five factors was used at the following levels: temperature (10 to 30 degrees C), acetic acid (0 to 4%), salt (0.5 to 16.5%), sucrose (0 to 8%), and pH (3.5 to 6.0) . A total of 1,820 treatment combinations were used to create the model, which correctly predicted 1,802 (99%) of the points, with 10 false positives and 8 false negatives . Concordance was 99.9%, discordance was 0.1%, and the maximum rescaled R2 value was 0.927 . Acetic acid was the factor having the most influence on the growth-no growth interface; addition of as little as 0.5% resulted in an increase in the observed minimum pH for growth from 4.0 to 5.5 . Increasing the salt concentration also had a significant effect on the interface; at all acetic acid concentrations, increasing salt increased the minimum temperature at which growth was observed . Using two literature data sets (26 conditions), the logistic model failed to predict growth in only one case . The results of this study suggest that the logistic regression model can be used to make conservative predictions of the growth-no growth interface of E . coli O157:H7.

J Food Prot, 2001 Dec, 64(12), 1899 - 903
Ecological relationships between the prevalence of cattle shedding Escherichia coli O157:H7 and characteristics of the cattle or conditions of the feedlot pen; Smith D et al.; This study was designed to describe the percentage of cattle shedding Escherichia coli O157:H7 in Midwestern U.S . feedlots and to discover relationships between the point prevalence of cattle shedding the organism and the characteristics of those cattle or the conditions of their pens . Cattle from 29 pens of five Midwestern feedlots were each sampled once between June and September 1999 . Feces were collected from the rectum of each animal in each pen . Concurrently, samples of water were collected from the water tank, and partially consumed feed was collected from the feedbunk of each pen . Characteristics of the cattle and conditions of each pen that might have affected the prevalence of cattle shedding E . coli O157:H7 were recorded . These factors included the number of cattle; the number of days on feed; and the average body weight, class, and sex of the cattle . In addition, the temperature and pH of the tank water were determined, and the cleanliness of the tank water and the condition of the pen floor were subjectively assessed . The samples of feces, feed, and water were tested for the presence of E . coli O157:H7 . E . coli O157:H7 was isolated from the feces of 719 of 3,162 cattle tested (23%), including at least one animal from each of the 29 pens . The percentage of cattle in a pen shedding E . coli O157:H7 did not differ between feedyards, but it did vary widely within feedyards . A higher prevalence of cattle shed E . coli O157:H7 from muddy pen conditions than cattle from pens in normal condition . The results of this study suggest that E . coli O157:H7 should be considered common to groups of feedlot cattle housed together in pens and that the condition of the pen floor may influence the prevalence of cattle shedding the organism.

Int J Food Microbiol, 2001 Dec 4, 71(1), 93 - 9
Factors influencing the detection and enumeration of Escherichia coli O157:H7 on alfalfa seeds; Wu FM et al.; Isolating Escherichia coli O157:H7 from batches of alfalfa seeds used to produce sprouts implicated in human illness has been difficult, perhaps due to nonhomogenous and very low-level contamination and inaccessibility of the pathogen entrapped in protected areas of the seed coat . We evaluated the effectiveness of various treatments in releasing E . coli O157:H7 from seeds . The influence of homogenization (blending or stomaching for 1 or 2 min), rinsing method (shaking for 5 min), soaking time (0 . 1, 3, 6, or 15 h), soaking temperature (4 or 21 degrees C), and the addition of surfactants (0.1%, 0.5%, or 1.0% Tween 80 or Span 20) to rinse water was determined . Blending or stomaching for 1 or 2 min, and soaking for 1 h or longer at 4 or 21 degrees C, respectively, resulted in maximum release of E . coli O157:H7 from seeds . Soaking seeds at 37 degrees C for 15 h increased cell populations of E . coli O157:H7 by approximately 3.6 log10 CFU/g, likely due to bacterial growth . The maximum number of cells released from seeds by rinse water containing 1.0% Span 20 was at 21 degrees C, whereas at 37 degrees C, 0.1% or 0.5% Tween 80 was more effective . Detection of E . coli O157:H7 on seeds stored at 37 degrees C for up to 13 weeks and on sprouts derived from these seeds was compared . E . coli O157:H7 inoculated on seeds at 2.0 log10 CFU/g was detected after storage of seeds for up to 8 weeks at 37 degrees C and in sprouts produced from the seeds . The pathogen was not detected on seeds stored for 13 weeks at 37 degrees C and was not isolated from sprouts produced from these seeds . Identifying seed treatment methods that enhance removal of E . coli O157:H7 from alfalfa seeds can aid the isolation and enumeration of the pathogen on seeds . With a combination of optimal conditions for detecting the pathogen, i.e . soaking seeds for 1 h and pummeling seeds for 1 min, an enrichment step in modified tryptic soy broth (TSB), and the use of immunomagnetic beads for separation of E . coli O157:H7 cells, E . coli O157:H7 was detected in alfalfa seeds incubated at 37 degrees C for up to 8 weeks as effectively as in sprouts produced from the seeds.

Emerg Infect Dis, 2002 Jan, 8(1), 54 - 62
Prevalence and genetic profiling of virulence determinants of non-O157 Shiga toxin-producing Escherichia coli isolated from cattle, beef, and humans, Calcutta, India; Khan A et al.; We investigated the prevalence of Shiga toxin-producing Escherichia coli (STEC) in hospitalized diarrhea patients in Calcutta, India, as well as in healthy domestic cattle and raw beef samples collected from the city's abattoir . Multiplex polymerase chain reaction using primers specific for stx1 and stx2 detected STEC in 18% of cow stool samples, 50% of raw beef samples, and 1.4% and 0.6% of bloody and watery stool samples, respectively, from hospitalized diarrhea patients . Various virulence genes in the STEC isolates indicated that stx1 allele predominated . Plasmid-borne markers, namely, hlyA, katP, espP, and etpD, were also identified . Bead enzyme-linked immunosorbent assay and Vero cell assay were performed to detect and evaluate the cytotoxic effect of the Shiga toxins produced by the strains . STEC is not an important cause of diarrhea in India; however, its presence in domestic cattle and beef samples suggests that this enteropathogen may become a major public health problem in the future.

Infect Immun, 2002 Jan, 70(1), 395 - 9
Variations in the csgD promoter of Escherichia coli O157:H7 associated with increased virulence in mice and increased invasion of HEp-2 cells; Uhlich GA et al.; Promoter alterations in the csgD gene of Escherichia coli O157:H7 strains ATCC 43894 and ATCC 43895 are associated with variations in curli expression and the ability to bind Congo red dye . Red variants of each strain were more invasive for cultured HEp-2 cells than were white variants . An ATCC 43895 red variant was more virulent than a white variant in a mouse model . However, there were no differences in Shiga toxin production between red and white variants.

Emerg Infect Dis, 2001 Nov-Dec, 7(6), 1049 - 51
Contact with farming environment as a major risk factor for Shiga toxin (Vero cytotoxin)-producing Escherichia coli O157 infection in humans; O'Brien SJ et al.; In a prospective, unmatched case-control study of sporadic Shiga toxin (Vero cytotoxin)-producing Escherichia coli O157 (STEC O157) infection in England, exposure to the farming environment emerged strongly as a risk factor (adjusted odds ratio = 2.45; 95% confidence intervals = 1.49-4.02; p=0.0004) posing further challenges and opportunities for prevention.

Emerg Infect Dis, 2001 Nov-Dec, 7(6), 977 - 82
A multistate outbreak of Escherichia coli O157:H7 infections linked to alfalfa sprouts grown from contaminated seeds; Breuer T et al.; A multistate outbreak of Escherichia coli O157:H7 infections occurred in the United States in June and July 1997 . Two concurrent outbreaks were investigated through independent case-control studies in Michigan and Virginia and by subtyping isolates with pulsed-field gel electrophoresis (PFGE) . Isolates from 85 persons were indistinguishable by PFGE . Alfalfa sprouts were the only exposure associated with E . coli O157:H7 infection in both Michigan and Virginia . Seeds used for sprouting were traced back to one common lot harvested in Idaho . New subtyping tools such as PFGE used in this investigation are essential to link isolated infections to a single outbreak.

Emerg Infect Dis, 2001 Sep-Oct, 7(5), 812 - 9
Factors contributing to the emergence of Escherichia coli O157 in Africa; Effler E et al.; In 1992, a large outbreak of bloody diarrhea caused by Escherichia coli O157 infections occurred in southern Africa . In Swaziland, 40,912 physician visits for diarrhea in persons ages >5 years were reported during October through November 1992 . This was a sevenfold increase over the same period during 1990-91 . The attack rate was 42% among 778 residents we surveyed . Female gender and consuming beef and untreated water were significant risks for illness . E . coli O157:NM was recovered from seven affected foci in Swaziland and South Africa; 27 of 31 patient and environmental isolates had indistinguishable pulsed-field gel electrophoresis patterns . Compared with previous years, a fivefold increase in cattle deaths occurred in October 1992 . The first heavy rains fell that same month (36 mm), following 3 months of drought . Drought, carriage of E . coli O157 by cattle, and heavy rains with contamination of surface water appear to be important factors contributing to this outbreak.

J Clin Apheresis, 2001, 16(3), 155 - 6
Consanguineous hemolytic uremic syndrome secondary to Escherichia coli O157:H7 infection treated with aggressive therapeutic plasma exchange; Downes KA et al.; We describe the successful management of an elderly husband and wife with Escherichia coli O157:H7 associated hemolytic uremic syndrome (HUS) treated with aggressive therapeutic plasma exchange (TPE) with replacement with fresh frozen plasma . Following twelve TPEs (three 1.5 volume; nine 1 volume), the husband's platelet count increased from 45 x 10(9)/L to 183 x 10(9)/L . Following ten 1.5 volume TPEs, the wife's platelet count increased from 30 x 10(9)/L to 193 x 10(9)/L . This is the first known occurrence of E . coli O157:H7 associated HUS in an elderly husband and wife successfully treated with aggressive TPE . We conclude that early, aggressive TPE should be considered and may be life-saving for E coli O157:H7 associated HUS in the elderly .

J Pediatr Surg, 2001 Dec, 36(12), 1838 - 40
Secondary sclerosing cholangitis and portal hypertension after O157 enterocolitis: Extremely rare complications of hemolytic uremic syndrome; Urushihara N et al.; The authors experienced an extremely rare case of secondary sclerosing cholangitis and portal hypertension developed as late complications of hemolytic uremic syndrome (HUS) owing to Escherichia coli O157:H7 in a 2-year-old boy . HUS after E coli O157 infection is the most frequent cause of acute renal failure in childhood and occasionally is accompanied by extrarenal complications such as encephalopathy, cardiomyopathy, ischemic colitis, and pancreatitis . Rarely, late colonic stenosis may develop secondary to the ischemic damage . Sclerosing cholangitis and subsequent cirrhosis with portal hypertension are very uncommon as late complications of HUS . To our knowledge, such a case has not been previously reported in the literature . J Pediatr Surg 36:1838-1840 .

FEMS Microbiol Lett, 2001 Nov 13, 204(2), 329 - 34
Sensitivity of Shiga toxin-producing Escherichia coli (STEC) strains for colicins under different experimental conditions; Jordi BJ et al.; Twenty Escherichia coli strains producing well-characterised colicins were tested for their inhibitory activity against five Shiga toxin-producing E . coli (STEC) strains using different media under aerobic and anaerobic conditions . The five STEC strains used were of serotype O26, O111, O128, O145 and O157:H7 which are frequently isolated serotypes associated with disease in humans . The main route of infection for humans is through the eating of badly cooked or handled beef . The major reservoir for STEC strains in cattle is the rumen . To mimic the situation in the rumen of cattle, overlay assays were also performed under anaerobic conditions in the presence of 30% rumen fluid . Colicins E1, E4, E8-J, K and S4 are most active against STEC strains under anaerobic conditions in the absence or presence of rumen fluid . These colicins will be used in future experiments with the aim to eradicate the presence of STEC in cattle.

J Food Prot, 2001 Nov, 64(11), 1820 - 3
Penetration of Escherichia coli O157:H7 into lettuce as influenced by modified atmosphere and temperature; Takeuchi K et al.; The effects of temperature and atmospheric oxygen concentration on the respiration rate of iceberg lettuce and Escherichia coli O157:H7 cells attachment to and penetration into damaged lettuce tissues were evaluated . Respiration rate of lettuce decreased as the temperature was reduced from 37 to 10 degrees C . Reducing the temperature further to 4 degrees C did not affect the respiration rate of lettuce . Respiration rate was also reduced by lowering the atmospheric oxygen concentration . Lettuce was submerged in E . coli O157:H7 inoculum at 4, 10, 22, or 37 degrees C under 21 or 2.7% oxygen . Attachment and penetration of E . coli O157:H7 were not related to the respiration rate . The greatest numbers of E . coli O157:H7 cells attached to damaged lettuce tissues at 22 degrees C at both oxygen concentrations . More cells were attached under 21% oxygen than under 2.7% oxygen at each temperature, but this difference was small . Penetration of E . coli O157:H7 into lettuce tissue was determined by immunostaining with a fluorescein isothiocyanate-labeled antibody . Under 21% oxygen, E . coli O157:H7 cells showed greatest penetration when lettuce was held at 4 degrees C, compared to 10, 22 . or 37 degrees C, and were detected at an average of 101 microm below the surfaces of cut tissues . However, under 2.7% oxygen, there were no differences in degree of penetration among four incubation temperatures . The degree of E . coli O157:H7 penetration into lettuce tissue at 4 or 22 degrees C was greater under 21% oxygen than under 2.7% oxygen; however, no difference was observed at 37 degrees C . Conditions that promote pathogen penetration into tissue could decrease the effectiveness of decontamination treatments.

J Biol Chem, 2002 Jan 25, 277(4), 2876 - 85 Epub 2001 Nov 09.
Cell surface-localized nucleolin is a eukaryotic receptor for the adhesin intimin-gamma of enterohemorrhagic Escherichia coli O157:H7; Sinclair JF et al.; Intimin-gamma is an outer membrane protein of enterohemorrhagic Escherichia coli (EHEC) O157:H7 that is required for the organism to adhere tightly to HEp-2 cells and to colonize experimental animals . Another EHEC O157:H7 protein, the Transferred intimin receptor (Tir), is considered the primary receptor for intimin-gamma . Nevertheless, Tir-independent binding of intimin-gamma to HEp-2 cells has been reported . This observation suggests the existence of a eukaryotic receptor(s) for intimin-gamma . In this study, we sought to identify that receptor(s) . First, we determined by equilibrium binding titration that the association of purified intimin-gamma with HEp-2 cells was specific and consistent with a single host cell receptor . Second, we isolated a protein from lysates of HEp-2 cells that bound intimin-gamma and subsequently identified this molecule as nucleolin, a protein involved in cell growth regulation that can be cell surface-expressed . Third, we established that purified intimin-gamma and nucleolin were co-localized on the surface of HEp-2 cells and that the site of EHEC O157:H7 attachment was associated with regions of nucleolin expression . Finally, we demonstrated that mouse anti-nucleolin sera significantly decreased the adherence of EHEC O157:H7 to HEp-2 cells . From this, we conclude that nucleolin is the HEp-2 cell receptor for intimin-gamma expressed by EHEC O157:H7.

Lett Appl Microbiol, 2001 Nov, 33(5), 352 - 6
Optimal growth temperature of O157 and non-O157 Escherichia coli strains; Gonthier A et al.; AIMS: There are several biological characteristics that differ between Escherichia coli O157:H7, a dangerous food-borne pathogen, and the other serotypes of E . coli . METHODS AND RESULTS: The optimal growth temperatures (T(opt)) were determined for 32 E . coli strains, whether each strain belonged to the O157 serotype or not . The mean values of T(opt) for the O157 and non-O157 groups were 40.2 and 41.2 degrees C, respectively . SIGNIFICANCE AND IMPACT OF THE STUDY: This difference is statistically significant (P=0.0002) but has no biological implication.

Lett Appl Microbiol, 2001 Nov, 33(5), 334 - 8
Detection of Shiga-toxigenic Escherichia coli (STEC) in fresh seafood and meat marketed in Mangalore, India by PCR; Sanath Kumar H et al.; AIMS: To study the incidence of Shiga-toxigenic Escherichia coli (STEC) in seafoods from India . METHODS AND RESULTS: Escherichia coli isolated from various seafoods such as fresh fish, clams and water were screened for the presence of stx, hlyA and rfbO157 genes by PCR; 5% of clams and 3% of fresh fish samples were positive for non-O157 STEC . CONCLUSIONS: STEC is prevalent in seafoods in India, and non-O157 serotype is more common . SIGNIFICANCE AND IMPACT OF THE STUDY: Seafood could be a vehicle for transmission of STEC even in tropical countries.

Microbiol Immunol, 2001, 45(9), 621 - 8
A rapid bioluminescent enzyme immunoassay (BLEIA) for the detection of Shiga toxin types 1 and 2; Yamazaki M et al.; In recent years, Escherichia coli O157: H7 has emerged as a global public health concern . Among the more important virulence characteristics of this strain is its ability to produce one or more Shiga toxins (Stx) . Traditional culture-based methods for assay of enteric toxins in foods and clinical samples are relatively slow and results can be ambiguous . In this work, we established a toxin-detection system based on bioluminescent enzyme immunoassay (BLEIA) using a simple and inexpensive device . The system could detect both Shiga toxin types 1 and 2 individually within 150 min with a detection limit for each toxin at 5 pg/ml . In our study of previously characterized Shigatoxigenic and all non-Shigatoxigenic E . coli and other bacterial species, we found all Shigatoxigenic strains to be positive and non-Shigatoxigenic E . coli and other bacterial species to be negative . This assay was also used to detect Stxs in milk and supernatant fluids from minced chicken and beef . For clinical stool samples we noted a tendency for the system to give unexpectedly high background level . Our results suggest the feasibility of using BLEIA methodology for the simple, rapid and sensitive detection of toxins from culture supernatant, various foods and clinical samples.

Epidemiol Infect, 2001 Oct, 127(2), 215 - 20
Risk factors for sporadic cases of Escherichia coli O157 infection: the importance of contact with animal excreta; Locking ME et al.; To determine environmental risk factors for sporadic E . coli O157 infection in Scotland we undertook a prospective, matched case-control study between 1 October 1996 and 31 March 1999 . One hundred and eighty-three cases and 545 matched controls were recruited . Contact with animal faeces (OR = 3.65; 95% CI 1.81, 7.34: P < 0.0005) and likely contact with animal faeces (OR = 4.8; 95% CI 2.42, 9.48; P < 0.0005) emerged as strong risk factors for infection . Certain exposures (mainly food-related) were inversely associated with infection i.e . were statistically protective . Most striking was the consumption of bottled water (OR = 0.28; 95% CI 0.15, 0.52; P < 0.0005) . Transmission of E . coli O157 does not occur simply through contaminated food . Members of the public need to be aware of the potential for acquiring E . coli O157 through contamination of the environment with animal faeces so that they may take measures to mitigate their risk.

