Appl Environ Microbiol, 2005 Jan, 71(1), 558 - 61 Transcriptional Analysis of Genes Encoding Shiga Toxin 2 and Its Variants in Escherichia coli; Zhang W et al.; Six of 37 non-O157 Escherichia coli strains possessing Shiga toxin (Stx) 2 gene variant stx(2d) or stx(2e) secreted no detectable Stx . These isolates produced significantly less stx mRNA than Stx2d, Stx2e, Stx2c, or Stx2 secretors did . Standard screening procedures miss a significant subset of E . coli harboring stx(2) variants. J Food Prot, 2004 Dec, 67(12), 2756 - 9 Optimization of a fluorescence sandwich enzyme-linked immunosorbent assay for detection of Escherichia coli O157:H7 in apple juice; Nyquist-Battie C et al.; Sandwich enzyme-linked immunosorbent assay, especially when coupled with biosensor technology, is a simple methodology that can rapidly screen juices for Escherichia coli O157:H7 contamination . However, sampling directly from apple juice and ciders has been postulated to reduce immunoassay sensitivity . In fluorescence sandwich enzyme-linked immunosorbent assays using commercially available polyclonal or monoclonal antibodies, sampling pasteurized apple juice spiked with E . coli O157:H7 compared to spiked phosphate-buffered saline shifted the range of detection . The spiked apple juice range of detection was 10(4) to 10(6) CFU/ml, whereas that for spiked phosphate-buffered saline was 10(6) to 10(8) CFU/ml, representing a hundredfold difference in sensitivity . Apple juice also increased background fluorescence intensity (P < 0.001) while reducing the net fluorescence intensity per CFU (P < 0.001) . The addition of the polymer polyvinylpyrrolidone to apple juice significantly improved assay performance by increasing sensitivity and net fluorescence intensity per CFU and by reducing background fluorescence . Adjusting pH of apple juice from 3.9 to 7.4 improved assay performance but not to the degree seen with phosphate-buffered saline or polyvinylpyrrolidone-treated apple juice samples . The apple juice polyphenol, epicatechin, reduced net fluorescence intensity in a concentration-dependent manner, a change that was reversed by polyvinylpyrrolidone . Taken all together, these results suggest that polyvinylpyrrolidone can improve detection of O157:H7 in juices by reducing the effect of polyphenols on fluorescence sandwich enzyme-linked immunosorbent assay performance. Kansenshogaku Zasshi, 2004 Nov, 78(11), 975 - 83 {Hazard evaluation of livestock-derived, verotoxin-producing Escherichia coli by enterohemolysin production assay and eaeA gene detection}; Kobayash T et al.; We examined enterohemolysin (Ehly) production, and detected the hlyA gene and the eaeA gene for the intestinal mucosal adherence factor intimin in 131 strains of human-derived verotoxin-producing Escherichia coli (VTEC) and 140 strains of livestock (cattle and swine) -derived VTEC to evaluate their hazards to humans . The hlyA gene was confirmed in 98.5% of human-derived, in 50.5% of cattle-derived, and in 10.3% of swine-derived VTEC strains . Ehly-positive rates were 96.2-97.7%, 45.9-55.0%, and 10.3-20.7% in human-, cattle-, and swine-derived VTEC strains, respectively . Thus, the positive rates differed among strains of different species origins . However, all 12 cattle-derived O157VTEC strains had hlyA, and were Ehly-positive . Although 97.7% of human-derived strains and all cattle-derived O157VTEC strains had eaeA, only 8.1% of cattle derived strains of serotypes other than O157 and 3.4% of swine-derived strains had eaeA . In human- and cattle-derived strains, the presence of eaeA was associated with Ehly: all eaeA-carrying strains had hlyA, and almost all of them were Ehly-positive . Cattle-derived eaeA-carrying strains accounted for 29.5-35.3% of Ehly-positive strains, compared to 100% in human-derived strains . Only 3-4% of Ehly-negative strains had eaeA, and none of the non-hlyA-carrying strains had eaeA . These findings suggest that 2 factors, eaeA and Ehly, serve as useful indicators for the evaluation of hazard to humans, and that Ehly is a useful indicator because cattle-derived Ehly-positive strains may have eaeA. Infect Immun, 2005 Jan, 73(1), 552 - 62 Cytolethal distending toxin from Shiga toxin-producing Escherichia coli O157 causes irreversible G2/M arrest, inhibition of proliferation, and death of human endothelial cells; Bielaszewska M et al.; Recently, cytolethal distending toxin V (CDT-V), a new member of the CDT family, was identified in Shiga toxin-producing Escherichia coli (STEC) O157 and particular non-O157 serotypes . Here we investigated the biological effects of CDT-V from STEC O157:H(-) (strain 493/89) on human endothelial cells, which are believed to be major pathogenetic targets in severe STEC-mediated diseases . CDT-V caused dose-dependent G(2)/M cell cycle arrest leading to distension, inhibition of proliferation, and death in primary human umbilical vein endothelial cells (HUVEC) and two endothelial cell lines, EA.hy 926 cells (HUVEC derived) and human brain microvascular endothelial cells (HBMEC) . The cell cycle effects of CDT-V were cell type specific . In HUVEC and EA.hy 926 cells, CDT-V caused a slowly developing but persistent G(2)/M block which resulted in delayed nonapoptotic cell death . In contrast, in HBMEC, CDT-V induced a rapidly evolving but transient G(2)/M block which was followed by progressive, mostly apoptotic cell death . In both HBMEC and EA.hy 926 cells, G(2)/M arrest was preceded by the early accumulation of a phosphorylated inactive form of cdc2 kinase . Significant G(2)/M arrest and inhibition of proliferation in both HUVEC and each of the endothelial cell lines were induced by 2 to 15 min of exposure to CDT-V, indicating that the effects of the toxin are irreversible . CDT-V-treated HBMEC and EA.hy 926 cells displayed fragmented nuclei and expressed phosphorylated histone protein H2AX, indicative of DNA damage followed by a DNA repair response . Our data demonstrate that CDT-V causes irreversible damage to human endothelial cells and thus may contribute to the pathogenesis of STEC-mediated diseases. Proc Natl Acad Sci U S A, 2005 Jan 4, 102(1), 221 - 6 Epub 2004 Dec 22. An unusual signal peptide facilitates late steps in the biogenesis of a bacterial autotransporter; Szabady RL et al.; Bacterial autotransporters are proteins that use a C-terminal porin-like domain to facilitate the transport of an upstream "passenger domain" across the outer membrane . Although autotransporters are translocated across the inner membrane (IM) via the Sec pathway, some of them contain exceptionally long signal peptides distinguished by a unique N-terminal sequence motif . In this study, we used the Escherichia coli O157:H7 autotransporter EspP as a model protein to investigate the function of the unusual signal peptides . We found that removal of the N-terminal motif or replacement of the EspP signal peptide did not affect translocation of the protein across the IM . Remarkably, modification of the signal peptide caused EspP to misfold in the periplasm and blocked transport of the passenger domain across the outer membrane . Further analysis suggested that the EspP signal peptide transits slowly through the Sec machinery . Based on these results, we propose that the unusual signal peptides not only function as targeting signals, but also prevent misfolding of the passenger domain in the periplasm by transiently tethering it to the IM. J Biotechnol, 2005 Jan 12, 115(1), 101 - 7 Alteration of tail fiber protein gp38 enables T2 phage to infect Escherichia coli O157:H7; Yoichi M et al.; Artificial control of phage specificity may contribute to practical applications, such as the therapeutic use of phages and the detection of bacteria by their specific phages . To change the specificity of phage infection, gene products (gp) 37 and 38, expressed at the tip of the long tail fiber of T2 phage, were exchanged with those of PP01 phage, an Escherichia coli O157:H7 specific phage . Homologous recombination between the T2 phage genome and a plasmid encoding the region around genes 37-38 of PP01 occurred in transformant E . coli K12 cells . The recombinant T2 phage, named T2ppD1, carried PP01 gp37 and 38 and infected the heterogeneous host cell E . coli O157:H7 and related species . On the other hand, T2ppD1 could not infect E . coli K12, the original host of T2, or its derivatives . The host range of T2ppD1 was the same as that of PP01 . Infection of T2ppD1 produced turbid plaques on a lawn of E . coli O157:H7 cells . The binding affinity of T2ppD1 to E . coli O157:H7 was weaker than that of PP01 . The adsorption rate constant (k(a)) of T2ppD1 (0.17x10(-9)(mlCFU(-1)min(-1)) was almost 1/6 that of PP01 (1.10x10(-9)(mlCFU(-1)min(-1))) . In addition to the tip of the long tail fiber, exchange of gene products expressed in the short tail fiber may be necessary for tight binding of recombinant phage. Vet Microbiol, 2005 Jan 5, 105(1), 37 - 45 Epub 2004 Dec 08. Phenotypic and genotypic characteristics of non-O157 Shiga toxin-producing Escherichia coli (STEC) from Swiss cattle; Zweifel C et al.; A total of 42 Shiga toxin-producing (STEC) strains from slaughtered healthy cattle in Switzerland were characterized by phenotypic and genotypic traits . The 42 sorbitol-positive, non-O157 STEC strains belonged to 26 O:H serotypes (including eight new serotypes) with four serotypes (O103:H2, O113:H4, O116:H-, ONT:H-) accounting for 38.1% of strains . Out of 16 serotypes previously found in human STEC (71% of strains), nine serotypes (38% of strains) were serotypes that have been associated with hemolytic-uremic syndrome (HUS) . Polymerase chain reaction (PCR) analysis showed that 18 (43%) strains carried the stx1 gene, 20 strains (48%) had the stx2 gene, and four (9%) strains had both stx1 and stx2 genes . Of strains encoding for stx2 variants, 63% were positive for stx2 subtype . Enterohemolysin (ehxA), intimin (eae), STEC autoagglutinating adhesin (saa) were detected in 17%, 21%, and 19% of the strains, respectively . Amongst the seven intimin-positive strains, one possessed intimin type beta1 (O5:H-), one intimin gamma1 (O145:H), one intimin gamma2/theta;, (O111:H21), and four intimin varepsilon (O103:H2) . The strains belonged to 29 serovirotypes (association between serotypes and virulence factors) . O103:H2 stx1eae-varepsilon ehxA, O116:H- stx2, and ONT:H- stx2c were the most common accounting for 29% of the strains . Only one strain (2.4%) of serovirotype O145:H- stx1stx2eae-gamma1ehxA showed a pattern of highly virulent human strains . This is the first study providing characterization data of bovine non-O157 STEC in Switzerland, and underlining the importance of the determination of virulence factors (including intimin types) in addition to serotypes to assess the potential pathogenicity of these strains for humans. J Biomed Sci, 2004 Nov-Dec, 11(6), 855 - 63 Identification of a negative regulator for the pathogenicity island of enterohemorrhagic Escherichia coli O157:H7; Lio JC et al.; Enterohemorrhagic Escherichia coli (EHEC) forms histological lesions termed attaching and effacing lesions (A/E lesions) on infected large intestine tissue . The major virulence factors involved in A/E lesions reside on a locus of enterocyte effacement (LEE), a pathogenicity island . The LEE comprises 41 specific open reading frames, of which most are organized in 5 major operons, LEE1, LEE2, LEE3, LEE4, and tir(LEE5) . The expression of LEE genes is regulated in a complicated manner by environmental factors such as temperature, osmolarity, and quorum sensing . Current knowledge is that regulation is hierarchical: a pivotal positive regulator, ler, is first stimulated, which in turn activates the expression of other operons . Herein, we report on the presence of a negative regulation protein located within the LEE . L0044 is 372 bp in length and is located outside of the 5 major operons . An isogenic L0044 deletion mutant displayed loss of the repression phenotype and increased synthesis of several LEE proteins when bacteria were cultured under repressive conditions that disfavor expression of LEE proteins . Reciprocally, trans expression of L0044 suppressed the expression of the LEE . Furthermore, mRNA of ler increased as a result of deleting L0044, and disrupting ler in a L0044-deleted background reversed the loss of the repression phenotype . Thus, L0044 plays a role in regulating the expression of virulence genes in EHEC by modulating the activation of ler . 