J Food Prot, 2000 Nov, 63(11), 1587 - 90 Analysis and modeling of the variability associated with UV inactivation of Escherichia coli in apple cider; Duffy S et al.; Raw data from validation studies of UV tubes used for nonthermal pathogen reduction in apple cider underwent comprehensive statistical analysis . Data from each tube that demonstrated at least a 5-log reduction of Escherichia coli ATCC 25922, a surrogate for E . coli O157:H7, in each of three trials were used in the analysis . The within- and between-tube variability was calculated for 70 tubes . The mean log reductions of the tubes fit a Beta distribution (Kolmogorov-Smirnov test, 0.0246), and the between-replicate variability followed a logistic distribution (Kolmogorov-Smirnov test, 0.0305) . These two distributions can be used together to model UV cider treatment as part of an overall E . coli O157:H7 in cider risk assessment . Examples of codes from @RISK and Analyticato describe these distributions, such as one would find in a quantitative risk assessment, are included. Lett Appl Microbiol, 2000 Nov, 31(5), 364 - 7 Survival of Escherichia coli O157:H7 in potato starch as affected by water activity, pH and temperature; Park CM et al.; A study was done to determine the survival characteristics of Escherichia coli O157:H7 in potato starch powder as affected by aw (0.24-0.26, 0.51-0.52 and 0.75-0.78), pH (4.1 and 6.7) and temperature (4, 20 and 37 degrees C) over a 33-week storage period . Survival was enhanced as the aw decreased . The rate of death was higher as the storage temperature increased . Survival did not appear to be affected by pH . Since the composition of foods greatly affects the viability of E . coli O157:H7 at reduced aw, development of models to predict patterns of inactivation in a given food should be carried out using data generated from that food or one with very similar composition, aw and pH. Lett Appl Microbiol, 2000 Nov, 31(5), 349 - 52 Survival of Escherichia coli O157 in faeces of experimentally infected rats and domestic pigeons; Cizek A et al.; In order to evaluate the role of some synanthropic animals in the spreading of Escherichia coli O157, laboratory rats and domestic pigeons were experimentally infected per os with E . coli O157 . Rats infected with 10(5) colony forming units (cfu) (n = 5) and 10(9) cfu (n = 5) shed E . coli O157 for 2 +/- 1.7 d and 9.8 +/- 1.3 d, respectively . In the faeces of infected rats stored at 4 degrees C in a moist environment, at 4 degrees C in a dry environment or at 20 degrees C in a moist environment, E . coli O157 survived for 34 weeks . When stored at 20 degrees C or - 20 degrees C in a dry environment, E . coli O157 survived for greater than or = 36 weeks . Pigeons infected with 10(5) cfu (n = 5) and 10(9) cfu (n = 5) shed the pathogen for 14.8 +/- 3.4 d and 20.2 +/- 5.2 d, respectively . Both species, rats and pigeons, can be important in spreading of the E . coli O157 infection in cattle. Lett Appl Microbiol, 2000 Oct, 31(4), 338 - 41 Improved isolation of Escherichia coli O157 using large enrichment volumes for immunomagnetic separation; Ogden ID et al.; The recovery of low numbers of Escherichia coli O157 in foods by immunomagnetic separation (IMS) was improved, on average, ninefold by increasing the enrichment volume tested from 1 to 10 ml while maintaining the volume of immunobeads constant at 0.02 ml . Although 50 ml volumes also gave improved recoveries (approximately threefold), the 50 ml volume cannot be recommended until a suitable magnetic separation apparatus has been developed . By testing 10 ml volumes, the improved sensitivity of the IMS procedure will reduce false-negative E . coli O157 tests and help improve epidemiological studies of this pathogen. Pediatr Int, 2000 Oct, 42(5), 523 - 7 Usefulness of serum fibrinogen degradation product-E in sporadic cases of classical hemolytic uremic syndrome; Aihara Y et al.; BACKGROUND: Hemolytic uremic syndrome (HUS) associated with Shiga toxin (Stx)-producing Escherichia coli O157:H7 infection is one of the diseases causing acute renal failure in young children . Although HUS is still a serious disease in children, no reliable predictive markers for HUS nor markers of disease severity are available so far . Recently, we experienced a sporadic case of typical HUS caused by Stx-producing E . coli O157:H7 and detected, at the prodromal stage, a high level of serum fibrin/fibrinogen degradation product-E (FDP-E) fraction . METHODS: To assess the usefulness of FDP-E for the treatment of HUS in clinical practice, we retrospectively examined serum levels of FDP-E in 22 patients with bloody diarrhea with or without HUS . RESULTS: There were significantly increased levels of serum FDP-E in patients with HUS, but not in those without HUS . Furthermore, serum levels of FDP-E may correlate with disease severity in patients with HUS . CONCLUSIONS: These results suggest that serum levels of FDP-E may be a useful marker of HUS in clinical practice. Epidemiol Infect, 2000 Aug, 125(1), 35 - 45 Estimation of the under-reporting rate for the surveillance of Escherichia coli O157:H7 cases in Ontario, Canada; Michel P et al.; Two models estimating the proportion of Escherichia coli O157:H7 cases not reported in the Ontario notifiable diseases surveillance system are described . The first model is a linear series of adjustments in which the total number of reported cases is corrected by successive underreporting coefficients . The structure of the second model is based on a relative difference in the proportion of E . coli O157:H7 cases which are hospitalized between the surveillance database and the underlying population . Based on this analysis, the rate of under-reporting of symptomatic cases of E . coli O157:H7 infection in Ontario ranges from 78 to 88% corresponding to a ratio of 1 reported case for approximately 4-8 symptomatic cases missed by the surveillance system . This study highlights the need to increase awareness among public health workers of the potential biases that may exist in the interpretation of routine surveillance data. Thromb Haemost, 2000 Oct, 84(4), 712 - 21 Verotoxin-1 induces tissue factor expression in human umbilical vein endothelial cells through activation of NF-kappaB/Rel and AP-1; Ishii H et al.; This study examined the effect of verotoxin-1 (VT-1), which is released from Escherichia coli O157:H7, on endothelial expression of tissue factor (TF), a cofactor required to initiate blood coagulation . In order to elucidate the molecular basis for development of hemolytic uremic syndrome (HUS) in patients infected with E . coli O157:H7, human umbilical vein endothelial cells (HUVECs) were exposed to purified VT-1 . VT-1 increased both TF activity and TF mRNA in HUVECs without loss of cell viability in a time- and dose-dependent manner from 0.1 to 10 ng/ml VT-1 . Nuclear proteins extracted from VT-1-stimulated HUVECs bound to the consensus NF-kappaB/Rel and AP-1 binding oligonucleotides in a dose-dependent manner within 2 h after the stimulation in electrophoretic mobility shift assays (EMSA) . Nuclear proteins from VT-1-stimulated HUVECs formed two complexes with the NF-kappaB/Rel binding motif in the human TF promoter (TF-kappaB motif) . The supershift assays, using antibodies for human p65, p50 or c-Rel, indicated that the lower complex was composed of p65/p50 and the higher complex was a p65 homo- or hetero-dimer with the Rel family, except c-Rel . The human TF promoter contains two AP-1 binding sites, the proximal and distal AP-1 binding sites . The supershift assays indicated that AP-1 containing mainly c-Jun and JunD, positively bound to the proximal AP-1 motif of TF (TF-AP-1) . The distal TF-AP-1 motif did not show positive binding with nuclear proteins from VT-1-stimulated HUVECs . Pretreatment of HUVECs with curcumin, an inhibitor of NF-kappaB/Rel activation, synthesis of c-Jun mRNA and binding of activated AP- I with AP-binding oligonucleotide, prevented the VT-1 induced increase in TF mRNA and activity in VT-1-stimulated HUVECs . Curcumin also inhibited NF-kappaB and AP-1 binding to TF-kappaB and proximal TF-AP-1 oligonucleotides, respectively, in a dose-dependent manner . The present work suggests that both the NF-kappaB/Rel and AP-1 activated in endothelial cells by stimulation with VT-1 binds to the TF-kappaB and proximal AP-1 binding sites, respectively, of the TF promoter. J Vet Med B Infect Dis Vet Public Health, 2000 Sep, 47(7), 551 - 9 Flow cytometry for the detection of enterohaemorrhagic Escherichia coli O157:H7 with latex beads sensitized with specific antibody; Kusunoki H et al.; To detect low concentrations of enterohaemorrhagic Escherichia coli O157:H7 rapidly, flow cytometry (FCM) was carried out with specific IgG-sensitized latex beads (IgG-Lx) . It was found that test samples for FCM can be prepared for much shorter periods by culturing E . coli O157:H7 in trypto-soya broth at 42 degrees C and by treatment with 0.5% formalin at 37 degrees C . FCM with IgG-Lx performed with E . coli O157:H7 prepared by such a procedure revealed that the lowest number of E . coli O157:H7 prepared in pure culture detected by FCM was 10(3)/ml . Because similar findings have already been reported by FCM with immunomagnetic beads, FCM with IgG-Lx is also suggested to be a valuable technique to detect low numbers of E . coli O157:H7 rapidly in food stuffs. Pediatr Nephrol, 2000 Oct, 14(12), 1092 - 7 Hemolytic uremic syndrome associated with Denys-Drash syndrome; Sherbotie JR et al.; The Denys-Drash syndrome is defined by the occurrence of combinations of pseudohermaphroditism, nephrotic syndrome with diffuse mesangial sclerosis, Wilms' tumor, and constitutional mutations in the WT1 suppressor gene . Most patients develop end-stage renal failure . Atypical hemolytic uremic syndrome (HUS) is defined by onset of acute hemolytic anemia with fragmented erythrocytes, thrombocytopenia, and renal failure in the absence of a gastrointestinal prodromal illness of bloody diarrhea . The purpose of this report is to describe the occurrence of features of atypical HUS and Denys-Drash syndrome in two African-American boys aged 13 and 16 months . Each had nephrotic syndrome, diffuse mesangial sclerosis, and WT1 point mutations . Both had grade III hypospadias and undescended testes . They had normal serum creatinine concentrations and hematology a month before presenting with HUS . Stool cultures for Escherichia coli O157:H7 were negative . Each patient has been transplanted with cadaver kidneys without recurrence of HUS. MMWR Morb Mortal Wkly Rep, 2000 Oct 13, 49(40), 911 - 3 Outbreak of Escherichia coli O157:H7 infection associated with eating fresh cheese curds--Wisconsin, June 1998; Escherichia coli O157:H7 infections in Wisconsin et al.; Communicable Disease Epidemiology Section, Wisconsin Division of Public Health, Madison 53701-2659, USA . proctme@dhfs.state.wi.us Escherichia coli O157:H7 infection became a reportable condition in Wisconsin on April 1, 2000; previously cases were voluntarily reported by physicians and laboratories . During 1992 through 1999, 1333 cases of E . coli O157:H7 infection occurred in Wisconsin residents and were reported to the Wisconsin Division of Public Health . During this interval, the highest age-specific mean annual incidence, 13.2 cases per 100,000 population, occurred in persons 3 to 5 years old . Only 28% of patients with reported cases identified bloody diarrhea among their signs and symptoms . Of reported cases, 17% (231/1333) were involved in the eight outbreaks investigated during this interval . Among case patient identifiable risk exposures, farm related (13.4%), recreational water related (8.1%), and unpasteurized milk/dairy product (7.0%) exposures were the most frequently noted . Relatively few infections involved raw/undercooked ground beef consumption (5.8%) . Recent use of pulsed-field gel electrophoresis has facilitated linkage of sporadically reported cases into recognized outbreaks . E . coli O157:H7 infections frequently occur in Wisconsin; acquisition of these infections in a wide variety of settings poses important challenges to their prevention and control. J Bacteriol, 2000 Nov, 182(21), 5962 - 8 Characterization of the distal tail fiber locus and determination of the receptor for phage AR1, which specifically infects Escherichia coli O157:H7; Yu SL et al.; Phage AR1 is similar to phage T4 in several essential genes but differs in host range . AR1 infects various isolates of Escherichia coli O157:H7 but does not infect K-12 strains that are commonly infected by T4 . We report here the determinants that confer this infection specificity . In T-even phages, gp37 and gp38 are components of the tail fiber that are critical for phage-host interaction . The counterparts in AR1 may be similarly important and, therefore, were characterized . The AR1 gp37 has a sequence that differs totally from those of T2 and T4, except for a short stretch at the N terminus . The gp38 sequence, however, has some conservation between AR1 and T2 but not between AR1 and T4 . The sequences that are most closely related to the AR1 gp37 and gp38 are those of phage Ac3 in the T2 family . To identify the AR1-specific receptor, E . coli O157:H7 was mutated by Tn10 insertion and selected for an AR1-resistant phenotype . A mutant so