Curr Microbiol, 2001 Nov, 43(5), 311 - 5
Resolution of Escherichia coli O157:H7 that contaminated radish sprouts in two outbreaks by two-dimensional gel electrophoresis; Yokoyama K et al.; Two-dimensional gel electrophoresis (2-DE) was performed to examine exoproteins and periplasmic proteins of Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains isolated from cases associated with radish sprouts in two outbreaks . We found that STEC O157:H7 released a large number of proteins into the medium during the stationary phase of growth, as observed with 2-DE . Although pulsed-field gel electrophoresis (PFGE) patterns of STECs NGY9 (RIMD0509894), a Sakai isolate; NGY33, a Gamagoori isolate; and NGY120, a Kanagawa isolate, were all the same, comparison of 2-DE patterns of exoproteins and periplasmic proteins clarified that NGY9 was distinct from NGY33, whereas NGY33 and NGY120 were of close lineage . We therefore suggest that 2-DE analysis of exoproteins and periplasmic proteins is a powerful epidemiological method with high resolution.

J Biochem (Tokyo), 2001 Nov, 130(5), 665 - 70
Demonstration of the pH sensitive binding of multivalent carbohydrate ligands to immobilized Shiga-like toxin 1 B subunits; Imai Y et al.; The cytotoxic effects of Shiga-like toxins from enterohemorrhagic Escherichia coli O157:H7 depend on the recognition of carbohydrate determinants by B subunits . As a specific carbohydrate ligand, globotriaosylceramide has been characterized . We developed an alternative binding assay using multivalent carbohydrate ligands . We prepared globotriose-conjugated poly-lysine, and measured their binding to immobilized recombinant B subunits by an ELISA format . The signals representing ligand binding were dependent on the amount of immobilized B subunits as well as on the concentration of the ligands . The ligand binding activity was lost in an acidic environment, in which changes in the local conformation of the B subunits have been reported . Furthermore, pH dependent dissociation of the ligands from the B subunits was observed . We also demonstrate that antiserum from mice immunized with the B subunits specifically interferes with ligand binding . This suggests further potential for an assay to screen for blocking antibodies that could inhibit toxin internalization into host cells.

Jpn J Infect Dis, 2001 Aug, 54(4), 140 - 3
Phage typing and DNA-based comparison of strains of enterohemorrhagic Escherichia coli O157 from apparently sporadic infections in Osaka City, Japan, 1996; Nishikawa Y et al.; A marked increase in sporadic cases of enteritis due to enterohemorrhagic Escherichia coli serogroup O157 occurred in Osaka City, Japan, during 1996 . To elucidate why the number of cases had increased, the isolates were classified using phage typing, random amplified polymorphic DNA analysis, and pulsed-field gel electrophoresis (PFGE) . Fifty-seven percent of the isolates (105/184) belonged to the same phage type (PT-32) and gave the same PFGE pattern; the clone had been isolated during a 3-week period, with a peak on July 15 . It was concluded that the majority of the cases identified in July 1996 formed an outbreak, although epidemiological links to a possible common source were not established . The possibility that this outbreak was part of a huge regional outbreak including children at primary schools in Sakai City was discussed.

Appl Environ Microbiol, 2001 Nov, 67(11), 5321 - 4
Rapid extraction of DNA From Escherichia coli and Cryptosporidium parvum for use in PCR; Higgins JA et al.; The Xtra Amp tube, Isocode paper, Instagene matrix, and PrepMan matrix methods were evaluated for their ability to rapidly extract PCR-quality DNAs from Escherichia coli O157:H7 and Cryptosporidium parvum . All methods provided satisfactory DNA from E . coli, and the Xtra Amp and Instagene reagents provided satisfactory DNA from C . parvum.

Int J Food Microbiol, 2001 Sep 28, 69(3), 199 - 208
Binding of collagen I to Escherichia coli O157:H7 and inhibition by carrageenans; Medina MB; Research at USDA attempts to eliminate or reduce Escherichia coli contamination in meat and poultry foods by understanding the attachment mechanisms . This study utilizes a surface plasmon resonance (SPR) biosensor to determine the interactions of the immobilized E . coli O157:H7 surface with collagen I and selected polysaccharides . The binding and dissociation kinetics of collagen I with E . coli surface molecules had a mean affinity constant (K) of 3 x l0(8) (M(-1)) while the dissociation rate was 4.4 x l0(-5) (S(-1)) . Using the SPR biosensor, carrageenan, sodium alginate and pectin were evaluated for their interactions with collagen I and the E . coli surface . Results showed 89% to 100% inhibition by carrageenans and about 50% by sodium alginate and less than 10% by pectin . The biosensor binding studies were augmented by the scanning electron microscopy studies, which also showed the attachment of E . coli to the collagen fibrils of the bovine tissues . These studies serve as the basis for developing new strategies to block bacterial attachment or detach pathogens from animal carcasses.

J Food Prot, 2001 Oct, 64(10), 1610 - 2
Screening bovine carcass sponge samples for Escherichia coli O157 using a short enrichment coupled with immunomagnetic separation and a polymerase chain reaction-based (BAX) detection stept; Kang DH et al.; A bovine carcass sponge sample screening protocol for detecting Escherichia coli O157:H7 was composed of a short selective enrichment followed by an immunomagnetic separation (IMS) and target detection using the BAX E . coli O157 polymerase chain reaction assay . This screening protocol was compared to a culture-based method for detection of the organism in carcass sponge samples . Enriched samples were subjected to IMS; the bead suspension was divided and plated on selected media or stored at -20 degrees C, then subjected to BAX analysis . The results showed a high degree of agreement between the plating method and the BAX system . Fifty-two of the 59 culture-positive samples were also positive using the BAX system (88.1% sensitivity) . Of the 76 samples that appeared negative for the presence of E . coli O157:H7 by the culture method, 66 were determined as negative using the BAX system (86.8% specificity) . Four of the 10 samples found negative by the initial culture method and positive by the BAX method were subsequently found to be culture positive upon reanalysis . Based on these data, the BAX system combined with a short, selective enrichment and IMS may be a rapid, reliable, and simple method to screen for E . coli O157:H7 in carcass sponge samples . Our data indicate that optimization and subsequent testing of this protocol for use as a carcass screening tool are warranted.

J Food Prot, 2001 Oct, 64(10), 1607 - 9
Chlorine inactivation of Escherichia coli O157:H7 in water; Zhao T et al.; Six human isolates of Escherichia coli O157:H7 and E . coli (ATCC 11229) were used to determine the concentrations of free chlorine and exposure times required for inactivation . Free chlorine concentrations of 0.25, 0.5, 1.0, and 2.0 ppm at 23 degrees C were evaluated, with sampling times at 0, 0.5, 1.0, and 2.0 min . Results revealed that five of six E . coli O157:H7 isolates and the E . coli control strain were highly susceptible to chlorine, with >7 log10 CFU/ml reduction of each of these strains by 0.25 ppm free chlorine within 1 min . However, comparatively, one of the seven strains was unusually tolerant to chlorine at 23 degrees C for 1 min, with a 4-, 5.5-, 5.8-, and >5.8-log CFU/ml reduction at free chlorine concentrations (ppm) of 0.25, 0.5, 1.0, and 2.0 . respectively . Based on these studies most isolates of E . coli O157:H7 have no unusual tolerance to chlorine; however, one strain was exceptional in being recovered after 1-min of exposure of 10(7) CFU/ml to 2.0 ppm of free chlorine . This isolate may be a useful reference strain for future studies on chlorine tolerance of E . coli O157:H7.

Infect Immun, 2001 Nov, 69(11), 6863 - 73
Identification and characterization of a novel genomic island integrated at selC in locus of enterocyte effacement-negative, Shiga toxin-producing Escherichia coli; Schmidt H et al.; The selC tRNA gene is a common site for the insertion of pathogenicity islands in a variety of bacterial enteric pathogens . We demonstrate here that Escherichia coli that produces Shiga toxin 2d and does not harbor the locus of enterocyte effacement (LEE) contains, instead, a novel genomic island . In one representative strain (E . coli O91:H(-) strain 4797/97), this island is 33,014 bp long and, like LEE in E . coli O157:H7, is integrated 15 bp downstream of selC . This E . coli O91:H(-) island contains genes encoding a novel serine protease, termed EspI; an adherence-associated locus, similar to iha of E . coli O157:H7; an E . coli vitamin B12 receptor (BtuB); an AraC-type regulatory module; and four homologues of E . coli phosphotransferase proteins . The remaining sequence consists largely of complete and incomplete insertion sequences, prophage sequences, and an intact phage integrase gene that is located directly downstream of the chromosomal selC . Recombinant EspI demonstrates serine protease activity using pepsin A and human apolipoprotein A-I as substrates . We also detected Iha-reactive protein in outer membranes of a recombinant clone and 10 LEE-negative, Shiga toxin-producing E . coli (STEC) strains by immunoblot analysis . Using PCR analysis of various STEC, enteropathogenic E . coli, enterotoxigenic E . coli, enteroaggregative E . coli, uropathogenic E . coli, and enteroinvasive E . coli strains, we detected the iha homologue in 59 (62%) of 95 strains tested . In contrast, espI and btuB were present in only two (2%) and none of these strains, respectively . We conclude that the newly described island occurs exclusively in a subgroup of STEC strains that are eae negative and contain the variant stx(2d )gene.

Infect Immun, 2001 Nov, 69(11), 6660 - 9
toxB gene on pO157 of enterohemorrhagic Escherichia coli O157:H7 is required for full epithelial cell adherence phenotype; Tatsuno I et al.; Adherence of enterohemorrhagic Escherichia coli (EHEC) to the intestinal epithelium is critical for initiation of a bacterial infection . An in vitro infection study previously indicated that EHEC bacteria initially adhere diffusely and then proliferate to develop MC, a process that is mediated by various secreted proteins, such as EspA, EspB, EspD, Tir, and intimin, as well as other putative adherence factors . In the present study, we investigated the role of a large 93-kb plasmid (pO157) in the adherence of O157:H7 (O157Sakai) and found the toxB gene to be involved in the full adherence phenotype . A pO157-cured strain of O157Sakai (O157Cu) developed microcolonies on Caco-2 cells; however, the number of microcolonies was lower than that of O157Sakai, as were the production and secretion levels of EspA, EspB, and Tir . Introduction of a mini-pO157 plasmid (pIC37) composed of the toxB and ori regions restored full adherence capacity to O157Cu, including production and secretion of the proteins . In contrast, introduction of a pO157 mutant possessing toxB::Km into O157Cu could not restore the full adherence phenotype . Expression of truncated versions of His-tagged ToxB also promoted EspB production and/or secretion by O157Cu . These results suggest that ToxB contributes to the adherence of EHEC to epithelial cells through promotion of the production and/or secretion of type III secreted proteins.

Arch Pediatr, 2001 Sep, 8 Suppl 4, 776s - 784s
{Post-diarrhea hemolytic-uremic syndrome: clinical aspects}; Loirat C; Every year in France, about 100 children, most of them less than 3 years old, have typical diarrhea-associated HUS (D + HUS) . Evidence of exposure to verotoxin producing E . coli (VTEC), mostly the O157: H7 serotype, is demonstrated in about 85% of cases . A prodromal illness of acute gastroenteritis with diarrhea, often bloody, precedes the HUS by 1 to 15 days . HUS onset is sudden, with the typical association of hemolytic anemia with fragmented red blood cells, thrombocytopenia and acute renal insufficiency . Involvement of other organs than the kidneys may occur, such as severe hemorrhagic colitis with rectal prolapse, bowel wall necrosis or secondary stenosis, acute pancreatitis, central nervous system involvement which determines the vital outcome . Early accurate supportive treatment allows a current mortality rate below 5%, with most deaths due to central nervous system involvement . Five to 10% of children develop end stage renal disease, rarely directly, more often after having recovered some renal function with chronic renal insufficiency during a few years . After 15 or more years follow-up, at least one third of patients have some degree of proteinuria and/or hypertension, and eventually chronic or end stage renal failure . Predictive features of poor renal outcome at the acute phase are severe gastrointestinal involvement, severe CNS involvement, polyncleosis over 20,000/mm3, and duration of initial anuria longer than one week . The role of VTEC in D + HUS makes the disease a public health problem . Preventive measures are essential.

J Microbiol Methods, 2001 Nov, 47(2), 249 - 53
Effects of preparation method, age, and plating technique of thin agar layer media on recovery of Escherichia coli O157:H7 injured by sodium chloride; Hajmeer MN et al.; The thin agar layer (TAL) method was experimentally tested to determine its ability to recover Escherichia coli O157:H7 injured by sodium chloride (NaCl) . Cells grown in Brain Heart Infusion broth with 0%, 5%, or 7.5% (w/v) NaCl were spread and spiral plated onto Tryptic Soy agar (TSA), MacConkey Sorbitol agar (MSA), and TSA/MSA TAL combinations . Generally, TSA recovered more injured cells than TAL (p < or =0.05), and TAL recovered more cells than MSA (p < or =0.05) . Preparation mode (two vs . three layers) and age (0, 1, or 7 days) of TAL had negligible effect on resuscitation of injured cells (p > 0.05) . TAL, which is conventionally used to recover heat, cold, and acid-injured foodborne pathogens, may be used to recover NaCl-injured E . coli O157:H7.

J Ind Microbiol Biotechnol, 2001 Jun, 26(6), 345 - 9
Detection of immunomagnetically captured Escherichia coli O157:H7 by antibody-conjugated alkaline phosphatase; Tu SI et al.; A rapid and sensitive detection process for Escherichia coli O157:H7 was developed using alkaline phosphatase (APase)-labeled anti-E . coli O157 antibodies to tag the targeted bacteria . Immunomagnetic beads or antibody-labeled streptavidin-coated magnetic beads were then used to capture the APase-tagged E . coli . Immunomagnetically captured bacteria were washed and distributed into microplates or optical cuvettes . The enzyme-catalyzed hydrolysis of p-nitro-phenol phosphate in alkaline solutions was then followed . Less than 1000 cfu/ml of E . coli O157:H7 could be detected . This approach was applied to detect the bacteria artificially spiked in beef hamburgers . Less than 1 cfu/g of E . coli O157:H7 produced a significant response after cultural enrichment for 4-6 h at 37 degrees C.

Appl Environ Microbiol, 2001 Oct, 67(10), 4914 - 8
Availability of glutamate and arginine during acid challenge determines cell density-dependent survival phenotype of Escherichia coli strains; Cui S et al.; The cell density-dependent acid sensitivity phenotypes of Escherichia coli strains K-12 and O157:H7 were examined with reference to three possible mechanisms of acid resistance . There was no evidence of any diffusible substance released from dead cells which could influence the cell density-dependent acid survival phenotype . Instead, cell density-dependent acid survival phenotype was associated with induction of glutamate- and arginine-decarboxylase acid survival pathways and concomitant availability of glutamate and arginine during acid challenge.

Appl Environ Microbiol, 2001 Oct, 67(10), 4901 - 7
Variation in resistance to high hydrostatic pressure and rpoS heterogeneity in natural isolates of Escherichia coli O157:H7; Robey M et al.; Several natural isolates of Escherichia coli O157:H7 have previously been shown to exhibit stationary-phase-dependent variation in their resistance to inactivation by high hydrostatic pressure . In this report we demonstrate that loss of the stationary-phase-inducible sigma factor RpoS resulted in decreased resistance to pressure in E . coli O157:H7 and in a commensal strain . Furthermore, variation in the RpoS activity of the natural isolates of O157:H7 correlated with the pressure resistance of those strains . Heterogeneity was noted in the rpoS alleles of the natural isolates that may explain the differences in RpoS activity . These results are consistent with a role for rpoS in mediating resistance to high hydrostatic pressure in E . coli O157:H7.

Can Vet J, 2001 Sep, 42(9), 714 - 20
Environmental sources and transmission of Escherichia coli O157 in feedlot cattle; Van Donkersgoed J et al.; A study was conducted in 2 feedlots in southern Alberta to identify environmental sources and management factors associated with the prevalence and transmission of Escherichia coli O157:H7 . Escherichia coli O157:H7 was isolated in preslaughter pens of cattle from feces (0.8%), feedbunks (1.7%), water troughs (12%), and incoming water supplies (4.5%), but not from fresh total mixed rations . Fresh total mixed rations did not support the growth of E . coli O157:H7 and E . coli from bovine feces following experimental inoculation . Within a feedlot, the feces, water troughs, and feedbunks shared a few indistinguishable subtypes of E . coli O157:H7 . A few subtypes were repeatedly isolated in the same feedlot, and the 2 feedlots shared a few indistinguishable subtypes . The prevalence of E . coli O157:H7 in water troughs of preslaughter cattle in 1 feedlot was associated with season, maximum climatic temperatures the week before sampling; total precipitation the week before sampling, and coliform and E . coli counts in the water trough.

J Food Prot, 2001 Sep, 64(9), 1346 - 51
Isolation and characterization of Escherichia coli O157:H7 from retail meats in Argentina; Chinen I et al.; Between February and May 2000, 279 meat samples were collected from 136 retail stores in Gualeguaychu City, Argentina . Samples were assayed for Escherichia coli O157:H7 by selective enrichment in modified EC broth containing novobiocin, followed by immunomagnetic separation (IMS) and plating onto both sorbitol MacConkey agar supplemented with cefixime and potassium tellurite and a chromogenic medium . Eleven E . coli O157:H7 isolates were detected in 6 (3.8%) of 160 ground beef samples, in 4 (4.8%) of 83 fresh sausages, and in 1 (3.3%) of 30 dry sausages . E . coli O157:H7 was not isolated from five hamburger patties or one barbecue-type fresh sausage assayed . The isolates were tested for virulence-related genes . Ten additional Shiga toxin-producing E . coli (STEC) O157:H7 isolates of food origin, recovered from different locations in Argentina, were included for comparison purposes . All 21 isolates harbored both eae and EHEC-hlyA genes, and 12 (57.1%) encoded stx2/stx2vh-a . The isolates were of phage types 87 (seven strains), 14 (four strains), 4 (three strains), and 26 (one strain) . Six strains were nontypable by phage typing . Pulsed-field gel electrophoresis (PFGE) revealed 19 XbaI-PFGE profiles . Fifteen (71%) strains were grouped in four clusters, which shared more than 80% of DNA restriction fragments . The enrichment culture method with IMS was a sensitive procedure to detect E . coli O157:H7 strains in retail meats . Some of the isolates from different stores presented a high clonal relatedness, as determined by XhaI-PFGE and phage typing, and harbored the virulence factors associated with human illness.

J Food Prot, 2001 Sep, 64(9), 1341 - 5
Hazard analysis of Escherichia coli O157:H7 contamination during beef slaughtering in Calvados, France; Guyon R et al.; To identify hazard points and critical points during beef slaughtering, which is a necessary first step toward developing a hazard analysis and critical control point system to control meat contamination by Escherichia coli O157:H7, samples (n = 192) from surfaces, work tops, worker's hands, and beef carcasses were collected from a slaughterhouse in Calvados, France . Five strains of E . coli O157:H7 were isolated from a footbridge and a worker's apron at the preevisceration post and from a worker's hand at the defatting post . Three isolates carried stx2c, eae, and EHEC-hlyA genes and showed similar molecular types by random amplified polymorphic DNA, polymerase chain reaction IS3, and XbaI pulsed-field gel electrophoresis . Thus, this study has shown that preevisceration and defatting post and associated worker's materials are critical points for carcasses contamination by E . coli O157:H7 during beef slaughtering.