2004 National Science Council, ROC and S . Karger AG, Basel J Med Microbiol, 2005 Jan, 54(Pt 1), 71 - 5 Characterization of Escherichia coli O157 : H7 isolates causing haemolytic uraemic syndrome in France; Bidet P et al.; Forty-seven non-epidemic Escherichia coli O157 : H7 isolates causing haemolytic uraemic syndrome in France were characterized . The isolates clustered into 36 clones using PFGE typing . All the isolates harboured eae and one or more copies of stx2 and belonged to phylogenetic group D . Nine per cent were resistant to amoxicillin. Biosens Bioelectron, 2005 Jan 15, 20(7), 1407 - 16 AFM and impedance spectroscopy characterization of the immobilization of antibodies on indium-tin oxide electrode through self-assembled monolayer of epoxysilane and their capture of Escherichia coli O157:H7; Yang L et al.; The microscopic surface molecular structures and macroscopic electrochemical impedance properties of the epoxysilane monolayer and anti-Escherichia coli antibody layer on an indium-tin oxide (ITO) electrode surface were studied in this paper . Characterization of stepwise changes in microscopic features of the surfaces and electrochemical properties upon the formation of each layer were carried out using both atomic force microscopy (AFM) and electrochemical impedance spectroscopy in the presence of {Fe(CN)(6)}(3-/4-) as a redox couple . AFM images of the self-assembled monolayer (SAM) evidenced the dense, complete, and homogeneous morphology of the epoxysilane monolayer on the ITO surface . The uniformity of the epoxysilane SAM allowed antibodies to attach to the epoxy surface groups of the silanes in a similarly uniform fashion . The effects of epoxysilane monolayer and the antibody layer on the electrochemical properties of the electrode were quantitatively analyzed in terms of double layer capacitance, electron transfer resistance, Warburg impedance and solution resistance using Randles model as the equivalent circuit . It was demonstrated that the epoxysilane monolayer and the antibody layer act as barriers for the electron transfer between the electrode surface and the redox species in the solution, resulting in most significant increases in the electron transfer resistance compared to all the electric elements . Immunoreaction with E . coli O157:H7 cells demonstrated specific recognition of the immobilized anti-E . coli antibodies as evidenced by AFM imaging and impedance spectroscopy . It was found that the binding of E . coli cells mainly affected the electron transfer resistance and Warburg impedance. J Clin Microbiol, 2004 Dec, 42(12), 5462 - 6 Development of a rapid PCR method using the insertion sequence IS1203 for genotyping Shiga toxin-producing Escherichia coli O157; Suzuki M et al.; We developed a rapid PCR method utilizing the diversity of the insertion site IS1203 for genotyping Shiga toxin-producing Escherichia coli (STEC) O157 (IS1203 PCR typing) . DNA fragments digested by PvuII, which cut IS1203 at one site, were ligated with themselves and detected by PCR with outward-facing primer pairs for IS1203 . To minimize nonspecific bands, nested PCR was also performed . Two fingerprinting patterns produced from the upstream or downstream regions of IS1203 were obtained within 1 or 2 days . By combining the two patterns, 79 STEC O157 isolates were classified into 39 types, which were then classified into 36 subtypes by pulsed-field gel electrophoresis (PFGE) . The discriminatory power of IS1203 PCR typing (D = 0.974) is similar to that of PFGE (D = 0.981) . This method can be used for rapid and simplified genotyping. Prev Vet Med, 2004 Dec 15, 66(1-4), 175 - 206 Associations between management, climate, and Escherichia coli O157 in the faeces of feedlot cattle in the Midwestern USA; Sargeant JM et al.; Our objective was to generate hypotheses for potential on-farm control strategies for Escherichia coli O157 by identifying associations between management practices and climate, and the presence of E . coli O157 in feedlot cattle . Faeces were obtained from 10,622 cattle in 711 pens on 73 feedlots between May and August 2001 . Management and climate information was obtained by questionnaire and observation at the time of sampling . The prevalence of E . coli O157 was 10.2% at the sample level, 52.0% at the pen-level, and 95.9% at the feedlot-level . The factors associated with the presence of E . coli O157 in cattle faeces were the frequency of observing cats in the pens or alleys (most common when observed daily), the presence of E . coli O157 in the water tanks (positive association), the historical use of injectable mass medication (positive association), the use of antibiotics in the ration or water (negative association), the wetness of the pen, number of cattle in the pen (negative association), wind velocity (positive association), and height of the feed bunk (positive association). Appl Environ Microbiol, 2004 Dec, 70(12), 7578 - 80 Association of Escherichia coli O157:H7 with houseflies on a cattle farm; Alam MJ et al.; The ecology of Escherichia coli O157:H7 is not well understood . The aims of this study were to determine the prevalence of and characterize E . coli O157:H7 associated with houseflies (HF) . Musca domestica L . HF (n = 3,440) were collected from two sites on a cattle farm over a 4-month period and processed individually for E . coli O157:H7 isolation and quantification . The prevalence of E . coli O157:H7 was 2.9 and 1.4% in HF collected from feed bunks and a cattle feed storage shed, respectively . E . coli O157:H7 counts ranged from 3.0 x 10(1) to 1.5 x 10(5) CFU among the positive HF . PCR analysis of the E . coli O157:H7 isolates revealed that 90.4, 99.2, 99.2, and 100% of them (n = 125) possessed the stx1, stx2, eaeA, and fliC genes, respectively . Large populations of HF on cattle farms may play a role in the dissemination of E . coli O157:H7 among animals and to the surrounding environment. Res Vet Sci, 2005 Apr, 78(2), 109 - 15 Naturally acquired attaching and effacing Escherichia coli in sheep; Wales AD et al.; In a series of experiments involving the inoculation of sheep with Escherichia coli O157:H7, and subsequent detailed histopathological examination of the intestinal mucosa, attaching-effacing (AE) lesions formed by elements of the natural flora were observed in 18% of animals . These incidental AE lesions typically were small and sparse, and were not associated with clinical disease . It was possible to identify further some of the lesional bacteria, revealing that E . coli O115 had formed lesions in one of the seven affected animals, and similarly E . coli O26 had formed some of the lesions in another . As AE strains, source flocks, housing and feed sources were diverse, a common source of lesion-forming bacteria appears to be unlikely . It is postulated that subclinical AE lesions are a mechanism of persistence of AE bacteria in sheep. Eur J Pediatr, 2005 Jan, 164(1), 37 - 43 Epub 2005 Jan. Colostrum from healthy Brazilian women inhibits adhesion and contains IgA antibodies reactive with Shiga toxin-producing Escherichia coli; Palmeira P et al.; Although Shiga toxin-producing Escherichia coli(STEC) has been isolated in Brazil, severe manifestations of the infection, such as haemorrhagic colitis and haemolytic-uraemic syndrome, are extremely rare in our population . Enteropathogenic Escherichia coli(EPEC) is the main aetiological agent of acute infantile diarrhoea in Brazil . There are many similarities between STEC and EPEC, such as the ability to produce attaching and effacing (A/E) lesions and some virulence-associated factors . Our aim was to investigate the presence of anti-STEC antibodies in healthy people living in an EPEC endemic area . Colostrum samples collected from 51 women living in low socio-economic conditions were analysed . Two STEC strains: O111:H- (Stx1) and O157:H7 (Stx2), and one EPEC strain (O111:H-) were used in the bacterial adhesion assays to HEp-2 cells, in the Stx1 and Stx2 cytotoxicity assays on Vero cells, in immunoblotting and in ELISA assays . All the samples strongly inhibited the adhesion of the three strains and contained SIgA antibodies reactive with antigens of EPEC O111:H-, STEC O111:H- and STEC O157:H7, mainly STEC and EPEC 94 kDa adhesin intimin . High titres of anti-LPS O111 antibodies were found in many samples . Nevertheless, the cytotoxic effect of both Stx1 and Stx2 on Vero cells was not neutralised by any sample . Conclusion: Our results suggest that Brazilian people may be exposed to Shiga toxin-producing Escherichia colimore frequently than previously thought or alternatively there may be a cross reactive immunity between enteropathogenic Escherichia coliand Shiga toxin-producing Escherichia coli. Lett Appl Microbiol, 2004, 39(6), 516 - 22 Comparison of fluorogenic and chromogenic assay systems in the detection of Escherichia coli O157 by a novel polymyxin-based ELISA; Blais BW et al.; AIMS: Different indicator enzymes and fluorogenic or chromogenic substrates were compared as detector systems in a novel polymyxin-based enzyme-linked immunosorbent assay (ELISA) for Escherichia coli O157 lipopolysaccharide (LPS) antigens . METHODS AND RESULTS: An ELISA system was developed using polymyxin immobilized in the wells of a microtitre plate as a high-affinity adsorbent for E . coli O157 LPS antigens, which were immunoenzymatically detected using anti-E . coli O157 antibody-enzyme conjugates . With peroxidase as the indicator enzyme the fluorogenic substrates Amplex Red and QuantaBlu produced only slight improvement in the performance characteristics of the polymyxin-ELISA compared with the use of the chromogenic substrate tetramethylbenzidine (TMB) . On the other hand, with alkaline phosphatase as the indicator enzyme a pronounced improvement in assay performance was noted using the fluorogenic substrate Attophos compared with the chromogenic substrate p-nitrophenylphosphate . CONCLUSIONS: The detection system exhibiting the best characteristics with respect to cost, ease of use and overall performance in the detection of E . coli O157 in enrichment cultures from a variety of solid foods was based on the use of peroxidase as the indicator enzyme with the chromogenic substrate TMB . SIGNIFICANCE AND IMPACT OF THE STUDY: The polymyxin-ELISA provides a rapid, simple and inexpensive assay system for the detection of E . coli O157 in foods. Am J Physiol Gastrointest Liver Physiol, 2004 Dec, 287(6), G1238 - 46 Adrenergic modulation of Escherichia coli O157:H7 adherence to the colonic mucosa; Green BT et al.; Enteric neurotransmitters can modulate the biodefensive functions of the intestinal mucosa, but their role in mucosal interactions with enteropathogens is not well defined . Here we tested the hypothesis that norepinephrine (NE) modulates interactions between enterohemorrhagic Escherichia coli O157:H7 (EHEC) and the colonic epithelium . Mucosal sheets from porcine distal colon were mounted in Ussing chambers . Drugs and an inoculum of either Shiga toxin-negative or -positive EHEC were added to the contraluminal and luminal bathing medium, respectively . After 90 min, adherent bacteria were quantified by an adherence assay and by immunohistochemical methods; short-circuit current (I(sc)) was measured continuously to assess changes in active ion transport . NE-treated tissues exhibited concentration-dependent increases in I(sc) and EHEC adherence . NE did not alter adherence of a rodent-adapted, noninfectious E . coli strain or two porcine-adapted non-O157 E . coli strains . The actions of NE on EHEC adherence but not I(sc) were prevented by the alpha-adrenergic antagonist yohimbine and the PKA activator Sp-8-bromoadenosine-3',5'-cyclic monophosphorothioate . Like NE, the PKA inhibitor Rp-8-bromoadenosine-3',5'-cyclic monophosphorothioate or indirectly acting sympathomimetic agents increased EHEC adherence . Nerve fibers immunoreactive for the NE-synthesizing enzymes tyrosine hydroxylase and dopamine beta-hydroxylase appeared to innervate the colonic epithelium . EHEC-like immunoreactivity on the colonic surface had the appearance of bacterial microcolonies and increased after NE treatment by a phentolamine-sensitive mechanism . Through interactions with alpha(2)-adrenergic receptors, NE appears to increase EHEC adherence to the colonic mucosa . Changes in sympathetic neural outflow may alter intestinal susceptibility to infection. J Clin Microbiol, 2004 Nov, 42(11), 5205 - 13 Development of a PCR-restriction fragment length polymorphism assay for the epidemiological analysis of Shiga toxin-producing Escherichia coli; Shima K et al.; Six characteristic regions (I to VI) were identified in Shiga toxin 2 (Stx2) phages (T . Sato, T . Shimizu, M . Watarai, M . Kobayashi, S . Kano, T . Hamabata, Y . Takeda, and S . Yamasaki, Gene 309:35-48, 2003) . Region V, which is ca . 