J Food Prot, 1998 Sep, 61(9), 1184 - 6 Thermotolerance of Escherichia coli O157:H7 ATCC 43,894, Escherichia coli B, and an rpoS-deficient mutant of Escherichia coli O157:H7 ATCC 43,895 following exposure to 1.5% acetic acid; Williams NC et al.; On a beef carcass, Escherichia coli may sequentially encounter acid- and heat-intervention steps . This study tested whether acid stress (1.5% {vol/vol} acetic acid, pH 4.0, 37 degrees C, 15 min) would enhance subsequent heat resistance of E . coli . Initially, cells (E . coli O157:H7 ATCC 43894, nonpathogenic E . coli B {strain FRIK-124}, and rpoS-deficient mutant 813-6 {derived from E . coli O157:H7 ATCC 43895}) were acid stressed and transferred to 54 degrees C trypticase soy broth (TSB), and survivors were immediately enumerated after at least three intervals of 12, 2, and 6 min, respectively, by plating . The ATCC 43894 and 813-6 strains survived the acid stress but strain FRIK-124 did not . Acid-stressed ATCC 43894 had significantly lower D values than the non-acid-stressed controls . Strain 813-6 had significantly lower D values than strain ATCC 43894, with no significant difference between acid-stressed and non-acid-stressed cells . In a second experiment, cooling of cells prior to plating resulted in an increased D value for acid-stressed ATCC 43894 cells, such that it was not significantly different from the D value for non-acid-stressed controls . Using this protocol, there was no significant difference in D values between acid-stressed and non-acid-stressed ATCC 43894 cells in prewarmed TSB (54, 58, and 62 degrees C), in prewarmed ground beef slurry (GBS; 58 degrees C), or in TSB and GBS inoculated at 5 degrees C and heated to 58 degrees C . The acid stress tested does not enhance subsequent heat resistance of E . coli. J Food Prot, 1998 Sep, 61(9), 1181 - 3 Survival of Escherichia coli O157:H7 after freezing and thawing in ground beef patties; Sage JR et al.; Survival of Escherichia coli O157:H7 strains QA 326, and ATCC 43889, 43894, and 43895 after freezing (-20 degrees C, 24 h) and thawing (4 degrees C for 12 h, 23 degrees C for 3 h, or microwave heating of 700 W for 120 s) in ground beef patties was determined by reference most probable number (MPN), hydrophobic grid membrane filter SD-39 agar, and sorbitol MacConkey agar (SMA) spread-plating methods . Populations decreased from 0.62 to 2.52 log10 CFU/g, with the extent varying significantly by strain . Strain QA 326 populations almost always decreased the most, up to 1.87 log10 CFU/g more than the least sensitive strain . Microwave heating was the most lethal thawing treatment for strain QA 326, and 4 degrees C thawing was the most lethal treatment for strain ATCC 43894 . Thawing treatments varied in relative lethality for the other two strains . For strain QA 326 (4 degrees C and microwave thaw treatments) and strain ATCC 43889 (4 and 23 degrees C thawing), the enumeration method significantly affected a population decrease . The SD-39 agar method best recovered strain QA 326 while the SD-39 agar method and the reference MPN method best recovered strain ATCC 43889 after 4 and 23 degrees C thawing, respectively . The greatest difference in population decrease measured by any two methods was 0.58 log10 CFU/g . Results showed (i) a wide range in freeze-thaw sensitivity among E . coli O157:H7 strains, (ii) no thawing method had consistently and significantly greater lethality, and (iii) the reference MPN, SD-39 agar, and SMA methods differed little in ability to enumerate E . coli O157:H7. J Food Prot, 1998 Sep, 61(9), 1098 - 102 Effectiveness of salt, pH, and diacetyl as inhibitors for Escherichia coli O157:H7 in dairy foods stored at refrigeration temperatures; Guraya R et al.; The behavior of Escherichia coli O157:H7 inoculated in 10% rehydrated nonfat dry milk adjusted to pH levels between 3.8 and 5.4 with lactic acid, salt levels of 0 to 6%, and diacetyl levels of 0, 5, and 10 micrograms/g was determined at 4 and 12 degrees