J Food Prot, 2001 Sep, 64(9), 1334 - 40
Survival of Escherichia coli O157:H7 on strawberry fruit and reduction of the pathogen population by chemical agents; Yu K et al.; Survival of Escherichia coli O157:H7 was studied on strawberry, a fruit that is not usually washed during production, harvest, or postharvest handling . Two strains of the bacteria were tested separately on the fruit surface or injected into the fruit . Both strains of E . coli O157:H7 survived externally and internally at 23 degrees C for 24 h and at 10, 5, and -20 degrees C for 3 days . The largest reduction in bacterial population occurred at -20 degrees C and on the fruit surface during refrigeration . In all experiments, the bacteria inside the fruit either survived as well as or better than bacteria on the surface, and ATCC 43895 frequently exhibited greater survival than did ATCC 35150 . Two strains of E . coli also survived at 23 degrees C on the surface and particularly inside strawberry fruit . Chemical agents in aqueous solution comprising NaOCl (100 and 200 ppm), Tween 80 (100 and 200 ppm), acetic acid (2 and 5%), Na3PO4 (2 and 5%), and H2O2 (1 and 3%) were studied for their effects on reduction of surface-inoculated (10(8) CFU/ml) E . coli O157:H7 populations on strawberry fruit . Dipping the inoculated fruit in water alone reduced the pathogen population about 0.8 log unit . None of the compounds with the exception of H2O2 exhibited more than a 2-log CFU/g reduction of the bacteria on the fruit surface . Three percent H202, the most effective chemical treatment, reduced the bacterial population on strawberries by about 2.2 log CFU/g.

J Food Prot, 2001 Sep, 64(9), 1328 - 33
Location of Escherichia coli O157:H7 on and in apples as affected by bruising, washing, and rubbing; Kenney SJ et al.; Confocal scanning laser microscopy (CSLM) was used to determine the location of Escherichia coli O157:H7 cells on the surface and in tissue of bruised Red Delicious cv . apples . Undamaged and bruised apples were inoculated by immersing in a suspension of E . coli O157:H7 cells transformed with a plasmid that encodes for the production of a green fluorescent protein . Apples were then washed in 0.1% (wt/vol) peptone water and/or rubbed with a polyester cloth and examined to determine if these treatments removed or introduced cells into lenticels, cutin, and cracks on the skin surface . Optical slices of the apples obtained using CSLM were examined to determine the depth at which colonization or attachment of cells occurred . Populations of E . coli O157:H7 on the surface of apples were determined to assess the effectiveness of washing and rubbing in physically removing cells . The location of cells on or in undamaged and bruised areas of apples that were not washed or rubbed did not differ significantly . However, washing apples resulted in an approximate 2-log reduction in CFU of E . coli O157:H7 per cm2 of apple surface . On unwashed apples, cells were detected at depths up to 30 microm below the surface . No E . coli O157:H7 cells were detected at locations more than 6 microm below the surface of washed apples . Cells that remained on the surface of rubbed apples appeared to be sealed within naturally occurring cracks and crevices in waxy cutin platelets . These cells may be protected from disinfection and subsequently released when apples are eaten or pressed for cider production.

Osaka City Med J, 2001 Jun, 47(1), 11 - 22
Induction of thymic apoptosis by Shiga toxin from Escherichia coli O157:H7 in vivo and in vitro; Iwata M et al.; It is not known how Shiga toxins (Stxs), which are major virulence factors of enterohemorrhagic Escherichia coli, can affect the host immune system . We investigated the effect of Stx2 on murine thymic cells in vivo and in vitro . After intraperitoneal administration of Stx2, the body weight of mice (BW) and the organ index of thymocytes (OIT) gradually decreased from day 1 to day 4 . Apoptosis of thymocytes, assessed by TUNEL staining, and DNA fragmentation assay, was marked on day 3 and day 4 . Decrease in BW and OIT due to Stx2 was antagonized by the simultaneous administration of murine anti-Stx2 IgG antibody with Stx2 . In vitro administration of Stx2 also induced apoptosis of cultured thymocytes on day 3 and day 4 in a dose-dependent fashion . These results showed that Stx2 directly caused apoptosis of thymocytes in vivo and in vitro . Our findings imply that StA2 may be intimately related to pathogenesis of E . coli O157:H7 infection, by causing apoptosis of thymus.

J Med Microbiol, 2001 Sep, 50(9), 752 - 8
Attaching and effacing lesions caused by Escherichia coli O157:H7 in experimentally inoculated neonatal lambs; Wales AD et al.; Four 6-day-old conventionally reared lambs were inoculated orally with a total of 10(9) cfu comprising equal numbers of four enterohaemorrhagic Escherichia coli (EHEC) O157:H7 strains . All animals remained clinically normal . Tissues were sampled under terminal anaesthesia at 12, 36, 60 and 84 h post inoculation (hpi) . EHEC O157:H7 was cultured from most gastrointestinal tract sites . Small, sparse attaching and effacing (AE) lesions were found in the caecum at 12 and 36 hpi and in the terminal colon and rectum at 84 hpi . Organisms in the lesions were labelled specifically by an O157 antiserum . The results indicate that the well-characterised mechanisms for intimate attachment encoded by the locus for enterocyte effacement (LEE) of EHEC O157:H7 may contribute to the initial events, at least, of colonisation of sheep.

Genome Res, 2001 Sep, 11(9), 1584 - 93
Shotgun optical maps of the whole Escherichia coli O157:H7 genome; Lim A et al.; We have constructed NheI and XhoI optical maps of Escherichia coli O157:H7 solely from genomic DNA molecules to provide a uniquely valuable scaffold for contig closure and sequence validation . E . coli O157:H7 is a common pathogen found in contaminated food and water . Our approach obviated the need for the analysis of clones, PCR products, and hybridizations, because maps were constructed from ensembles of single DNA molecules . Shotgun sequencing of bacterial genomes remains labor-intensive, despite advances in sequencing technology . This is partly due to manual intervention required during the last stages of finishing . The applicability of optical mapping to this problem was enhanced by advances in machine vision techniques that improved mapping throughput and created a path to full automation of mapping . Comparisons were made between maps and sequence data that characterized sequence gaps and guided nascent assemblies.

Clin Infect Dis, 2001 Oct 1, 33(7), 923 - 31 Epub 2001 Sep 05.
The central Scotland Escherichia coli O157:H7 outbreak: risk factors for the hemolytic uremic syndrome and death among hospitalized patients; Dundas S et al.; Little is known about risk factors for complications of Escherichia coli O157:H7 infection in adults . The 1996 outbreak in central Scotland involved the largest number of adult case patients in whom hemolytic uremic syndrome (HUS) developed and, ultimately, the largest number of deaths associated with E . coli O157:H7 infection that has yet been recorded . We investigated risk factors for HUS in a retrospective study of all hospitalized case patients in this outbreak . Of 120 case patients, 34 had HUS develop, 28 of whom were adults . Sixteen adults died . Significant risk factors for HUS were age <15 years or >65 years (odds ratio {OR}, 4.4; 95% confidence interval {CI}, 1.3-14.4), hypochlorhydria (OR, 6.7; 95% CI, 1.9-24.0), and coincidental antibiotics (OR, 4.7; 95% CI 1.4-16.5) . Factors associated with HUS were as follows: white blood cell count >20 x 10(9) cells/L (OR, 8.25; 95% CI, 1.1-60.3), neutrophil count >15 x 10(9) cells/L (OR, 8.5; 95% CI, 1.5-50.1), and serum albumin level <35 g/L (OR, 7.2; 95% CI, 1.2-42.5) < or =3 days after symptom onset . Deaths were confined to case patients >65 years of age . Early identification of risk factors for HUS is vital and could select case patients for trials of preventative and treatment therapies.

Appl Environ Microbiol, 2001 Sep, 67(9), 3810 - 8
Genotypic analyses of Escherichia coli O157:H7 and O157 nonmotile isolates recovered from beef cattle and carcasses at processing plants in the Midwestern states of the United States; Barkocy-Gallagher GA et al.; Escherichia coli O157:H7 and O157 nonmotile isolates (E . coli O157) previously were recovered from feces, hides, and carcasses at four large Midwestern beef processing plants (R . O . Elder, J . E . Keen, G . R . Siragusa, G . A . Barkocy-Gallagher, M . Koohmaraie, and W . W . Laegreid, Proc . Natl . Acad . Sci . USA 97:2999-3003, 2000) . The study implied relationships between cattle infection and carcass contamination within single-source lots as well as between preevisceration and postprocessing carcass contamination, based on prevalence . These relationships now have been verified based on identification of isolates by genomic fingerprinting . E . coli O157 isolates from all positive samples were analyzed by pulsed-field gel electrophoresis of genomic DNA after digestion with XbaI . Seventy-seven individual subtypes (fingerprint patterns) grouping into 47 types were discerned among 343 isolates . Comparison of the fingerprint patterns revealed three clusters of isolates, two of which were closely related to each other . Remarkably, isolates carrying both Shiga toxin genes and nonmotile isolates largely fell into specific clusters . Within lots analyzed, 68.2% of the postharvest (carcass) isolates matched preharvest (animal) isolates . For individual carcasses, 65.3 and 66.7% of the isolates recovered postevisceration and in the cooler, respectively, matched those recovered preevisceration . Multiple isolates were analyzed from some carcass samples and were found to include strains with different genotypes . This study suggests that most E . coli O157 carcass contamination originates from animals within the same lot and not from cross-contamination between lots . In addition, the data demonstrate that most carcass contamination occurs very early during processing.

J Food Prot, 2001 Aug, 64(8), 1244 - 8
Ascorbic acid enhances destruction of Escherichia coli O157:H7 during home-type drying of apple slices; Burnham JA et al.; Destruction of Escherichia coli O157:H7 was evaluated on inoculated apple slices dehydrated at two temperatures with and without application of predrying treatments . Half-ring slices (0.6 cm thick) of peeled and cored Gala apples were inoculated by immersion for 30 min in a four-strain composite inoculum of E . coli O157:H7 . The inoculated slices (8.7 to 9.4 log CFU/g) either received no predrying treatment (control), were soaked for 15 min in a 3.4% ascorbic acid solution, or were steam blanched for 3 min at 88 degrees C immediately prior to drying at 57.2 or 62.8 degrees C for up to 6 h . Samples were plated on tryptic soy (TSA) and sorbitol MacConkey (SMAC) agar media for direct enumeration of surviving bacterial populations . Steam blanching changed initial inoculation levels by +0.3 to -0.7 log CFU/g, while immersion in the ascorbic acid solution reduced the inoculation levels by 1.4 to 1.6 log CFU/g . Dehydration of control samples for 6 h reduced mean bacterial populations by 2.9 log CFU/g (TSA or SMAC) at 57.2 degrees C and by 3.3 (SMAC) and 3.5 (TSA) log CFU/g at 62.8 degrees C . Mean decreases from initial inoculum levels for steam-blanched slices after 6 h of drying were 2.1 (SMAC) and 2.0 (TSA) log CFU/g at 57.2 degrees C, and 3.6 (TSA or SMAC) log CFU/g at 62.8 degrees C . In contrast, initial bacterial populations on ascorbic acid-pretreated apple slices declined by 5.0 (SMAC) and 5.1 (TSA) log CFU/g after 3 h of dehydration at 57.2 degrees C, and by 7.3 (SMAC) and 6.9 (TSA) log CFU/g after 3 h at 62.8 degrees C . Reductions on slices treated with ascorbic acid were in the range of 8.0 to 8.3 log CFU/g after 6 h of drying, irrespective of drying temperature or agar medium used . The results of immersing apple slices in a 3.4% ascorbic acid solution for 15 min prior to drying indicate that a predrying treatment enhances the destruction of E . coli O157:H7 on home-dried apple products.

J Food Prot, 2001 Aug, 64(8), 1235 - 9
Direct microscopic observation of lettuce leaf decontamination with a prototype fruit and vegetable washing solution and 1% NaCl-NaHCO3; Takeuchi K et al.; Efficacy of a prototype, food-grade alkaline surfactant washing solution and 1% NaCl-NaHCO3 (pH 10.0) against Escherichia coli O157:H7 cells on lettuce leaves was evaluated . Lettuce was inoculated with 10(9) CFU/ml of E . coli O157:H7 for 24 +/- 1 h at 4 degrees C . Samples were rinsed and treated with the prototype washing solution containing lauryl sodium sulfate or NaCl-NaHCO3 for 3 min at 22 degrees C . Viability of E . coli O157:H7 cells was examined by plate counts at the surface and cut edge, and by confocal scanning microscopic (CSLM) observation of samples stained with Sytox green and Alexa 594 conjugated antibody against E . coli O157:H7 at intact leaf surface, stomata, and damaged tissue (0 to 10, 30 to 40, and 0 to 40 microm from the cut surface) . Although both treatments caused significant log reductions in CFU at the surface and cut edge, log reductions were greater for the prototype washing solution (0.7 to 1.1 log CFU/cm2) than for NaCl-NaHCO3 (0.2 to 0.4 log CFU/cm2) (P < 0.05) . Percentage of viability determined by CSLM for prototype washing solution was significantly greater at 30 to 40 microm from cut surfaces than at 0 to 10 and 0 to 40 microm from cut surfaces and intact surfaces (P < 0.05) . Stomata provided moderate protection . NaCl-NaHCO3 was less effective than the prototype washing solution, and high percentages of E . coli O157:H7 cells remained viable at all sites except at the surface . The percent viabilities determined by CSLM were not significantly different from those determined by plate counts for NaCl-NaHCO3 treatment (P > 0.05) . However, CSLM indicated significantly greater percent viability than plate counts for lettuce treated with the prototype washing solution (P < 0.05) . Surfactant-containing washing solutions warrant additional testing for decontamination of fresh produce.

J Food Prot, 2001 Aug, 64(8), 1199 - 205
Acid phosphatase activity and color changes in consumer-style griddle-cooked ground beef patties; Lyon BG et al.; The U.S . Department of Agriculture and the Food and Drug Administration have issued temperature requirements to help consumers cook beef patty products that are free of pathogens . Verification of end-point temperature (EPT) is needed in cooked meat products due to concerns over outbreaks of Escherichia coli O157:H7 . Acid phosphatase (ACP) activity was studied as a potential method for determination of EPT in ground beef patties cooked nonfrozen, patties frozen 7 days and thawed at room temperature 4 h in a refrigerator or by microwave, and patties made from ground beef frozen in store packages, then thawed in a refrigerator overnight . Pressed-out meat juices were analyzed from patties (n = 314) cooked to 57.2 degrees C (135 degrees F) . 65.6 degrees C (150 degrees F), 71.1 degrees C (160 degrees F), and 79.4 degrees C (175 degrees F) target EPTs . Expressed meat juice and internal meat patty color decreased in redness as EPT increased . Freezing whole packs with slow refrigerator or room temperature thawing caused significantly greater loss of redness in expressed cooked meat juice than did other handling methods . Log10 ACP had a significant linear (R2 = 0.99) response to EPT . Results show that the 3- to 5-min ACP test could be used to verify EPT in griddle-cooked hamburger patties.

J Infect Dis, 2001 Oct 1, 184(7), 918 - 21 Epub 2001 Aug 15.
Genetic and evolutionary analysis of mutations in the gusA gene that cause the absence of beta-glucuronidase activity in Escherichia coli O157:H7; Monday SR et al.; Escherichia coli serotype O157:H7 do not exhibit beta-glucuronidase (GUD) activity but carry the gusA gene (uidA) that encodes for GUD . In trans-complementation, the gusA gene cloned from the GUD-positive variant strain 493-89 effectively restored GUD activity in O157:H7 strain 35150 . Comparison of gusA sequences from the GUD-negative 35150 strain to that of 493-89 revealed several base mutations, including a guanosine (G) dinucleotide insertion that caused a frameshift in the 35150 gusA gene and introduced a predicted premature termination codon . This explains the absence of GUD activity in O157:H7 . A 35150 gusA construct from which the G-G insertion was deleted restored activity in GUD-negative O157:H7 transformants . The G-G insertion was present in all GUD-negative O157:H7 strains but was absent in their GUD-positive variants . The G-G insertion that produced the characteristic GUD-negative phenotype to O157:H7 strains appeared later than the other gusA mutations in the evolutionary emergence of O157:H7.

Microbiology, 2001 Aug, 147(Pt 8), 2341 - 53
Association between intimin (eae) and EspB gene subtypes in attaching and effacing Escherichia coli strains isolated from diarrhoeic lambs and goat kids; Cid D et al.; Attaching and effacing Escherichia coli (AEEC) strains isolated from diarrhoeic lambs and goat kids were characterized for intimin (eae) and EspB (espB) gene subtypes by PCR and sequencing, and for genetic relatedness by PFGE . Fifty (23 ovine and 27 caprine) AEEC strains of 398 (246 ovine and 152 caprine) analysed were detected by colony blot hybridization . These strains were epidemiologically unrelated since they were isolated from different outbreaks of neonatal diarrhoea over a long period . Ovine AEEC strains belonged to serogroups O2, O4, O26, O80, O91 or were untypable, and caprine strains belonged to serogroups O3, O153 and O163 . Two intimin subtypes were detected among the ovine and caprine strains studied . Most of the strains (43/50) had the beta type intimin gene, but seven ovine strains possessed a variant gamma type intimin gene (gamma(V)) . Analysis of deduced amino acid sequences of the eae gene revealed that the sequences of beta intimin of ovine and caprine strains were virtually identical to those of beta intimin of rabbit EPEC, human EPEC clone 2 and swine AEEC, whereas the gamma(V) intimin present in seven ovine strains had 75-76% identity with gamma intimin of human EHEC clone 1 strains, and 96% of identity with intimin of the human EHEC strain 95NR1 of serotype O111:H- . A PCR test was developed to identify the three different espB gene subtypes, espB of human EPEC clone 1 (espBalpha), espB of human EHEC clone 1 (espBgamma) and espB of rabbit EPEC and human EPEC clone 2 (espBbeta) . There was close correlation between the intimin beta type and the espBbeta gene subtype in the ovine and caprine AEEC strains . The seven ovine strains possessing the gamma(V) intimin gene possessed the espBalpha gene subtype . None of the strains studied possessed the espBgamma gene found in human O157:H7 EHEC strains . PFGE analysis of genomic DNA of selected strains showed a great diversity among strains . Cluster analysis of PFGE patterns showed greater divergence between strains with the gamma(V) intimin gene than between strains with the beta intimin gene . This study showed that most of the AEEC strains isolated from diarrhoeic lambs and goat kids possessed beta intimin and espB genes identical to those of rabbit EPEC, and they may be associated with enteric disease in small ruminants.

J Bacteriol, 2001 Sep, 183(17), 5187 - 97
Quorum sensing is a global regulatory mechanism in enterohemorrhagic Escherichia coli O157:H7; Sperandio V et al.; Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries . EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells . We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene . In this study we hybridized an E . coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenic luxS mutant . We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in the luxS mutant . Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes . Consistent with this pattern of gene expression, the luxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium) . Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis . Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation . We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant . The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 microm/s, respectively . We also observed an increase in the production of Stx due to quorum sensing . Genes encoding Stx, which are transcribed along with lambda-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing . These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E . coli as well as for virulence factors.