10 kb in size and is located in the upstream region of the Stx operons, includes the most distinctive region among six Stx phages whose genome sequences have been determined . In this study, we developed a PCR-restriction fragment length polymorphism (RFLP) assay for the epidemiological analysis of Shiga toxin-producing Escherichia coli (STEC) on the basis of the diversity of region V . When region V was amplified by long and accurate-PCR (LA-PCR) with five control E . coli strains carrying six different Stx phages such as E . coli strains C600 (Stx1 phage), C600 (933W phage), C600 (Stx2 phage-I), C600 (Stx2 phage-II), and O157:H7 Sakai strain RIMD0509952 (VT1-Sakai phage and VT2-Sakai phage), an expected size of the band was obtained . Restriction digest of each PCR product with BglI or EcoRV also gave the expected sizes of banding patterns and discriminated the RFLPs of five control strains . When a total of 204 STEC O157 strains were analyzed by LA-PCR, one to three bands whose sizes ranged from 8.2 to 14 kb were obtained . Two STEC O157 strains, however, did not produce any bands . Subsequent restriction digest of the PCR products with BglI or EcoRV differentiated the RFLPs of 202 STEC O157 strains into 24 groups . The RFLP patterns of pulsed-field gel electrophoresis (PFGE) of representative strains of STEC O157 divided into 24 groups were well correlated with those of PCR-RFLP when STEC O157 strains were isolated in the same time period and in the close geographic area . To evaluate the PCR-RFLP assay developed here, ten strains, each isolated from four different outbreaks in different areas in Japan (Tochigi, Hyogo, Aichi, and Fukuoka prefecture), were examined to determine whether the strains in each group showed the same RFLP patterns in the PCR-RFLP assay . In accordance with the results of PFGE except for strains isolated in an area (Fukuoka), which did not produce any amplicon, ten strains in each group demonstrated the same RFLP pattern . Taken together, these data suggest that the PCR-RFLP based on region V is as useful as PFGE but perhaps more simple and rapid than PFGE for the molecular epidemiological analysis of STEC strains during sporadic and common source outbreaks. Am J Vet Res, 2004 Oct, 65(10), 1367 - 76 Distribution of Escherichia coli O157:H7 within and among cattle operations in pasture-based agricultural areas; Renter DG et al.; OBJECTIVE: To determine the distribution of Escherichia coli O157:H7 in pasture-based cattle production areas . SAMPLE POPULATION: Two 100-km2 agricultural areas consisting of 207 pasture, 14 beef-confinement, and 3 dairy locations within 24 cattle operations . PROCEDURE: 13,726 samples from cattle, wildlife, and water sources were obtained during an 11-month period . Escherichia coli O157:H7 was identified by use of culture and polymerase chain reaction assays and characterized by pulsed-field gel electrophoresis (PFGE) . RESULTS: Odds of recovering E coli O157:H7 from feeder-aged cattle were > 4 times the odds for cow-calf or dairy cattle . There was no difference in prevalence for pastured versus confined cattle after controlling for production age group . Number of samples collected (37 to 4,829), samples that yielded E coli O157:H7 (0 to 53), and PFGE subtypes (0 to 48) for each operation varied and were highly correlated . Although most PFGE subtypes were only detected once, 17 subtypes were detected on more than 1 operation . Ten of 12 operations at which E coli O157:H7 was detected had at least 1 subtype that also was detected on another operation . We did not detect differences in the probability of having the same subtype for adjacent operations, nonadjacent operations in the same study area, or operations in the other study area . CONCLUSIONS AND CLINICAL RELEVANCE: Strategies aimed at controlling E coli O157:H7 and specific subtypes should account for the widespread distribution and higher prevalence in feeder-aged cattle regardless of production environment and the fact that adjacent and distant cattle operations can have similar subtypes. J Food Prot, 2004 Oct, 67(10), 2107 - 16 Survival and growth of Escherichia coli O157:H7 in roast beef and salami after exposure to an alkaline cleaner; Sharma M et al.; Survival and growth of wild-type (EDL 933) and rpoS-deficient (FRIK 816-3) strains of Escherichia coli O157:H7 after exposure to an alkaline cleaner for 2 min and inoculating into roast beef (pH 6.3) and hard salami (pH 4.9) at low (0.003 to 0.52 CFU/g) and high (0.69 to 31.5 CFU/g) populations were determined . Roast beef was stored at 4 and 12 degrees C; salami was stored at 4, 12, and 20 degrees C . At 4 degrees C, untreated cells of both strains showed greater reductions in populations in salami than in roast beef during a 21-day storage period . Populations of treated and untreated cells recovered from roast beef and salami stored at 4 degrees C on tryptic soy agar were significantly (P < or = 0.05) higher than on sorbitol MacConkey agar, indicating that a portion of the cells was injured . Treated and untreated cells grew in roast beef at 12 degrees C . Growth of treated cells of the FRIK 816-3 strain in roast beef at 12 degrees C was significantly slower than that of the EDL 933 strain . Populations of both strains decreased at different rates in salami stored at different temperatures (20 degrees C > 12 degrees C > 4 degrees C) . E . coli O157:H7 strain EDL 933 grew more rapidly at 20 degrees C in a slurry (pH 5.97) prepared from stored salami (17 days at 20 degrees C) on which Penicillium chrysogenum had grown than in a slurry (5.23) prepared from salami showing no mold growth . Within 2 to 3 days, populations were ca . 3 log CFU/ml higher in slurry made from infected salami than in control salami . Results indicate that treatment of E . coli O157: H7 with an alkaline cleaner for 2 min does not impair resuscitation and growth of surviving cells in roast beef at 12 degrees C . Cross protection of cells exposed to an alkaline cleaner against subsequent stress conditions imposed by roast beef and salami stored at 4 degrees C was not evident in either of the test strains. J Food Prot, 2004 Oct, 67(10), 2099 - 106 Effect of simulated spray chilling with chemical solutions on acid-habituated and non-acid-habituated Escherichia coli O157:H7 cells attached to beef carcass tissue; Stopforth JD et al.; Samples (10 by 20 by 2.5 cm) of beef carcass tissue were inoculated (10(4) to 10(5) CFU/cm2) with Escherichia coli O157: H7 that was either non-acid habituated (prepared by incubating at 15 degrees C for 48 h in inoculated filter-sterilized composite {1:1} of hot and cold water meat decontamination runoff fluids, pH 6.05) or acid habituated (prepared in inoculated water fluids mixed with filter-sterilized 2% lactic acid {LA} runoff fluids in a proportion of 1/99 {vol/vol}, pH 4.12) . The inoculated surfaces were exposed to conditions simulating carcass chilling (- 3 degrees C for 10 h followed by 38 h at 1 degree C) . Treatments applied to samples (between 0 and 10 h) during chilling included the following: (i) no spraying (NT) or spraying (for 30 s every 30 min) with (ii) water, (iii) cetylpyridinium chloride (CPC; 0.1 or 0.5%), (iv) ammonium hydroxide (AH; 0.05%), (v) lactic acid (LA; 2%), (vi) acidified sodium chlorite (ASC; 0.12%), (vii) peroxyacetic acid (PAA; 0.02%), (viii) sodium hydroxide (SH; 0.01%), or (ix) sodium hypochlorite (SC; 0.005%) solutions of 4 degrees C . Samples were taken at 0, 10, 24, 36, and 48 h of the chilling process to determine changes in E . coli O157:H7 populations . Phase 1 tested water, SH, PAA, LA, and 0.5% CPC on meat inoculated with non-acid-habituated pathogen populations, whereas phase 2 tested water, SC, AH, ASC, LA, and 0.1% CPC on meat inoculated with acid- and non-acid-habituated populations . Reductions in non-acid-habituated E . coli O157:H7 populations from phase 1 increased in the order NT = water = SH < PAA < LA < CPC . Reductions from phase 2 for acid-habituated cells increased in the order NT = water = SC < ASC = LA = AH < CPC, whereas on non-acid-habituated cells the order observed was NT = water = SC < AH = ASC < LA < CPC . Previous acid habituation of E . coli O157:H7 inocula rendered the cells more resistant to the effects of spray chilling, especially with acid; however, the trend of reduction remained spray chilling with water = non-spray chilling < spray chilling with chemical solutions. J Food Prot, 2004 Oct, 67(10), 2092 - 8 Irradiation and chlorination effectively reduces Escherichia coli O157:H7 inoculated on cilantro (Coriandrum sativum) without negatively affecting quality; Foley D et al.; Cilantro (Coriandrum sativum) inoculated with Escherichia coli O157:H7 at levels approximating 10(7) CFU/g was dipped in 200 ppm chlorine solution followed by low-dose gamma irradiation . Samples were plated on tryptic soy agar containing 50 microg/ml nalidixic acid (TSAN) as well as TSAN plates with two 7-ml layers of basal yeast extract agar (TSAN-TAL) . Levels of E . coli O157:H7 recovered from both types of media were determined over 11 days . Chlorination alone reduced counts by just over 1.0 log cycle, whereas irradiation at 1.05 kGy resulted in a 6.7-log reduction, and a combination of irradiation and chlorination reduced counts more than 7 log cycles . Trained panels performed analytical sensory tests at time intervals for 14 days to detect changes in yellowing, tip burn, browning, black rot, sliminess, off-aroma, and off-flavor . Sensory tests found no significant differences among attributes over time or dose in samples irradiated at 1.08 to 3.85 kGy . This study showed that combination treatments of chlorination and low-dose irradiation can significantly reduce levels of E . coli O157:H7 in fresh cilantro while maintaining product quality. Emerg Infect Dis, 2004 Oct, 10(10), 1856 - 8 Escherichia coli O157 cluster evaluation; Gupta A et al.; We investigated a multistate cluster of Escherichia coli O157:H7 isolates; pulsed-field gel electrophoresis subtyping, using a single enzyme, suggested an epidemiologic association . An investigation and additional subtyping, however, did not support the association . Confirmating E . coli O157 clusters with two or more restriction endonucleases is necessary before public health resources are allocated to follow-up investigations. Semin Pediatr Infect Dis, 2004 Oct, 15(4), 260 - 5 The role of Shiga-toxin-producing Escherichia coli in hemorrhagic colitis and hemolytic uremic syndrome; Cleary TG; The Shiga-toxin-producing E . coli represent a major class of pathogens that have been defined over the last twenty years . They cause distinctive clinical manifestations such as afebrile bloody diarrhea with severe abdominal pain (hemorrhagic colitis) and microangiopathic hemolytic anemia with renal failure (hemolytic uremic syndrome) . The most common Shiga-toxin-producing E . coli is serotype O157:H7, although at least one hundred different serotypes share the virulence traits and clinical manifestations with this organism . Understanding the pathophysicology, improving diagnostic tools, and developing a treatment strategy are important areas of ongoing investigations. J Bacteriol, 2004 Nov, 186(21), 7290 - 301 Role of hha and ler in transcriptional regulation of the esp operon of enterohemorrhagic Escherichia coli O157:H7; Sharma VK et al.; The locus of enterocyte effacement (LEE), which includes five major operons (LEE1 through LEE4 and tir), enables enterohemorrhagic Escherichia coli (EHEC) O157:H7 to produce attaching and effacing lesions on host cells . Expression of LEE2, LEE3, and tir is positively regulated by ler, a gene located in LEE1 . Transcriptional regulation of the esp operon (LEE4), however, is not well defined . Transposon mutagenesis was used to identify transcriptional regulators of the esp operon by screening for mutants with increased beta-galactosidase activity in an EHEC O157:H7 strain harboring an esp::lac transcriptional fusion . All mutants with significant increases in beta-galactosidase activity had transposon insertions in hha (hha::Tn) . Specific complementation of the hha::Tn mutation with a plasmid-encoded copy of hha reduced beta-galactosidase activity to the level expressed in the parental esp::lac strain . Purified Hha, however, bound poorly to the esp promoter, suggesting that Hha might repress the transcription of a positive regulator of esp . Transposon mutagenesis of a Deltahha esp::lac strain expressing elevated levels of beta-galactosidase resulted in ler mutants with reduced beta-galactosidase activity . Purified Hha bound to the ler promoter with a higher affinity, and complementation of a Deltahha mutation in a Deltahha ler::lac strain repressed beta-galactosidase activity to the level expressed in a ler::lac strain . A positive regulatory role of ler in esp expression was demonstrated by specific binding of Ler to the esp promoter, reduced expression of beta-galactosidase in Deltaler esp::lac strains with and without hha, and severalfold-increased transcription of ler and espA in strains lacking hha . These results indicate that hha-mediated repression of ler causes reduced expression of the esp operon. J Appl Microbiol, 2004, 97(5), 1045 - 53 Intermittent and persistent shedding of Escherichia coli O157 in cohorts of naturally infected calves; Robinson SE et al.; AIMS: We conducted two short-term studies of cohorts of naturally infected calves to determine the prevalence and concentrations of Escherichia coli O157 shed in faeces . METHODS AND RESULTS: Two cohorts of calves were sampled; in the first study 14 calves were sampled up to five times a day for 5 days; in the second study a group of 16 separate calves were sampled once or twice a day for 15 days . All cattle within the two cohorts shed E . coli O157 at some point during the respective studies . In 18% of samples, E . coli O157 could only be isolated using immunomagnetic separation after an enrichment period, suggesting concentrations <250 CFU g(-1) . The highest concentrations recorded were 6.7 x 10(5) and 1.6 x 10(6) CFU g(-1) for studies 1 and 2 respectively . CONCLUSIONS: Persistent, high shedders (shedding >10(3) CFU g(-1)) were evident in both studies but, in the majority of calves, the pathogen was isolated intermittently . SIGNIFICANCE AND IMPACT OF THE STUDY: The variable patterns of shedding have important implications for the design of appropriate sampling protocols and for gaining meaningful estimates of parameters used in mathematical models of transmission. Mol Microbiol, 2004 Oct, 54(2), 337 - 52 Co-ordinate single-cell expression of LEE4- and LEE5-encoded proteins of Escherichia coli O157:H7; Roe AJ et al.; Escherichia coli O157:H7 is a zoonotic pathogen that can express a type III secretion system (TTSS) considered important for