obtained has an insertion occurring at ompC that encodes an outer membrane porin . To confirm the role of OmpC in the AR1 infection, homologous replacement was used to create an ompC disruption mutant (RM) . When RM was complemented with OmpC originated from an O157:H7 strain, but not from K-12, its AR1 susceptibility was fully restored . Our results suggest that the host specificity of AR1 is mediated at least in part through the OmpC molecule. Southeast Asian J Trop Med Public Health, 2000 Mar, 31(1), 77 - 9 Rapid isolation and detection of Escherichia coli O157:H7 by use of rainbow agar O157 and PCR assay; Radu S et al.; This study has evaluated the use of a commercially available Rainbow agar O157 and polymerase chain reaction (PCR) assays for the detection of Shiga-like toxin producing Escherichia coli and to serotype E . coli O157:H7 from raw meat . The Rainbow agar O157 was found to be selective and sensitive for the screening of the E . coli O157 from artificially and naturally contaminated meat samples . Shiga-like toxin producing E . coli were identified with two primer pairs that amplified fragments of the SLT-I (384 bp) and SLT-II (584 bp) . E . coli O157:H7 was serotyped with a primer pair specified for the H7 flagellar gene, which amplify specific DNA fragments (625 bp) from all E . coli O157:H7 strains . The use of Rainbow agar O157 described allows for the presumptive isolation of E . coli O157 in 24 hours . Identification and confirmation of the presumptive isolates as E . coli O157:H7 by PCR assays require additional 6-8 hours . The above-mentioned screening and identification procedures should prove to be a very useful method since it allows for the specific detection of E . coli O157:H7. Int J Food Microbiol, 2000 Sep 15, 60(1), 107 - 10 Detection of freeze-injured Escherichia coli O157:H7 cells from foods by resuscitation prior to selective enrichment; Nakagawa H et al.; We tried to detect Escherichia coli O157:H7 in food samples artificially contaminated with freeze-injured E . coli O157:H7 using an enrichment method with modified EC broth supplemented with novobiocin (mEC + n) . When the samples were cultured for enrichment immediately after inoculation of freeze-injured cells, E . coli O157:H7 was not detected in 13 out of 18 samples . However, allowing the food samples to stand for 3 h at room temperature prior to enrichment in mEC + n remarkably improved recovery of E . coli O157:H7 except for some acidic foods . E . coli O157:H7 was detected in the acidic foods by introducing a resuscitation step of 3-h of incubation in a non-selective broth at room temperature prior to enrichment with mEC + n. Commun Dis Public Health, 2000 Sep, 3(3), 201 - 7 Investigation and management of sporadic gastrointestinal infections with potentially Vero cytotoxin producing Escherichia coli in Scotland; Evans CJ et al.; Recognition of the potential of Escherichia coli O157 and other Vero cytotoxin producing E . coli (VTEC) organisms to cause serious disease led to the recommendation that all diarrhoeal stool specimens be examined for E . coli O157 . National guidelines exist for the testing and exclusion of cases and contacts of VTEC infection . A survey was conducted to discover the extent to which these recommendations are followed in Scotland by asking about current practices for public health management of identified cases and laboratory investigation of E . coli infection . About two thirds of Scottish health boards followed national guidelines for testing and exclusion of cases and contacts of VTEC O157 infection . Most laboratories tested all diarrhoeal stools for E . coli O157 but detection methods varied and a minority tested selected stools for non-O157 E . coli serogroups . Standardisation of policies for laboratory testing of VTEC infection would improve national surveillance . Adherence to evidence based guidelines would standardise public health management of VTEC infections in Scotland. Am J Kidney Dis, 2000 Oct, 36(4), 687 - 94 Soluble Fas and soluble Fas-ligand in children with Escherichia coli O157:H7-associated hemolytic uremic syndrome; Masri C et al.; We measured soluble Fas-ligand (sFas-L) and soluble Fas (sFas) levels by sandwich enzyme-linked immunosorbeny assay and compared them among (1) healthy controls (n = 11), (2) children with hemorrhagic colitis (HC) caused by a non-verotoxin-producing pathogen (n = 23), (3) patients with uncomplicated Escherichia coli O157:H7 HC (n = 14), and (4) children with O157:H7-associated hemolytic uremic syndrome (HUS) (n = 24) . Children with uncomplicated E coli O157:H7 HC and HUS were matched for duration of enteric prodrome before blood sample collection . We also compared sFas-L and sFas levels among patients with HUS according to severity of renal dysfunction; abnormally increased sFas-L levels were noted in only 4% of the children (n = 3) . Abnormally high concentrations of sFas were noted in 9% of the children with HC caused by a non-verotoxin-producing pathogen, 29% of the patients with uncomplicated E coli O157:H7 HC, and 69% of the children with O157:H7-associated HUS . Compared with healthy controls, patients with HUS had twofold greater concentrations of sFas (P: < 0.0001) . Levels of sFas were not statistically different between 14 patients with uncomplicated O157:H7 HC and 14 children with HUS (8.2 +/- 4.7 versus 11.0 +/- 4.6 U/mL, respectively; P: < 0.07) when matched for time after onset of enteritis (7.0 +/- 3.7 versus 7.3 +/- 3.8 days, respectively) . Greater concentrations of sFas were noted in patients with HUS who developed oligoanuria (n = 10; P: < 0.007), required peritoneal dialysis (n = 10; P: < 0.007), or had a decreased glomerular filtration rate (n = 5; P: < 0.002) 1 year later . Our data show that plasma concentrations of sFas but not sFas-L are abnormally increased in children with O157:H7 infections . Levels of sFas are associated with severity of renal dysfunction during HUS . Further studies are needed to clearly determine the role and origin of circulating sFas among children with infections caused by E coli O157:H7. FEMS Microbiol Lett, 2000 Oct 1, 191(1), 7 - 10 Inducible stx2 phages are lysogenized in the enteroaggregative and other phenotypic Escherichia coli O86:HNM isolated from patients; Iyoda S et al.; We characterized two Shiga toxin-producing Escherichia coli (STEC) O86:HNM isolates from a patient with hemolytic uremic syndrome (HUS) or bloody diarrhea . Both of them did not possess the eaeA gene . However, the isolate from a HUS patient carried genetic markers of enteroaggregative E . coli (EAEC) and showed aggregative adherence pattern to HEp-2 cells . The other isolate from bloody diarrhea, which was negative with EAEC markers, was diffusely adhered to HEp-2 cells . The stx2 gene in both E . coli O86:HNM strains was encoded in each infectious phage, which was partially homologous to that of strain EDL933, a STEC O157:H7 . These results will help to explain the genotypic divergences of STEC. Emerg Infect Dis, 2000 Sep-Oct, 6(5), 530 - 3 Prevalence of non-O157:H7 shiga toxin-producing Escherichia coli in diarrheal stool samples from Nebraska; Fey PD et al.; We determined the prevalence of Shiga toxin-producing Escherichia coli (STEC) in diarrheal stool samples from Nebraska by three methods: cefixime-tellurite sorbitol MacConkey (CT- SMAC) culture, enterohemorrhagic E . coli (EHEC) enzyme immunoassay, and stx1,2 polymerase chain reaction (PCR) . Fourteen (4.2%) of 335 specimens were positive by at least one method (CT-SMAC culture {6 of 14}, EHEC enzyme immunoassay {13 of 14}, stx1,2 PCR {14 of 14}) . Six contained serogroup O157, while non-O157 were as prevalent as O157 serogroups. J Bacteriol, 2000 Oct, 182(19), 5381 - 90 Gene conservation and loss in the mutS-rpoS genomic region of pathogenic Escherichia coli; Herbelin CJ et al.; The extent and nature of DNA polymorphism in the mutS-rpoS region of the Escherichia coli genome were assessed in 21 strains of enteropathogenic E . coli (EPEC) and enterohemorrhagic E . coli (EHEC) and in 6 strains originally isolated from natural populations . The intervening region between mutS and rpoS was amplified by long-range PCR, and the resulting amplicons varied substantially in length (7.8 to 14.2 kb) among pathogenic groups . Restriction maps based on five enzymes and sequence analysis showed that strains of the EPEC 1, EPEC 2, and EHEC 2 groups have a long mutS-rpoS region composed of a approximately 6.0-kb DNA segment found in strain K-12 and a novel DNA segment ( approximately 2.9 kb) located at the 3' end of rpoS . The novel segment contains three genes (yclC, pad1, and slyA) that occur in E . coli O157:H7 and related strains but are not found in K-12 or members of the ECOR group A . Phylogenetic analysis of the common sequences indicates that the long intergenic region is ancestral and at least two separate deletion events gave rise to the shorter regions characteristic of the E . coli O157:H7 and K-12 lineages. Arch Microbiol, 2000 Jul-Aug, 174(1-2), 28 - 34 Identification of H-specific determinants in flagellin of four Escherichia coli strains; Seah JN et al.; The H serogroup of Escherichia coli is determined by the flagellar antigen, flagellin . Sequence analysis of the flagellin gene, fliC, reveals a central variable region and the highly conserved N- and C-termini . This variable region has been shown to encode both H-specific and cross-reactive epitopes . Using polyclonal antibodies, we mapped the linear H-specific determinants in flagellin from four E . coli serotypes O157:H10, 0138:H14, O157:H42 and O157:H43 . The specificity of all potential fragments was verified with 52 ECRC (Escherichia coli Reference Center) H-specific antisera . Our results indicated that: (a) a specific determinant of H10 flagellin (1263 bp long) maps to the region covering amino acid residues 305-331; (b) a specific determinant of H14 flagellin (1653 bp long) maps to the region covering amino acid residues 430-461; (c) a specific determinant of H42 flagellin (1281 bp long) maps to a region covering amino acid residues 171-201; and (d) a specific determinant of H43 flagellin (1506 bp long) maps to a region covering amino acid residues 200-260. J Food Prot, 2000 Sep, 63(9), 1173 - 8 Studies on the growth of Escherichia coli O157:H7 strains at 45.5 degrees C; Ferenc J et al.; The objectives of the present report were to examine the ability of 18 strains of Escherichia coli O157:H7 to grow in EC broth at 42.4, 43.5, 44.5, and 45.5 degrees C, and to document the incidence of phenotypic variants present in low numbers that are capable of growth at 45.5 degrees C in EC broth . Among the 18 strains of E . coli O157:H7 studied, only 3 were capable of producing turbid growth with gas formation in EC broth at 45.5 degrees C with 1 x 10(2) initial CFU/ml . Higher initial densities of CFU resulted in turbid growth and gas formation in EC broth at 45.5 degrees C with all strains . The presence of bile salts #3 in EC broth was found to be inhibitory at 45.5 degrees C . All 18 strains were found to be capable of growth at 45.5 degrees C in nonselective media . The ability of at least one sensitive strain to grow in EC broth at 45.5 degrees C was found to be dependent on the initial number of CFU/ml . Prior growth of cells of a sensitive strain in EC broth at 45.5 degrees C from a cell density of 2.0 x 10(7) to 8.0 x 10(7) CFU/ml followed by removal of cells and reinoculation at a cell density of 2.0 x 10(6) CFU/ml resulted in growth at 45.5 degrees C that did not occur without such conditioning of the inhibitory medium . These results indicate that the ability of most strains of E . coli O157:H7 to grow in EC broth at 45.5 degrees C is dependent on the initial density of CFU and that at low densities of CFU the ability to initiate growth is dependent on either low numbers of phenotypic variants tolerant to the presence of bile salts #3 in EC broth at 45.5 degrees C or to conditioning of the medium with prior elevated numbers of cells. J Food Prot, 2000 Sep, 63(9), 1167 - 72 Use of a Shiga toxin (Stx)-enzyme-linked immunosorbent assay and immunoblot for detection and isolation of Stx-producing Escherichia coli from naturally contaminated beef; Atalla HN et al.; The purpose of this study was to evaluate an enzyme-linked immunosorbent assay (ELISA) and an immunoblot procedure for detection and isolation of Shiga toxin-producing Escherichia coli (STEC) from beef, and to correlate the presence of STEC in beef with E . coli and total coliform counts . A total of 120 samples of boneless beef supplied to a meat processor in southern Ontario were tested for the presence of STEC, E . coli, and total coliforms . Following enrichment in modified tryptic soy broth, samples were screened for Shiga toxin (Stx) by a Stx-ELISA and a Vero cell assay (VCA) . Samples that were positive in the Stx-ELISA were subjected to the Stx-immunoblot for STEC isolation . Overall, 33.3% of samples were positive in the VCA, and 34.2% were positive in the Stx-ELISA . There was almost complete agreement between the Stx-ELISA and the VCA results (kappa = 0.98) . The sensitivity and specificity of the Stx-ELISA with respect to the VCA were 100% and 98.75%, respectively . STEC were isolated by the Stx-immunoblot from 87.8% of the samples that were positive in the Stx-ELISA . The STEC isolates belonged to 19 serotypes, with serotype O113:H21 accounting for 10 of 41 isolates . No STEC of serotype O157:H7 were isolated . There was a significant correlation between E . coli counts and total coliform counts (Spearman correlation coefficient = 0.68, P < 0.01) . The E . coli count was positively correlated with detection of STEC by both the Stx-ELISA and the VCA (P < 0.01). Folia Microbiol (Praha), 1999, 44(4), 455 - 6 Isolation of verotoxigenic Escherichia coli O157 from poultry; Pilipcinec E et al.; Results obtained by examination of cloacal swabs from poultry for the presence of verotoxigenic strains of E . coli O157:H7 are presented . Twenty samples (9.2%) of 216 samples examined were positive for E . coli O157 . Out of 20 E . coli O157, 19 strains were positive for the production of both verotoxins (VT1 and VT2) . However, none of them was positive for the presence of H7 antigen. Epidemiol Infect, 2000 Jun, 124(3), 343 - 9 Predictors for the development of haemolytic uraemic syndrome with Escherichia coli O157:H7 infections: with focus on the day of illness; Ikeda K et al.; A large outbreak of Escherichia coli O157 infections via school lunches occurred at primary schools in 1996 in Sakai City, Japan . As many as 10,000 patients suffered from diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome (HUS) . Using data on 288 inpatient school children affected by this outbreak, of whom 36 presented complete HUS and the remaining 252 tested positive for E . coli O157 culture, we attempted to identify predictors for the progression to HUS . Within the first 5 days of illness, clinical features associated with inpatients who developed HUS compared with those without HUS included a C reactive protein (CRP) level higher than 1.2 mg/dl (OR 44.26; 95% CI 5.83-336.23), a white blood cell (WBC) count greater than 11.0 x 10(9)/l (OR 5.03; 95% CI 2.13-11.87) and a temperature higher than 38.0 degrees C (OR 5.00; 95% CI 2.25-11.08) . It can be concluded that these three factors are predictive factors for the development of HUS in patients with E . coli O157 infection, and patients who have two or all of these factors should be observed closely. Proc Natl Acad Sci U S A, 2000 Sep 12, 97(19), 10325 - 9 Cattle lack vascular receptors for Escherichia coli O157:H7 Shiga toxins; Pruimboom-Brees IM et al.; Escherichia coli O157:H7 causes Shiga toxin (Stx)-mediated vascular damage, resulting in hemorrhagic colitis and the hemolytic uremic syndrome in humans . These infections are often foodborne, and healthy carrier cattle are a major reservoir of E . coli O157:H7 . We were interested in knowing why cattle are tolerant to infection with E . coli O157:H7 . Cattle tissues were examined for the Stx receptor globotriaosylceramide (Gb(3)), for receptivity to Stx binding in vitro, and for susceptibility to the enterotoxic effects of Stx in vivo . TLC was used to detect Gb(3) in tissues from a newborn calf . Gb(3) was detected by TLC in kidney and brain, but not in the gastrointestinal tract . Immunohistochemistry was used to define binding of Stx1 and Stx2 overlaid onto sections from cattle tissues . Stx1 and Stx2 bound to selected tubules in the cortex of the kidney of both newborn calves (n = 3) and adult cattle (n = 3) . Stx did not bind to blood vessels in any of the six gastrointestinal and five extraintestinal organs examined . The lack of Gb(3) and of Stx receptivity in the gastrointestinal tract raised questions about the toxicity of Stx in bovine intestine . We found that neither viable E . coli O157:H7 nor Stx-containing bacterial extracts were enterotoxic (caused fluid accumulation) in ligated ileal loops in newborn calves . The lack of vascular receptors for Stx provides insight into why cattle are tolerant reservoir hosts for E . coli O157:H7. Lett Appl Microbiol, 2000 Aug, 31(2), 139 - 42 Characterization of shiga toxin type 2 variant B-subunit in Escherichia coli strains from asymptomatic human carriers by PCR-RFLP; Stephan R et al.; Subtyping of shiga toxin type 2 variant B-subunit in 35 non-O157 and two O157 strains isolated from 37 asymptomatic human carriers yielded two strains with stx2, 10 strains with stx2c and 24 strains with stx2d genes . One isolate harboured stx2 and stx2c . The high Stx2d prevalence in asymptomatic carriers was conspicuous and may indicate a reduced pathogenicity of these toxin variants . Therefore, in order to appraise a positive STEC laboratory result, the strain must be isolated in every case . Shiga toxin types and further virulence-associated factors have to be investigated. Appl Environ Microbiol, 2000 Sep, 66(9), 4149 - 51 Determination of the sensitivity of a rapid Escherichia coli O157:H7 assay for testing 375-gram composite samples; Tsai WL et al.; Both 25-g single-size ground beef samples and 375-g composite ground beef samples were tested by a method combining an immunomagnetic separation (IMS) technique with a sandwich enzyme-linked immunosorbent assay (ELISA) system (IMS-ELISA) . The results demonstrated that IMS-ELISA could detect the target, Escherichia coli O157:H7, at the level of 10(-1) CFU/g of sample in either the 25- or 375-g sample size. Appl Environ Microbiol, 2000 Sep, 66(9), 3911 - 6 Contribution of dps to acid stress tolerance and oxidative stress tolerance in Escherichia coli O157:H7; Choi SH et al.; An Escherichia coli O157:H7 dps::nptI mutant (FRIK 47991) was generated, and its survival was compared to that of the parent in HCl (synthetic gastric fluid, pH 1.8) and hydrogen peroxide (15 mM) challenges . The survival of the mutant in log phase (5-h culture) was significantly impaired (4-log(10)-CFU/ml reduction) compared to that of the parent strain (ca . 1.0-log(10)-CFU/ml reduction) after a standard 3-h acid challenge . Early-stationary-phase cells (12-h culture) of the mutant decreased by ca . 4 log(10) CFU/ml while the parent strain decreased by approximately 2 log(10) CFU/ml . No significant differences in the survival of late-stationary-phase cells (24-h culture) between the parent strain and the mutant were observed, although numbers of the parent strain declined less in the initial 1 h of acid challenge . FRIK 47991 was more sensitive to hydrogen peroxide challenge than was the parent strain, although survival improved in stationary phase . Complementation of the mutant with a functional dps gene restored acid and hydrogen peroxide tolerance to levels equal to or greater than those exhibited by the parent strain . These results demonstrate that decreases in survival were from the absence of Dps or a protein regulated by Dps . The results from this study establish that Dps contributes to acid tolerance in E . coli O157:H7 and confirm the importance of Dps in oxidative stress protection. Scand J Infect Dis, 2000, 32(4), 385 - 94 Escherichia coli 'O' group serology of a haemolytic uraemic syndrome (HUS) epidemic; Goldwater PN et al.; This is the first comprehensive serological analysis of a haemolytic uraemic syndrome (HUS) outbreak . A wide range of 'O' group Escherichia coli antibody responses in patients and controls was examined . The study provides a unique insight into the epidemiology of such epidemics, points a way to the most appropriate investigation of these and indicates possible answers to a number of issues related to severity of disease . In order to be able to test for a wide variety of E . coli 'O' antigens, a microagglutination assay was used to examine E . coli 'O' group serological responses of 22 children admitted to hospital with HUS and 14 contemporaneous age-matched controls . A total of 51 'O' serogroup strains were used . These included 'O' groups reported to be associated with cases of HUS, with 6 isolates from patients associated with the Adelaide outbreak (O26, O111, O123 and O157), environmental Verocytotoxigenic/Shiga-toxin producing Escherichia coli (VTEC/STEC) strains and common human commensal strains . Sixteen clinically confirmed HUS cases (72.7%) of 22 seroconverted to 1 or more serogroups of which 11 (50%) seroconverted to O111 (the serogroup isolated from 16 patients) . In addition, 11 (50%) and 10 (45.5%) developed antibody to O137 and O145, respectively, although no stool isolates of these serogroups were made . Seventeen (77.3%) of 22 HUS patients had antibody to serogroup O157, with 11 (50%) seroconversions, however, O157:H- was isolated from only 2 of these . Overall, titres ranged from 100 to 6400, some of the highest in 3 patients were against O157, whose faeces yielded only Enterohaemorrhagic E . coli (EHEC) O111, and only 1 developed O111 antibody . Mixed infection was demonstrated serologically by microagglutination (confirmed by Western blot) and was consistent with the findings of 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 also involved . In HUS associated with EHEC infection, multiple strain infection may be the rule rather than the exception . A relationship with clinical severity deserves further investigation . Non-O157 EHEC (in addition to O157) should be sought in all future outbreaks of EHEC disease. Vet Rec, 2000 Jul 15, 147(3), 65 - 8 Natural and experimental infection of normal cattle with Escherichia coli O157; Wray C et al.; The objective of the study was to determine the effects of inoculating cattle orally with a strain of Escherichia coli O157:H7 (A84/92) . However, before they were challenged two of the six calves were found to be infected naturally with a wild-type strain of E coli O157 and two more of them became infected later . The number of daily faeces samples from which the wild-type E coli O157 was isolated ranged from one to 10 . After they were inoculated, A84/92 was detected in all the calves' faeces on one to six of the next 14 days, and later from the faeces samples of three calves on two, three, and 11 occasions, the last occasion being between 19 and 51 days after inoculation . Two calves were redosed with A84/92, and the organism was isolated on a further five and 15 occasions, the last being after 20 and 58 days . In three dry cows, A84/92 was isolated from the faeces on three to 11 of the 14 days after they were inoculated . Two of the cows were redosed and from one of them it was isolated on 15 occasions, the last being 44 days after the initial infection; in the other cow no further isolation was made . In three lactating cows, it was detected on three to four of the 14 days after they were inoculated, and similar results were obtained after they were reinoculated . None of the animals showed clinical signs and no lesions were detected in the intestines of the calves . Three calves had a serological response to E coli O157 but, with the exception of one cow which had a slight increase to IgM levels, no serological changes were observed in the adult cattle. Infect Immun, 2000 Sep, 68(9), 5344 - 53 Up-regulation of both intimin and eae-independent adherence of shiga toxigenic Escherichia coli O157 by ler and phenotypic impact of a naturally occurring ler mutation; Ogierman MA et al.; Shiga toxigenic Escherichia coli (STEC) strains are important human pathogens which are capable of causing diarrhea, hemorrhagic colitis, and the potentially fatal hemolytic-uremic syndrome (HUS) . An important virulence trait of certain STEC strains, such as those belonging to serogroup O157, is the capacity to produce attaching and effacing (A/E) lesions on enterocytes, a property encoded by the locus for enterocyte effacement (LEE) . LEE contains the eae gene, which encodes intimin, an outer membrane protein which mediates the intimate attachment of bacteria to the host epithelial cell surface, and eae is routinely used as a marker for LEE-positive STEC strains . However, the O157:H(-) STEC strain 95SF2 carries eae but did not produce A/E lesions on HEp-2 cells, as judged by a fluorescent actin staining assay . In this assay, 95SF2 adhered poorly to the HEp-2 cells, and those that did bind exhibited abnormal cell division . In contrast, the O157:H7 STEC strain EDL933 adhered strongly and produced typical A/E lesions . We have demonstrated that 95SF2 carries a defective LEE regulatory gene, ler, with a single base change with respect to that published for ler of EDL933, resulting in an Ile(57)-to-Thr substitution . Ler shows homology to H-NS-like regulators, which are modulators of transcription, and the mutation occurs in a domain implicated in oligomerization . 