Int J Food Microbiol, 2001 Jul 20, 67(1-2), 89 - 96
The effects of freezing and thawing on the survival of Escherichia coli O157:H7 in apple juice; Yamamoto SA et al.; Unpasteurized apple juice, adjusted to pH 3.6 to 7.0 was inoculated (10(7) CFU/ml) with single strains of E . coli O157:H7 to evaluate the effect of frozen storage on the viability of this organism . Samples were stored under frozen conditions (-20+/-2 degrees C) for up to 16 days . Cell populations were determined at regular intervals by plating onto tryptic soy agar with added pyruvate (TSAP) or onto sorbitol MacConkey agar (SMA) . Populations in the neutralized juice remained unchanged during frozen storage . Populations in non-neutralized juice decreased by 1-3 log10 CFU/ml depending on the strain tested and the pH of the juice . The greatest population decrease was observed with the first freeze/thaw cycle of frozen storage (24 h) and a slow decline in survival occurred thereafter . Injury was observed after 2 weeks of storage when juice pH was at or below pH 4.2 . When samples were subjected to multiple freeze/thaw cycles, loss of viability and injury increased with each freeze/thaw cycle.

Int J Food Microbiol, 2001 Jul 20, 67(1-2), 71 - 80
Detecting and genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays; Call DR et al.; Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance . As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E . coli O157:H7 . The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A) . Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification . The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg) . Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFU ml(-1) (E . coli O157:H7) from chicken rinsate without the aid of pre-enrichment . Four isolates of E . coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array . Glass-based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products; the system is amenable to automation.

Lett Appl Microbiol, 2001 Aug, 33(2), 95 - 9
Growth of Escherichia coli O157:H7 during sprouting of alfalfa seeds; Stewart D et al.; AIMS: Escherichia coli O157:H7 was monitored daily during sprouting of alfalfa seeds inoculated at high (3.92 log10 cfu g(-1)) and low (1.86 log10 cfu g(-1)) levels to assess the extent of pathogen growth during production . METHODS AND RESULTS: Sprouts and rinse water were tested by direct and membrane filter plating on modified sorbitol MacConkey agar and BCM O157:H7(+) agar; the antibody-direct epifluorescent filter technique; and rapid immunoassays . The pathogen reached maximum populations after one and two days of sprouting seeds inoculated at high and low levels, respectively; in either case, populations of 5-6 log10 cfu g(-1) were reached . Detection limits of two rapid immunoassays, Reveal and VIP, without enrichment were determined to be 5-7 log10 cfu ml(-1) . CONCLUSION: These results show the ability of E . coli O157:H7 to grow to high levels during sprouting; however, because these levels may be below detection limits, it is necessary to include enrichment when monitoring sprout production for E . coli O157:H7 by the rapid test kits . SIGNIFICANCE AND IMPACT OF THE STUDY: The data indicate that sprouts may harbor high levels of pathogens . The appropriate use of rapid test methods for pathogen monitoring during sprouting is indicated.

J Vet Med B Infect Dis Vet Public Health, 2001 Jun, 48(5), 321 - 30
Detection of enterohemorrhagic Escherichia coli in meat foods using DNA probes, enzyme-linked immunosorbent assay and polymerase chain reaction; Alexandre M et al.; Enterohemorrhagic Escherichia coli (EHEC) is an important cause of diarrhoea with blood and haemolytic uremic syndrome (HUS) in children and elderly people . Infections with EHEC are a world-wide public health problem, related to consumption of contaminated ground beef . The aim of this study was to establish whether different meat foods sold in Santiago, Chile pose an infection risk by EHEC and to evaluate three different diagnostic techniques in foods, to determine which is most applicable for use in Chile . A parallel analysis was performed on 64 samples of meat foods (23 refrigerated ground meat, 23 refrigerated long pork sausages and 18 frozen hamburgers) sold in Santiago, Chile using DNA probes, enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) . Twenty-four samples (24 of 64 = 37.5%) were positive by DNA probes, ELISA or PCR . The positive and negative predictive values, sensitivity and specificity of ELISA were 26.7, 81.6, 30.8 and 78.4%, respectively . The positive and negative predictive values, sensitivity and specificity of PCR were 91.7, 96.2, 84.6 and 98%, respectively . The EHEC serogroups most frequently isolated were O158, O157, O119, O125 and O114 . These results show that, although molecular techniques such as enzyme immunoassays are useful for EHEC detection in meat foods, PCR has advantages in terms of sensitivity, specificity, cost and ease of implementation in Chile.

Blood Coagul Fibrinolysis, 2001 Jun, 12(4), 283 - 8
Thrombogenic alleles, Escherichia coli O157:H7 infections, and hemolytic uremic syndrome; Sprouse JT et al.; Hemolytic uremic syndrome (HUS) of childhood most commonly follows gastrointestinal infection with Escherichia coli O157:H7 . This pathogen elaborates Shiga toxins that are believed to cause microvascular injury and to trigger a thrombogenic response . The exact mechanisms leading to variable disease manifestations are unknown . Allelic variation in genes encoding selected coagulation factors and inhibitors of fibrinolysis were examined to determine whether or not a causal relationship exists between hypercoagulability and the development of HUS . No correlation between the thrombogenic factor V (G1691A), factor II (G20210A), methylenetetrahydrofolate reductase (C677T), or the plasminogen activator inhibitor (PAI)-1 promotor (4G/5G) genotypes and the risk of infection with E . coli O157:H7, or the risk of development of HUS among infected patients, was found . Serum PAI-1 levels did not correlate with the PAI-1 genotype . We conclude that the alleles studied are not major risk factors for the acquisition of E . coli O157:H7 infection, or of E . coli O157:H7-related HUS.

J Food Prot, 2001 Jul, 64(7), 970 - 4
Acid stress, starvation, and cold stress affect poststress behavior of Escherichia coli O157:H7 and nonpathogenic Escherichia coli; Leenanon B et al.; The effects of acid shock, acid adaptation, starvation, and cold stress of Escherichia coli O157:H7 (ATCC 43895), an rpoS mutant (FRIK 816-3), and nonpathogenic E . coli (ATCC 25922) on poststress heat resistance and freeze-thaw resistance were investigated . Following stress, heat tolerance at 56 degrees C and freeze-thaw resistance at -20 to 21 degrees C were determined . Heat and freeze-thaw resistance of E . coli O157:H7 and nonpathogenic E . coli was enhanced after acid adaptation and starvation . Following cold stress, heat resistance of E . coli O157:H7 and nonpathogenic E . coli was decreased, while freeze-thaw resistance was increased . Heat and freeze-thaw resistance of the rpoS mutant was enhanced only after acid adaptation . Increased or decreased tolerance of acid-adapted, starved, or cold-stressed E . coli O157:H7 cells to heat or freeze-thaw processes should be considered when processing minimally processed or extended shelf-life foods.

Microb Pathog, 2001 Aug, 31(2), 59 - 67
Localization of Shiga toxins of enterohaemorrhagic Escherichia coli in kidneys of paediatric and geriatric patients with fatal haemolytic uraemic syndrome; Chaisri U et al.; Haemolytic uraemic syndrome (HUS) is characterized by haemolytic anaemia, thrombocytopenia and renal failure . Infection with enterohaemorrhagic Escherichia coli (EHEC), mainly O157:H7, has been strongly implicated as the major cause of HUS in children . The pathogenesis of HUS caused by the infection is not well understood and the defined sites of Stx in kidney of EHEC-infected humans has not been clearly demonstrated . The aim of this study was to investigate and compare the locations of Stx deposition in kidneys of paediatric and geriatric patients who died from enterohaemorrhagic E . coli O157 (EHEC) associated HUS, using an immunoperoxidase staining of the tissues . The study revealed that binding of Stx was relatively less and limited only to the renal tubules of an adult case (81 years old), while more binding was found at both renal tubules and glomeruli of an infant case (21 months old) . The Stx binding in the infant's glomeruli was at podocytes, mesangial and endothelial cells . It has been known that young children are more susceptible than adults to HUS . One possibility for this is that the more extensive binding of the Stx to the kidney tissue of the paediatric patient might be due to the higher synthesis and expression of Stx receptors, i.e . Gb(3), in infants and less so in the aged individuals . However, other alternatives are possible, for example, the difference in stage of HUS in individual patients . Thus it is too early to draw any conclusion on this enigma and further investigation is required .

Infect Immun, 2001 Aug, 69(8), 5107 - 14
Differences in levels of secreted locus of enterocyte effacement proteins between human disease-associated and bovine Escherichia coli O157; McNally A et al.; Ongoing extensive epidemiological studies of verotoxin-carrying Escherichia coli O157 (stx(+) eae(+)) have shown this bacterial pathogen to be common in cattle herds in the United States and the United Kingdom . However, the incidence of disease in humans due to this pathogen is still very low . This study set out to investigate if there is a difference between strains isolated from human disease cases and those isolated from asymptomatic cattle which would account for the low disease incidence of such a ubiquitous organism . The work presented here has compared human disease strains from both sporadic and outbreak cases with a cross-section, as defined by pulsed-field gel electrophoresis, of E . coli O157 strains from cattle . Human (n = 22) and bovine (n = 31) strains were genotyped for carriage of the genes for Shiga-like toxin types 1, 2, and 2c; E . coli secreted protein genes espA, espB, and espP; the enterohemolysin gene; eae (intimin); ast (enteroaggregative E . coli stable toxin {EAST}); and genes for common E . coli adhesins . Strains were also phenotyped for hemolysin, EspP, Tir, and EspD expression as well as production of actin and cytoskeletal rearrangement associated with attaching and effacing (A/E) lesions on HeLa cells . The genotyping confirmed that there was little difference between the two groups, including carriage of stx(2) and stx(2c), which was similar in both sets . ast alleles were confirmed to all contain mutations that would prevent EAST expression . espP mutations were found only in cattle strains (5 of 30) . Clear differences were observed in the expression of locus of enterocyte effacement (LEE)-encoded factors between strains and in different media . EspD, as an indicator of LEE4 (espA, -B, and -D) expression, and Tir levels in supernatants were measured . Virtually all strains from both sources could produce EspD in Luria-Bertani broth, although at very different levels . Standard trichloroacetic acid precipitation of secreted proteins from tissue culture medium produced detectable levels of EspD from the majority of strains of human origin (15 of 20) compared with only a few (4 of 20) bovine strains (P < 0.001), which is indicative of much higher levels of protein secretion from the human strains . Addition of bovine serum albumin carrier protein before precipitation and enhanced detection techniques confirmed that EspD could be detected after growth in tissue culture medium for all strains, but levels from strains of human origin were on average 90-fold higher than those from strains of bovine origin . In general, levels of secretion also correlated with ability to form A/E lesions on HeLa cells, with only the high-level protein secretors in tissue culture medium exhibiting a localized adherence phenotype . This research shows significant differences between human- and bovine-derived E . coli O157 (stx(+) eae(+)) strains and their production of certain LEE-encoded virulence factors . These data support the recent finding of Kim et al . (J . Kim, J . Nietfeldt, and A . K . Benson, Proc . Natl . Acad . Sci . USA 96:13288-13293, 1999) proposing different E . coli O157 lineages in cattle and humans and extend the differential to the regulation of virulence factors . Potentially only a subset of E . coli O157 isolates (stx(+) eae(+)) in cattle may be capable of causing severe disease in humans.

Semin Thromb Hemost, 2001 Jun, 27(3), 207 - 13
The role of virulence factors in enterohemorrhagic Escherichia coli (EHEC)--associated hemolytic-uremic syndrome; Karch H; Enterohemorrhagic Escherichia coli (EHEC) are the most common cause of postdiarrheal hemolytic-uremic syndrome (HUS) . The most important EHEC serotype implicated worldwide is O157:H7 . However, several so-called non-O157 EHEC serotypes have emerged . After a mean incubation period of 3 days, patients develop watery diarrhea accompanied by cramping abdominal pain . During the next days, in most patients watery diarrhea changes to bloody diarrhea . One week after the onset of diarrhea, in about 15% of infected patients under 10 years of age EHEC infection results in a systemic complication, HUS . Shiga toxins (Stxs) are considered the major virulence factors of EHEC involved in the pathogenesis of HUS . It is generally believed that after intestinal infection with EHEC, Stxs cross the intestinal barrier and bind to endothelial cells . At this point they presumably injure the host cell by inhibition of protein synthesis, stimulation of prothrombotic messages, or induction of apoptosis . The B subunit of Stx binds to the membrane receptor globotriaosylceramide (Gb3) . Gb3 facilitates the endocytosis and intracellular trafficking of the toxin . The Stx A subunit hydrolyzes a specific adenine residue of the 60S ribosomal subunit of mammalian cells . As a consequence, Stx shuts down the protein machinery of the susceptible cell . The HUS is the net effect of a variety of interacting factors, including background risk of acquisition, host factors (such as age), virulence characteristics of the infecting EHEC strain, and exogenous factors . All known EHEC virulence determinants are located on mobile genetic elements, and this has an important impact on the evolution of these pathogens . The evolution of EHEC has a dynamic component that includes different genetic mechanisms . The recent progress in understanding the pathogenesis and epidemiology of EHEC infections forms a basis for the development of future strategies to prevent EHEC infections in humans.

Lett Appl Microbiol, 2001 Jul, 33(1), 36 - 9
Immunomagnetic separation of Escherichia coli O26, O111 and O157 from vegetables; Safarikova M et al.; AIMS: Raw fruits and vegetables have been increasingly associated with human infections caused by Shiga toxin-producing Escherichia coli . This study evaluates the isolation and detection of E . coli O26, O111 and O157 from vegetable samples using immunomagnetic particles . METHODS AND RESULTS: Standard cultivation and immunomagnetic separation (IMS) procedures were compared . It was found that immunomagnetic particles could efficiently concentrate E . coli cells, detecting significantly more bacteria than with standard cultivation procedures . CONCLUSION: Bacteria were detected in 93-100% of the inoculated samples using the IMS procedure, but only 36-93% samples tested by standard cultivation procedures were found to be positive . SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that E . coli O26, O111 and O157 immunomagnetic particles can be a very useful and efficient tool for the detection of E . coli strains in raw vegetables, and could probably be used with samples of animal origin.

Arch Med Res, 2001 Jul-Aug, 32(4), 251 - 7
Lectins in fruits having gastrointestinal activity: their participation in the hemagglutinating property of Escherichia coli O157:H7; Coutino-Rodriguez R et al.; BACKGROUND: In fruits with therapeutic properties for antidiarrheal and laxative uses, the presence of lectins may be the bioactive properties that interfere with bacterial adhesion, thought to be competition for glycoside signal sites in the attachment . METHODS: This study identifies lectins in crude extracts from fruits such as Tamarindus indica (tamarind), Spontia vulgaris (plum), Psidium guava (guava), Mangifera indica (mango), Cydonia vulgaris (quince), and Crataegus mexicanus (tejocote) . To verify the procedures, extracts from Ricinus communis (castor bean), Glycine max (soybean), Phaseolus vulgaris (beans), Vicia fava (fava bean), and Solanum tuberosum (potato) were used as controls for lectin activity . Both sources of lectins were analyzed to determine their participation in the host-parasite interaction, using as a model the hemagglutinating properties of Escherichia coli O157:H7 (EHA) . RESULTS: All extracts showed hemagglutination to group O erythrocytes test (HA) with the exception of mango . Two new galactose-specific lectins were identified from tamarind and guava . When analyzed for participation in EHA, only guava lectins inhibited this, while soybean lectin induced hemolysis; as both lectins bind to galactose, it is probable that their recognition occurs in different domains . Sugars involved in the attachment between Escherichia coli O157:H7 and red cells were identified and again, galactose in addition to mannose was found to be related in EHA . On the other hand, guava lectins also agglutinated E . coli O157:H7, perhaps due to the same galactose-specific lectin or to another type of lectin . CONCLUSIONS: In summary, guava has a galactose-specific lectin that prevents adhesion of E . coli O157:H7 to red cells; this lectin is mediated by galactose . Prevention could also be due to their capacity of agglutinating E . coli by guava lectins . Soybean lectin induced hemolysis only when bacteria was present, but not with floating secretions . This finding showed that guava is a source of lectin that can be explored to prevent adhesion of E . coli to epithelial intestinal cells; contrariwise, soya must be studied to see its participation in the uremia caused during E . coli O157:H7 pathogenesis.

Thromb Haemost, 2001 Jun, 85(6), 975 - 8
von Willebrand factor-cleaving protease in childhood diarrhoea-associated haemolytic uraemic syndrome; Hunt BJ et al.; A deficiency of von Willebrand factor (vWF)-cleaving protease, either due to a congenital deficiency or to the presence of a protease inhibitor of vWF-cleaving protease has been associated with thrombotic thrombocytopenic purpura (TTP) . We have studied vWF-cleaving protease in diarrhoea-associated haemolytic uraemic syndrome (D+ HUS), which shares clinical features with TTP . 29 children with acute D+ HUS and 13 control children were studied . vWF-cleaving protease activity was normal (range 50-150%) in 39 of 42 plasma samples . Levels of protease activity between 25 and 50% were noted in plasma from two D+ HUS patients . One D+HUS patient, who had clinical features of TTP, had a vWF-cleaving protease inhibitor producing a severe deficiency of vWF-cleaving protease . Thus a deficiency of vWF-cleaving protease appears to be atypical in D+HUS . The detection of a vWF-cleaving protease inhibitor in one patient suggests it may be associated with infection such as E . coli O157.

Int J Hyg Environ Health, 2001 May, 203(4), 347 - 61
Review of epidemiological surveys on the prevalence of contamination of healthy cattle with Escherichia coli serogroup O157:H7; Meyer-Broseta S et al.; This paper gathers and critically analyses the results of 26 published epidemiological surveys on the prevalence of contamination of cattle with verocytotoxin-producing E . coli (VTEC) serogroup O157:H7 . These surveys have been conducted since 1986 on farms in North America (10 studies), on farms in Europe (6 studies) and at slaughterhouses prior to or just after slaughter (7 studies) or after skinning and evisceration (3 studies) . The purpose of this review is to understand the first stages of the epidemiology of the infection in animals and humans (the infection process being obscure in many points) and to prepare herd-based control measures to reduce the risk of O157:H7 human infection . The different statistical methods employed in these surveys, as well as the various laboratory screening methods used for detecting positive animals are presented . The observed frequencies of infected animals (animal prevalence) and herds (herd prevalence) are given as a function of localisation, year, type of industry (beef or dairy) and age . From these measured prevalence values, the risk of contamination of ground beef by E . coli O157:H7 in the first stages of the farm-to-fork continuum is assessed . First, we follow the evolution of contamination frequencies from the living animal on-farm to carcasses before transformation . Then, within each set of measurements (i.e., on farm or at slaughterhouse), we identify the effects of the following factors: target population, sampling strategies and laboratory procedures . We argue that the prevalence values inferred from these measurements are very likely underestimated, due to insufficient sampling and not enough sensitive laboratory procedures (one exception being the immunomagnetic bead separation technique) . No firm conclusion can be drawn as to the effects of geographical localisation and season . In those surveys, the effect of hygiene level at slaughterhouse on prevalence values is not quantitatively assessed . In addition, there is growing evidence of other sources of E . coli O157:H7 than live cattle in the farm environment, such as feed, water and water-troughs.