colonization and persistence in ruminants . E . coli O157:H7 strains have been shown to vary markedly in levels of protein secreted using the TTSS and this study has confirmed that a high secretion phenotype is more prevalent among isolates associated with human disease than isolates shed by healthy cattle . The variation in secretion levels is a consequence of heterogeneous expression, being dependent on the proportion of bacteria in a population that are actively engaged in protein secretion . This was demonstrated by indirect immunofluorescence and eGFP fusions that examined the expression of locus of enterocyte effacement (LEE)-encoded factors in individual bacteria . In liquid media, the expression of EspA, tir::egfp, intimin, but not map::egfp were co-ordinated in a subpopulation of bacteria . In contrast to E . coli O157:H7, expression of tir::egfp in EPEC E2348/69 was equivalent in all bacteria although the same fusion exhibited variable expression when transformed into an E . coli O157:H7 background . An E . coli O157:H7 strain deleted for the LEE demonstrated weak but variable expression of tir::egfp indicating that the elements controlling the heterogeneous expression lie outside the LEE . The research also demonstrated the rapid induction of tir::egfp and map::egfp on contact with bovine epithelial cells . This control in E . coli O157:H7 may be required to limit exposure of key surface antigens, EspA, Tir and intimin during colonization of cattle but allow their rapid production on contact with bovine gastrointestinal epithelium at the terminal rectum. Shokuhin Eiseigaku Zasshi, 2004 Jun, 45(3), 113 - 9 {Detection method of injured Escherichia coli O157 in noodles and vegetables}; Tanaka K et al.; An enrichment procedure and a polymerase chain reaction (PCR) method for the detection of injured Escherichia coli O157 in foods were examined . Freeze-injured E . coli O157 inoculated in boiled spaghetti could be detected in 6-h culture within 12 h by the PCR method . Cells injured by heating in boiled spaghetti and cells injured by chlorine treatment in raw lettuce and carrot did not grow sufficiently to be detected in 6-h culture but were detected in 18-h culture using selective agar media . The injured cells could be also detected in 18-h culture within 24 h by the PCR method . Enrichment at 42 degrees C in trypticase soy broth (TSB) was more effective than that at 42 degrees C in modified EC broth with novobiocin . These results indicated that the usage of enrichment in TSB for 18 h at 42 degrees C in combination with the PCR method is suitable for screening for E . coli O157 in boiled or chlorinated foods, even if the O157 cells are injured. Appl Environ Microbiol, 2004 Oct, 70(10), 6061 - 5 Influence of apple cultivars on inactivation of different strains of Escherichia coli O157:H7 in apple cider by UV irradiation; Basaran N et al.; This study examined the effect of different apple cultivars upon the UV inactivation of Escherichia coli O157:H7 strains within unfiltered apple cider . Apple cider was prepared from eight different apple cultivars, inoculated with approximately 10(6) to 10(7) CFU of three strains of E . coli O157:H7 per ml (933, ATCC 43889, and ATCC 43895), and exposed to 14 mJ of UV irradiation per cm(2) . Bacterial populations for treated and untreated samples were then enumerated by using nonselective media . E . coli O157:H7 ATCC 43889 showed the most sensitivity to this disinfection process with an average 6.63-log reduction compared to an average log reduction of 5.93 for both strains 933 and ATCC 43895 . The highest log reduction seen, 7.19, occurred for strain ATCC 43889 in Rome cider . The same cider produced the lowest log reductions: 5.33 and 5.25 for strains 933 and ATCC 43895, respectively . Among the apple cultivars, an average log reduction range of 5.78 (Red Delicious) to 6.74 (Empire) was observed, with two statistically significant (alpha < or = 0.05) log reduction groups represented . Within the paired cultivar-strain analysis, five of eight ciders showed statistically significant (alpha < or = 0.05) differences in at least two of the E . coli strains used . Comparison of log reductions among the E . coli strains to the cider parameters of (o)Brix, pH, and malic acid content failed to show any statistically significant relationship (R(2) > or = 0.95) . However, the results of this study indicate that regardless of the apple cultivar used, a minimum 5-log reduction is achieved for all of the strains of E . coli O157:H7 tested. Anim Health Res Rev, 2004 Jun, 5(1), 15 - 33 Escherichia coli 0157:H7: an update on intestinal colonization and virulence mechanisms; Moxley RA; Cattle are a major reservoir of Escherichia coli 0157:H7, an important zoonotic pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome (HUS) . Colonization of cattle occurs predominantly in the large intestine, and may especially target follicle-associated epithelium (FAE) in the terminal rectum . Bacterial colonization involves induction of attaching-effacing (A/E) lesions, mediated by type III secreted proteins and an outer membrane protein called intimin . ToxB, encoded on plasmid pO157, contributes to adherence of E . coli O157:H7 through promotion of the production and/or secretion of type III secreted proteins . Production of type III secreted proteins and intestinal colonization appear to involve quorum-sensing mechanisms . In the human host, E . coli O157:H7 may have a preference for FAE in the distal small intestine . The H7 flagellum induces production of chemokines such as interleukin 8, and neutrophilic infiltration of the intestinal mucosa, which in turn may enhance Shiga toxin (Stx) uptake across the intestinal epithelium . Both Stx and cytokine responses play critical roles in the induction of the vascular lesions that underlie hemorrhagic colitis and HUS . In cattle, Stx binds to intestinal crypt cells and submucosal lymphocytes but not vascular endothelium . The role played by Stx in cattle may be to suppress mucosal immunity, yet enhance other effects that promote intestinal colonization. New Microbiol, 2004 Jul, 27(3), 255 - 61 Detection and characterization of verocytotoxin-producing Escherichia coli (vtec) O157 and non-O157 in cattle at slaughter; Bonardi S et al.; Between September 2001 to June 2002, 145 samples of bovine caecal content were collected at slaughter for verocytotoxin-producing Escherichia coli (VTEC) serogroups O157 and non-O157 detection . For E . coli O157 the immunomagnetic-separation technique was performed . The enterohaemolytic phenotype was the target for non-O157 VTEC identification . The vero cell assay (VCA) was performed for toxic activity detection . The genomic sequence for VT1, VT2 and intimin (vt1, vt2, eae genes) were identified by PCR analysis . Eight VTEC O157 and eight non-O157 VTEC isolates were detected . VTEC O157, eae-positive strains were shed by 9.7% of feedlot cattle and by 2.5% of dairy cows . Non-O157 VTEC, eae-negative isolates were detected in the intestinal content of 12.5% dairy cows and of 2.1% feedlot cattle . VTEC-shedding cattle came from 18.1% of the farms included in the study . From cattle faeces, VTEC O91:H- (VT2-positive, eae-negative), responsible of human diarrhoeal disease in Europe, was recovered . Other VTEC serogroups identified in the present study were O74, O109, O110, O116, and O117. J Vet Med B Infect Dis Vet Public Health, 2004 Aug, 51(6), 288 - 92 Characterization of pathogenic Escherichia coli isolated from humans in Austria: phenotypes, toxin gene types and epidemiology; Wagner M et al.; One hundred and ten clinical Escherichia coli isolates of serovar O157 (n = 102) and O26 (n = 8) were characterized for the presence of putative virulence genes by PCR . All but one of these isolates contained the eae gene . The EHEC-hly gene could be detected in all E . coli O157 and in 50% of E . coli O26 isolates . Forty-five (40.9%) of the 110 E . coli were positive for both stx(1) and stx(2) genes, 2 (1.8%) isolates were positive for stx(1) and 57 isolates (51.8%) were positive for stx(2) only . Among the 102 stx(2) positive isolates, 14 (13.7%) E . coli O157 contained also the stx(2c) variant gene . No other stx(2) variant was identified . Six clinical isolates (five E . coli O157:H7 and one E . coli O26) did not contain stx genes . Ten non-pathogenic E . coli isolates which were amplified as controls didn't contain any stx and eae gene but two of the ten strains contained the EHEC-hly gene . By their growth on chromogenic media, all but two of 50 E . coli O157 could be differentiated from eight E . coli O26 and 10 non-pathogenic E . coli . Sixty-one of the O157:H7 isolates were further subjected to pulsed-field gel electrophoresis (PFGE) which identified 49 distinguishable patterns . In five cases where contact infection among family members was suspected, indistinguishable PFGE patterns confirmed the epidemiological relatedness of the isolates . Moreover, two PFGE clusters were identified which comprised five and three strains, respectively . These findings indicate the occurrence of both family and diffuse outbreaks of E . coli O157 infections in Austria during recent years and demonstrate the need for molecular subtyping of these pathogens. J Food Prot, 2004 Sep, 67(9), 1991 - 9 Draft risk assessment of the public health impact of Escherichia coli O157:H7 in ground beef; Ebel E et al.; An assessment of the risk of illness associated with Escherichia coli O157:H7 in ground beef was drafted in 2001 . The exposure assessment considers farm, slaughter, and preparation factors that influence the likelihood of humans consuming ground beef servings containing E . coli O157:H7 and the number of cells in a contaminated serving . Apparent seasonal differences in prevalence of cattle infected with E . coli O157:H7 corresponded to seasonal differences in human exposure . The model predicts that on average 0.018% of servings consumed during June through September and 0.007% of servings consumed during the remainder of the year are contaminated with one or more E . coli O157:H7 cells . This exposure risk is combined with the probability of illness given exposure (i.e., dose response) to estimate a U.S . population risk of illness of nearly one illness in each 1 million (9.6 x 10(-7)) servings of ground beef consumed . Uncertainty about this risk ranges from about 0.33 illness in every 1 million ground beef servings at the 5th percentile to about two illnesses in every 1 million ground beef servings at the 95th percentile. Infect Immun, 2004 Oct, 72(10), 6168 - 71 Long polar fimbriae contribute to colonization by Escherichia coli O157:H7 in vivo; Jordan DM et al.; The contribution of long polar fimbriae to intestinal colonization by Escherichia coli O157:H7 was evaluated in sheep, conventional pigs, and gnotobiotic piglets . E . coli O157:H7 strains with lpfA1 and lpfA2 mutated were recovered in significantly lower numbers and caused fewer attachment and effacement lesions than the parent strain. J Toxicol Environ Health A, 2004 Oct 22-Nov 26, 67(20-22), 1667 - 77 Vulnerability of waterborne diseases to climate change in Canada: a review; Charron D et al.; This project addresses two important issues relevant to the health of Canadians: the risk of waterborne illness and the health impacts of global climate change . The Canadian health burden from waterborne illness is unknown, although it presumably accounts for a significant proportion of enteric illness . Recently, large outbreaks with severe consequences produced by E . coli O157:H7 and Cryptosporidium have alarmed Canadians and brought demands for political action . A concurrent need to understand the health impacts of global climate changes and to develop strategies to prevent or prepare for these has also been recognized . There is mounting evidence that weather is often a factor in triggering waterborne disease outbreaks . A recent study of precipitation and waterborne illness in the United States found that more than half the waterborne disease outbreaks in the United States during the last half century followed a period of extreme rainfall . Projections of international global climate change scenarios suggest that, under conditions of global warming most of Canada may expect longer summers, milder winters, increased summer drought, and more extreme precipitation . Excess precipitation, floods, high temperatures, and drought could affect the risk of waterborne illness in Canada . The existing scientific information regarding most weather-related adverse health impacts and on the impacts of global climate change on health in Canada is insufficient for informed decision making . The results of this project address this need through the investigation of the complex systemic interrelationships between disease incidence, weather parameters, and water quality and quantity, and by projecting the potential impact of global climate change on those relationships. J Clin Microbiol, 2004 Sep, 42(9), 4007 - 15 Phage types and genotypes of shiga toxin-producing Escherichia coli O157:H7 isolates from humans and animals in spain: identification and characterization of two predominating phage types (PT2 and PT8); Mora A et al.; Phage typing and DNA macrorestriction fragment analysis by pulsed-field electrophoresis (PFGE) were used for the epidemiological subtyping of a collection of Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains isolated in Spain between 1980 and 1999 . Phage typing distinguished a total of 18 phage types among 171 strains isolated from different sources (67 humans, 82 bovines, 12 ovines, and 10 beef products) . However, five phage types, phage type 2 (PT2; 42 strains), PT8 (33 strains), PT14 (14 strains), PT21/28 (11 strains), and PT54 (16 strains), accounted for 68% of the study isolates . PT2 and PT8 were the most frequently found among strains from both humans (51%) and bovines (46%) . Interestingly, we detected a significant association between PT2 and PT14 and the presence of acute pathologies . A group of 108 of the 171 strains were analyzed by PFGE, and 53 distinct XbaI macrorestriction patterns were identified, with 38 strains exhibiting unique PFGE patterns . In contrast, phage typing identified 15 different phage types . A total of 66 phage type-PFGE subtype combinations were identified among the 108 strains . PFGE subtyping differentiated between unrelated strains that exhibited the same phage type . The most common phage type-PFGE pattern combinations were PT2-PFGE type 1 (1 human and 11 bovine strains), PT8-PFGE type 8 (2 human, 6 bovine, and 1 beef product strains), PT2-PFGE subtype 4A (1 human, 3 bovine, and 1 beef product strains) . Nine (29%) of 31 human strains showed phage type-PFGE pattern combinations that were detected among the bovine strains included in this study, and 26 (38%) of 68 bovine strains produced phage type-PFGE pattern combinations observed among human strains included in this study, confirming that cattle are a major reservoir of strains pathogenic for humans . PT2 and PT8 strains formed two groups which differed from each other in their motilities, stx genotypes, PFGE patterns, and the severity of the illnesses that they caused. Int J Food Microbiol, 2004 Nov 1, 96(2), 189 - 98 Shiga toxin-producing Escherichia coli in healthy young beef steers from Argentina: prevalence and virulence properties; Meichtri L et al.; Between July 1999 and December 2000, the prevalence of Shiga toxin-producing Escherichia coli (STEC) was established in 200 Argentine healthy young beef steers (14-16 months old) grown under local production systems with a feed grain period of 3-4 months, and the STEC strains isolated were examined in regard to their phenotypic and genotypic characteristics . Stool samples (n = 70) and rectal swabs (n = 130) were taken at the slaughterhouse level . By polymerase chain reaction (PCR), Shiga toxin (stx) gene sequences were detected in 69% of the samples . Eighty-six STEC strains were isolated from 39% of the animals . Serogroups identified, in order of frequency, were: O8 (16 strains), O113 (14), O103 (5), O91 (4), O171 (3), O174 (3), O25 (2), O112 (2), O145 (2), O2, O11, O104, O121, O128, O143, O146, O157 . The most frequent serotype isolated was O8:H19 (12.9%) . A total of 17 serotypes, including E . coli O157:H7 found in one animal (0.5%), have been previously associated with hemolytic uremic syndrome (HUS), bloody and non-bloody diarrhea in different countries, including Argentina . The prevalent genotype isolated was stx2 (51 of 86, 59.3%) . Subtyping of stx2 variants showed the prevalence of stx2vh-b (25.6%) and stx2vh-a types (24.4%), and revealed the presence of an atypical stx2-v . Only 7.0% of STEC strains carried eae, and 33.7% harbored EHEC-hlyA gene . The full virulent genotype (stx/eae/EHEC-hlyA) was found to be present in 4 of the 86 (4.7%) STEC strains isolated . This research indicates that young steers from the main beef-producing area of Argentina are an important reservoir of STEC strains; however, its importance as agents of human diseases in our country has still to be established. J Med Microbiol, 2004 Oct, 53(Pt 10), 1037 - 43 Phenotypic and genotypic characterization of beta-D-glucuronidase-positive Shiga toxin-producing Escherichia coli O157:H7 isolates from deer; Nagano H et al.; Beta-glucuronidase-positive (GUD+) Shiga toxin-producing Escherichia coli (STEC) O157:H7 was isolated from both an asymptomatic woman and uncooked deer meat in her possession in Hokkaido, Japan . The phenotypic and genotypic characteristics of the two isolates were identical or closely related, indicating probable transmission of the deer isolate to the woman . Moreover, several other GUD+ STEC O157:H7 strains investigated belonged to the distinct atypical GUD+ STEC O157:H7 group that has been identified previously . This is the first report that deer can be a reservoir of GUD+ STEC O157:H7 in Japan. Int J Food Microbiol, 2004 Oct 1, 96(1), 103 - 9 Attachment of Escherichia coli O157:H7 grown in tryptic soy broth and nutrient broth to apple and lettuce surfaces as related to cell hydrophobicity, surface charge, and capsule production; Hassan AN et al.; This study investigated the effect of growth in tryptic soy broth (TSB) and nutrient broth (NB) on the ability Escherichia coli O157:H7 to attach to lettuce and apple surfaces . In addition, cell surface hydrophobicity, charge and capsule production were determined on cells grown in these media . Cells grown in NB attached less to lettuce and apple surfaces than did those grown in TSB . TSB, but not NB, supported capsule production by E . coli O157:H7 . Cells grown in TSB were more hydrophilic than those grown in NB . No difference was found in the electrokinetic properties of cells grown in these media . Electrostatic and hydrophobic interactions and surface proteins did not appear to play an important role in the attachment of E . coli O157:H7 to these surfaces . Of the factors studied, only capsule production was associated with attachment ability . J Pediatr, 2004 Sep, 145(3), 412 - 4 Hemolytic uremic syndrome in a child with laboratory-acquired Escherichia coli O157:H7; Salerno AE et al.; A 6-year-old girl touched an agar plate containing Escherichia coli O157:H7 while visiting a hospital laboratory, and subsequently, colitis and hemolytic uremic syndrome developed . Pulsed-field gel electrophoresis patterns of the isolate cultured from her stool and that from the laboratory were identical. J Bacteriol, 2004 Sep, 186(18), 6179 - 85 The small noncoding DsrA RNA is an acid resistance regulator in Escherichia coli; Lease RA et al.; DsrA RNA is a small (87-nucleotide) regulatory RNA of Escherichia coli that acts by RNA-RNA interactions to control translation and turnover of specific mRNAs . Two targets of DsrA regulation are RpoS, the stationary-phase and stress response sigma factor (sigmas), and H-NS, a histone-like nucleoid protein and global transcription repressor . Genes regulated globally by RpoS and H-NS include stress response proteins and virulence factors for pathogenic E . coli . Here, by using transcription profiling via DNA arrays, we have identified genes induced by DsrA . Steady-state levels of mRNAs from many genes increased with DsrA overproduction, including multiple acid resistance genes of E . coli . Quantitative primer extension analysis verified the induction of individual acid resistance genes in the hdeAB, gadAX, and gadBC operons . E . coli K-12 strains, as well as pathogenic E . coli O157:H7, exhibited compromised acid resistance in dsrA mutants . Conversely, overproduction of DsrA from a plasmid rendered the acid-sensitive dsrA mutant extremely acid resistant . Thus, DsrA RNA plays a regulatory role in acid resistance . Whether DsrA targets acid resistance genes directly by base pairing or indirectly via perturbation of RpoS and/or H-NS is not known, but in either event, our results suggest that DsrA RNA may enhance the virulence of pathogenic E . coli. Medicina (B Aires), 2004, 64(4), 352 - 6 {Enterohemorrhagic Escherichia coli and hemolytic-uremic syndrome in Argentina}; Rivero MA et al.; The hemolytic-uremic syndrome (HUS) is a multisystemic disorder that is characterized by the onset of acute renal failure, microangiopathic hemolytic anemia and thrombocytopenia . It is the most common cause of acute renal failure and the second cause of chronic renal failure and renal transplantation in children in Argentina . Our country has the highest incidence of HUS in the world, with approximately 420 new cases observed each year with an incidence of 12.2 cases per 100,000 children in the age group 0-5 years . Numerous etiologic factors have been associated with HUS but the infection with enterohemorrhagic Escherichia coli (EHEC) is considered the most common cause . The majority of outbreaks and sporadic cases in humans have been associated with serotype O157:H7, although other O:H serotypes have been isolated, and they are a subgroup of Verocytotoxin-producing Escherichia coli (VTEC) . Cattle are the principal reservoir of VTEC . Infections in humans are a consequence of consumption of undercooked meat, raw milk and other contaminated food or water . Direct contact with animals or people infected is another source of infection. J Food Prot, 2004 Aug, 67(8), 1760 - 4 Influence of freezing and freezing plus acidic calcium sulfate and lactic acid addition on thermal inactivation of Escherichia coli O157:H7 in ground beef; Zhao T et al.; Undercooked ground beef is a leading vehicle for acquiring Escherichia coli O157:H7 infections through consumption of foods . Studies have been performed to determine the effect of freezing and the combined effect of freezing and addition of a mixture of 20% acidic calcium sulfate (final concentration of 0.4% in ground beef) and 10% lactic acid (final concentration of 0.2% in ground beef) (ACS-LA) on the thermal sensitivity of E . coli O157:H7 in ground beef . Five strains of E . coli O157: H7 were separately inoculated into ground beef and held at 5 degrees C for up to 10 days or -20 degrees C for up to 3 weeks then heated at 57, 60, 62.8, 64.3, and 68.3 degrees C to determine rates of thermal inactivation . Results revealed that D-values (decimal reduction times) at equivalent temperatures for four of five E . coli O157:H7 strains were less in the previously frozen than in the refrigerated ground beef and that strains isolated from ground beef in 1993 and 1994 were generally more sensitive to thermal inactivation than those isolated in 1999 and 2000 . Only one strain of E . coli O157:H7 was used to determine the effect of ACS-LA in previously frozen or refrigerated ground beef on rates of thermal inactivation . The addition of ACS-LA to ground beef at 20 ml/kg increased the thermal sensitivity of E . coli O157:H7 in both previously frozen and refrigerated ground beef, with greatest rates of inactivation occurring in previously frozen ground beef containing ACS-LA . D-values at 57 degrees C obtained for E . coli O157:H7 in previously refrigerated and frozen ground beef containing ACS-LA and ACS-LA diluted by half were significantly (P < 0.05) less than those obtained in ground beef with no ACS-LA added . D-values at 60 and 62.8 degrees C were consistently less in ACS-LA treated ground beef, but for most treatments the results were not significantly (P > 0.05) different than the controls . Results revealed that the addition of ACS-LA to ground beef, whether frozen or refrigerated, can reduce the temperature or time required to kill E . coli O157:H7 during heating. J Food Prot, 2004 Aug, 67(8), 1755 - 9 Process control and sampling for Escherichia coli O157:H7 in beef trimmings; Murphy RY et al.; Raw beef producers currently face the problem of Escherichia coli O157:H7 surface contamination of beef carcasses that can lead to product adulteration . Although carcass interventions are in place, elimination of E . coli O157:H7 from every potential hiding place on the surfaces of a beef carcass is not technologically feasible . Therefore, E . coli O157:H7 on beef carcasses might further contaminate the surfaces of beef trimmings . With the use of case scenarios from nine commercial processing facilities, we present a process control and statistical sampling approach for monitoring beef trimmings to divert contaminated lots of the trimmings from the raw ground beef supply chain. J Food Prot, 2004 Aug, 67(8), 1591 - 6 Influence of animal origin and lineage on survival of Escherichia coli O157:H7 strains in strong and weak acid challenges; Saridakis CE et al.; Twenty-five strains of Escherichia coli O157:H7 isolated from humans, cattle, and pigs were maintained in HCl (pH 2.5) and in a volatile fatty acid (VFA) mixture (pH 4.0) for up to 6 h at 37 degrees C to assess their ability to survive in acidic conditions that simulate those of the stomach and ileum, respectively . In HCl, the average group survival of bovine strains was significantly higher than that of porcine and human strains, whereas in VFAs, porcine strains were significantly more resistant than bovine and human strains . Bovine strains exhibited significantly higher average survival in HCl than in VFAs . The average survival of strains classified as octamer-based genome scanning (OBGS) lineage II was significantly superior to that of strains classified as OBGS lineage I in HCl . The group of lineage I strains was more resistant in VFAs compared with lineage II, but only after 6 h of challenge . The possible involvement of urease in acid resistance of E . coli O157:H7 was also examined . Although the strains possessed the ureC gene, as shown by PCR, this gene did not appear to contribute to acid resistance under the conditions tested . The