95SF2 was able to adhere and produce A/E lesions on HEp-2 cells when EDL933 ler was expressed from a multicopy plasmid . Conversely, introduction of a plasmid carrying 95SF2 ler into EDL933 abolished adherence and capacity to form A/E lesions . Studies with eae deletion derivatives of 95SF2 and EDL933 demonstrated that the ler-mediated adherence to HEp-2 cells is largely independent of intimin . We have also demonstrated that EDL933 ler, but not 95SF2 ler, increases the level of intimin in O157 STEC. Infect Immun, 2000 Sep, 68(9), 5090 - 5 Human response to Escherichia coli O157:H7 infection: antibodies to secreted virulence factors; Li Y et al.; Vaccination has been proposed for the prevention of disease due to enterohemorrhagic Escherichia coli (EHEC), but the immune response following human infection, including the choice of potential antigens, has not been well characterized . To study this, sera were obtained from five pediatric patients with acute diarrhea caused by E . coli O157:H7 0, 8, and 60 days after hospitalization . These sera were used to examine the immune response to four different EHEC virulence factors: Tir (translocated intimin receptor, which is inserted into the host cell membrane), intimin (bacterial outer membrane protein which binds to Tir), EspA (secreted protein which forms filamentous structures on EHEC surface), and EspB (inserted into the host membrane and cytoplasm) . The response to O157:H7 lipopolysaccharide was also examined . Sera were assayed against purified recombinant proteins using immunoblot analysis and by enzyme-linked immunosorbent assay to determine the sera's titers to each of the antigens in all patients . We found that there was little reaction to EspA, EspB, and intimin in the acute-phase sera, although there was some reactivity to Tir . By day 8, titers of antibody to all four virulence factors were present in all patients, with a very strong response against Tir (up to a titer of 1:256,000), especially in hemolytic-uremic syndrome patients, and lesser strong responses to the other three antigens . The titer to the antigens 60 days after hospitalization was decreased but was still highest for Tir . These results suggest that there is a strong immune response to Tir, and to a lesser extent to the other three virulence factors, following EHEC disease, indicating that these bacterial molecules are potential vaccine candidates for preventing EHEC disease . They also suggest that bacterial virulence factors that are inserted into host cells during infection by type III secretion systems (Tir or EspB) are still recognized by the host immune response. Paediatr Drugs, 2000 Jul-Aug, 2(4), 243 - 52 Haemolytic uraemic syndrome; Robson WL; Diarrhoea-associated haemolytic uraemic syndrome develops in about 5 to 10% of children with haemorrhagic colitis due to Escherichia coli (E . coli) O157:H7 and is a common cause of acute renal failure in childhood . Endothelial cell damage, white blood cell activation and platelet-endothelial cell interactions are important in the pathogenesis . Meticulous supportive care, with attention to nutrition and fluid, and electrolyte balance, is important . Dialysis is necessary in many children . Public health follow-up is important to minimise the spread of E . coli O157:H7, which is transmitted by person-to-person, as well as through contaminated food products . 20-year follow-up studies report that 75% of children recover without any clinically significant long term sequelae . Chronic renal failure is reported in about 5% of children. New Microbiol, 2000 Jan, 23(1), 47 - 53 Comparison of Vero cell assay, polymerase chain reaction and an enzyme immunoassay for identification of verocytotoxin-producing Escherichia coli O157:H7; Bonardi S et al.; This study evaluated three different analytical methods for identification of Verocytotoxin-producing E . coli O157:H7 (VTEC) strains . A total of 34 E . coli O157:H7 strains isolated from bovine faeces and bovine carcasses were comparatively tested with Vero cell assay (VCA), PCR and the sandwich ELISA "RIDASCREEN Verotoxin" test . The VCA, performed without a neutralization assay, gave a false positive result because a VCA-positive E . coli O157:H7 strain did not possess the VT-coding genes when tested with PCR . The lack of specificity of the VCA could be avoided by testing for neutralization of cytotoxicity . The commercial ELISA system was as sensitive and specific as PCR, with the advantages of being a more rapid and easier procedure which could be employed in all first level diagnostic laboratories. J Appl Microbiol, 2000 Jul, 89(1), 49 - 55 A comparison of methods for the detection of Escherichia coli O157:H7 from artificially-contaminated dairy products using PCR; McKillip JL et al.; Rapid nucleic acid-based methods to detect human pathogens in foods are dependent on the reliability of the DNA or RNA extraction method used . Skim milk, non-fat dry milk, Cheddar and Brie cheese, and reconstituted whey powder were seeded with serially diluted (10(0)-10(7) cfu 10 ml(-1)) Escherichia coli O157:H7 and subjected to DNA extraction (i) directly from the food product using a solvent-based procedure and (ii) using a guanidinium isothiocyanate (GITC) procedure after previous bacterial concentration . Both the efficiency of DNA extraction and the overall PCR detection limits were evaluated . In almost all instances, the total DNA yield using the solvent method was greater than that obtained for the concentration method . However, the purity of the DNA obtained after bacterial concentration was significantly better than that obtained after organic extraction alone . PCR detection limits after each DNA recovery method varied with the specific food, ranging from 10(1) to 10(4) cfu ml(-1) for all products except whey powder . DNA yields and subsequent PCR detection limits for reconstituted whey powder were extremely poor, and neither procedural changes nor the addition of PCR enhancement agents were able to improve recovery and/or detection . It is concluded that the efficiency of DNA extraction is an extremely important and frequently overlooked variable impacting the overall detection limits of PCR-based detection strategies. J Food Prot, 2000 Aug, 63(8), 1026 - 31 Heat inactivation of Escherichia coli O157:H7 in apple cider containing malic acid, sodium benzoate, and potassium sorbate; Dock LL et al.; The effect of pH modification and preservative addition in apple cider on the heat resistance of Escherichia coli O157:H7 was investigated . E . coli O157:H7 and various amounts of potassium sorbate (0 to 0.2%), sodium benzoate (0 to 0.2%), and malic acid (0 to 1%) were added to apple cider . Thermal inactivation experiments were performed at 47, 50, and 53 degrees C, and D- and z-values were calculated . In apple cider without additives, the D-value at 50 degrees C (D50) was about 65 min, but addition of preservatives and malic acid significantly (P < 0.01) decreased D-values . D50-values decreased to 13.9 min in cider with 0.5% malic acid, 13.2 min with 0.1% sorbate, and 7.0 min with 0.1% benzoate added . Addition of both sorbate and malic acid had similar effects as either one alone, thus additive effects were not present . However, addition of both 0.2% benzoate and 1% malic acid did show additive effects, lowering D50 to 0.3 min . Addition of all three components (0.2% sorbate, 0.2% benzoate, and 1% malic acid) resulted in a D50 = 18 s . The z-value of cider without additives was about 6 degrees C, whereas z-values of cider containing malic acid, benzoate, and/or sorbate ranged from about 6 degrees C to 26 degrees C . This increase may result in a longer 5-log reduction time at higher temperatures (i.e., 70 degrees C) in cider with benzoate as compared to cider without additives. J Food Prot, 2000 Aug, 63(8), 1021 - 5 Heat inactivation of Escherichia coli O157:H7 in apple juice exposed to chlorine; Folsom JP et al.; Exposure of Escherichia coli O157:H7 to chlorine before heat treatment results in increased production of heat shock proteins . Current heating regimens for pasteurizing apple cider do not account for chlorine exposure in the wash water . This research determined the effect of sublethal chlorine treatment on thermal inactivation of E . coli O157:H7 . D58-values were calculated for stationary-phase cells exposed to 0.6 mg/liter of total available chlorine and unchlorinated cells in commercial shelf-stable apple juice (pH 3.6) . D58-values for unchlorinated and chlorine-exposed cells in buffer were 5.45 and 1.65 min, respectively (P < 0.01) . Death curves of chlorine-exposed and unchlorinated cells in apple juice were not completely linear . Unchlorinated cells heated in apple juice exhibit a 3-min delay before onset of linear inactivation . Chlorine treatment eliminated this shoulder, indicating an overall loss of thermotolerance . The linear portion of each curve represented a small fraction of the total population . D58-values calculated from these populations are 0.77 min for unexposed cells and 1.19 min for chlorine-exposed cells (P = 0.05) . This indicates that a subpopulation of chorine-treated cells is possibly more resistant to heat because of chlorine treatment . The effect of chlorine treatment, however, is insignificant when compared with the effect of losing the shoulder . This is illustrated by the time required to kill the initial 90% of the cell population . This is observed to be 3.14 min for unchlorinated versus 0.3 min for chlorine-exposed cells (P < 0.001) . These observations indicate that current heat treatments need not be adjusted for the effect of chlorine treatment. J Food Prot, 2000 Aug, 63(8), 1015 - 20 UV inactivation, liquid-holding recovery, and photoreactivation of Escherichia coli O157 and other pathogenic Escherichia coli strains in water; Sommer R et al.; Drinking water, water used in food production and for irrigation, water for fish farming, waste water, surface water, and recreational water have been recently recognized as a vector for the transmission of pathogenic Escherichia coli, especially serotype O157:H7 . We investigated the UV (253.7 nm) inactivation behavior and the capability of dark repair (liquid-holding recovery) and photoreactivation of seven pathogenic (including three enterohemorrhagic E . coli) strains and one nonpathogenic strain of E . coli (ATCC 11229) with respect to the use of UV light for water disinfection purposes . Because most bacteria and yeast are known to be able to repair UV damage in their nucleic acids, repair mechanisms have to be considered to ensure safe water disinfection . We found a wide divergence in the UV susceptibility within the strains tested . A 6-log reduction of bacteria that fulfills the requirement for safe water disinfection was reached for the very most susceptible strain O157:H7 (CCUG 29199) at a UV fluence of 12 J/m2, whereas for the most resistant strain, O25:K98:NM, a UV fluence of about 125 J/m2 was needed . Except for one strain (O50:H7) liquid-holding recovery did not play an important role in recovery after UV irradiation . By contrast, all strains, particularly strains O25:K98:NM, O78:K80:H12, and O157:H7 (CCUG 29193), demonstrated photorepair ability . For a 6-log reduction of these strains, a UV fluence (253.7 nm) up to 300 J/m2 is required . The results reveal that the minimum fluence of 400 J/m2 demanded in the Austrian standard for water disinfection is sufficient to inactivate pathogenic E . coli . A fluence of 160 J/m2 (recommendation in Norway) or 250 J/m2 (recommendation in Switzerland) cannot be regarded as safe in that respect. J Infect, 2000 Jul, 41(1), 45 - 9 Does blood type B protect against haemolytic uraemic syndrome? An analysis of the 1996 Sakai outbreak of Escherichia coli O157:H7 (VTEC O157) infection . The Osaka HUS Critical Care Study Group; Shimazu T et al.; OBJECTIVES: Expression of the P1 blood type antigen is suggested to have a protective effect against post-enteropathic haemolytic uraemic syndrome (HUS) . The B blood type may also protect against HUS, since terminal trisaccharide sequences similar to those of the B blood type determinants are reported to have an affinity to Vero cytotoxin that is 23% as strong as that of the P1 determinants . Thus, we studied whether ABO blood types were related to the occurrence or severity of HUS . METHODS: We obtained clinical and laboratory data of 49 HUS patients treated in 14 critical care facilities during the 1996 Escherichia coli O157:H7 outbreak in Sakai, Japan . We retrospectively studied whether ABO blood types were related to the occurrence or severity of HUS . RESULTS: The numbers of patients with blood types A, B, O or AB were 29, 8, 12, and 0, respectively . For each blood type, the number of patients with severe renal complications was 16, 6, 9, and 0, respectively . The distribution of blood types among the HUS patients deviated from a population-based distribution of blood types (P<0.05, Chi-squared test); i.e., the frequency of the A blood phenotype was significantly higher among our HUS patients . However, there was no significant difference in the frequency of patients with the A antigen (A and AB blood groups) among our HUS patients, whereas the frequency of B antigen expression was significantly lower (P<0.05, Chi-squared test) . The risk of severe renal complications did not appear to be related to ABO blood types . CONCLUSIONS: Our data suggest that expression of the B antigen has a protective effect against the onset of HUS, but that it does not affect the severity of the disease . Arch Pediatr, 2000 Jun, 7 Suppl 3, 544s - 550s {Verotoxin-producing Escherichia coli infections: study of its prevalence in children in the Auvergne region}; Pradel N et al.; Verotoxin producing Escherichia coli (VTEC) have been associated with disease outbreaks of diarrhea hemorrhagic colitis and hemolytic-uremic syndrome in humans . Contamination occurs mainly by ingestion of beef and dairy products, but water and person to person transmission have also been described . Most of the clinical signs are due to the production of Stx1 and/or Stx2 Shiga toxins, also called verotoxins . Other virulence factors include enterohemolysin, and the product of the eae gene, intimin, involved in the attaching