MMWR Morb Mortal Wkly Rep, 2001 Jun 15, 50(23), 489 - 91
University outbreak of calicivirus infection mistakenly attributed to Shiga toxin-producing Escherichia coli O157:H7--Virginia, 2000; The emerging nosocomial pathogens Cryptosporidium et al.; Department of Medicine, University of North Carolina at Chapel Hill, 27599-7030, USANew and emerging infectious diseases pose a threat to public health and may be responsible for nosocomial outbreaks . Cryptosporidium parvum and Escherichia coli are gastrointestinal pathogens that have caused nosocomial infections via person-to-person transmission, environmental contamination, or contaminated water or food . Helicobacter pylori has been transmitted via inadequately disinfected endoscopes . Finally, hepatitis C may be acquired by healthcare personnel by percutaneous or mucous membrane exposure to blood or between patients by use of contaminated blood products or via environmental contamination . Rigorous adherence to Standard Precautions, Contact Precautions for patients with infectious diarrhea, disinfection of environmental surfaces, and appropriate disinfection of endoscopes are adequate to prevent nosocomial acquisition of these pathogens.

Appl Environ Microbiol, 2001 Jul, 67(7), 2908 - 15
Quantitative detection of Escherichia coli O157 in surface waters by using immunomagnetic electrochemiluminescence; Shelton DR et al.; A protocol for the quantitative detection of Escherichia coli O157 in raw and concentrated surface waters using immunomagnetic electrochemiluminescence (IM-ECL) was developed and optimized . Three antibody sandwich formats were tested: commercial anti-O157:H7 IM beads, IM beads made in-house with a polyclonal anti-O157:H7 immunoglobulin G (IgG), or IM beads made in-house with a monoclonal anti-O157:H7 IgG coupled with a polyclonal anti-O157:H7 IgG to which an electrochemiluminescent label (TAG) was attached . The monoclonal IM bead-polyclonal TAG format was chosen for optimization because it gave lower background levels and linear regression slopes of ca . 1.0, indicative of a constant ECL signal per cell . The dynamic range was ca . 10(1) to 10(5) cells ml(-1) in phosphate-buffered saline and in raw water samples . The monoclonal IM beads selectively captured E . coli O157 cells in the presence of ca . 10(8) cells of a non-O157 strain of E . coli ml(-1) . Background ECL signals from concentrated (100-fold) water samples were substantially higher and more variable than raw water samples . The background signal was partially eliminated by the addition of polyvinylpolypyrrolidone . Successive cell capture incubations, termed sequential bead capture (SBC), were optimized for establishing baseline ECL values for individual water samples . The linear dynamic range with SBC was ca . 10(2) to 10(5) E . coli O157 cells ml of concentrated water(-1) . To validate the protocol, 10-liter surface water samples were spiked with ca . 5,000 E . coli O157 (Odwalla) cells and concentrated by vortex filtration, and 1- or 3-ml aliquots were analyzed by IM-ECL . Differential ECL signals (SBC) from 1- and 3-ml samples were statistically significant and were generally consistent with standard curves for these cell concentrations . Enrichments were conducted with aliquots of spiked raw water and concentrated water using EC broth and minimal lactose broth (MLB) . All tubes with concentrated water became turbid and gave a positive ECL response for E . coli O157 (>10,000 ECL units); MLB gave a somewhat higher detection rate with spiked raw water . The potential sensitivity of the IM-ECL assay is ca . 25 E . coli O157 cells ml of raw water(-1), 25 cells 100 ml of 100-fold concentrated water(-1), or 1 to 2 viable cells liter(-1) with concentration and enrichment . The IM-ECL assay appears suitable for routine analysis and screening of water samples.

J Infect Dis, 2001 Jul 15, 184(2), 206 - 10 Epub 2001 Jun 18.
A monkey model for enterohemorrhagic Escherichia coli infection; Kang G et al.; Adult Macaca radiata (n=22) were infected intragastrically with 10(12) Escherichia coli O157:H7 strain 84-01, which produces Shiga toxins 1 and 2 . Clinical symptoms and bacterial excretion were documented in each monkey for a specified time period before they were killed . At necropsy, samples were obtained for culture and histologic and ultrastructural examination . Seventeen monkeys had diarrhea: E . coli O157 was isolated from postinfection stool samples from all monkeys and from autopsy cultures for 14 of 22 monkeys . Histologic examination showed attaching-effacing lesions, which appeared at 12 h and persisted for 7 days, in 12 monkeys . Widening of the intercellular spaces, degeneration and vacuolization of the epithelial cells, epithelial tufting, extrusion of epithelial cells, and neutrophilic infiltration were characteristic features seen in 20 of the 22 infected monkeys but not in 4 control monkeys . This monkey model closely parallels the early stages of the disease produced by E . coli O157:H7 and would be useful in the further study of pathogenic mechanisms and prevention methods in enterohemorrhagic E . coli infections.

J AOAC Int, 2001 May-Jun, 84(3), 752 - 60
Detex for detection of Escherichia coli O157 in raw ground beef and raw ground poultry; Henry YM et al.; A method for detection of Escherichia coli O157 in beef and poultry is presented . The method is antibody-based and uses a patented antibody-specific metal-plating procedure for the detection of E . coli O157 in enriched meat samples . Both raw ground beef and raw ground poultry were tested as matrixes for the organism . The sensitivity and specificity of the assay were 98 and 90%, respectively . The accuracy of the assay was 96% . Overall, the method agreement between the E . coli O157 Detex assay and the U.S . Department of Agriculture/Food Safety Inspection Service method was 96% . Sample temperature upon loading of the apparatus was critical to the observed false-positive rate of the system.

Biotechniques, 2001 Jun, 30(6), 1262 - 4, 1266-7
Use of multiple primers in RAPD analysis of clonal organisms provides limited improvement in discrimination; Hopkins KL et al.; Randomly amplified polymorphic DNA (RAPD) analysis using two or more primers has been reported to provide additional discriminatory ability over one primer used individually . This may be of particular application in epidemiological typing of clonal organisms, such as Shiga toxin-producing E . coli O157, where strain differentiation can be difficult . Using four arbitrary primers individually, and in all possible permutations, E . coli O157 isolates and other arbitrarily chosen E . coli strains were typed using RAPD analysis . For most nonclonal strains, the use of two primers resulted in increased differentiation between isolates; however, more than two primers did not increase further the discriminatory capacity . E . coli O157 isolates that produced virtually identical profiles using one primer did not show increased differentiation when using two or more primers, demonstrating that in some cases, where strains of an organism are highly related, there is limited advantage to using more than one primer in RAPD analysis.

Int J Food Microbiol, 2001 May 21, 66(1-2), 85 - 93
A field survey of Escherichia coli O157 ecology on a cattle farm in Italy; Conedera G et al.; A field survey was performed in a heifer raising operation in Northern Italy to study the introduction, maintenance and dissemination of Escherichia coli O157 in the herd and to identify possible control measures at the farm level . Rectal swabs from two different groups of animals (surveys 1 and 2) were tested for E . coli O157 by an immunomagnetic separation technique . In survey 1, a group of female calves (341 animals initially) introduced from 30 dairy herds during April 1996 to March 1997 were tested for E . coli O157 on arrival from the original herd when housed in individual hutches, 2-3 days after completion of weaning (which was associated with grouping) and 2 months after weaning . No statistically significant difference between excretion rates (3.8%, 4.2%, 4.4%, respectively) was found . Calves from which E . coli O157 was isolated on arrival came from 6 of the 30 dairy herds . Strains isolated during survey 1 belonged to seven different pulsed field gel electrophoresis (PFGE) profiles . In survey 2, a group of young animals aged, at the beginning of the study, between 2 1/2 and 7 1/2 months (median = 124 days) was tested monthly for E . coli O157 for 11-15 months from May 1996 to July 1997 . The group included 92 animals for 11 months and then gradually decreased to 59 animals . Overall, E . coli O157, belonging to six different PFGE profiles, were isolated from 138 (10.7%) of 1293 rectal swabs . Monthly excretion rates ranged from 2.7% to 23.7%, with summer peaks in both years . Fifty-nine (64.1%) of the 92 heifers were positive at least once: of these 59 animals, 22 (37.3%) were positive on only one occasion, 23 (39%) were positive on two occasions and 14 (23.7%) were positive on three or more occasions . From two heifers positive on 9 out of the 15 sampling visits, strains with the same PFGE profile were isolated, respectively, on seven and eight occasions while strains with only one band difference were isolated on the remaining occasions . E . coli O157 was also isolated from 6 of 16 samples of bedding, two of two samples of slurry and one of five samples from water troughs collected during survey 2.

Int J Food Microbiol, 2001 May 21, 66(1-2), 63 - 9
A survey of the prevalence of Escherichia coli O157 in raw meats, raw cow's milk and raw-milk cheeses in south-east Scotland; Coia JE et al.; 2429 samples of foodstuffs were examined for the presence of verocytotoxigenic Escherichia coli O157 (VTEC O157) by means of immunomagnetic separation (IMS) over a 2-year period commencing April 1997 . Specimens comprised 1190 raw meats, 500 raw milks and 739 raw-milk cheeses . The meat and cheese samples were purchased from retail premises in south-east Scotland; raw milk samples were obtained directly from farms . In addition, total E . coli counts were performed on milk and cheese samples, and the pH of cheese specimens measured . The water activity (Aw) was also measured for a representative sample of each cheese type, and for all of the samples with high levels of E . coli . VTEC O157 was isolated from two samples of beef burger, both manufactured on the premises of the same butchers shop . Control studies with artificially inoculated foodstuffs demonstrated a sensitivity of detection of < 5 organisms 25 g(-1) . These findings, which contrast with the results of similar studies elsewhere in the UK, suggest that other sources of infection may be important in explaining the high rates of infection with this organism in south-east Scotland.

Int J Food Microbiol, 2001 May 21, 66(1-2), 111 - 7
The fate of Escherichia coli O157 in soil and its potential to contaminate drinking water; Ogden LD et al.; The survival and transport of Escherichia coli and E . coli O157 after cattle slurry application were studied on drained plots in both grassland and arable stubble at three sites in Scotland . Leaching losses were between 0.2% and 10% of total E . coli and were dependent on rainfall . Recovery of E . coli in grass and soil declined with approximately first order kinetics . Residual numbers, in excess of background declined more slowly . The pattern was similar for both grass and arable plots . Laboratory incubations of soil cores, with applied slurry containing E . coli and E . coli O157 were performed in soils with different moisture contents at two temperatures for clay loam and sandy loam soils . Both E . coli populations were measured over a 4-week period . Using a dual population approach, the die off of the susceptible pool was linear with a half-life of 3-4 days, and was faster at the higher temperature and lowest moisture content . The resistant pool was not strongly affected by temperature or moisture and had a half-life for die off of between 18 and 24 days . After a 4-week period, < 100 cfu g/soil of E . coli and E . coli O157 remained . The die off rate of E . coli O157 was the same or slightly faster than that of the commensal E . coli population, indicating that the field behaviour of E . coli O157 can be studied by monitoring the total population of E . coli applied with slurry . The risk of significant pollution of water by E . coli is highest immediately after application of slurry, and the first increments of drainflow carry significant concentrations . Thereafter, the risk of pollution is very low . If weather conditions are dry after application on well-drained sandy soils, it is unlikely that any significant losses of organisms to drains will occur . Such data can be used to control and minimise the risk of E . coli O157 contaminating drinking water.

J Food Prot, 2001 Jun, 64(6), 767 - 9
Sublethal sanitizer stress and adaptive response of Escherichia coli O157:H7; Zook CD et al.; The effect of sublethal exposure to peroxyacetic acid (PAA) sanitizer on adaptation to peroxidative stress and development of thermal cross-resistance was investigated in Escherichia coli O157:H7 . Acute sublethal PAA sanitizer exposure was used to represent a contact scenario . Cultures were grown in Trypticase soy-yeast extract broth . Acute treatment cultures were pretreated with 0.1% PAA, then all cultures were challenged at either 80 mM H202 or 54 degrees C . Acute and peroxide control cultures showed substantially increased peroxidative tolerance (D80mM > 2 h) versus negative control cultures not exposed to sanitizer (D80mM = 0.19+/-0.03 h) . The inactivation rate of the acetic acid control (D80mM = 0.21+/-0.05 h) was similar to the negative control rate . Acute (D54 degrees C = 0.55+/-0.07 h) cultures did not exhibit increased thermal resistance versus the control (D54 degrees C = 0.54+/-0.07 h) . Thermal injury was determined as difference in D54 degrees C value (deltaD54 degrees c) obtained on pyruvate and deoxycholate media . Thermal-induced injury was not observed in either control (deltaD54 degrees C = 0.04 h) or acute (deltaD54 degrees C = 0.05 h) cultures.

Bratisl Lek Listy, 2001, 102(2), 59 - 65
Is it possible to influence the mortality in children with hemolytic uremic syndrome?
Dolezel Z, Kopecna L, Starha J, Dostalkova D.
THE CURRENT STATE: Hemolytic uremic syndrome (HUS) is the most frequent cause of acute renal failure in children . In our geographic conditions D(+)HUS prevails, in its etiology E . coli O157:H7 is represented most . In the course of HUS there can occur extrarenal damage to some organs, the manifestation of multiple organ failure is then possible . Delayed diagnosis of HUS, its complicated course and therapeutical strategy influence greatly the mortality rate of affected children . SUBJECTIVE: The optimal therapeutical procedure was elaborated on the base of evaluating the causes of death in children with HUS, and, simultaneously, there were revealed further reserves that can decrease the mortality rate . METHODS AND MATERIAL: A retrospective analysis of mortality within 1982-2000 was carried out in one of three centres dealing with the therapy for HUS in children in the Czech Republic . The total number of HUS children was 69 (40 girls, 29 boys, mean age 4.5 years) . 9 patients out of the analyzed group died . RESULTS: Out of 9 children (4 girls, 5 boys) died, D(+)HUS was present in 7 (77.8%, 4 girls, 3 boys, mean age 2.1 years), D(-)HUS occurred in 2 subjects (22.2%, boys, mean age 7.5 years) . Dialysis therapy was needed in all 9 children (peritoneal dialysis and/or hemodialysis) . The mortality rate in our group of children was 13% . An autopsy carried out in dead children showed dominant severe affection of kidneys/brain/heart . CONCLUSION: In spite of marked decrease in the number of HUS children died particularly in developed countries, permanent attention must be paid to this disease . Besides early diagnosis, corresponding therapy is necessary . That should be performed at a specialized centre . The elaborated algorithm can be used in the therapy for HUS . This is one of the ways for further decrease of both mortality and chronic morbidity in children suffering from HUS . (Tab . 3, Fig . 1, Ref . 15.).

Int J Food Microbiol, 2001 May 10, 65(3), 193 - 200
Escherichia coli O157:H7 in faeces from cattle, sheep and pigs in the southwest part of Norway during 1998 and 1999; Johnsen G et al.; During a 2-year period from January 1998 to December 1999, intestinal content from 1541 cattle, 665 sheep and 1976 pigs were analysed for Escherichia coli O157:H7 using the immunomagnetic separation procedure . The animals originated from 848, 605 and 832 herds from the southwest part of Norway, respectively . E . coli O157:H7 was present in three samples from cattle from different herds, giving a herd prevalence of 0.35% and an animal prevalence of 0.19% . From pigs, E . coli O157:H7 was isolated from two pigs from different herds, giving a herd prevalence of 0.24% and an animal prevalence of 0.1% . A follow-up study revealed another positive testing pig from one of these herds . E . coli O157:H7 was not found from any of the 665 investigated sheep . By PCR analysis, all six E . coli O157:H7 isolates were shown to contain the genes encoding Shiga toxin 2 (stx2), the intimin protein (eae) and the H7 flagellum (fliC-H7) . One of the cattle isolates also harboured the Shiga toxin 1 encoding (stx1) gene . The six isolates were differentiated into three pulse-field gel electrophoresis profiles . The results indicate that the occurrence of E . coli O157:H7 in cattle, sheep and pigs in the southwest part of Norway is low compared to other European countries.

J Clin Microbiol, 2001 Jun, 39(6), 2272 - 9
Antibody response to Shiga toxins Stx2 and Stx1 in children with enteropathic hemolytic-uremic syndrome; Ludwig K et al.; A Western blot (immunoblot) assay (WBA) for the detection of immunoglobulin G antibodies to Shiga toxins Stx2 and Stx1 in sera from 110 patients with enteropathic hemolytic-uremic syndrome (53 culture confirmed to have Shiga toxin-producing Escherichia coli {STEC} infection) and 110 age-matched controls was established by using a chemiluminescence detection system . Thirty-nine (74%) of the 53 culture-confirmed cases were infections with STEC serotype O157, and 14 (26%) were associated with infection by other STEC serotypes . The frequency of an anti-Stx2 response following infection by a Stx2-producing strain (34 of 48 cases; 71%) was higher than that of an anti-Stx1 response following Stx1-producing STEC infection (4 of 10) . Furthermore, the frequency of an anti-Stx2 response in 110 control sera (10%) was significantly higher than the frequency of an anti-Stx1 response (1.8%) (P = 0.0325) . For STEC O157 culture-confirmed cases WBA for toxin detection had a diagnostic sensitivity of 71% and a specificity of 90% . Because of its high specificity the assay might be a helpful tool for diagnosing suspected STEC infection when tests of stool samples or serological tests against various lipopolysaccharide antigens are negative . Furthermore, the prevalence of anti-Stx antibodies in healthy controls probably reflects the population immunity to systemic Stx-associated disease . It can thus serve as a basis for comparing immunity levels in different populations and for considering future Stx toxoid immunization strategies.

Appl Environ Microbiol, 2001 Jun, 67(6), 2863 - 6
Survival of Escherichia coli O157:H7 ATCC 43895 in a model apple juice medium with different concentrations of proline and caffeic acid; Reinders RD et al.; The effects of proline and caffeic acid on the survival of Shiga toxin-producing Escherichia coli (STEC) O157:H7 strain ATCC 43895 in a model apple juice medium were studied . It is hypothesized that the inhibitory effect of caffeic acid may explain why almost all outbreaks of STEC O157:H7 infections linked to apple juice or cider have occurred in October or November.

Appl Environ Microbiol, 2001 Jun, 67(6), 2460 - 8
Heterogeneity of Shiga toxin-producing Escherichia coli strains isolated from hemolytic-uremic syndrome patients, cattle, and food samples in central France; Pradel N et al.; A detailed analysis of the molecular epidemiology of non-O157:H7 Shiga toxin-producing Escherichia coli (STEC) was performed by using isolates from sporadic cases of hemolytic-uremic syndrome (HUS), animal reservoirs, and food products . The isolates belonged to the O91 and OX3 serogroups and were collected in the same geographical area over a short period of time . Five typing methods were used; some of these were used to explore potentially mobile elements like the stx genes or the plasmids (stx(2)-restriction fragment length polymorphism {RFLP}, stx(2) gene variant, and plasmid analyses), and others were used to study the whole genome (ribotyping and pulsed-field gel electrophoresis {PFGE}) . The techniques revealed that there was great diversity among the O91 and OX3 STEC strains isolated in central France . A close relationship between strains of the same serotype having the same virulence factor pattern was first suggested by ribotyping . However, stx(2)-RFLP and stx(2) variant analyses differentiated all but 5 of 21 isolates, and plasmid analysis revealed further heterogeneity; a unique combination of characteristics was obtained for all strains except two O91:H21 isolates from beef . The latter strains were shown by PFGE to be the most closely related isolates, with >96% homology, and hence may be subtypes of the same strain . Overall, our results indicate that the combination of stx(2)-RFLP, stx(2) variant, and plasmid profile analyses is as powerful as PFGE for molecular investigation of STEC diversity . Finally, the non-O157:H7 STEC strains isolated from HUS patients were related to but not identical to those isolated from cattle and food samples in the same geographical area . The possibility that there are distinct lineages of non-O157:H7 STEC, some of which are more virulent for humans, should be investigated further.