C . Cell populations were determined by surface plating on tryptic soy agar after 7 and 35 days of incubation . Survival was also determined using retail cultured diary products . E . coli O157:H7 did not survive in skim milk at pH 3.8 and was reduced by 3 log cycles at pH 4.1, regardless of salt, diacetyl, and temperature levels . At pH levels above 4.4, survival was observed at lower salt concentrations for up to 35 days at both 12 and 4 degrees C . The organism grew (up to a 2.2-log increase) at pH 5.0 at 2% salt levels after 35 days of storage at 12 degrees C . Diacetyl at a concentration of 10 ppm had no effect on survival and growth . In all but one case, E . coli O157:H7 was inactivated in yogurt, sour cream, and buttermilk at a rate similar to or greater than what was consistent with the acidified skim milk data . Also consistent with the skim milk data, growth occurred in two of the three cottage cheese samples at 12 degrees C after 7 days but not after 35 days or at 4 degrees C, when a 1- to 2-log decline was observed. J Food Prot, 1998 Sep, 61(9), 1093 - 7 An improved direct plate method for the enumeration of stressed Escherichia coli O157:H7 from food; McCarthy J et al.; The use of sorbitol MacConkey agar (SMAC) performed poorly in supporting growth of stressed Escherichia coli O157:H7 cells . Up to a 3-log difference was observed between counts on SMAC and tryptone soy agar (TSA) . It is critical in the risk assessment of certain foods to be able to enumerate stressed and healthy E . coli O157:H7 in a background of potentially healthy competing bacteria . Investigations carried out to overcome the inhibitory effect of SMAC included the reduction of the selective agent concentration, inclusion of a recovery stage in broth prior to plating out, addition of recovery agents, and delayed exposure to the selective agent . The only successful approach was delayed exposure to the selective agent . This was achieved by resuscitating the stressed cells on a membrane placed on the surface of a TSA plate and, after a defined time period sufficient for full resuscitation, transferring the membrane to the surface of a SMAC plate . The choice of membrane material was critical for maintaining the positive sorbitol color change used to identify wild-type E . coli . Track-etched polycarbonate membranes allowed the typical color reactions to be visualized, whereas cellulose acetate did not . The method was validated with E . coli O157:H7 cells stressed by low pH and high salt conditions, whereby all cells that would previously be undetectable on direct inoculation of SMAC were countable. Lett Appl Microbiol, 1998 Aug, 27(2), 76 - 8 Escherichia coli O157 serology: false-positive ELISA results caused by human antibodies binding to bovine serum albumin; Chart H et al.; An analysis of farm workers and rural dwellers for serum antibodies to the lipopolysaccharide (LPS) of Escherichia coli O157 detected sera with antibodies binding to bovine serum albumin (BSA) by ELISA . These antibodies were not specific for BSA when examined by immunoblotting, and the ELISA values were reduced to a background level when plates were blocked with normal rabbit serum. J Appl Microbiol, 1998 Sep, 85(3), 425 - 8 Reliability of CHROMagar O157 for the detection of enterohaemorrhagic Escherichia coli (EHEC) O157 but not EHEC belonging to other serogroups; Bettelheim KA; CHROMagar O157 is designed for the rapid isolation and identification of enterohaemorrhagic Escherichia coli (EHEC), particularly O157, characterized by pink colonies . Five hundred and eighty-five E . coli strains, including O157, O111 and O113 serogroups, from many sources were examined on CHROMagar O157 . Enterohaemorrhagic E . coli O157 could readily be isolated and recognized uniquely by typical pink colonies . Some other EHEC also produced pink colonies, whereas O113 and many other EHEC strains were blue and indistinguishable from Shiga-like toxin-negative strains of E . coli. Pediatr Nephrol, 