and effacing adherence phenotype . The predominant serotype is O157:H7, but VTEC strains of more than one hundred serotypes can cause human disease . In order to determine the prevalence of VTEC infections among children in the central part of France, stool samples from hospitalized children were examined for stx1 and stx2 genes by using a polymerase chain reaction (PCR) technique . From October 1997 to September 1998, 658 stool samples were analysed: among them 19 (3%) were stx-PCR positive . Only 8 children out of 19 had diarrhea, and for 5 of them, an enteric pathogen other than VTEC was isolated . VTEC strains were isolated from 10 samples: most of the isolates did not produce verotoxins at a high level, and they did not belong to serotypes associated with pathogenicity, which might explain the absence of relationship between VTEC isolation and pathogenicity in our study. Gut, 2000 Sep, 47(3), 377 - 81 Enterohaemorrhagic Escherichia coli O157:H7 target Peyer's patches in humans and cause attaching/effacing lesions in both human and bovine intestine; Phillips AD et al.; BACKGROUND: Enterohaemorrhagic Escherichia coli (EHEC) constitute a significant risk to human health worldwide, and infections, particularly with serogroup O157:H7, are associated with consumption of a variety of food and water vehicles, particularly food of bovine origin . EHEC cause acute gastroenteritis, bloody diarrhoea, and haemorrhagic colitis; up to 10% of cases develop severe complications, including the haemolytic uraemic syndrome, with a 5% case fatality . A virulence characteristic of enteropathogenic E coli, the attaching/effacing lesion, is considered to be important in EHEC . However, although EHEC produce this lesion on cultured human cells, this has not been demonstrated on human intestinal mucosal surfaces . In addition, the initial site(s) of colonisation of EHEC in humans is not known . AIMS: To assess the association of EHEC O157:H7 with paediatric and bovine intestine using in vitro organ culture and determine if attaching/effacing lesions occur . METHODS: Ultrastructural analysis of in vitro intestinal organ cultures of human small and large intestine was used to investigate adhesion of O157:H7 EHEC to intestinal surfaces . Bovine intestinal organ culture was used to examine the pathology produced by the same EHEC strain in cattle . RESULTS: The study showed that EHEC O157:H7 adhered to human intestinal mucosa . Binding and attaching/effacing lesion formation of O157:H7 in humans was restricted to follicle associated epithelium of Peyer's patches . The same strain caused attaching/effacing lesions on bovine mucosa . CONCLUSIONS: O157:H7 targets follicle associated epithelium in humans where it causes attaching/effacing lesions . The same human isolate can cause attaching/effacing lesions in cattle, indicating that similar pathogenic mechanisms operate across human and bovine species Rev Latinoam Microbiol, 1997 Jul-Dec, 39(3-4), 159 - 65 Hemolysins and verotoxin (VT) in enteric Escherichia coli isolated from pigs; Zamora J et al.; One hundred and three E . coli strains isolated from the intestinal contents of pigs were examined for hemolysis and verotoxin production on Vero monolayer cells . Hemolysins were produced by 18 (17.5%) strains;--hemolysin was produced by 6 strains of which 4 belonged to serotype O149:K91, K88ac;--hemolysin was produced by 8 strains which could no be serotyped; 1 strain produced-hemolysin and possessed fimbrial antigens K88; the remaining 3 strains were enterohemolytic and VT producers . Among the 85 non-hemolytic strains, 11 showed some antigenic properties . Only 3 could be serotyped (O64:K 'V142'; O157:K 'V17' and O149:K91); 4 had fimbrial antigens K88 and 1 strain was K99, and 3 possessed capsular antigen (K89) . According to these results it is possible to conclude that both hemolytic and non-hemolytic E . coli strains could be pathogenic. Arch Intern Med, 2000 Aug 14-28, 160(15), 2380 - 5 Where's the beef? The role of cross-contamination in 4 chain restaurant-associated outbreaks of Escherichia coli O157:H7 in the Pacific Northwest; Jackson LA et al.; BACKGROUND: From March through August 1993, outbreaks of Escherichia coli O157:H7 occurred at 4 separate Oregon and Washington steak and salad bar restaurants affiliated with a single national chain . OBJECTIVE: To determine the cause of outbreaks of E coli O157:H7 at 4 chain restaurants . METHODS: Independent case-control studies were performed for each outbreak . Available E coli O157:H7 isolates were subtyped by pulse-field gel electrophoresis and by phage typing . RESULTS: Infection was not associated with beef consumption at any of the restaurants . Implicated foods varied by restaurant but all were items served at the salad bar . Among the salad bar items, no single item was implicated in all outbreaks, and no single item seemed to explain most of the cases at any individual restaurant . Molecular subtyping of bacterial isolates indicated that the first 2 outbreaks, which occurred concurrently, were caused by the same strain, the third outbreak was caused by a unique strain, and the fourth was multiclonal . CONCLUSIONS: Independent events of cross-contamination from beef within the restaurant kitchens, where meats and multiple salad bar items were prepared, were the likely cause of these outbreaks . Meat can be a source of E coli O157:H7 infection even if it is later cooked properly, underscoring the need for meticulous food handling at all stages of preparation. Pediatr Infect Dis J, 2000 Jul, 19(7), 642 - 7 Leukocytosis in children with Escherichia coli O157:H7 enteritis developing the hemolytic-uremic syndrome; Buteau C et al.; BACKGROUND: Fewer than 10% of children with Escherichia coli O157:H7 enteritis develop hemolytic-uremic syndrome (HUS) . OBJECTIVE: To determine whether circulating leukocytes are independent risk markers of developing HUS during E . coli O157:H7 enteritis . METHODS: We reviewed the charts of all children with culture-proved E . coli O157:H7 infections seen at Sainte-Justine Hospital between 1987 and 1997 . Epidemiologic data, laboratory indices and circulating leukocytes counts were noted . HUS diagnosis was validated with independent HUS patient lists from the pediatric nephrology services of tertiary care hospitals in the Montreal metropolitan area . The date of onset of enteritis was determined by two independent observers . Leukocyte counts were compared among the following independent groups: (1) uncomplicated O157:H7 enteritis (Group 1); (2) O157:H7 enteritis with the subsequent development of HUS (Group 2); (3) HUS already present at the time of medical consultation (Group 3) . RESULTS: There were 369 children with E . coli O157:H7 infection . A complete blood count was not performed in 114 (31%) patients . Observers disagreed on the date of onset of gastroenteritis in 34 (9%) children only (kappa 0.92) . The study population thus included 221 patients: Group 1, n = 161; Group 2, n = 27; and Group 3, n = 33 . Patients developing HUS (Group 2) presented greater total leukocyte (P < 0.008), polymorphonuclear (P < 0.008) and monocyte (P < 0.07) counts than those with an uncomplicated course (Group 1) . Logistic regression analysis showed that young age {odds ratio (OR), 0.98; 95% confidence interval (CI), 0.96 to 0.99}, duration of enteric prodrome < or =3 days (OR 4.8, 95% CI 1.13 to 20.7) and initial leukocytosis (OR 1.22, 95% CI, 1.11 to 1.35) were independent predictors of HUS . CONCLUSIONS: Based on the variables identified above, further studies are needed to determine whether the inflammatory response of the host represents only a marker of the severity of gastrointestinal infection or whether, alternatively, it is a pathophysiologic factor that leads to HUS. J Food Prot, 2000 Jul, 63(7), 855 - 9 Molecular beacon polymerase chain reaction detection of Escherichia coli O157:H7 in milk; McKillip JL et al.; A fluorescently labeled oligonucleotide probe (molecular beacon) was applied to detect Escherichia coli O157:H7 in artificially contaminated skim milk during polymerase chain reaction (PCR) amplification of extracted DNA . The probe was designed to hybridize with a region of the slt-II gene coding for the A subunit and to fluoresce when the hairpin-stem conformation was linearized upon hybridization to the target sequence . The molecular beacon was incorporated into PCR reactions containing DNA extracted from artificially contaminated skim milk . The degree of fluorescence was monitored in PCR reactions containing 10(3), 10(5), and 10(7) CFU of E . coli O157:H7 per ml and was found to correlate with the amount of template in each reaction . Fluorescence significantly increased above background levels by cycle 8, 14, or 14 in reactions containing DNA from the 10(7)-, 10(5)-, or 10(3)-CFU/ml template, respectively (P < 0.05) . Molecular beacon PCR demonstrated positive results more rapidly than traditional agarose gel electrophoresis analysis of PCR products . Use of molecular beacons allows real-time monitoring of PCR reactions, and the closed-tube format allows simultaneous detection and confirmation of target amplicons without the need for agarose gel electrophoresis and/or Southern blotting . This is the first report of a stem-and-loop molecular beacon being applied for direct detection of a pathogen in food. Int J Food Microbiol, 2000 Jun 30, 58(1-2), 73 - 82 Efficacy of novel organic acid and hypochlorite treatments for eliminating Escherichia coli O157:H7 from alfalfa seeds prior to sprouting; Lang MM et al.; This study investigated novel two-step organic acid/hypochlorite treatments as alternatives to 20000 ppm active chlorine (from calcium hypochlorite) for eliminating Escherichia coli O157:H7 from alfalfa seeds prior to sprouting . Commercially available alfalfa seeds were inoculated with a five-strain E . coli O157:H7 mixture and dried to attain ca . 10(6) CFU/g of seeds . Seeds then underwent one of several soak treatments including: (1) 5% (v/v) lactic acid for 10 min at 42 degrees C; (2) 5% acetic acid (v/v) for 10 min at 42 degrees C; (3) 2.5% lactic acid for 10 min at 42 degrees C followed by 2000 ppm active chlorine (from calcium hypochlorite) for 15 min at 25 degrees C; (4) 5% lactic acid for 10 min at 42 degrees C followed by 2000 ppm active chlorine for 15 min at 25 degrees C; or (5) 20000 ppm active chlorine for 15 min at 25 degrees C . Each treatment reduced numbers of inoculum cells by about 6.0 log10 CFU/g as determined by plating on Sorbitol MacConkey agar (SMac) . Plating on non-selective brain heart infusion agar (BHI) showed that treatments 1-4 reduced counts by 2.3-4.1 log10 CFU/g, thus indicating a large proportion of injured cells . Successive lactic acid and hypochlorite treatments (3 and 4) were more lethal than either organic acid alone (1 and 2) . No surviving cells were detected on SMac or BHI following treatment with 20000 ppm active chlorine (treatment 5) . Regardless of the previous treatment, E . coli O157:H7 counts increased to 10(7)-10(8) CFU/g during sprouting . Germination of seeds was not adversely affected by any of the treatments (germination > 90%) . Results of this study show that: (a) non-lethal cell injury must be considered when evaluating intervention treatments against E . coli O157:H7 on alfalfa seeds; (b) reductions of 2-4 log10 CFU/g can be attained without using 20000 ppm active chlorine; (c) successive lactic acid and hypochlorite treatments have greater lethality than organic acid treatments alone; and (d) none of the treatments tested can prevent regrowth of surviving E . coli O157:H7 during sprouting. Nature, 2000 Jun 29, 405(6790), 1073 - 7 Crystal structure of enteropathogenic Escherichia coli intimin-receptor complex; Luo Y et al.; Intimin and its translocated intimin receptor (Tir) are bacterial proteins that mediate adhesion between mammalian cells and attaching and effacing (A/E) pathogens . Enteropathogenic Escherichia coli (EPEC) causes significant paediatric morbidity and mortality world-wide . A related A/E pathogen, enterohaemorrhagic E . coli (EHEC; O157:H7) is one of the most important food-borne pathogens in North America, Europe and Japan . A unique and essential feature of A/E bacterial pathogens is the formation of actin-rich pedestals beneath the intimately adherent bacteria and localized destruction of the intestinal brush border . The bacterial outer membrane adhesin, intimin, is necessary for the production of the A/E lesion and diarrhoea . The A/E bacteria translocate their own receptor for intimin, Tir, into the membrane of mammalian cells using the type III secretion system . The translocated Tir triggers additional host signalling events and actin nucleation, which are essential for lesion formation . Here we describe the the crystal structures of an EPEC intimin carboxy-terminal fragment alone and in complex with the EPEC Tir intimin-binding domain, giving insight into the molecular mechanisms of adhesion of A/E pathogens. Rinsho Shinkeigaku, 2000 Mar, 40(3), 237 - 42 {A case of hemolytic uremic syndrome caused by verotoxin producing enteropathogenic Escherichia coli (O157:H7) with prolonged cerebrovascular dysfunction}; Yaginuma M et al.; A 56-year-old woman was admitted to our hospital because of bloody watery diarrhea . Stool cultures on admission