Anal Biochem, 2001 Jun 1, 293(1), 22 - 30
Detection of analytes by immunoassay using up-converting phosphor technology; Niedbala RS et al.; Up-Converting Phosphor Technology (UPT) is based on lanthanide-containing, submicrometer-sized, ceramic particles that can absorb infrared light and emit visible light . Biological matrices do not up-convert; hence, there is no contribution to test background from sample autofluorescence . Up-converting phosphors do not photobleach and are inert to common assay interferants such as hemoglobin . A reader called UPlink has been developed to interrogate lateral flow test strips that utilize UPT labels . The reader contains a miniaturized, 1-W, infrared laser with peak emission at 980 nm . Preliminary assays that use up-converting phosphor labels, including tests for a drugs of abuse panel and Escherichia coli O157:H7, have been developed . In a "sandwich" assay format, 10(3) org/mL E . coli O157:H7 organisms were detectable in a negative control background of 10(9) other organisms per milliliter of culture medium . Coefficients of variation in concentrations tested from 0 to 10(7) org/mL were all < or =10% . In a competitive inhibition assay format, a multiplexed test simultaneously detected amphetamine, methamphetamine, phencyclidine, and opiates in saliva . For all assays, the percent displacement at 10 ng/mL was > or =40% demonstrating performance comparable with lab-based, commercially available EIAs . All assays were complete in 10 min . The development of rapid tests using UPT creates new applications for on-site testing with sensitivity not available using other label technologies .

J Bacteriol, 2001 Jun, 183(12), 3811 - 5
Insertion mutagenesis of wca reduces acid and heat tolerance of enterohemorrhagic Escherichia coli O157:H7; Mao Y et al.; Strains of enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 produce under stress copious amounts of exopolysaccharide (EPS) composed of colanic acid (CA) . Studies were performed to evaluate the association of production of CA with survival of EHEC under adverse environmental conditions . A CA-deficient mutant, M4020, was obtained from a CA-proficient parental strain, E . coli O157:H7 W6-13, by inserting a kanamycin resistance gene cassette (kan) into wcaD and wcaE, 2 of the 21 genes required for CA biosynthesis . M4020 was defective in CA production as determined from the ratio of uronic acid to protein (UA/P) of cells grown from 1 to 4 days at 25 degrees C on minimal glucose agar (MGA), MacConkey agar, and sorbitol-MacConkey agar, and by colony morphology on MGA . The results of stress treatment revealed that M4020 was substantially less tolerant to acid (pH 4.5 and 5.5) and heat (55 and 60 degrees C) in comparison to W6-13, indicating that CA confers on E . coli O157:H7 a protective effect from the environmental stresses of acid and heat.

Res Vet Sci, 2001 Apr, 70(2), 175 - 7
Molecular characterisation of Shiga toxin-producing Escherichia coli O157 strains isolated in Poland; Osek J; Ten Escherichia coli O157 strains isolated from cattle and children in Poland were investigated by the use of molecular biological methods . All strains possessed the intimin and enterohaemolysin genes and harboured the genetic determinants for Stx2 toxin (five isolates), Stx1 toxin (two strains) or both (three isolates) . The genetic relatedness of the strains was examined by restriction fragment length polymorphism (RFLP) of chromosomal DNA digested with Xbal and Notl . Nine closely related RFLP patterns were observed . Comparison of bovine and human E coli O157 isolates based on the analysis of Xbal and Notl digested profiles showed that all strains belonged to one genetic cluster . These results indicate that cattle must be considered as a possible source of human E coli O157 infection in Poland .

J Food Prot, 2001 May, 64(5), 599 - 605
Modeling the survival of Escherichia coli O157:H7 in apple cider using probability distribution functions for quantitative risk assessment; Duffy S et al.; Outbreaks of foodborne illness from apple cider have prompted research on the survival of Escherichia coli O157:H7 in this food . Published results vary widely, potentially due to differences in E . coli O157:H7 strains, enumeration media, and other experimental considerations . We developed probability distribution functions for the change in concentration of E . coli O157:H7 (log CFU/day) in cider using data from scientific publications for use in a quantitative risk assessment . Six storage conditions (refrigeration {4 to 5 degrees C}; temperature abuse {6 to 10 degrees C}; room temperature {20 to 25 degrees C}; refrigerated with 0.1% sodium benzoate, 0.1% potassium sorbate, or both) were modeled . E . coli survival rate data for all three unpreserved cider storage conditions were highly peaked, and these data were fit to logistic distributions: ideal refrigeration, logistic (-0.061, 0.13); temperature abuse, logistic (-0.0982, 0.23); room temperature, logistic (-0.1, 0.29) and uniform (-4.3, -1.8), to model the very small chance of extremely high log CFU reductions . There were fewer published studies on refrigerated, preserved cider, and these smaller data sets were modeled with beta (4.27, 2.37) x 2.2 - 1.6, normal (-0.2, 0.13), and gamma (1.45, 0.6) distributions, respectively . Simulations were run to show the effect of storage on E . coli O157:H7 during the shelf life of apple cider . Under every storage condition, with and without preservatives, there was an overall decline in E . coli O157:H7 populations in cider, although a small fraction of the time a slight increase was seen.

Eur J Epidemiol, 2000, 16(10), 885 - 9
Evidence of persisting serum antibodies to Escherichia coli O157 lipopolysaccharide and Verocytotoxin in members of rural communities in England; Evans J et al.; The techniques of enzyme-linked immunosorbent assay (ELISA) and immunoblotting were used to examine a total of 1667 sera, from apparently healthy members of rural communities in England, for antibodies to the lipopolysaccharide (LPS) of Escherichia coli O157 and Verocytotoxins (VT) . Twenty-nine sera from 22 individuals were shown to have antibodies specific for E . coli O157 LPS . Some of these lived on livestock farms and had occupational contact with cattle, suggesting that personnel working with farm animals may produce serum antibodies to the O157 LPS antigens . Fifteen people had IgG class antibodies to O157 LPS, suggesting long-term exposure to E . coli O157 and five people had serum antibodies on more than one occasion showing evidence of persistent antibodies to O157 LPS . Thirteen sera from 12 of 22 individuals also contained antibodies to VT1, VT2 or both toxins . Ten sera contained antibodies to VT1 and VT2, three sera contained antibodies to VT2 only.

MMWR Morb Mortal Wkly Rep, 2001 Apr 20, 50(15), 293 - 7
Outbreaks of Escherichia coli O157:H7 infections among children associated with farm visits--Pennsylvania and Washington, 2000; Type 1 fimbriation and its phase switching in diarrheagenic Escherichia coli strains; Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Fukuoka 812-8582, JapanType 1 fimbriae can be expressed by most Escherichia coli strains and mediate mannose-sensitive (MS) adherence to mammalian epithelial cells . However, the role of type 1 fimbriae in enteric pathogenesis has been unclear . Expression of type 1 fimbriae in E . coli is phase variable and is associated with the inversion of a short DNA element (fim switch) . Forty-six strains of diarrheagenic E . coli were examined for the expression of type 1 fimbriae . Only four of these strains were originally type 1 fimbriated . Seventeen strains, originally nonfimbriated, expressed type 1 fimbriae in association with off-to-on inversion of the fim switch, after serial passages in static culture . The switching frequencies of these strains, from fimbriate to nonfimbriate, were greater than that of the laboratory strain E . coli K-12 . None of the 16 strains of serovar O157:H7 or O157:H(-) expressed type 1 fimbriae after serial passages in static culture . The nucleotide sequence analysis of the fim switch region revealed that all of the O157:H7 and O157:H(-) strains had a 16-bp deletion in the invertible element, and the fim switch was locked in the "off" orientation . The results suggest that expression of type 1 fimbriae may be regulated differently in different E . coli pathogens causing enteric infections.

Pediatr Res, 2001 May, 49(5), 653 - 9
von Willebrand factor and von Willebrand factor-cleaving metalloprotease activity in Escherichia coli O157:H7-associated hemolytic uremic syndrome; Tsai HM et al.; Hemolytic uremic syndrome (HUS) usually occurs after infection with Shiga toxin-producing bacteria . Thrombotic thrombocytopenic purpura, a disorder with similar clinical manifestations, is associated with deficient activity of a circulating metalloprotease that cleaves von Willebrand factor at the Tyr842-Met843 peptide bond in a shear stress-dependent manner . We analyzed von Willebrand factor-cleaving metalloprotease activity and the status of von Willebrand factor in 16 children who developed HUS after Escherichia coli O157:H7 infection and in 29 infected children who did not develop this complication . Von Willebrand factor-cleaving metalloprotease activity was normal in all subjects, but von Willebrand factor size was decreased in the plasma of each of 16 patients with HUS . The decrease in circulating von Willebrand factor size correlated with the severity of thrombocytopenia and was proportional to an increase in von Willebrand factor proteolytic fragments in plasma . Immunohistochemical studies of the kidneys in four additional patients who died of HUS demonstrated glomerular thrombi in three patients, and arterial and arteriolar thrombi in one patient . The glomerular thrombi contained fibrin but little or no von Willebrand factor . A decrease in large von Willebrand factor multimers, presumably caused by enhanced proteolysis from abnormal shear stress in the microcirculation, is common in HUS.

Microbios, 2001, 104(409), 167 - 75
Acid tolerance of Escherichia coli O157:H7 and its survival in apple juice; Koodie L et al.; Outbreaks of diarrhoea and haemolytic uraemic syndrome have been associated with the consumption of apple cider and apple juice . The organism implicated in these outbreaks has been Escherichia coli O157:H7, indicating the resistance of the serotype to acidic pH . On comparing the growth of this serotype with a control strain of E . coli, it was found that strain O157:H7 grew well in trypticase soy broth at pH levels ranging from 2.0 to 9.0, while control strains failed to grow at pH levels below 4.0 and above 9.0 . The growth of both strains were inhibited by adding 0.05% of either benzoic acid or sorbic acid . Similarly, O157:H7 grew well in both natural (unpasteurized) as well as in pasteurized apple juice and the growth was inhibited by adding 0.1% of either benzoic acid or sorbic acid . Control strains of E . coli failed to grow in either types of apple juice . The possible sources of contamination of natural apple juice with O157:H7 serotype are discussed.

Tohoku J Exp Med, 2001 Jan, 193(1), 73 - 7
Thrombotic stroke in a child with diarrhea-associated hemolytic-uremic syndrome with a good recovery; Nakahata T et al.; A boy aged 3.5 years with post-diarrheal hemolytic-uremic syndrome (HUS) was referred to our hospital because of convulsion and stupor . He had been admitted to a regional hospital with a 3-day history of bloody diarrhea, colic abdominal pain and fever . Two days later, he had complained of generalized seizures and oliguria . On admission, he developed anuria, and serum blood nitrogen and creatinine increased to 56 mg/100 ml and 2.8 mg/100 ml, respectively . Platelets decreased to 42,000/microl . Under the diagnosis of HUS, a continuous hemodiafiltration treatment had to be instituted . Computed tomography of his head at hospital day 5 revealed abnormal low density area of infarction with edema in both the basal ganglia involving with the posterior limb of internal capsule . Serum titer of IgM antibody to Escherichia coli O157 showed positive value . Although his anuria and stupor persisted over 10 days, he recovered without serious complications . These clinical observations may indicate that patients with similar lesions do not necessarily have serious morbidity.

Appl Environ Microbiol, 2001 May, 67(5), 2367 - 70
Mutations in the csgD promoter associated with variations in curli expression in certain strains of Escherichia coli O157:H7; Uhlich GA et al.; Single-base-pair csgD promoter mutations in human outbreak Escherichia coli O157:H7 strains ATCC 43894 and ATCC 43895 coincided with differential Congo red dye binding from curli fiber expression . Red phenotype csgD::lacZ promoter fusions had fourfold-greater expression than white promoter fusions . Cloning the red variant csgDEFG operon into white variants induced the red phenotype . Substrate utilization differed between red and white variants.

FEMS Microbiol Lett, 2001 Apr 13, 197(2), 195 - 201
Use of representational difference analysis to identify Escherichia coli O157-specific DNA sequences; Allen NL et al.; Whereas several important virulence factors in Escherichia coli O157 have been identified, studies suggest they are not always essential and are probably insufficient to account for the severe clinical manifestation of E . coli O157 infection . Identification of putative virulence determinants is crucial to the understanding of bacterial pathogenesis and genomic comparison analysis may aid the characterisation of unidentified virulence attributes . In this study, representational difference analysis (RDA) was used for genomic comparison of E . coli O157 with the proposed ancestral strain, E . coli O55 . Unique E . coli O157 gene sequences were isolated and one, termed RDA-1, taken forward for further analysis . Southern blotting with labelled RDA-1 as a probe showed it to be present in 77% of E . coli O157 isolates and absent in all non-E . coli O157 screened . Sequence flanking RDA-1 was obtained from a genomic clone identified by hybridisation, and contained an open reading frame predicted to encode a novel iron-regulated outer membrane protein.

Biosci Biotechnol Biochem, 2001 Feb, 65(2), 414 - 9
Inhibition of Vero cell cytotoxic activity in Escherichia coli O157:H7 lysates by globotriaosylceramide, Gb3, from bovine milk; Watarai S et al.; In order to clarify the presence and verotoxin (VT) inhibitory activity of globotriaosylceramide (Gb3) in bovine milk, we analyzed neutral glycosphingolipids (GSLs) from bovine milk and investigated the inhibitory effect of bovine milk Gb3 on the cytotoxicity of VT2 . Five species of neutral GSLs, designated as N-1, N-2, N-3, N-4, and N-5, were separated on thin-layer chromatography (TLC) . N-1, N-2, and N-3 showed the same mobility as glucosylceramide, lactosylceramide, and Gb3 on the TLC plate, respectively . N-4 and N-5 GSLs migrated below globoside on the TLC plate . N-3 GSL having the same TLC mobility as Gb3 from bovine milk was immunologically identified as Gb3 by monoclonal antibody against Gb3, anti-CD77 monoclonal antibody . Furthermore, the effect of bovine milk Gb3 on VT2-induced cytotoxicity was investigated . We found that treatment of VT2 with bovine milk Gb3 can reduce the cytotoxic effect of VT2.

Appl Environ Microbiol, 2001 Apr, 67(4), 1983 - 5
Enhanced acid sensitivity of pressure-damaged Escherichia coli O157 cells; Pagan R et al.; Pressure-damaged Escherichia coli O157 cells were more acid sensitive than native cells and were impaired in pH homeostasis . However differences in acid sensitivity were not related to differences in cytoplasmic pH (pH(i)) . Cellular beta-galactosidase was more acid labile in damaged cells . Sensitization to acid may thus involve loss of protective or repair functions.

J Food Prot, 2001 Feb, 64(2), 147 - 51
Quantitative determination of the role of lettuce leaf structures in protecting Escherichia coli O157:H7 from chlorine disinfection; Takeuchi K et al.; Viability of Escherichia coli O157:H7 cells on lettuce leaves after 200 mg/liter (200 ppm) chlorine treatment and the role of lettuce leaf structures in protecting cells from chlorine inactivation were evaluated by confocal scanning microscopy (CSLM) . Lettuce samples (2 by 2 cm) were inoculated by immersing in a suspension containing 10(9) CFU/ml of E . coli O157: H7 for 24+/-1 h at 4 degrees C . Rinsed samples were treated with 200 mg/liter (200 ppm) chlorine for 5 min at 22 degrees C . Viability of E . coli O157:H7 cells was evaluated by CSLM observation of samples stained with Sytox green (dead cell stain) and Alexa 594 conjugated antibody against E . coli O157:H7 . Quantitative microscopic observations of viability were made at intact leaf surface, stomata, and damaged tissue . Most E . coli O157:H7 cells (68.3+/-16.2%) that had penetrated 30 to 40 microm from the damaged tissue surface remained viable after chlorine treatment . Cells on the surface survived least (25.2+/-15.8% survival), while cells that penetrated 0 to 10 microm from the damaged tissue surface or entered stomata showed intermediate survival (50.8 +/-13.5 and 45.6+/-9.7% survival, respectively) . Viability was associated with the depth at which E . coli O157:H7 cells were in the stomata . Although cells on the leaf surface were mostly inactivated, some viable cells were observed in cracks of cuticle and on the trichome . These results demonstrate the importance of lettuce leaf structures in the protection of E . coli O157:H7 cells from chlorine inactivation.

Vet Microbiol, 2001 Apr 19, 79(4), 323 - 35
Characterisation and clonal relationships of Shiga-toxigenic Escherichia coli (STEC) isolated from Australian dairy cattle; Cobbold R et al.; A total of 136 Shiga toxin-producing Escherichia coli (STEC) isolated during a longitudinal survey of three Australian dairy farms were examined to determine their virulence factors, serotype and genomic relationships . This study aimed to assess the potential of these STEC to cause disease in humans and to analyse the on-farm ecology of STEC . Virulence factors (stx, eae, ehxA) were used as determinants of potential to be enterohaemorrhagic E . coli (EHEC) and were examined using polymerase chain reaction (PCR) . Among the cattle groups tested, calves, both before and during weaning, shed the most putative EHEC and were the main source of serotypes commonly associated with human disease . E . coli O157:H7 and E . coli O26:H11 represented 9.4 and 7.8% of cattle STEC isolates respectively, with other putative EHEC serotypes reported for the first time from cattle . Based on serotype and virulence factors, 20% of STEC were putative EHEC . Pulsed-field gel electrophoresis (PFGE) was used to compare the genomic profiles of STEC from dairy farms . Isolates common to cattle and the farm environment were identified . Multiple strains of STEC with high clonal turnover were detected in the faeces of cattle, and isolates appeared to be specific to individual farms . To fully assess the pre-slaughter EHEC risk factors on-farm, examination of STEC virulence is as important as determination of STEC prevalence.

Lett Appl Microbiol, 2001 Mar, 32(3), 152 - 5
The survival of Escherichia coli O157:H7 in slurry from cattle fed different diets; McGee P et al.; AIMS: The survival characteristics of Escherichia Coli O157:H7 were investigated in bovine slurry from cattle fed two different diets: (i) silage and (ii) silage + concentrates . METHODS AND RESULTS: Slurry samples collected from freshly-agitated tanks were inoculated at a level of log10 6.0 cfu g(-1) and stored in the laboratory at 10 degrees C . Over a 12 week storage period, a 3.5 and 5.5 log reduction was observed in slurry from cattle fed a silage and silage plus concentrate diet, respectively . CONCLUSIONS: The persistence of E . coli O157:H7 in slurry over a 3 month storage period indicates its potential for transmitting the organism back into the environment . SIGNIFICANCE AND IMPACT OF THE STUDY: The discussion concludes however, that despite pathogen survival in slurry, it may not represent a major source of transmission in the farm environment.