data indicate that there is a relationship between acid resistance and source or lineage of O157:H7 strains. J Food Prot, 2004 Aug, 67(8), 1568 - 73 Modified immunoliposome sandwich assay for the detection of Escherichia coli O157:H7 in apple cider; Park S et al.; Detection of Escherichia coli O157:H7 in fruit juices such as apple cider is necessary for diagnosis of infection and epidemiological investigations . However, inhibitors in the apple cider, such as endogenous polyphenols and acids, often decrease the sensitivity of PCR assays and immunoassays, thus routinely requiring laborious cell separation steps to increase the sensitivity . In the current study, polyethylene glycol (PEG)-derivatized liposomes encapsulating sulforhodamine B were tagged with anti-E . coli O157:H7 antibodies and used in an immunoliposome sandwich assay for the detection of E . coli O157:H7 in apple cider . Even without prior separation, this assay can detect E . coli O157:H7 in apple cider samples inoculated with as few as 1 CFU/ml after an 8-h enrichment period . The lower limit of detection in pure cultures without enrichment was 7 x 10(3) CFU/ml (280 CFU/40-microl sample) . PEGylated immunoliposomes are suitable as an analytical reagent for the detection of E . coli O157:H7 in fruit juices containing polyphenols. Vnitr Lek, 2004 Jul, 50(7), 519 - 25 {Hemolytic-uremic syndrome}; Blahova K; Hemolytic-uremic syndrome (HUS) is the most common cause of acute renal failure in children below 3 years of age . It is defined by a triad of symptoms which associates hemolytic anemia with fragmented erythrocytes, thrombocytopenia and acute renal failure . Three types of HUS can be distinguished: typical HUS, also called diarrhoea-associated (D+HUS), very rare atypical HUS (D-HUS) and secondary HUS (drug induced, C+HUS, in patients receiving marrow transplantation, etc.) . The common event among these entities appears to be vascular endothelial cell injury, which induces mechanical destruction of erythrocytes, activation of platelet aggregation and local intravascular coagulation, especially in the renal microvasculature . D+HUS represents 90% of HUS in children . Evidence of exposure to verotoxin (VT), shiga toxin (ST) producing Escherichia coli (VTEC or STEC) has been demonstrated in many countries in about 85% of cases . Serotype O157:H7 is the most frequent . Early and accurate supportive treatment and early start of dialysis is the major importance and allows a current mortality rate below 5%-10% . Vital prognosis is compromized in cases with multivisceral involvement . After 15 years or more of apparent recovery, 20 to 60% of patients have residual renal symptoms, with up to 20% having chronic renal insufficiency (CRI) or end-stage renal disease (ESRD) . Atypical HUS represents less than 10% of HUS in children . Some of these cases (familial) are associated with low C3 levels, hereditary deficiency of factor H or with mutations in factor H gene . The deficiency of von Willebrand factor cleaving protease, as reported in adults with thrombotic thrombocytopenic purpura (TTP), is not present in D+HUS. Infect Immun, 2004 Sep, 72(9), 5452 - 9 Transcriptome of enterohemorrhagic Escherichia coli O157 adhering to eukaryotic plasma membranes; Dahan S et al.; Using a DNA microarray, we determined changes in enterohemorrhagic Escherichia coli O157:H7 gene expression during binding to plasma membranes . Analysis of the complete transcriptomes of the bound bacteria revealed increased levels of stress-associated mRNAs and decreased levels of mRNA encoding proteins involved in translation and type III secretion. Infect Immun, 2004 Sep, 72(9), 5446 - 51 The neuroendocrine stress hormone norepinephrine augments Escherichia coli O157:H7-induced enteritis and adherence in a bovine ligated ileal loop model of infection; Vlisidou I et al.; The role of the neuroendocrine environment in the pathogenesis of enteric bacterial infections is increasingly being recognized . Here we report that norepinephrine augments Escherichia coli O157:H7-induced intestinal inflammatory and secretory responses as well as bacterial adherence to intestinal mucosa in a bovine ligated ileal loop model of infection . Norepinephrine modulation of enteritis and adherence was dependent on the ability of E . coli O157:H7 to form attaching and effacing lesions. Am J Clin Pathol, 2004 Jun, 121 Suppl, S81 - 8 Hemolytic uremic syndrome revisited: Shiga toxin, factor H, and fibrin generation; Blackall DP et al.; The hemolytic uremic syndrome (HUS) is a disease characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal failure . These features reflect the underlying histopathologic lesion: fibrin-rich thrombi that predominate in the renal microvasculature . HUS most commonly affects children younger than 5 years and is associated with Shiga toxin-producing enteric bacteria, the most important of which is Escherichia coli O157:H7 . In this setting, HUS is epidemic and also might affect adults, particularly elderly people . Sporadic cases of HUS more commonly occur in adults and are associated with a wide variety of inciting agents and conditions . Although the disease manifestations might be similar and endothelial activation or injury likely represents a common etiologic event, differing responses to therapy suggest different pathogenic mechanisms . As more is understood about the underlying pathogenesis of the diseases that we now lump together as HUS, more efficacious and rational treatment and prevention strategies are likely to follow. J AOAC Int, 2004 Jul-Aug, 87(4), 856 - 60 Toward an international standard for PCR-based detection of foodborne Escherichia coli O157: validation of the PCR-based method in a multicenter interlaboratory trial; Abdulmawjood A et al.; The performance of a polymerase chain reaction (PCR) method for detection of Escherichia coli O157, previously validated on DNA extracted from pure cultures, was evaluated on spiked cattle swabs through an interlaboratory trial, including 12 participating laboratories from 11 European countries . Twelve cattle swab samples, spiked at 4 levels (0, 1-10, 10-100, and 100-1000 colony-forming units, in triplicate) with E . coli O157 were prepared centrally in the originating laboratory; the receiving laboratories performed pre-PCR treatment followed by PCR . The results were reported as positive when the correct amplicons were present after gel electrophoresis . The statistical analysis, performed on 10 sets of reported results, determined the diagnostic sensitivity to be 92.2% . The diagnostic specificity was 100% . The accordance (repeatability) was 90.0%, calculated from all positive inoculation levels . The concordance (reproducibility) was 85.0%, calculated from all positive inoculation levels . The concordance odds ratio (degree of interlaboratory variation calculated from all positive inoculation levels) was 1.58, indicating the robustness of the PCR method . Thus, the interlaboratory variation due to personnel, reagents, minor temperature or pH fluctuations and, not least, thermal cyclers, did not affect the performance of the method, which is currently being considered as part of an intenational PCR standard. Appl Environ Microbiol, 2004 Aug, 70(8), 4792 - 9 Acid resistance systems required for survival of Escherichia coli O157:H7 in the bovine gastrointestinal tract and in apple cider are different; Price SB et al.; Escherichia coli O157:H7 is a highly acid-resistant food-borne pathogen that survives in the bovine and human gastrointestinal tracts and in acidic foods such as apple cider . This property is thought to contribute to the low infectious dose of the organism . Three acid resistance (AR) systems are expressed in stationary-phase cells . AR system 1 is sigma(S) dependent, while AR systems 2 and 3 are glutamate and arginine dependent, respectively . In this study, we sought to determine which AR systems are important for survival in acidic foods and which are required for survival in the bovine intestinal tract . Wild-type and mutant E . coli O157:H7 strains deficient in AR system 1, 2, or 3 were challenged with apple cider and inoculated into calves . Wild-type cells, adapted at pH 5.5 in the absence of glucose (AR system 1 induced), survived well in apple cider . Conversely, the mutant deficient in AR system 1, shown previously to survive poorly in calves, was susceptible to apple cider (pH 3.5), and this sensitivity was shown to be caused by low pH . Interestingly, the AR system 2-deficient mutant survived in apple cider at high levels, but its shedding from calves was significantly decreased compared to that of wild-type cells . AR system 3-deficient cells survived well in both apple cider and calves . Taken together, these results indicate that E . coli O157:H7 utilizes different acid resistance systems based on the type of acidic environment encountered. J Bacteriol, 2004 Aug, 186(16), 5506 - 12 Escherichia coli serogroup O107/O117 lipopolysaccharide binds and neutralizes Shiga toxin 2; Gamage SD et al.; The AB(5) toxin Shiga toxin 2 (Stx2) has been implicated as a major virulence factor of Escherichia coli O157:H7 and other Shiga toxin-producing E . coli strains in the progression of intestinal disease to more severe systemic complications . Here, we demonstrate that supernatant from a normal E . coli isolate, FI-29, neutralizes the effect of Stx2, but not the related Stx1, on Vero cells . Biochemical characterization of the neutralizing activity identified the lipopolysaccharide (LPS) of FI-29, a serogroup O107/O117 strain, as the toxin-neutralizing component . LPSs from FI-29 as well as from type strains E . coli O107 and E . coli O117 were able bind Stx2 but not Stx1, indicating that the mechanism of toxin neutralization may involve inhibition of the interaction between Stx2 and the Gb(3) receptor on Vero cells. Luminescence, 2004 Jul-Aug, 19(4), 193 - 8 Chemiluminescence detection of Escherichia coli in fresh produce obtained from different sources; Mathew FP et al.; A chemiluminescence-based assay is developed for the rapid detection of Escherichia coli in fresh produce . The assay was based on the reaction of beta-galactosidase enzyme from E . coli with a phenylgalactosidase-substituted dioxetane substrate . Light emitted from the reaction was measured in a luminometer and data correlated with counts of E . coli enumerated on sorbitol-MacConkey agar plates . A strain of E . coli O157:H7 was used to inoculate samples of fresh produce to differentiate the inoculum from the natural E . coli potentially present on the produce . Fresh market samples were tested for generic E . coli and E . coli O157:H7 . Significant differences in light emission were found in samples with high initial E . coli counts when market samples were compared to respective heat-treated samples . The assay was able to detect E . coli in all produce tested, particularly at higher contamination or inoculation levels . The sensitivity of the assay ranged between 10(2)-10(5) CFU within 30 min . The chemiluminescence assay provides a simple and rapid method for detection of viable E . coli, an important step towards enhancing food safety . J Food Prot, 2004 Jul, 67(7), 1365 - 70 Persistence of enterohemorrhagic Escherichia coli O157:H7 in soil and on leaf lettuce and parsley grown in fields treated with contaminated manure composts or irrigation water; Islam M et al.; Outbreaks of enterohemorrhagic Escherichia coli O157:H7 infections associated with lettuce and other leaf crops have occurred with increasing frequency in recent years . Contaminated manure and polluted irrigation water are probable vehicles for the pathogen in many outbreaks . In this study, the occurrence and persistence of E . coli O157:H7 in soil fertilized with contaminated poultry or bovine manure composts or treated with contaminated irrigation water and on lettuce and parsley grown on these soils under natural environmental conditions was determined . Twenty-five plots, each 1.8 by 4.6 m, were used for each crop, with five treatments (one without compost, three with each of the three composts, and one without compost but treated with contaminated water) and five replication plots for each treatment . Three different types of compost, PM-5 (poultry manure compost), 338 (dairy manure compost), and NVIRO-4 (alkaline-stabilized dairy manure compost), and irrigation water were inoculated with an avirulent strain of E . coli O157:H7 . Pathogen concentrations were 10(7) CFU/g of compost and 10(5) CFU/ml of water . Contaminated compost was applied to soil in the field as a strip at 4.5 metric tons per hectare on the day before lettuce and parsley seedlings were transplanted in late October 2002 . Contaminated irrigation water was applied only once on the plants as a treatment in five plots for each crop at the rate of 2 liters per plot 3 weeks after the seedlings were transplanted . E . coli O157:H7 persisted for 154 to 217 days in soils amended with contaminated composts and was detected on lettuce and parsley for up to 77 and 177 days, respectively, after seedlings were planted . Very little difference was observed in E . coli O157:H7 persistence based on compost type alone . E . coli O157:H7 persisted longer (by > 60 days) in soil covered with parsley plants than in soil from lettuce plots, which were bare after lettuce was harvested . In all cases, E . coli