revealed the verotoxin producing enteropathogenic Escherichia coli (VTEC) (O157:H7) . Hemolytic uremic syndrome (HUS) appeared on the 4th hospital day . Plasma exchange was performed from the 4th day to the 11th day . Mental confusion appeared on the 6th day, and was aggravated until the 18th day, and slowly improved afterwards . Serial cranial CT in series demonstrated low density areas in the bilateral parietooccipital lobes on the 26th day, and the left one diminished but the right one became more evident on the 49th day . The series of single photon emission computed topography (SPECT) revealed that in the region of the temporoparietooccipital lobe cerebral blood flow decreased on the left side on the 30th day, on both sides on the 39th day, and on the right side on the 82nd day . There are few reports of prolonged cerebrovascular dysfunction observed in serial studies of SPECT in a patient of HUS by VTEC (O157:H7) . We should be aware, however, of the appearance of the CNS involvement in the recovery stage because the prolonged cerebrovascular dysfunction may occur as our case. Appl Environ Microbiol, 2000 Jul, 66(7), 3117 - 8 Modification of sorbitol MacConkey medium containing cefixime and tellurite for isolation of Escherichia coli O157:H7 from radish sprouts; Fujisawa T et al.; A modified version of sorbitol MacConkey medium containing cefixime and tellurite (CT-SMAC medium) was produced by adding salicin and 4-methylumbelliferyl-beta-D-galactopyranoside to CT-SMAC medium; this medium was designated CT-SSMAC medium and was used to isolate Escherichia coli O157:H7 from radish sprouts . Of 101 non-E . coli bacteria isolated from radish sprouts that produced colorless colonies similar to colonies of E . coli O157:H7 grown on CT-SMAC medium, 92 (91%) formed colonies that were red to pink or were beta-galactosidase negative and colorless on CT-SSMAC medium . On the other hand, colonies of E . coli O157:H7 strains were colorless and beta-galactosidase positive on CT-SSMAC medium . Our results suggest that CT-SSMAC medium is more selective than CT-SMAC medium for isolating E . coli O157:H7. Appl Environ Microbiol, 2000 Jul, 66(7), 2866 - 72 Selective enrichment with a resuscitation step for isolation of freeze-injured Escherichia coli O157:H7 from foods; Hara-Kudo Y et al.; We studied injury of Escherichia coli O157:H7 cells in 11 food items during freeze storage and methods of isolating freeze-injured E . coli O157:H7 cells from foods . Food samples inoculated with E . coli O157:H7 were stored for 16 weeks at -20 degrees C in a freezer . Noninjured and injured cells were counted by using tryptic soy agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite . Large populations of E . coli O157:H7 cells were injured in salted cabbage, grated radish, seaweed, and tomato samples . In an experiment to detect E . coli O157:H7 in food samples artificially contaminated with freeze-injured E . coli O157:H7 cells, the organism was recovered most efficiently after the samples were incubated in modified E . coli broth without bile salts at 25 degrees C for 2 h and then selectively enriched at 42 degrees C for 18 h by adding bile salts and novobiocin . Our enrichment method was further evaluated by isolating E . coli O157:H7 from frozen foods inoculated with the organism prior to freezing . Two hours of resuscitation at 25 degrees C in nonselective broth improved recovery of E . coli O157:H7 from frozen grated radishes and strawberries, demonstrating that the resuscitation step is very effective for isolating E . coli O157:H7 from frozen foods contaminated with injured E . coli O157:H7 cells. Zh Mikrobiol Epidemiol Immunobiol, 2000 Jan-Feb, (1), 48 - 50 {A selective nutrient medium for isolating clinical strains of Escherichia coli O157:H7}; Sultanov ZZ et al.; A dried selective culture medium, electrolyte-deficient sorbitol agar (EDS agar), for the isolation and preliminary identification of E . coli O157:H7 from clinical material has been developed . The medium is not inferior in its quality to analogous foreign media and requires no scarce ingredients for its manufacture. Symp Ser Soc Appl Microbiol, 2000, (29), 99S - 105S Molecular epidemiological investigation of enterohaemorrhagic Escherichia coli isolates in Japan; Terajima J et al.; We have established several measures for control and prevention of EHEC infection including designation of the disease as notifiable since there was the sudden increase in the incidence of infection with EHEC O157:H7 in Japan in 1996, involving multiple outbreaks . Improvements in methodologies for isolation of these organisms and enhanced laboratory screening have revealed a variety of sources in food and animals . Although there seems to be a bovine reservoir for O157 EHEC in Japan as well as North America and UK, different foods have been linked to EHEC infection including salads, radish sprouts and salmon roe . There is clear evidence that divergent clones of EHEC O157:H7 are prevalent throughout Japan based on laboratory surveillance, however, we still need to better define the role of EHEC serogroups other than Escherichia coli O157 as important causes of human infection. Symp Ser Soc Appl Microbiol, 2000, (29), 90S - 98S VTEC: lessons learned from British outbreaks; Pennington TH; Important Escherichia coli O157 outbreaks in England and Scotland since 1982-83 are reviewed . The scientific lessons learned from them are described and their legal consequences outlined . The light shed by them on relationships between law and science is discussed, and questions of blame are analysed in the context of Reason's 'resident pathogen' metaphor and Vaughan's study of the 1986 Challenger Space Shuttle disaster. Symp Ser Soc Appl Microbiol, 2000, (29), 79S - 89S Thermal inactivation of Escherichia coli O157:H7; Stringer SC et al.; Verotoxin-producing Escherichia coli O157:H7 is a cause of serious foodborne illness . It has a very small infectious dose and so it is vital to eliminate this pathogen from food . As heat treatment is the method of bacterial destruction most frequently used in food processing, accurate prediction of thermal death rates is necessary to achieve desired safety margins whilst minimizing processing . In most studies thermal inactivation has been described using first-order reaction kinetics and D-values . Whilst this approach does not seem justified on a theoretical basis, and may increase inaccuracy, there is no doubt that it is convenient and in many cases provides an adequate description of thermal death . A review of published data on the measured thermal inactivation of E . coli O157:H7 shows no strong evidence that a heat treatment of 70 degrees C for 2 min (or equivalent) fails to deliver a 6-decimal reduction in cell numbers. Symp Ser Soc Appl Microbiol, 2000, (29), 71S - 78S Survival of verocytotoxigenic Escherichia coli O157 in soil, water and on surfaces; Maule A; Cattle and sheep are major reservoirs of Escherichia coli O157 and consequently these and certain other farm animals can pass out large numbers of this organism in their faeces . Thus the ability of the organism to survive in faeces, on pastureland and in associated water systems has important implications for its spread to crops by direct application of manure, by irrigation with infected water or directly to man by contact with animals or contaminated soil . Model systems were used to determine the persistence of the organism in river water, cattle faeces, soil cores and on stainless steel work surfaces . Survival of the organism was found to be greatest in soil cores containing rooted grass . Under these conditions viable numbers were shown to decline from approximately 10(8) g(-1) soil to between 10(6) and 10(7) g(-1) soil after 130 d . When the organism was inoculated into cattle faeces it remained detectable at high levels for more than 50 d . In contrast the organism survived much less readily in cattle slurry and river water where it fell in numbers from more than 10(6) ml(-1) to undetectable levels in 10 and 27 d, respectively . The survival of E . coli O157 was also investigated on stainless steel surfaces, where as air-dried deposits, it was shown to survive for periods in excess of 60 d . It was most stable at chill temperatures (4 degrees C) and viability was only partially reduced at 18 degrees C . In addition to stainless steel, the organism was shown to survive for extended periods on domestic (plastic) cutting boards, both at room and chill temperatures . Sanitizing agents, such as hypochlorites and a compound comprising both cationic and anionic-based active ingredients were found to be effective in killing various VTEC on stainless steel surfaces. Symp Ser Soc Appl Microbiol, 2000, (29), 38S - 50S Role of non-O157 VTEC; Bettelheim KA; Non-O157 VTEC are typical Escherichia coli that differ only in their ability to produce verocytotoxins (VT) . The transmission of VTEC is discussed in relation to the transmission of commensal E . coli . The emergence over the last few decades of a great variety of VTEC serotypes from healthy and diseased humans and animals is described . Particular attention is given to the distribution of the more important serogroups pathogenic for humans that have been described from around the world, particularly serogroups O26, O111, O128 and O103 . The possible role of ruminants as reservoirs is discussed . The problems of laboratory diagnosis of non-O157 VTEC are considered and various laboratory methods are assessed . Evidence is presented that the particular E . coli serotypes now known to be VTEC were present in humans and animals many years ago, but have acquired the ability to produce VT and probably other virulence factors . Finally, predictions are made of the possible increase in problems associated with these emerging pathogens. Symp Ser Soc Appl Microbiol, 2000, (29), 31S - 37S Verocytotoxin-producing Escherichia coli: a veterinary view; Synge BA; This overview places verocytotoxin-producing Escherichia coli (VTEC) in perspective with other E . coli types that cause disease in animals . VTEC O157 and other verocytotoxin-producing serotypes cause severe disease in man but to date, although other VTEC are found in animals, zoonosis appears to be associated with E . coli O157 only . The epidemiology of E . coli O157 in cattle has been studied in Scotland, and this work is described alongside current knowledge. Symp Ser Soc Appl Microbiol, 2000, (29), 24S - 30S Clinical presentation, complications and treatment of infection with verocytotoxin-producing Escherichia coli . Challenges for the clinician; Dundas S et al.; Seventeen years after its recognition, outbreaks and sporadic infections attributed to Escherichia coli O157 continue to increase . Acute gastrointestinal, and the systemic complications haemolytic uraemic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP), are frequent and severe . Current challenges that face clinicians are the early recognition of infection, identification of risk factors for poor prognosis, determination of appropriate monitoring for the development of complications, establishment of a therapeutic strategy and, finally, advice for patients about their long-term prognosis . Clinical features which, in combination, have been shown to distinguish E . coli O157 infection from other enteric pathogens are a history of bloody diarrhoea, visibly bloody stools, absence of fever, a leucocyte count greater than 10 x 10(9) l(-1) and abdominal tenderness on physical examination . The most consistent risk factors for the development of HUS/TTP are the extremes of age and a raised leucocyte count . Bloody diarrhoea and 'antimotility' drugs are also likely to be important risk factors . Recent evidence from the central Scotland outbreak suggests that individuals who are taking drugs that reduce gastric acidity or antibiotics at the time of infection with E . coli O157, or who have a short incubation period, may also be at increased risk of progression to HUS/TTP . Clinical management, in particular the role of antibiotics in gastrointestinal infection, remains controversial, and retrospective assessment of the 1996 outbreaks from central Scotland and Japan only adds to this controversy . Therapeutic plasma exchange is a promising treatment for adults who develop HUS/TTP, but its role has yet to be determined definitively, either in a randomized controlled trial or by an international register of cases . Significant chronic sequelae of infection occur, particularly irritable bowel syndrome after uncomplicated gastrointestinal infection, and renal failure after HUS/TTP . Their frequency and severity are likely to become evident over the next decade. Anal Biochem, 2000 Jul 1, 282(2), 218 - 26 Rapid SLT gene detection on polyethylene-coacrylic acid film without molecular labels or surface-fouling agents; Zhang P et al.; The commercially available copolymer of 10 mol% acrylic acid and polyethylene is easily formed into a nonfluorescing, non-polynucleotide-adsorbing film . The film has surface carboxylate functions whose concentration can be increased by heating to 80 degrees C in 30% NaOH . The carboxylate groups will react at pH approximately 7 with commercially available, oligo-DNA, 2-8 ng/microl, that has been synthesized with a C(12)-alkylamino tail on the 5'-end . The