Lett Appl Microbiol, 2001 Mar, 32(3), 126 - 30
Optimization of random amplification of polymorphic DNA analysis for molecular subtyping of Escherichia coli O157; Hopkins KL et al.; Random amplification of polymorphic DNA (RAPD) analysis using the polymerase chain reaction has proved to be a useful technique in the epidemiological investigation of micro-organisms but may suffer from a lack of reproducibility in poorly optimized protocols . In this study a method of obtaining reproducible genomic fingerprints using RAPD analysis of Escherichia coli O157 is described . By systematic optimization of reaction conditions and selection of suitable primers, reproducible and discriminatory profiles could be obtained from all E . coli O157 strains tested . In addition, two other methods of obtaining reproducible profiles from E . coli O157 strains without the need to purify genomic DNA are described.

Infect Immun, 2001 Apr, 69(4), 2107 - 15
Complete nucleotide sequence and analysis of the locus of enterocyte Effacement from rabbit diarrheagenic Escherichia coli RDEC-1; Zhu C et al.; The pathogenicity island termed the locus of enterocyte effacement (LEE) is found in diverse attaching and effacing pathogens associated with diarrhea in humans and other animal species . To explore the relation of variation in LEE sequences to host specificity and genetic lineage, we determined the nucleotide sequence of the LEE region from a rabbit diarrheagenic Escherichia coli strain RDEC-1 (O15:H-) and compared it with those from human enteropathogenic E . coli (EPEC, O127:H6) and enterohemorrhagic E . coli (EHEC, O157:H7) strains . Differing from EPEC and EHEC LEEs, the RDEC-1 LEE is not inserted at selC and is flanked by an IS2 element and the lifA toxin gene . The RDEC-1 LEE contains a core region of 40 open reading frames, all of which are shared with the LEE of EPEC and EHEC . orf3 and the ERIC (enteric repetitive intergenic consensus) sequence present in the LEEs of EHEC and EPEC are absent from the RDEC-1 LEE . The predicted promoters of LEE1, LEE2, LEE3, tir, and LEE4 operons are highly conserved among the LEEs, although the upstream regions varied considerably for tir and the crucial LEE1 promoter, suggesting differences in regulation . Among the shared genes, high homology (>95% identity) between the RDEC-1 and the EPEC and EHEC LEEs at the predicted amino acid level was observed for the components of the type III secretion apparatus, the Ces chaperones, and the Ler regulator . In contrast, more divergence (66 to 88% identity) was observed in genes encoding proteins involved in host interaction, such as intimin (Eae) and the secreted proteins (Tir and Esps) . A comparison of the highly variable genes from RDEC-1 with those from a number of attaching and effacing pathogens infecting different species and of different evolutionary lineages was performed . Although RDEC-1 diverges from some human-infecting EPEC and EHEC, most of the variation observed appeared to be due to evolutionary lineage rather than host specificity . Therefore, much of the observed hypervariability in genes involved in pathogenesis may not represent specific adaptation to different host species.

J Vet Med B Infect Dis Vet Public Health, 2001 Feb, 48(1), 67 - 72
Prevalence of eae and shiga toxin genes among Escherichia coli strains isolated from healthy calves; Osek J et al.; Strains of Escherichia coli (n = 390) isolated from 132 healthy, 4-8-week old calves, were tested by polymerase chain reaction (PCR) for the eae (intimin) gene and shiga toxin genes (stx1 and stx2) . All strains were also analysed for F5, F17 and F41 fimbriae and for the heat-labile (LT) and heat-stable (STI and STII) genetic markers . Overall, the eae gene was detected in 84 (21.5%) of the strains tested . Only 21 (5.4%) isolates were positive for stx1 (18 strains) or stx2 (three strains); nine of the stx1-positive isolates also possessed the eae gene . A high percentage (29.2%) of the isolates tested expressed F17 but no enterotoxin genes were detected . None of the eae- or stx-positive strains belonged to the O157 serogroup.

Int J Food Microbiol, 2001 Feb 28, 64(1-2), 127 - 38
Survival of Escherichia coli O157:H7 in frozen foods: impact of the cold shock response; Bollman J et al.; The survival of Escherichia coli O157:H7 strains in both frozen foods and trypticase soy broth (TSB) was investigated following cold shocking at 10 degrees C for 1.5 h . Using both trypticase soy agar (TSA) and violet red bile agar (VRBA) as recovery media, it was demonstrated that survival levels between cold shocked (CS) and non-cold shocked (NS) E . coli in ground beef or pork were not significantly different (P < or = 0.05) . In contrast, cold shocking E . coli in either milk, whole egg or sausage resulted in a significant(P < or = 0.05) enhancement in survival . For milk, survival levels of CS E . coli, by 28 days of frozen storage, were 1.89 and 1.66 log10 cfu/ml higher on TSA and VRBA, respectively, when compared to NS cells . In egg these values were 0.64 and 1.31, while in sausage, values of 0.74 and 1.19 were obtained . In TSB (pH 7) survival of CS E . coli for some strains was about 3 log10 cfu/ml higher when compared to NS cells . Acidification of TSB (pH 5), however, appeared to negate the protective effects of the cold shock treatment . In milk, increasing the differential between the growth and cold shock temperatures resulted in higher numbers of survivors . In this regard the growth-cold shock temperature protocol giving optimum protection was 37-10 degrees C . In contrast, growth of E . coli at 20 degrees C followed by cold shocking at 10 degrees C did not result in any significant freeze protection . In addition, increased protection due to cold shocking was correlated with the appearance of a novel protein appearing at pI 4.8 following isoelectric focusing analysis, thus demonstrating an alteration of protein synthesis.

J Food Prot, 2001 Mar, 64(3), 305 - 9
Survival and growth of Escherichia coli O157:H7 inoculated onto cut lettuce before or after heating in chlorinated water, followed by storage at 5 or 15 degrees C; Li Y et al.; This study determined the effects of mild heat and chlorine treatment followed by storage for up to 18 days at 5 degrees C or 7 days at 15 degrees C on the survival and growth of Escherichia coli O157:H7 inoculated onto fresh-cut iceberg lettuce . The efficacy of treatment with 20 ppm chlorine in killing the pathogen on lettuce at 50 degrees C was determined . Treatment of lettuce with 20 ppm chlorine at either 20 or 50 degrees C did not result in significantly greater reductions in populations of E . coli O157:H7 compared to respective treatments in water without chlorine . The pathogen steadily decreased in viability on treated lettuce throughout subsequent storage at 5 degrees C for 18 days . The population increased by 2.3 to 3.2 log10 CFU/g within 2 days, then continued to increase at a slower rate through 7 days of storage at 15 degrees C . At 4 and 7 days, significantly (alpha = 0.05) higher populations were reached on lettuce that had been treated at 50 degrees C, compared to respective samples that had been treated at 20 degrees C, regardless of the presence of 20 ppm chlorine in the treatment water . Treatment of lettuce with 20 ppm chlorine at 50 or 20 degrees C before or after inoculation with E . coli O157:H7 did not have a marked influence on behavior of the pathogen during subsequent storage at 5 or 15 degrees C.

J Food Prot, 2001 Mar, 64(3), 298 - 304
Expression of red-shifted green fluorescent protein by Escherichia coli O157:H7 as a marker for the detection of cells on fresh produce; Takeuchi K et al.; Escherichia coli O157:H7 was transformed with a plasmid vector red-shifted green fluorescence protein (pEGFP) to express red-shifted green fluorescence protein (EGFP) from Aequorea victoria . The EGFP expression among total cells and nonviable cells was determined at the cellular level by microscopic observation of immunostained and membrane-impermeable, dye-stained cultures, respectively . E . coli O157:H7 retained pEGFP during frozen storage at -80 degrees C . The percentage of EGFP expression was improved by repeated subculturing, reaching 83.4 +/- 0.1%, although the fluorescence intensity varied among cells . A low percentage of EGFP-expressing cells was nonviable . The percentage of EGFP decreased when the culture plate was kept at 4 degrees C, suggesting that some cells lost pEGFP during refrigeration . The storage of the culture suspension in sterile deionized water at 4 degrees C for 24 h reduced the percentage of EGFP expression, indicating that some EGFP was denatured . The application of EGFP as a marker for E . coli O157:H7 on green leaf lettuce, cauliflower, and tomato was evaluated using confocal scanning laser microscopy . EGFP-transformed cells were readily visible under confocal scanning laser microscopy on all produce types . The numbers of E . coli O157:H7 cells detected with EGFP were equivalent to those detected with immunostaining for green leaf lettuce and cauliflower but less for tomato . E . coli O157:H7 attached preferentially to damaged tissues of green leaf lettuce and tomato over intact tissue surfaces and to flowerets of cauliflower than to stem surfaces . EGFP can serve as a marker to characterize E . coli O157:H7 attachment on green leaf lettuce and cauliflower but may not be suitable on tomato.

J Clin Microbiol, 2001 Mar, 39(3), 1161 - 4
Particular biochemical profiles for enterohemorrhagic Escherichia coli O157:H7 isolates on the ID 32E system; Leclercq A et al.; The ability of the ID 32E system to identify and discriminate 74 Escherichia coli O157 isolates among 106 E . coli non-O157 isolates was evaluated . The results showed atypical biochemical reactions but accurate identification at the species level and no unique biochemical profile numbers for E . coli O157, although these numbers were distinct from those of other serotypes.

J Clin Microbiol, 2001 Mar, 39(3), 1140 - 3
Phage types and genotypes of Shiga toxin-Producing Escherichia coli O157 in Finland; Saari M et al.; This study examined Shiga toxin-producing Escherichia coli (STEC) O157, using phage typing, pulsed-field gel electrophoresis, and typing of Shiga toxin variant genes by PCR with restriction fragment length polymorphism in an epidemiological survey of STEC O157 isolated from humans in Finland between 1990 and 1999.

Rev Med Chil, 2000 Dec, 128(12), 1335 - 41
{Prevalence of enterohemorrhagic Escherichia coli in a cattle area of Argentina . Genotypic characterization of the strains of animal origin}; Notario R et al.; BACKGROUND: There is a high prevalence of infection by Enterohemorrhagic Escherichia coli (EHEC) and patients with hemolytic uremic syndrome (HUS) in Argentina . AIM: To study cattle and pigs as a possible reservoir of EHEC in Argentina . MATERIAL AND METHODS: One hundred two healthy animals (68 cattles and 31 pigs) from a livestock in Argentina, were studied . Stool samples were obtained with a rectal swab . The strains were identified by DNA hybridization with specific gene probes detecting Shiga-like toxin 1 and 2 (Stx1, Stx2), and hly gen related to fimbrial adhesin-associated plasmid . EHEC strains were serogrouped using commercial antisera . RESULTS: EHEC was isolated from 30 out of 68 bovines cultures (44.1%) and from 25 out of 31 pigs (58.1%) . Isolates carrying genes codifying both Stx1 and Sxt2, were observed in 50% of cattle and 63.9% of pigs . The gene which codifies for hemolysin (associated to fimbrial adhesin) was observed in about 41% of EHEC isolates . Strains belonging to serogroups O26, O111, and O157 were isolated from cattle, and O111, and O157 from pigs . CONCLUSIONS: The high percentage of EHEC in both cattle and pigs and the presence of human infection-associated serogroups, suggests that these animals are a reservoir of EHEC associated with disease in humans.

J Bacteriol, 2001 Mar, 183(6), 2081 - 5
Role for a phage promoter in Shiga toxin 2 expression from a pathogenic Escherichia coli strain; Wagner PL et al.; Shiga toxins (Stxs), encoded by the stxA and stxB genes, are important contributors to the virulence of Escherichia coli O157:H7 and other Stx-producing E . coli (STEC) strains . The stxA and stxB genes in STEC strains are located on the genomes of resident prophages of the lambda family immediately downstream of the phage late promoters (p(R')) . The phage-encoded Q proteins modify RNA polymerase initiating transcription at the cognate p(R') promoter which creates transcription complexes that transcend a transcription terminator immediately downstream of p(R') as well as terminator kilobases distal to p(R') . To test if this Q-directed processive transcription plays a role in stx(2)AB expression, we constructed a mutant prophage in an O157:H7 clinical isolate from which p(R') and part of Q were deleted but which has an intact pStx, the previously described stx(2)AB-associated promoter . We report that production of significant levels of Stx2 in this O157:H7 isolate depends on the p(R') promoter . Since transcription initiating at p(R') ultimately requires activation of the phage lytic cascade, expression of stx(2)AB in STEC depends primarily on prophage induction . By showing this central role for the prophage in stx(2)AB expression, our findings contradict the prevailing assumption that phages serve merely as agents for virulence gene transfer.

Epidemiol Infect, 2000 Dec, 125(3), 537 - 48
Genomic fingerprinting of shigatoxin-producing Escherichia coli (STEC) strains: comparison of pulsed-field gel electrophoresis (PFGE) and fluorescent amplified-fragment-length polymorphism (FAFLP); Heir E et al.; For epidemiological studies of shigatoxin-producing Escherichia coli (STEC) infections, rapid, reproducible and highly discriminative methods are required . In this study, we examined the performance of the fluorescent amplified-fragment-length polymorphism (FAFLP) technique for epidemiological fingerprinting of STEC isolates and compared it to the acknowledged fingerprinting method pulsed-field gel electrophoresis (PFGE) . A total of 88 STEC isolates, including 82 of serotype O157:H7 or O157:H-, were subjected to fingerprinting by both PFGE and FAFLP . The isolates included sporadic and epidemiologically related strains of both animal and human origin from widespread geographical locations . The FAFLP fingerprint patterns confirmed the clonal nature of STEC O157 strains . Among the 82 O157:H7/H- isolates belonging to 49 distinct groups of epidemiological unrelated isolates, 24 FAFLP profiles and 51 PFGE patterns were obtained . Thus, PFGE had a higher discriminatory power than FAFLP and overall correlated better to available epidemiological data . Consequently, the PFGE technique remains the method of choice in epidemiological investigations of STEC infections.

Zh Mikrobiol Epidemiol Immunobiol, 2000 Nov-Dec, (6), 90 - 4
{PCR test systems for genetic identification of enterohemorrhagic Escherichia coli}; Vorob'ev AA et al.; The analysis of modern data on the development of amplification test systems for the gene indication of enterohemorrhagic E . coli (EHEC) is presented . In this work the emphasis is laid on the importance of using specific primers whose nucleotide sequence is closely linked with genes controlling the key factors of EHEC pathogenicity; these factors include the determinants of the synthesis of adhesins and invasins (bfp, eae, tir), shiga-like toxins (stx1, stx2), enterohemolysin (ehx), serine protease (epsA) and specific LPS of O-antigen (rfb) . The problem of using primers whose sequence is not linked with virulence genes, but which may also be used for the gene indication of E . coli O157:H7 (uid, fliC) is discussed.

J Med Microbiol, 2001 Jan, 50(1), 90 - 5
Restriction endonuclease analysis of RAPD-PCR amplicons derived from Shiga-like toxin-producing Escherichia coli O157 isolates; Hopkins KL et al.; Shiga-like toxin-producing Escherichia coli O157 isolates were characterised by random amplification of polymorphic DNA by PCR (RAPD-PCR) analysis developed to allow robust epidemiological typing of E . coli . Amplification with primer 1247 or 1290 generated a reproducible profile, but was not capable of distinguishing sufficiently between epidemiologically unrelated strains . Subsequent digestion of the amplicons with selected restriction endonucleases improved the discriminatory ability of this method for strains showing limited differentiation following RAPD-PCR analysis alone . Restriction endonuclease analysis of RAPD-PCR fragments generated from closely related strains has the potential to provide additional discriminatory information without loss of specificity.

Lett Appl Microbiol, 2001 Feb, 32(2), 118 - 22
Occurrence and virulence factors of non-O157 Shiga toxin-producing Escherichia coli in retail meat in Dunedin, New Zealand; Brooks HJ et al.; Retail raw meat was sampled for the presence of Shiga toxin-producing Escherichia coli (STEC) using enrichment culture and Vero cell assay . The STEC obtained were serotyped and tested for enterohaemolysin (Ehly) production and the eae gene . The presence of Shiga toxin genes (stx) was confirmed by polymerase chain reaction . A total of 18 STEC were isolated accounting for 12% of beef, 17% of lamb and 4% of pork samples . Five isolates produced Ehly but none possessed the eae gene . Five isolates were identified which possessed the stx2 gene and belonged to serotypes associated with severe infection.

Anal Biochem, 2001 Feb 15, 289(2), 281 - 8
Use of real-time polymerase chain reaction and molecular beacons for the detection of Escherichia coli O157:H7; Fortin NY et al.; Molecular beacons (MBs) are oligonucleotide probes that fluoresce upon hybridization . In this paper, we described the development of a real-time PCR assay to detect the presence of Escherichia coli O157:H7 using these fluorogenic reporter molecules . MBs were designed to recognize a 26-bp region of the rfbE gene, coding for an enzyme necessary for O-antigen biosynthesis . The specificity of the MB-based PCR assay was evaluated using various enterohemorrhagic (EHEC) and Shiga-like toxin-producing (STEC) E . coli strains as well as bacteria species that cross-react with the O157 antisera . All E . coli serotype O157 tested was positively identified while all other species, including the closely related O55 were not detected by the assay . Positive detection of E . coli O157:H7 was demonstrated when >10(2) CFU/ml was present in the samples . The capability of the assay to detect E . coli O157:H7 in raw milk and apple juice was demonstrated . As few as 1 CFU/ml was detected after 6 h of enrichment . These assays could be carried out entirely in sealed PCR tubes, enabling rapid and semiautomated detection of E . coli O157:H7 in food and environmental samples .

Microbiology, 2001 Jan, 147(Pt 1), 145 - 52
Analysis of type 1 fimbriae expression in verotoxigenic Escherichia coli: a comparison between serotypes O157 and O26; Roe AJ et al.; Previous research has shown that verotoxin-producing Escherichia coli (VTEC) O157 strains appear unable to express type 1 fimbriae although other serotypes such as O26 and O118 can . This study has investigated the molecular basis of this difference . The study confirmed the presence of a 16 bp deletion within the regulatory region of fimA (fim switch) in 63 VTEC O157 strains but not in other VTEC serotypes tested . The fim switch was shown to be detectable only in the phase off orientation in VTEC O157, but detection of the switch in the phase on orientation correlated with the degree of mannose-sensitive yeast agglutination in VTEC O26 . Repair of the 16 bp deletion in the VTEC O157 fim switch region restored phase-variable expression of fimA in a permissive background . Non-O157 VTEC, especially O26 and O118, can be pathogenic in cattle; the role of type 1 fimbriae in this and colonization is discussed.

J Med Microbiol, 2000 Apr, 49(4), 383 - 6
Identification and characterisation of Escherichia coli strains of O157 and non-O157 serogroups containing three distinct Shiga toxin genes; Furst S et al.; Three Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from patients with diarrhoea were identified, each of which contained three distinct stx genes (stx1, stx2 and stx2c) . The strains belonged to the serotypes O52:H19, O75:H- and O157:H- and harboured eae and EHEC-hly sequences . Colony-blot immunoassay was used to demonstrate that both major types of Stx were expressed . The association of stx genes with either phage or phage DNA was demonstrated in all three strains . Isolated phage DNA from all strains contained stx1 sequences, but stx2 sequences were found only in phage DNA of two of these strains . The presence of three distinct stx genes may enhance the virulence of STEC strains and should be monitored . The observations demonstrate not only the potential of stx genes to spread within different serotypes, but also their capacity to accumulate within a single strain.