O157:H7 in soil, regardless of source or crop type, persisted for > 5 months after application of contaminated compost or irrigation water. Microbiology, 2004 Jul, 150(Pt 7), 2357 - 571 Positive effects of multiple pch genes on expression of the locus of enterocyte effacement genes and adherence of enterohaemorrhagic Escherichia coli O157 : H7 to HEp-2 cells; Iyoda S et al.; Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) genomes contain a pathogenicity island, termed the locus of enterocyte effacement (LEE), which encodes genes involved in the formation of attaching and effacing lesions on epithelial cells . To elucidate the regulatory mechanism of the LEE genes in EHEC, an EHEC O157 genomic library was screened for clones which modulated expression of the LEE genes . From more than 5000 clones, a DNA fragment was obtained containing a perC homologue as a positive regulator for the LEE genes . In EPEC, perC is known to be part of the per operon, along with perA and perB, located on the EPEC adherence factor plasmid, which is not found in EHEC . However, the complete genome sequence of EHEC O157 Sakai strain reveals that there are five perC-like sequences, but no perA and perB, on the chromosome . These five perC homologues were characterized, and it was found that three of the homologues (renamed perC homologue pchA, pchB and pchC) encoded 104 aa proteins, and when expressed on a multicopy plasmid enhanced the expression of LEE genes . In contrast, perC homologues encoding proteins of 89 and 90 aa, renamed pchD and pchE, respectively, had no significant effect . Deletion mutants of the pch genes were constructed, and the effect on the expression of LEE-encoded type III effector proteins, such as EspA, B and D, and adhesion phenotype to HEp-2 cells was examined . Deletion of pchA or pchB, but not pchC, decreased the expression of Esp proteins and adhesion to HEp-2 cells . Such effects were more apparent with mutants carrying double deletions of pchA/pchB or pchA/pchC, suggesting that pchA/B/C are all necessary for full expression of the LEE genes and adhesion to HEp-2 cells . Further study demonstrated that the positive effect of pchA/B/C was caused by enhanced transcription of the LEE-encoded regulatory gene, ler . Introduction of a multicopy plasmid carrying each pchA/B/C gene significantly induced microcolony formation by EHEC O157 on HEp-2 cells . These results suggest that the pchABC genes are necessary for full virulence of EHEC O157. Int J Food Microbiol, 2004 Aug 15, 95(1), 19 - 27 Serotypes and virulence genes of ovine non-O157 Shiga toxin-producing Escherichia coli in Switzerland; Zweifel C et al.; Sixty ovine STEC strains were examined with the aim (i) to serotype the strains, (ii) to characterize virulence factors, and (iii) to discuss possible associations between these factors and to assess the potential pathogenicity of these strains for humans . The 60 sorbitol-positive, non-O157 STEC strains belonged to 19 O:H serotypes, whereas 68% were of five serotypes (O87:H16, O91:H-, O103:H2, O128:H2, O176:H4) . 52% belonged to serotypes reported in association with HUS . Five serotypes were not previously reported in sheep strains . Of the 47 strains encoding for stx1 variants, 57% were stx1c- and of the 45 encoding for stx2 variants, 80% were stx2d-positive . Eighty-two percent of the strains showed further putative virulence factors: 13% were eae-, 60% ehxA- and 67% saa-positive . The associations between harboring (i) eae and stx1, stx2, ehxA or no saa and (ii) saa and stx1c or stx2d were significant (P<0.05) . The strains belonged to 27 seropathotypes (association between serotypes and virulence factors), but 57% belonged to only six and O91:H-stx1 stx2d saa and O128:H2 stx1c stx2d ehxA saa were the most common . Seven of the eight intimin-positive strains harbored eae . Four strains of serotype O103:H2 and O121:H10 harboring stx2, eae and ehxA showed virulence factors typical for strains associated with severe human disease . However, according to the virulence factors, the majority of the ovine non-O157 STEC strains are assumed low-virulence variants . Nevertheless, as long as the contribution and interaction of these factors in milder disease remains unclear P, a certain risk for humans cannot be excluded. J Microbiol Methods, 2004 Aug, 58(2), 223 - 31 Estimation of viable Escherichia coli O157 in surface waters using enrichment in conjunction with immunological detection; Shelton DR et al.; The use of a minimal lactose enrichment broth (MLB) in conjunction with immunomagnetic electrochemiluminescence detection (IM-ECL) was evaluated for the estimation of viable Escherichia coli O157 populations in surface water samples . In principle, E . coli O157 populations (C(initial E . coli O157)) can be derived from enrichment data according to the equation: C(initial E . coli O157) = C(initial coliforms) x C(final E . coli O157)/C(final coliforms)), assuming that the growth rates and lag times of water-borne E . coli O157 and collective coliforms are sufficiently comparable, or at least consistent . We have previously described a protocol for determining C(final E . coli O157) in MLB-enriched water samples . In the present study, 80% of coliforms (red/pink colonies on MacConkey Agar) grew in MLB, indicating that this provides reasonably accurate estimates of C(initial coliforms) . Estimates of C(final coliforms) were determined from turbidity data . Initial E . coli O157 populations (C(initial E . coli O157)) were calculated for 33 Baltimore watershed samples giving a positive IM-ECL response . The majority of samples contained E . coli O157 concentrations of < 1 cell per 100 ml . These data indicate that E . coli O157 are present in surface water samples but at very low levels . Growth rates for MLB-enriched coliforms were highly variable (k= 0.47 +/- 0.13 h(-1), n= 72) . There was no correlation between growth rates and any measured water parameter, suggesting that coliform populations in water samples are spatially and temporally unique . Although variability in growth rates was expected to yield some low values, the fact that most E . coli O157 concentrations were < 1 suggests that other factor(s) were also responsible . Studies with E . coli O157:H7 and wild-type E . coli suggest that increased lag times due to starvation were at least partially responsible for the observed data . Based on estimates of C(initial coliforms) and k(coliforms), MLB was evaluated for sensitivity and quantitativeness . Simulated populations of E . coli O157:H7 at stationary phase varied from ca . 10(3) to 10(8) cells ml(-1) enrichment culture . Although not suitable for quantitation, MLB enrichment in conjunction with IM-ECL can detect as few as one viable water-borne E . coli O157 cell per 100 ml surface water . Experiments are in progress to evaluate alternative media for sensitivity and quantitative detection of enterohemorrhagic E . coli. J Microbiol Methods, 2004 Aug, 58(2), 213 - 22 Multiple-Locus Variable-Number Tandem-Repeats Analysis of Escherichia coli O157 using PCR multiplexing and multi-colored capillary electrophoresis; Lindstedt BA et al.; The Multiple-Locus Variable-Number Tandem-Repeats Analysis (MLVA) method is currently being used as the primary typing tool for Shiga-toxin-producing Escherichia coli (STEC) O157 isolates in our laboratory . The initial assay was performed using a single fluorescent dye and the different patterns were assigned using a gel image . Here, we present a significantly improved assay using multiple dye colors and enhanced PCR multiplexing to increase speed, and ease the interpretation of the results . The different MLVA patterns are now based on allele sizes entered as character values, thus removing the uncertainties introduced when analyzing band patterns from the gel image . We additionally propose an easy numbering scheme for the identification of separate isolates that will facilitate exchange of typing data . Seventy-two human and animal strains of Shiga-toxin-producing E . coli O157 were used for the development of the improved MLVA assay . The method is based on capillary separation of multiplexed PCR products of VNTR loci in the E . coli O157 genome labeled with multiple fluorescent dyes . The different alleles at each locus were then assigned to allele numbers, which were used for strain comparison. J Food Prot, 2004 Jun, 67(6), 1234 - 7 Occurrence of Shiga toxin-producing Escherichia coli O157 in selected dairy and meat products marketed in the city of Rabat, Morocco; Benkerroum N et al.; Samples of meat and dairy products taken from the city of Rabat, Morocco, were examined for the presence of Escherichia coli O157 by the selective enrichment procedure followed by plating on cefixime-tellurite-sorbitol MacConkey agar and a latex agglutination test . The ability of isolates to produce Shiga toxins (ST1 or ST2) was also tested by an agglutination test using sensitized latex . Dairy samples (n = 44) included different products commonly consumed in the country . Meat samples (n = 36) were taken from traditional butchers because these products are generally marketed in this way . Random samples were taken from each product during the period of January through May . Of the 80 samples tested, 8 (10%) harbored E . coli O157 . Four dairy and four meat samples were contaminated (9.1 and 11.1%, respectively) . Of 10 E . coli O157 isolates from contaminated samples demonstrating true antigen-antibody agglutination, 5 (50%) produced either ST2 alone or ST2 plus ST1 . Four of the five strains (80%) were meat isolates and produced ST2 with or without ST1, and the fifth was a dairy isolate producing ST2. J Food Prot, 2004 Jun, 67(6), 1153 - 6 Modeling of Escherichia coli inactivation by UV irradiation at different pH values in apple cider; Quintero-Ramos A et al.; This study examined the effects and interactions of UV light dose (1,800 to 20,331 microJ/cm2) and apple cider pH (2.99 to 4.41) on the inactivation of Escherichia coli ATCC 25922, a surrogate for E . coli O157:H7 . A predictive model was developed to relate the log reduction factor of E . coli ATCC 25922 to the UV dose . Bacterial populations for treated and untreated samples were enumerated with the use of nonselective media . The results revealed that UV dose was highly significant in the inactivation of E . coli, whereas pH showed no significant effect at higher UV doses . Doses of 6,500 microJ/cm2 or more were sufficient to achieve a greater than 5-log reduction of E . coli . Experimental inactivation data were fitted adequately by a logistic regression model . UV irradiation is an attractive alternative to conventional methods for reducing bacteria in unpasteurized apple cider. Cell Struct Funct, 1999 Oct, 24(5), 247 - 53 Exocytotic secretion of toxins from macrophages infected with Escherichia coli O157; Shimada O et al.; This study examined whether macrophages are involved in the development of pathogenicity in Shiga-like toxin (SLT)-producing enterohemorrhagic Escherichia coil (EHEC) O157:H7 . Macrophages were infected with the bacteria, after which the macrophage culture medium showed a clear increase in toxicity in rats in vivo as well as in rat aortic endothelial cells in vitro . The increased toxicity resulted mainly from a rapid increase in the concentrations of SLT type I (SLT-I) and type II (SLT-II) and partly from an increase in concentrations of the proinflammatory cytokines, tumor necrosis factor alpha (TNFalpha) and interleukin-1 (IL-1), in the culture medium . Most of the EHEC O157 added to the macrophage culture were quickly incorporated to form phagosomes, which then fused with lysosomes to become phagolysosomes . During this intracellular digestion process, the EHEC O157 remained alive for about 15 min, and continued synthesizing and secreting the toxins SLT-1 and SLT-II . The bacteria were then killed and digested in the phagolysosomes with significant amounts of the toxins retained . Subsequently, the contents of the phagolysosomes were exocytotically secreted from the macrophage cell membrane into the surrounding culture medium . Such a sequence of events in macrophages may occur in vivo, suggesting the active involvement of macrophages in the rapid increase in pathogenicity, such as seen in the onset of hemolytic-uremic syndrome (HUS) in patients infected with EHEC O157 . The exocytotic secretion is considered to be one of the most basic cellular functions in macrophages. Clin Infect Dis, 2004 Jul 1, 39(1), 1 - 7 Epub 2004 Jun 11. A multistate outbreak of Escherichia coli O157:H7 infection linked to consumption of beef tacos at a fast-food restaurant chain; Jay MT et al.; We investigated a multistate outbreak of Escherichia coli O157:H7 infections . Isolates from 13 case patients from California, Nevada, and Arizona were matched by pulsed-field gel electrophoresis subtyping . Five case patients (38%) were hospitalized, and 3 (23%) developed hemolytic uremic syndrome; none died . The median age was 12 years (range, 2-75 years), and 10 (77%) were female . Case-control studies found an association between illness and eating beef tacos at a national Mexican-style fast-food restaurant chain (88% of cases versus 38% of controls; matched OR, undefined; 95% confidence interval, 1.49 to infinity; P=.009) . A trace-back investigation implicated an upstream supplier of beef, but a farm investigation was not possible . This outbreak illustrates the value of employing hospital laboratory-based surveillance to detect local clusters of infections and the effectiveness of using molecular subtyping to identify geographically dispersed outbreaks . The outbreak investigation also highlights the need for a more efficient tracking system for food products. Anal Biochem, 2004 Jul 15, 330(2), 342 - 9 Liposome-based microcapillary immunosensor for detection of Escherichia coli O157:H7; Ho JA et al.; Our group has previously reported a sandwich-based strip immunoassay for rapid detection of Escherichia coli O157:H7 {Anal . Chem . 