reaction is mediated with water-soluble carbodiimide reagent and is assumed to result in a primary amide bond between the polymer film and the modified oligo-DNA . The tethered oligo-DNA retains its hybridization activity, and its surface concentration is sufficient to permit qualitative, labelless detection of hybridized target by fluorescence after brief staining with ethidium bromide . The film is used to detect Shiga-like toxin gene II (SLT-II) from Escherichia coli O157:H7 after asymmetric, capillary, PCR amplification, and a 4-h hybridization . Captured target may be removed from the film using distilled water, after which the film can be used again without noticeable loss of activity . The method provides relatively rapid detection of PCR amplimers without having to use molecular labels, or surface-fouling agents . Am J Kidney Dis, 2000 Jul, 36(1), 42 - 6 Platelet-activating factor acetylhydrolase gene mutation in Japanese children with Escherichia coli O157-associated hemolytic uremic syndrome; Xu H et al.; Platelet-activating factor (PAF) may be involved in the pathogenesis of Escherichia coli O157-associated hemolytic uremic syndrome (HUS) . PAF is degraded to inactive products by PAF acetylhydrolase . In this study, we investigated whether a PAF acetylhydrolase gene mutation (G-->T transversion at position 994) is involved in HUS in Japanese children . A point mutation in the PAF acetylhydrolase gene (G994T) was identified using polymerase chain reaction in 50 Japanese children with E coli O157-associated HUS and 100 healthy Japanese . We then determined the relationship between the PAF acetylhydrolase G994T gene mutation and clinical features of HUS . There was no difference in genotype and allele frequencies between patients with HUS and healthy controls . The mean duration of oligoanuria was significantly longer in patients with the GT genotype than in those with the GG genotype (P = 0.012) . Although 11 of 15 patients (73%) heterozygous for the mutant allele (GT) required dialysis, only 13 of the 35 wild-type homozygotes (GG; 37%) required dialysis (P = 0 . 030) . Mean plasma PAF acetylhydrolase activity was significantly less in patients with the GT genotype than in those with the GG genotype (P < 0.0001) . In conclusion, we have shown an association between the G994T PAF acetylhydrolase gene mutation and the severity of renal damage in E coli O157-associated HUS . Our study suggests that analysis of the PAF acetylhydrolase gene mutation in Japanese children with E coli O157-associated HUS may allow the prediction of the severity of HUS. Int J Food Microbiol, 2000 May 25, 56(1), 13 - 20 Efficacy of allyl isothiocyanate in killing enterohemorrhagic Escherichia coli O157:H7 on alfalfa seeds; Park CM et al.; Volatile compounds occurring in the essential oil of plants were tested for their efficacy in killing Escherichia coli O157:H7 . Experiments using an agar disk assay revealed that exposure of the pathogen to 50 microl of eugenol, carvacrol, linalool, or methyl jasmonate in a 950-cc jar at 20, 37 or 47 degrees C for up to 48 h failed to inhibit colony formation . However, exposure to 8 microl of allyl isothiocyanate (AIT) (equivalent to 8.4 ppm in the air within the jar, if completely volatilized) resulted in more than a 7-log10 reduction in population of E . coli O157:H7 at 37 degrees C within 48 h; significant (P < or = 0.05) reduction in populations also occurred in the presence of 4 microl of AIT compared to 2 microl, which had no lethal affect . At 20 degrees C, the lethality of AIT was substantially less, although significant reduction occurred when disks were exposed to 8 or 10 microl of AIT compared to 4 or 6 microl and when exposed to 4 or 6 microl compared to 2 microl . Treatment with 10 microl of AIT for 5 h at 47 degrees C resulted in death of 6 log10 of E . coli O157:H7 . The efficacy of AIT in killing E . coli O157:H7 on dry and wet alfalfa seeds was investigated . The pathogen, at an initial population of 2.7 log10 cfu/g of seed, was not recovered by direct plating (< 0.7 log10 cfu/g) or enrichment of wet seeds exposed to 50 microl of AIT/950-cc jar for 24 h at 37 or 47 degrees C . Exposure of dry seeds containing 2.9 log10 cfu of E . coli O157:H7 per g to an atmosphere containing 100 microl of AIT/950-cc jar (ca . 105 ppm AIT if completely volatilized) for 24 h at 47 degrees C did not eliminate viable E . coli O157:H7 cells . Unfortunately, the enhanced effectiveness of AIT in killing the pathogen on wet alfalfa seeds is offset by a dramatic reduction in seed viability . Nevertheless, the use of AIT as an alternative to chlorine for the purpose of killing E . coli O157:H7 and perhaps other pathogens on alfalfa seed holds promise . Factors that may influence conditions rendering increased sensitivity of E . coli O157:H7 to AIT without compromising seed viability should be investigated. J Food Prot, 2000 Jun, 63(6), 819 - 21 How does Escherichia coli O157:H7 testing in meat compare with what we are seeing clinically? Acheson DW. Escherichia coli O157:H7 is but one of a group of Shiga toxin-producing E . coli (STEC) that cause both intestinal disease such as bloody and nonbloody diarrhea and serious complications like hemolytic uremic syndrome (HUS) . While E . coli O157: H7 is the most renowned STEC, over 200 different types of STEC have been documented in meat and animals, at least 60 of which have been linked with human disease . A number of studies have suggested that non-O157 STEC are associated with clinical disease, and non-O157 STEC are present in the food supply . Non-O157 STEC, such as O111 have caused large outbreaks and HUS in the United States and other countries . The current policy in the United States is to examine ground beef for O157:H7 only, but restricting the focus to O157 will miss other important human STEC pathogens. J Food Prot, 2000 Jun, 63(6), 703 - 8 Reduction of Escherichia coli O157:H7 counts on whole fresh apples by treatment with sanitizers; Wisniewsky MA et al.; The objectives of this study were to determine if washing of whole apples with solutions of three different sanitizers (peroxyacetic acid, chlorine dioxide, or a chlorine-phosphate buffer solution) could reduce a contaminating nonpathogenic Escherichia coli O157:H7 population by 5 logs and at what sanitizer concentration and wash time such a reduction could be achieved . Sanitizers were tested at 1, 2, 4, 8, and 16 times the manufacturer's recommended concentration at wash times of 5, 10, and 15 min . Whole, sound Braeburn apples were inoculated with approximately 1 x 108 or 7 x 106 CFU per apple, stored for 24 h, then washed with sterile water (control) or with sanitizers for the prescribed time . Recovered bacteria were enumerated on trypticase soy agar . Washing with water alone reduced the recoverable population by almost 2 logs from the starting population; this can be attributed to physical removal of organisms from the apple surface . No sanitizer, when used at the recommended concentration, reduced the recovered E . coli population by 5 logs under the test conditions . The most effective sanitizer, peroxyacetic acid, achieved a 5-log reduction when used at 2.1 to 14 times its recommended concentration, depending on the length of the wash time . The chlorine-phosphate buffer solution reduced the population by 5 logs when used at 3 to 15 times its recommended concentration, depending on wash time . At no concentration or wash time tested did chlorine dioxide achieve the 5-log reduction. J Med Microbiol, 2000 Jun, 49(6), 565 - 74 Genotypic variations of Shiga toxin-converting phages from enterohaemorrhagic Escherichia coli O157: H7 isolates; Osawa R et al.; Pulsed-field gel electrophoresis (PFGE) analysis revealed that enterohaemorrhagic Escherichia coli (EHEC) O157:H7 strains had considerable variations in their genomes . This study investigated whether or not the molecular profile of Shiga toxin (Stx) 1- and Stx2-converting phages isolated from EHEC O157:H7 strains, derived from various sources in the USA and Japan, corresponded to the variations of host strains' genotypes as determined by PFGE . A total of 51 Stx-converting phages including 12 Stx1-converting phages and 37 Stx2-converting phages was isolated from seven USA isolates and 20 Japanese isolates . The average Dice coefficient values showed 44% similarity between phage DNAs in Stx2-converting phages digested with SmaI and 55% in Stx1-converting phages digested with HindIII, indicating considerable variation among phage DNA . In particular, restriction fragment length polymorphism (RFLP) patterns of Stx2-converting phage DNA varied according to the PFGE type of their host strain, which suggests that the phage genomes have altered their genotypic characteristics with those of host genomes . However, there are several exceptions: the RFLP patterns of some Stx2-converting phages were quite similar irrespective of the different genotypes of the host strains, indicating that horizontal transfer of Stx2-converting phage may also occur under some circumstances. J Reprod Med, 2000 May, 45(5), 442 - 4 Immunologic evaluation of an Escherichia coli O157-infected pregnant woman . A case report; Tanaka H et al.; BACKGROUND: To the best of our knowledge, this is the first case of pregnancy complicated by hemorrhagic enterocolitis due to Escherichia coli O157 . CASE: A woman with hemorrhagic enterocolitis due to E coli O157 was seen at 32 weeks of gestation . We investigated her immune response to 0157 lipopolysaccharide and to Shiga toxin in the sera and breast milk . CONCLUSION: IgM and IgA to 0157 lipopolysaccharide in the breast milk of this patient might protect her infant after the disappearance of serum IgM. Kansenshogaku Zasshi, 2000 Apr, 74(4), 372 - 7 {Molecular investigation of pathogenic factors of suspected-diarrheogenic Escherichia coli isolates from patients feces}; Okazaki M et al.; One hundred fifty-one O serotypable Escherichia coli strains which were assumed diarrheogenic E . coli among 2,240 strains of E . coli isolated from the in- and outpatients stools with or without gastrointestinal symptoms at Kyorin University Hospital from February 1994 to September 1996 were examined for the relationship between the possession of eight pathogenic factor-related genes and gastrointestinal symptoms of the patients using the polymerase chain reaction (PCR) for these strains . The rate of possession of pathogenic factor-related genes in the E . coli examined was 20.5% (31 strains) and gastrointestinal symptoms were found in all the patients with these strains except one . In the patients without gastrointestinal symptoms, E . coli isolates that possesses these genes was detected in only one case during 61 cases . The respective genes detected were eaeA and astA in each 14 strains, VT1 in 6, VT2 in 5, ST1b in 4, aggR in 3 and LT in 2, ST1a and invE gene was not detected . In particular, the O157 strains were found in 55.6% (5/9 strains) for these genes, and individual strains had VT1, VT2, eaeA and astA genes simultaneously . In contrast, none of these related genes was found in 9 strains of enteroinvasive serotype but enteropathogenic E . coli-related genes were found in 3 strains . The rate of possession of the genes related to enterotoxigenic E . coli, O159 which was most frequently isolated was low as 2.3% (1/43 strains, astA gene) and there were strains showing low correlation to the state of possession of the genes with the O serotype . Since the prevalence of the gastrointestinal symptoms is clearly high for the case which possesses the strain of which the pathogenic factor-related gene was detected, it was suggested that detection of pathogenic factor-related genes in E . coli isolates from feces using the PCR could be an effective means to decide whether the bacteria concerned was a causal bacteria or not in clinical practice. J Clin Microbiol, 2000 Jun, 38(6), 2440 - 2 Relationship of genetic type of Shiga toxin to manifestation of bloody diarrhea due to enterohemorrhagic Escherichia coli serogroup O157 isolates in Osaka City, Japan; Nishikawa Y et al.; One hundred sixty-nine strains of enterohemorrhagic Escherichia coli serogroup O157 were examined for the correlation between the genotype of their Shiga toxin genes (stx) and manifestation of bloody diarrhea (BD) . It was shown that the strains carrying only stx2vha were probably less virulent and caused BD less frequently. J Clin Microbiol, 2000 Jun, 38(6), 2366 - 8 Epidemiologic subtyping of Escherichia coli serogroup O157 strains isolated in Ontario by phage typing and pulsed-field gel electrophoresis; Preston MA et al.; Phage typing and DNA macrorestriction fragment analysis by pulsed-field gel electrophoresis (PFGE) were evaluated for use in the epidemiological subtyping of Escherichia coli serogroup O157 strains isolated in Ontario, Canada . Among 30 strains isolated from patients with sporadic cases of infection, 22 distinct XbaI macrorestriction patterns were identified and 17 strains exhibited unique PFGE patterns . In contrast, phage typing identified only seven different phage types and 17 strains belonged to the same phage type . A total of 25 phage type-macrorestriction pattern combinations were identified among the strains from patients with sporadic cases of infection . PFGE subtyping differentiated