Eur J Epidemiol, 2000, 16(8), 757 - 62
Virulence genotypes and serotypes of verotoxigenic Escherichia coli isolated from cattle and foods in Argentina . Importance in public health; Parma AE et al.; Virulence factors of Verotoxin-producing Escherichia coli (VTEC) strains isolated from hamburgers and ground beef were studied in Argentina by PCR . Their virulence profiles were correlated with those corresponding to strains isolated from calves and adult cattle . Most virulent profiles (VTs+ eae+ Mp+) were present in E . coli from healthy and diarrheic calves corresponding to O5:H-, O5:H27, O20:H?, O26:H11, O38:H?, O103:H-, O103:H2, O111:H-, O118:H16, O165:H-serotypes . The presence of the eae gene was significantly more frequent among VTEC strains isolated from calves (20/26; 76%) than from adult cattle (1/39; 2.5%) (p < 0.005) . VT2+ eae- E . coli was prevalent in foods and adult cattle at slaughterhouse . The prevalence of the eae gene was similar between VTEC strains isolated from meat (0/21) and adult cattle (1/39; 2.5%) which constitutes the main population processed at slaughterhouses in Argentina . Serotyping showed that VTEC strains were distributed among 31 serotypes, some of which (O20:H19, O91:H21, O113:H21, O116:H21, O117:H7, O171:H2, OX3:H21) were shared between bovine and food strains . These O serogroups have been isolated from cases of haemorrhagic colitis (HC) and haemolyticuraemic syndrome (HUS) in humans in several continental European countries . This study confirms the role of cattle as a reservoir of many VTEC serotypes other than O157:H7 and represents a base for future diagnostic, prevention and control strategies of EHEC in this country . In addition, this study affirms the advantages of PCR-based screening of E . coli isolates given the finding of so many verotoxin-producing strains.

J Clin Microbiol, 2001 Jan, 39(1), 24 - 8
Genetic analysis for virulence factors in Escherichia coli O104:H21 that was implicated in an outbreak of hemorrhagic colitis; Feng P et al.; Isolates of enterohemorrhagic Escherichia coli (EHEC) of serotype O104:H21 implicated in a 1994 outbreak of hemorrhagic colitis in Montana were analyzed for the presence of trait EHEC virulence markers . By using a multiplex PCR that specifically amplifies several genes, the O104:H21 strains were found to carry only the Shiga toxin 2 gene (stx2) and to express Stx2 . They did not have the eaeA gene for gamma-intimin, which is typically found in O157:H7, or the alpha- or beta-intimin derivatives, which are common in other EHEC and enteropathogenic E . coli serotypes . Results of the multiplex PCR also indicated that the ehxA gene for enterohemolysin was absent from O104:H21 . This, however, was not consistent with the results of a phenotypic assay that showed them to be hemolytic or a PCR analysis with another set of ehxA-specific primers, which indicated the presence of ehxA . To resolve this discrepancy, the ehxA region in O104:H21 and O157:H7 strains, to which the multiplex PCR primers anneal, was cloned and sequenced . Comparison of the sequences showed that the upstream primer binding site in the ehxA gene of O104:H21 was not identical to that of O157:H7 . Specifically, there were several base mutations, including an A-to-G substitution at the 3' end of the primer binding site . These base mutations are presumably not unique to O104:H21, since other enterohemolytic serotypes were also not detected with the ehxA primers used in the multiplex PCR . Comparison of the ehxA sequences of O104:H21 strains with those of other Stx-producing E . coli strains showed that they more closely resembled those of O8:H19 strains, which have cluster II ehxA genes, than those of O157:H7 strains, which have cluster I ehxA sequences . By modifying the upstream ehxA primer, the multiplex PCR was able to detect ehxA genes in both O157:H7 and O104:H21 strains.

Appl Environ Microbiol, 2001 Jan, 67(1), 133 - 41
Modeling of combined processing steps for reducing Escherichia coli O157:H7 populations in apple cider; Uljas HE et al.; Probabilistic models were used as a systematic approach to describe the response of Escherichia coli O157:H7 populations to combinations of commonly used preservation methods in unpasteurized apple cider . Using a complete factorial experimental design, the effect of pH (3 . 1 to 4.3), storage temperature and time (5 to 35 degrees C for 0 to 6 h or 12 h), preservatives (0, 0.05, or 0.1% potassium sorbate or sodium benzoate), and freeze-thaw (F-T; -20 degrees C, 48 h and 4 degrees C, 4 h) treatment combinations (a total of 1,600 treatments) on the probability of achieving a 5-log(10)-unit reduction in a three-strain E . coli O157:H7 mixture in cider was determined . Using logistic regression techniques, pH, temperature, time, and concentration were modeled in separate segments of the data set, resulting in prediction equations for: (i) no preservatives, before F-T; (ii) no preservatives, after F-T; (iii) sorbate, before F-T; (iv) sorbate, after F-T; (v) benzoate, before F-T; and (vi) benzoate, after F-T . Statistical analysis revealed a highly significant (P < 0.0001) effect of all four variables, with cider pH being the most important, followed by temperature and time, and finally by preservative concentration . All models predicted 92 to 99% of the responses correctly . To ensure safety, use of the models is most appropriate at a 0.9 probability level, where the percentage of false positives, i.e., falsely predicting a 5-log(10)-unit reduction, is the lowest (0 to 4.4%) . The present study demonstrates the applicability of logistic regression approaches to describing the effectiveness of multiple treatment combinations in pathogen control in cider making . The resulting models can serve as valuable tools in designing safe apple cider processes.

J Infect Dis, 2001 Feb 1, 183(3), 435 - 43 Epub 2000 Dec 27.
Immunoprophylactic potential of cloned Shiga toxin 2 B subunit; Marcato P et al.; The Shiga toxins Stx1 and Stx2 contribute to the development of enterohemorrhagic O157:H7 Escherichia coli-mediated colitis and hemolytic-uremic syndrome in humans . The Stx2 B subunit, which binds to globotriaosylceramide (GB3) receptors on target cells, was cloned . This involved replacing the Stx2 B subunit leader peptide nucleotide sequences with those from the Stx1 B subunit . The construct was expressed in the TOPP3 E . coli strain . The Stx2 B subunits from this strain assembled into a pentamer and bound to a GB3 receptor analogue . The cloned Stx2 B subunit was not cytotoxic to Vero cells or apoptogenic in Burkitt's lymphoma cells . Although their immune response to the Stx2 B subunit was variable, rabbits that developed Stx2 B subunit-specific antibodies, as determined by immunoblot and in vitro cytotoxicity neutralization assays, survived a challenge with Stx2 holotoxin . This is thought to be the first demonstration of the immunoprophylactic potential of the Stx2 B subunit.

Acta Med Okayama, 2000 Dec, 54(6), 265 - 73
Epidemiological studies of coincidental outbreaks of enterohemorrhagic Escherichia coli O157:H7 infection and infectious gastroenteritis in Niimi City; Tsumagari K et al.; A sharp rise in the number of patients with infectious gastroenteritis was observed in the 25th week of year 1996 in the Takahashi-Ashin district by researchers with the Infectious Disease Surveillance Program for tuberculosis and other infectious diseases in the Okayama Prefecture . This sharp rise occurred coincidentally with an outbreak of enterohemorrhagic Escherichia coli O157:H7 (EHEC O157) infection in Niimi City of the Takahashi-Ashin district . However, this phenomenon of coincidental outbreaks was not observed during the outbreak of EHEC O157 infection in Oku Cho . By reviewing outpatients' charts in a sentinel hospital in Niimi City for the Infectious Disease Surveillance Program, it was noted that patients with acute gastrointestinal infection visiting the hospital during the increased incidence of infectious gastroenteritis may have been included as misclassified cases of EHEC O157 infection . On the other hand, the exponential probability plotting of symptomatic patients with EHEC O157 infection in Niimi City revealed a breaking point which suggested a dual exposure to contaminated food or an overlap with other acute gastrointestinal infections . The latter possibility was discounted, because stool culture-positive patients with EHEC O157 infection also exhibited a similar breaking point, and furthermore, the coincidental increase in infectious gastroenteritis in the same area was attributable to the EHEC O157 infection . The present study demonstrates the association between the sharp rise in gastroenteritis and the outbreak of EHEC O157 in the Takahashi-Ashin district . A careful analysis of the cases of infectious gastroenteritis by the Infectious Disease Surveillance Program would have predicted the outbreak of EHEC O157.

J AOAC Int, 2000 Nov-Dec, 83(6), 1349 - 56
Sensitivity and specificity of the test kit BAX for screening/E . coli O157:H7 in ground beef: independent laboratory study; Hochberg AM et al.; An independent laboratory study of the BAX for Screening/E . coli O157:H7 kit was conducted at the National Food Laboratory, Inc., Dublin, CA, to complete AOAC Performance Tested Method certification . The BAX system kit was compared with the BAM culture method and a modified BAM culture method for detection of E . coli O157:H7 in ground beef . The BAX system kit detected the target organism at levels approximately 10-fold lower than those that gave positive BAM results . This study validated product claims, and Performance Tested Method status was granted.

Microbiol Immunol, 2000, 44(10), 857 - 61
Functional analysis of the type III secretion system in enteropathogenic Escherichia coli O157:H45; Abe A et al.; A mass outbreak of Escherichia coli O157:H45 was first reported in Japan in 1998 . This pathogen was classified as an enteropathogenic E . coli (EPEC) O157 because it was characterized by the Shiga toxin gene (stx)-negative and bundle-forming pilus (bfp) gene-positive genotypes . In this study, we investigated the type III secretion system in EPEC O157 . Although no type III secreted proteins, Esps (E . colisecreted proteins), in EPEC O157:H45 were detectable in culture supernatant, secreted proteins were induced by the introduction of an EPEC plasmid-encoded regulator, per . In further contrast to EHEC O157:H7, EPEC O157:H45 triggered the accumulation of tyrosine phosphorylated proteins beneath the adherent bacteria . These results suggest that regulation of the type III secretion apparatus and host signal transduction events between E . coli O157:H45 and O157:H7 are completely different.

J Med Entomol, 2000 Nov, 37(6), 945 - 9
Epidemiological potential of excretion and regurgitation by Musca domestica (Diptera: Muscidae) in the dissemination of Escherichia coli O157: H7 to food; Sasaki T et al.; We previously reported that enterohemorrhagic Escherichia coli O157: H7 (EHEC) proliferates in the mouthparts of the house fly Musca domestica vicina Macquart and are excreted for at least 3 d after ingestion . However, the role of the crop and excretory behavior of the house fly in contamination of human food with EHEC is not known . In the current experiments, EHEC persisted in the crop of house flies for at least 4 d . The number of EHEC in an excreted droplet was approximately 10(4) 1 h after bacterial feeding, > 1.8 x 10(5) 3 h after feeding, and then drastically decreased after 24 h . Excretion is one of the major mechanisms for decreasing number of EHEC in the crop and gut of the house fly . The frequency of excretion by females with developing eggs in their ovary was clearly higher (6.5 min per drop) than for males or females with mature eggs . Minute eosin-sign around a container filled with eosin-supplemented trypticase soy broth might be derived from frequent contact by house fly contaminated mouthparts . These results show that frequent excretion potentially enhances the dissemination of EHEC to foods, particularly during the first 24 h after ingestion of the bacteria.

FEMS Immunol Med Microbiol, 2000 Dec, 29(4), 283 - 8
Detection of PCR products of Escherichia coli O157:H7 in human stool samples using surface plasmon resonance (SPR); Kai E et al.; A method for the rapid detection of verotoxin-producing Escherichia coli O157:H7 in stools was evaluated . Strains possessing Shiga toxin-2 (stx-2) genes were isolated from stool samples and amplified using oligonucleotide primers . Stools spiked with cultured E . coli O157:H7 (strain 298 or strain 1646) were detected to be polymerase chain reaction (PCR) positive at 10(2) cfu per 0.1 g of stool . Stool samples from patients and healthy carriers showed a high correlation between positive results for a PCR and the presence of verotoxin-producing E . coli O157:H7, confirmed by isolation of serotype O157:H7 on sorbitol MacConkey medium (10 of 10 stool samples) . These PCR products could be detected using a BIAcore 2000 surface plasmon resonance device using peptide nucleic acid as a sensor probe . In this report we use this method for the rapid detection of DNA from significant pathogenic organisms.

Syst Appl Microbiol, 2000 Oct, 23(3), 315 - 24
Comparative analysis of the whole set of rRNA operons between an enterohemorrhagic Escherichia coli O157:H7 Sakai strain and an Escherichia coli K-12 strain MG1655; Ohnishi M et al.; Two primer sets for direct sequence determination of all seven rRNA operons (rrn) of Escherichia coli have been developed; one is for specific-amplification of each rrn operon and the other is for direct sequencing of the amplified operons . Using these primer sets, we determined the nucleotide sequences of seven rrn operons, including promoter and terminator regions, of an enterohemorrhagic E . coli (EHEC) O157:H7 Sakai strain . To elucidate the intercistronic or intraspecific variation of rrn operons, their sequences were compared with those for the K-12 rrn operons . The rrn genes and the internal transcribed spacer regions showed a higher similarity to each other in each strain than between the corresponding operons of the two strains . However, the degree of intercistronic homogeneity was much higher in the EHEC strain than in K-12 . In contrast, promoter and terminator regions in each operons were conserved between the corresponding operons of the two strains, which exceeded intercistronic similarity.

J Clin Microbiol, 2000 Dec, 38(12), 4616 - 20
Genotyping of verocytotoxin-producing Escherichia coli O157: comparison of isolates of a prevalent phage type by fluorescent amplified-fragment length polymorphism and pulsed-field gel electrophoresis analyses; Smith D et al.; We applied the high-resolution genotyping technique fluorescent amplified-fragment length polymorphism (FAFLP) analysis to 71 isolates of a single phage type (PT8) of pulsed-field gel electrophoresis (PFGE)-characterized verocytotoxin-producing Escherichia coli O157 . Twenty-seven similar, but not identical, groupings were defined by both FAFLP analysis and the PFGE profiles . Given the FAFLP analysis conditions described here, these two methods exhibited equivalent discriminatory powers.

Appl Environ Microbiol, 2000 Dec, 66(12), 5540 - 3
Loss of O157 O antigenicity of verotoxin-producing Escherichia coli O157:H7 surviving under starvation conditions; Hara-Kudo Y et al.; Verotoxin (VT)-producing Escherichia coli O157:H7 was culturable on agar media after being left in water for 21 months . However, there were a number of colonies which had lost O157 O antigenicity . These colonies produced VTs, which are pathogenic to humans . These observations suggest that the immunologic methods based on O157 O antigenicity are unable to detect and isolate VT-producing E . coli in foods and other environments if the organism has been under starvation conditions for a long period.

Appl Environ Microbiol, 2000 Dec, 66(12), 5536 - 9
Does enterohemorrhagic Escherichia coli O157:H7 enter the viable but nonculturable state in salted salmon roe?
Makino SI, Kii T, Asakura H, Shirahata T, Ikeda T, Takeshi K, Itoh K.
An outbreak caused by salted salmon roe contaminated with enterohemorrhagic Escherichia coli O157 occurred in Japan in 1998 . Since about 0.75 to 1.5 viable cells were estimated to cause infection, we presumed that O157 might enter the viable but nonculturable (VNC) state in salted salmon roe and consequently that viable cell numbers might be underestimated . Although patient-originating O157 cells could not grow on agar plates after 72 h of incubation in 13% NaCl, they were resuscitated in yeast extract broth, and more than 90% of the cells were shown to be viable by fluorescent staining, suggesting that almost all of them could enter the VNC state in NaCl water . Roe-originating O157 was resistant to NaCl because it could grow on agar after 72 h of incubation in NaCl water, but about 20% of cells appeared to enter the VNC state . Therefore, germfree mice were infected with O157 to examine the resuscitation of cells in the VNC state and the retention of pathogenicity . O157 that originated in roe, but not patients, killed mice and was isolated from the intestine . However, these isolates had become sensitive to NaCl . O157 cells of roe origin incubated in normal media also killed mice and were isolated from the intestine, but they also became transiently NaCl sensitive . We therefore propose that bacterial cells might enter the VNC state under conditions of stress, such as those encountered in vivo or in high salt concentrations, and then revive when those conditions have eased . If so, the VNC state in food is potentially dangerous from a public health viewpoint and may have to be considered at the time of food inspection . Finally, the establishment of a simple recovery system for VNC cells should be established.

Curr Infect Dis Rep, 2000 Feb, 2(1), 61 - 67
Shiga Toxin-Producing Escherichia coli; Jaeger JL et al.; Shiga toxin-producing Escherichia coli (STEC) are emerging as a significant source of foodborne infectious disease in the developed world . Multistate outbreaks of E . coli O157 and non-O157 serogroups in the United States are facilitated by the centralization of food processing and distribution . Our ability to recognize the clonality of these clusters has been advanced by developments in molecular detection techniques and in the establishment of active surveillance practices . These studies have helped identify important risk factors for both sporadic and outbreak STEC infection, allowing us to develop appropriate prevention strategies . Identification of these factors is of critical importance because of the lack of adequate treatments available . This brief review of the literature discusses major developments in the epidemiology, pathogenesis, diagnosis, treatment, and prevention of STEC disease published in the past few years.

Mol Cell Probes, 2000 Dec, 14(6), 333 - 7
Multiplex PCR for detection of trait and virulence factors in enterohemorrhagic Escherichia coli serotypes; Feng P et al.; A multiplex PCR assay was developed which allowed the simultaneous detection of five trait genes or virulence markers in enterohemorrhagic Escherichia coli (EHEC) serotypes . A primer pair, designed to detect a single base-pair mutation in the uidA gene, is specific only for the prototypic EHEC of O157:H7 serotype and its toxigenic, non-motile variants . In a similar way, primers to the eaeA gene of the gamma-intimin derivative specifically detects strains in the EHEC 1 clonal group, which consists mostly of O157:H7 and some O55:H7 serotypes . The other three primer pairs, specific for stx1, stx2 and both variants of ehxA genes, will detect the presence of these virulence genes in all EHEC serotypes . Analysis of 34 strains, including various serotypes of EHEC, Shiga toxin-producing E . coli and enteropathogenic E . coli, confirmed that the multiplex PCR assay detected the presence of these genes in a manner consistent with the known genotype of each respective strains . Copyright 2000 Academic Press.

What Is Listeria Monocytogenes?, What Is Salmonella?, What Is Dna?, What Is Amino Acid?, What Is Anthrax?, e, Bacteria, r, Microorganism, r, Microorganisms, c, Microbe, e, Microbiology, c, Haemophilus, a, Cell suspensions, c, Antibiotics, c, Salmonella typhimurium, r, Escherichia coli, e, Pseudomonas, o, Bacteriological, a, Bacillus, s, Pseudomonas aeruginosa, e, P. fluorescens, s, Prokaryotes, i, S. cerevisiae, i, Lactobacilli, o, Bacteriophages, e, Corynebacter, c, Antibiotics, n, Streptococcal, e, Bacteria, o, Escherichia coli, a, Pseudomonas aeruginosa, n, S. cerevisiae

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005