75 (2003) 4330} . In the present study, a microcapillary flow injection liposome immunoanalysis (mFILIA) system was developed for the detection of heat-killed E . coli O157:H7 . A fused-silica microcapillary with anti-E . coli O157:H7 antibodies chemically immobilized on the internal surface via protein A served as an immunoreactor/immunoseparator for the mFILIA system . Liposomes tagged with anti-E . coli O157:H7 and encapsulating a fluorescent dye were used as the detectable label . In the presence of E . coli O157:H7, sandwich complexes were formed between the immobilized antibodies in the column, the sample of E . coli O157:H7 and the antibody-tagged sulforhodamine-dye-loaded liposomes . Signals generated by lysing the bound liposomes with 30 mM n-octyl-beta-D-glucopyranoside were measured by a fluorometer . The detected signal was directly proportional to the amount of E . coli O157:H7 in the test sample . The mFILIA system successfully detected as low as 360 cells/mL (equivalent to 53 heat-killed bacteria in the 150 microL of the sample solution injected) . MeOH (30%) was used for the regeneration of antibody binding sites in the capillary after each measurement, which allowed the immunoreactor/immunoseparator to be used for at least 50 repeated assays . The calibration curve for heat-killed E . coli O157:H7 has a working range of 6 x 10(3)-6 x 10(7)cells, and the total assay time was less than 45 min . A coefficient of variation for triplicate measurements was < or =8.9%, which indicates an acceptable level of reproducibility for this newly developed method. Emerg Infect Dis, 2004 May, 10(5), 928 - 31 Hemolytic uremic syndrome incidence in New York; Chang HG et al.; A comparison of New York's traditional communicable disease surveillance system for diarrhea-associated hemolytic uremic syndrome with hospital discharge data showed a sensitivity of 65% . Escherichia coli O157:H7 was found in 63% of samples cultured from hemolytic uremic syndrome patients, and samples were more likely to be positive when collected early in illness. Emerg Infect Dis, 2004 May, 10(5), 842 - 7 Virulence factors for hemolytic uremic syndrome, Denmark; Ethelberg S et al.; We present an analysis of strain and patient factors associated with the development of bloody diarrhea and hemolytic uremic syndrome (HUS) among Shiga toxin-producing Escherichia coli (STEC) patients registered in Denmark in a 6-year period . Of 343 STEC patients, bloody diarrhea developed in 36.4% and HUS in 6.1% . In a multivariate logistic regression model, risk factors for bloody diarrhea were the eae and stx2 genes, O groups O157 and O103, and increasing age . Risk factors for HUS were presence of the stx2 (odds ratio {OR} 18.9) and eae (OR undefined) genes, being a child, and having bloody diarrhea . O group O157, although associated with HUS in a univariate analysis (OR 4.0), was not associated in the multivariate analysis (OR 1.1) . This finding indicates that, rather than the O group, the combined presence of the eae and stx2 genes is an important predictor of HUS. J Vet Sci, 2004 Jun, 5(2), 119 - 24 Escherichia coli O157:H7 adherence to HEp-2 cells is implicated with curli expression and outer membrane integrity; Kim SH et al.; Escherichia coli (E . coli) has ability to express thin aggregative fimbriae, known as curli, on the cell surface . Previously, a few example of curli expression in serogroup O157:H7 of enterohemorrhagic E . coli (EHEC) were reported, compared to other E . coli groups . However, significance of curliation in the EHEC pathobiology has not been described well in the literature . A highly curliated O157:H7 strain was used in this study in order to elucidate role of curliation in EHEC adherence to cultured HEp-2 cells . The expression of curli in the EHEC isolate was consistent with strong positive indication of Congo-red (CR) binding and formation of clumps in the bottom of the tube containing Luria-Bertani (LB) broth when cultured overnight at 37 degrees C . A few CR-binding negative (CR-) colonies occurred spontaneously within the population of CR+ isolate . The CR+ EHEC showed massive aggregative adhesion pattern, whereas the spontaneous CR- strain showed typical localized adherence on HEp-2 cells . Electron microscopy confirmed highly curliated bacteria in the CR+ EHEC sample . Interestingly, the curliation disappeared in a msbB1 and msbB2 double mutant derived from the CR+ EHEC . These results suggest that the compromised outer membrane integrity caused by msbB mutations may abrogate curli production in the CR+ EHEC harbouring penta-acylated lipid A structure in their outer membrane. Epidemiol Infect, 2004 Jun, 132(3), 495 - 505 GIS-supported investigation of human EHEC and cattle VTEC O157 infections in Sweden: geographical distribution, spatial variation and possible risk factors; Kistemann T et al.; This article describes the spatial and temporal distribution of verotoxin-producing Escherichia coli among humans (EHEC) and cattle (VTEC) in Sweden, in order to evaluate relationships between the incidence of EHEC in humans, prevalence of VTEC O157 in livestock and agricultural structure by an ecological study . The spatial patterns of the distribution of human infections were described and compared with spatial patterns of occurrence in cattle, using a Geographic Information System (GIS) . The findings implicate a concentration of human infection and cattle prevalence in the southwest of Sweden . The use of probability mapping confirmed unusual patterns of infection rates . The comparison of human and cattle infection indicated a spatial and statistical association . The correlation between variables of the agricultural structure and human EHEC incidence was high, indicating a significant statistical association of cattle and farm density with human infection . The explained variation of a multiple linear regression model was 0.56. Epidemiol Infect, 2004 Jun, 132(3), 467 - 84 Disease burden in The Netherlands due to infections with Shiga toxin-producing Escherichia coli O157; Havelaar AH et al.; Surveys carried out between 1990 and 2000 indicated that the incidence of STEC O157-associated gastroenteritis in The Netherlands was 1250 cases/year (median), of which 180 visited a general practitioner, 40 are reported and 0.6 are fatal, mainly in the elderly . There are approximately 20 cases of STEC O157-associated haemolytic-uraemic syndrome (HUS) per year, mainly in children . There are 2.5 HUS patients per year who develop end-stage renal disease (ESRD) . There are an estimated 2 HUS-related and 0.5 ESRD-related fatalities per year . The mean disease burden associated with STEC O157 in the Dutch population is 116 (90% confidence interval 85-160) Disability Adjusted Life Years (DALYs) per year . Mortality due to HUS (58 DALYs), and ESRD (21 DALYs) and dialysis due to ESRD (21 DALYs) constitute the main determinants of disease burden . Sensitivity analysis indicates that uncertainty associated with model assumptions did not have a major effect on these estimates. J Vet Med Sci, 2004 May, 66(5), 585 - 7 High prevalence of enterohemorrhagic Escherichia coli (EHEC) O157 from cattle in selected regions of Japan; Ezawa A et al.; The prevalence of enterohemorrhagic Escherichia coli (EHEC) O157 was examined in bovine faeces . EHEC O157 was isolated from the faeces of 42 (13.0%) of 324 cattle . Of the 4 farms and the facilities tested, the 3 farms and the facilities were found positive for EHEC O157 . The highest isolation rate among the farms was 33.7% . The prevalence of EHEC O157 in heifers was higher than that in calves and other cattle . No cattle positive for EHEC O157 showed any clinical signs except 2 calves with diarrhea in a veterinary hospital . Almost all isolates possessed the stx gene, and Stx-positive strains carrying both stx(1) and stx(2) genes were predominant . These results indicate that EHEC O157 are distributed in bovine faeces, and that dairy and beef farms in selected regions of Japan are heavily contaminated with the organisms. J Clin Microbiol, 2004 Jun, 42(6), 2388 - 97 Insertions, deletions, and single-nucleotide polymorphisms at rare restriction enzyme sites enhance discriminatory power of polymorphic amplified typing sequences, a novel strain typing system for Escherichia coli O157:H7; Kudva IT et al.; Polymorphic amplified typing sequences (PATS) for Escherichia coli O157:H7 (O157) was previously based on indels containing XbaI restriction enzyme sites occurring in O-island sequences of the O157 genome . This strain-typing system, referred to as XbaI-based PATS, typed every O157 isolate tested in a reproducible, rapid, straightforward, and easy-to-interpret manner and had technical advantages over pulsed-field gel electrophoresis (PFGE) . However, the system was less discriminatory than PFGE and was unable to differentiate fully between unrelated isolates . To overcome this drawback, we enhanced PATS by using another infrequently cutting restriction enzyme, AvrII (also known as BlnI), to identify additional polymorphic regions that could increase the discriminatory ability of PATS typing . Referred to as AvrII-based PATS, the system identified seven new polymorphic regions in the O157 genome . Unlike XbaI, polymorphisms involving AvrII sites were caused by both indels and single-nucleotide polymorphisms occurring in O-island and backbone sequences of the O157 genome . AvrII-based PATS by itself provided poor discrimination of the O157 isolates tested . However, when primer pairs amplifying the seven polymorphic AvrII sites were combined with those amplifying the eight polymorphic XbaI sites (combined PATS), the discriminatory power of PATS was enhanced . Combined PATS matched related O157 isolates better than PFGE while differentiating between unrelated isolates . PATS typed every O157 isolate tested and directly targeted polymorphic sequences responsible for differences in the restriction digest patterns of O157 genomic DNA, utilizing PCR rather than relying on gel electrophoresis . This enabled PATS to resolve the ambiguity in PFGE typing, including that arising from the "more distantly related" and "untypeable" profiles. Appl Environ Microbiol, 2004 Jun, 70(6), 3500 - 5 Adaptation of Escherichia coli O157:H7 to pH alters membrane lipid composition, verotoxin secretion, and resistance to simulated gastric fluid acid; Yuk HG et al.; The influence of adaptation to pH (from pH 5.0 to 9.0) on membrane lipid composition, verotoxin concentration, and resistance to acidic conditions in simulated gastric fluid (SGF) (pH 1.5, 37 degrees C) was determined for Escherichia coli O157:H7 (HEC, ATCC 43895), an rpoS-deficient mutant of ATCC 43895 (HEC-RM, FRIK 816-3), and nonpathogenic E . coli (NPEC, ATCC 25922) . Regardless of the strain, D values (in SGF) of acid-adapted cells were higher than those of non-acid-adapted cells, with HEC adapted at pH 5.0 having the greatest D value, i.e., 25.6 min . Acid adaptation increased the amounts of palmitic acid (C16:0) and decreased cis-vaccenic acid (C18:1 omega 7c) in the membrane lipids of all strains . The ratio of cis-vaccenic acid to palmitic acid increased at acidic pH, causing a decrease in membrane fluidity . HEC adapted to pH 8.3 and HEC-RM adapted to pH 7.3 exhibited the greatest verotoxin concentrations (2,470 and 1,460 ng/ml, respectively) at approximately 10(8) CFU/ml . In addition, the ratio of extracellular to intracellular verotoxin concentration decreased at acidic pH, possibly due to the decrease of membrane fluidity . These results suggest that while the rpoS gene does not influence acid resistance in acid-adapted cells it does confer decreased membrane fluidity, which may increase acid resistance and decrease verotoxin secretion. Dev Comp Immunol, 2004 May 17, 28(6), 635 - 45 Gene expression profiling of bovine macrophages in response to Escherichia coli O157:H7 lipopolysaccharide; Chitko-McKown CG et al.; The aim of this study was to identify changes in bovine macrophage gene expression in response to treatment with Escherichia coli 0157:H7 lipopolysaccharide (LPS), utilizing a human gene microarray . Bovine cDNA from control and LPS-treated primary macrophages hybridized to greater than 5644 (79.8%) of the non-control gene targets on a commercially available microarray containing greater than 7075 targets (Incyte Genomics, St . Louis, MO) . Of these target sequences, 44 were differentially expressed upon exposure to LPS, including 18 genes not previously reported to exist in cattle . These included a pentaxin-related gene, CASP8, TNF-induced genes, interferon-induced genes, and inhibitors of apoptosis . Using the human microarray, cDNA from bovine LPS-treated and control macrophages consistently hybridized to t