between unrelated strains that exhibited the same phage type, and in one group of strains, phage typing differentiated between strains of the same PFGE subtype . Both typing procedures correctly identified outbreak-related isolates as belonging to the same type in four separate outbreaks . Each outbreak strain was characterized by a distinct macrorestriction pattern, while phage typing subdivided the outbreak strains into only three different types . A small percentage of outbreak-related isolates had PFGE patterns that differed slightly (one or two DNA fragment differences) from that of the outbreak strain . On the other hand, each isolate from the same outbreak belonged to the same phage type as that of the outbreak strain . We conclude that phage typing and PFGE fingerprinting represent complementary procedures for the subtyping of E . coli serogroup O157 and that the combined use of these procedures provides optimal discrimination. J Clin Microbiol, 2000 Jun, 38(6), 2134 - 40 Molecular characteristics and epidemiological significance of Shiga toxin-producing Escherichia coli O26 strains; Zhang WL et al.; Fifty-five Shiga toxin (Stx)-producing Escherichia coli (STEC) O26:H11 and O26:H(-) strains isolated from humans between 1965 and 1999 in Germany and the Czech Republic were investigated for their chromosomal and plasmid characteristics . All motile (n = 23) and nonmotile (n = 32) STEC O26 strains were shown to possess the identical flagellin subunit-encoding gene (fliC) . We observed a striking recent shift of the stx genotype from stx(1) to stx(2) among the STEC O26 isolates . While stx(1) was the exclusive genotype identified in our collection until 1994, 94% of the isolates obtained after 1997 possessed stx(2) either alone (71%) or together with stx(1) (23%) . Plasmid profiling demonstrated a remarkable heterogeneity with respect to plasmid sizes and combinations . Southern blot analysis of plasmid DNA with probes specific to potential accessory virulence genes revealed considerable additional variability in gene composition and arrangement . Pulsed-field gel electrophoresis (PFGE) differentiated 16 subgroups among the 55 STEC O26 strains . Using these techniques we demonstrate the emergence of a new clonal subgroup characterized by PFGE pattern A and a unique combination of virulence markers including stx(2) and a single, approximately 90-kb plasmid harboring the enterhemorrhagic E . coli hlyA and etp genes . The proportion of PFGE subgroup A strains among STEC O26 isolates rose from 30% in 1996 to more than 50% in 1999 . Four clusters of infections with the clonal subgroup A were identified . We conclude that the STEC serogroup O26 is diverse and that pathogenic clonal subgroups can rapidly emerge during short intervals . The extensive genetic diversity of STEC O26 provides a basis for molecular subtyping of this important non-O157 STEC serogroup. Microbiol Immunol, 2000, 44(4), 271 - 4 Release of membrane vesicles containing endotoxic lipopolysaccharide in Escherichia coli O157:H7 clinical isolates; Meno Y et al.; Membrane vesicles released by E . coli O157:H7 strains were investigated by immuno-electron microscopy using anti-O157 antibody . Anti-O157 antibody enhanced the negative-staining of vesicles and we found numerous small vesicles clearly formed around bacterial cells . An immunogold-electron microscopic examination confirmed that lipopolysaccharide (LPS) including the O-side chain is present on the surface of the vesicles . Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the purified vesicles showed that the vesicles contained LPS consisting of a lipid-A and an O polysaccharide . In addition, the endotoxic activity of the vesicle was confirmed by a limulus test . These results suggest that the vesicles may play an important role in the pathogenesis of Escherichia coli O157:H7. Appl Environ Microbiol, 2000 Jun, 66(6), 2513 - 9 Restriction-site-specific PCR as a rapid test to detect enterohemorrhagic Escherichia coli O157:H7 strains in environmental samples; Kimura R et al.; Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important food-borne pathogen in industrialized countries . We developed a rapid and simple test for detecting E . coli O157:H7 using a method based on restriction site polymorphisms . Restriction-site-specific PCR (RSS-PCR) involves the amplification of DNA fragments using primers based on specific restriction enzyme recognition sequences, without the use of endonucleases, to generate a set of amplicons that yield "fingerprint" patterns when resolved electrophoretically on an agarose gel . The method was evaluated in a blinded study of E . coli isolates obtained from environmental samples collected at beef cattle feedyards . The 54 isolates were all initially identified by a commonly used polyclonal antibody test as belonging to O157:H7 serotype . They were retested by anti-O157 and anti-H7 monoclonal antibody enzyme-linked immunosorbent assay (ELISA) . The RSS-PCR method identified all 28 isolates that were shown to be E . coli O157:H7 by the monoclonal antibody ELISA as belonging to the O157:H7 serotype . Of the remaining 26 ELISA-confirmed non-O157:H7 strains, the method classified 25 strains as non-O157:H7 . The specificity of the RSS-PCR results correlated better with the monoclonal antibody ELISA than with the polyclonal antibody latex agglutination tests . The RSS-PCR method may be a useful test to distinguish E . coli O157:H7 from a large number of E . coli isolates from environmental samples. Emerg Infect Dis, 2000 May-Jun, 6(3), 293 - 7 Costs and benefits of a subtype-specific surveillance system for identifying Escherichia coli O157:H7 outbreaks; Elbasha EH et al.; We assessed the societal costs and benefits of a subtype-specific surveillance system for identifying outbreak-associated Escherichia coli O157:H7 infections . Using data from Colorado, we estimated that if it averted five cases annually, the system would recover all its costs. J Infect Dis, 2000 May, 181(5), 1825 - 9 Epub 2000 May 09. Escherichia coli O157:H7 strains that express Shiga toxin (Stx) 2 alone are more neurotropic for gnotobiotic piglets than are isotypes producing only Stx1 or both Stx1 and Stx2; Donohue-Rolfe A et al.; Infection with Escherichia coli O157:H7 can lead to hemolytic uremic syndrome (HUS) in some children . Epidemiologic data suggest that Shiga toxin (Stx) 2-producing strains are more frequently associated with HUS than are Stx1-producing strains . Less clear is whether strains that express Stx2 alone are more frequently associated with HUS than strains that express Stx1 and Stx2 . Isogenic mutants 933stx1- and 933stx2- were produced from strain 933 (Stx1 and Stx2 producer), and 86-24stx2- was produced from strain 86-24 (Stx2 producer) . Neurologic lesions or symptoms developed in 18 (90%) of 20 gnotobiotic piglets orally infected with strain 86-24, in 15 (85%) of 18 infected with mutant 933stx1-, in 9 (31%) of 29 infected with strain 933, in 0 of 5 infected with mutant 86-24stx2-, and in 0 of 6 infected with mutant 933stx2- . It was concluded that strains expressing Stx2 alone are more neurotropic for piglets when fed orally than are those strains expressing Stx1 and 2, whereas Stx1-producing strains induce only diarrhea . It is also conceivable that strains that produce Stx2 may constitute a significant predictive risk factor for HUS in humans. Epidemiol Infect, 2000 Apr, 124(2), 207 - 13 A one year study of Escherichia coli O157 in raw beef and lamb products; Chapman PA et al.; Between April 1996 and March 1997 we examined 5093 samples of raw beef and lamb products for the presence of E . coli O157 . Samples were purchased from 81 small butchers' shops in south Yorkshire . In March 1997 we also examined five samples of dried mint for the presence of E . coli O157 . Strains of E . coli O157 were isolated by enrichment culture in modified buffered peptone water followed by immunomagnetic separation and culture of magnetic beads onto cefixime tellurite sorbitol MacConkey agar . Strains were characterized by phage typing, toxin genotyping and plasmid analysis . Strains of E . coli O157 were isolated from 72 (1.4%) of 5093 samples; it was isolated from 36 (1.1%) of 3216 samples of beef products and from 29 (2.9%) samples of lamb products . The highest prevalence was found in lamb sausages and lamb burgers where E . coli O157 was isolated from 3 (4.1%) of 73 and 18 (3.7%) of 484 samples respectively . Strains of E . coli O157 were isolated most frequently during early summer . Strains of E . coli O157 were also isolated from 2 of 5 samples of dried mint although we did not determine how the mint had become contaminated . All isolates of E . coli O157 were Verocytotoxin-producing as determined by both Vero cell assay and DNA hybridization for the genes encoding Verocytotoxin and all were positive for the eaeA gene . A combination of phage typing, toxin genotyping and plasmid profile subdivided the 72 strains of E . coli isolated into 20 different subtypes, of which 18 were indistinguishable from strains isolated previously from cattle and sheep; of these 18 strains, 8 were indistinguishable from strains isolated from human cases of infection during the study period. Anal Biochem, 2000 Apr 10, 280(1), 151 - 8 Immunoliposome sandwich assay for the detection of Escherichia coli O157:H7; Park S et al.; We describe the development of a field-portable colorimetric immunoassay for the detection of Escherichia coli O157:H7, using antibody-directed liposomes (immunoliposomes) encapsulating dye as an analytical reagent . Antibodies (anti-E . coli O157:H7) thiolated by 2-iminothiolane were coupled to malemide-tagged liposomes encapsulating the marker dye, sulforhodamine B . Transmission electron microscopy showed that the immunoliposomes bound only to the serotype without any cross-reactivity with tested negative controls . A wicking reagent containing immunoliposomes and the test sample and a plastic-backed nitrocellulose strip with a measurement zone were used in a sandwich (noncompetitive) assay format . During the capillary migration of the wicking reagent, E . coli, with surface-bound immunoliposomes, was captured at the measurement zone on which antibodies to E . coli O157:H7 were immobilized . The color density of the measurement zone was directly proportional to the amount of E . coli O157:H7 in the sample . The detection limit of the current assay with pure cultures of the serotype was ca . 10(4) colony-forming units (CFU)/mL . The assay, which does not need washing and incubation steps, can be completed in 8 min . These results demonstrate the feasibility of using dye-encapsulating immunoliposomes in microporous membranes for the rapid detection of molecules with multivalent antigenic sites. FEMS Microbiol Lett, 2000 May 1, 186(1), 79 - 84 A sensitive microsphere coagulation ELISA for Escherichia coli O157:H7 using Russell's viper venom; Strachan NJ et al.; A microsphere coagulation enzyme-linked immunosorbent assay (MC-ELISA) using Russell's viper venom factor X activator (RVV-XA) is described for the detection of Escherichia coli O157:H7 . This microtitre plate assay comprises a standard sandwich immunoassay incorporating RVV-XA as the enzyme label . Coagulation substrate together with polystyrene microspheres are added to the wells of the microtitre plate . RVV-XA initiates the coagulation cascade causing formation of an artificial clot of polystyrene microspheres bound together with fibrin . As few as 10(2) E . coli O157 in a well (10(3) per ml) can be detected within 3 h . The assay is two orders of magnitude more sensitive than a standard ELISA and is a generic technique with the potential for widespread use in sandwich immunoassays. J Public Health Med, 2000 Mar, 22(1), 99 - 107 Escherichia coli O157:H7; an economic assessment of an outbreak; Roberts JA et al.; BACKGROUND: The aim of the study was to assess the impact of an outbreak of Escherichia coli O157:H7 that occurred in 1994 in a rural community, with a population of approximately 107,000, to the west of Edinburgh . METHODS: The impact of the outbreak was assessed during the acute phase of the illness and in the subsequent 12 months . The method involved three surveys of confirmed cases using general practice notes, hospital records and interviews with cases . Key persons involved in the investigation and control of the outbreak were also interviewed . The impact of the illness on cases and their families was estimated and the resources used to treat cases and to control the outbreak were costed and long-term costs projected . RESULTS: There were 71 cases whose ages ranged from 7 months to 84 years . The mortality rate was 1.4 per hundred cases . There were 10 cases of haemolytic uraemic syndrome (HUS) and one case of thrombotic thrombocytopenia purpura (TTP) . Two children were on long-term dialysis . Co-morbidity involving the immune system was associated with hospital admission . The illness lasted on average 6.9 weeks . Twenty-six per cent of cases reported symptoms 12 months later . The average cost per HUS case was 62,353 pound sterlings, the TTP case cost 21,422 pound sterlings, non-HUS and non-TTP cases cost 1,030 pound sterlings . The costs of investigating and controlling the outbreak were 171,848 pound sterlings . The costs of cases proje