1998 Aug, 12(6), 485 - 8 Renal function in Inuit survivors of epidemic hemolytic-uremic syndrome; Ogborn MR et al.; We undertook a case-control study to evaluate the renal health of survivors of hemolytic-uremic syndrome (HUS) from the 1991 Arctic epidemic of Escherichia coli O157:H7 gastroenteritis 4 years after the epidemic . Eighteen children who developed HUS during the 1991 epidemic and 18 age- and sex-matched controls from the same community who had uncomplicated gastroenteritis were compared in 1995 for height, weight, blood pressure, urinalysis, and glomerular filtration rate (GFR), measured using continuous subcutaneous infusion of non-radioactive iothalamate . HUS survivors did not differ from controls in height, weight, systolic (HUS 118 mmHg, control 117 mmHg) or diastolic (HUS 64 mmHg, control 62 mmHg) blood pressures . Hematuria was detected more frequently in HUS survivors (11/18 vs . 4/18, P<0.05), but no child had proteinuria . Mean GFR did not differ between the two groups (HUS 159 ml/min per 1.73 m2, control 147 ml/min per 1.73 m2) . Survivors of post-enteritic HUS from the 1991 Arctic E . coli 0157:H7 outbreak have excellent renal function 4 years after the epidemic. Bull World Health Organ, 1998, 76(3), 245 - 55 Prevention and control of enterohaemorrhagic Escherichia coli (EHEC) infections: memorandum from a WHO meeting . WHO Consultation on Prevention and Control of Enterohaemorrhagic Escherichia coli (EHEC) Infections; Reilly A; Escherichia coli is a commonly occurring inhabitant of the intestine of humans and other animals, but there are several pathogenic types of E . coli which cause a variety of human diseases . One of these pathogenic types, E . coli O157:H7, belongs to the group of enterohaemorrhagic E . coli (EHEC) which produce potent toxins and cause a particularly severe form of disease, haemorrhagic colitis (HC) . About 10% of patients with HC can go on to develop haemolytic uraemic syndrome (HUS), a life-threatening complication of E . coli O157:H7 infection that is characterized by acute renal failure, haemolytic anaemia, and thrombocytopenia . These sequelae are particularly serious in young children and older people . On average, 2-7% of patients with HUS die, but in some outbreaks among the elderly the mortality rate has been as high as 50% . This Memorandum reviews the growing importance of E . coli O157:H7 as a foodborne pathogen and reports on the issues of surveillance, outbreak investigation, and control strategies with respect to EHEC infections that were discussed at the WHO Consultation on Prevention and Control of EHEC Infections, held in Geneva on 28 April to 1 May 1997 . Recommended measures for prevention and control include the following: use of potable water in food production; presentation of clean animals at slaughter; improved hygiene throughout the slaughter process; appropriate use of food processing measures; thorough cooking of foods; and the education of food handlers, abattoir workers, and farm workers on the principles and application of food hygiene. South Med J, 1998 Sep, 91(9), 798 - 804 Escherichia coli and the hemolytic-uremic syndrome; Begue RE et al.; BACKGROUND: The hemolytic-uremic syndrome (HUS) comprises hemolytic anemia, acute renal failure, and thrombocytopenia . It is the most frequent cause of acute renal failure in children . METHODS: This review is based on an extensive overview of the literature dealing with the HUS in children . RESULTS: HUS is the most common cause of acute renal failure in infants and young children and follows a diarrheal prodrome approximately 90% of the time . Nearly all postdiarrheal cases are caused by enterohemorrhagic E coli infections, in particular serotype O157:H7 . Mortality is around 5%, and approximately 50% of survivors manifest some types of sequelae . CONCLUSION: Surveillance and contact investigation are important to control outbreaks, as well as early and aggressive treatment of symptomatic subjects to prevent mortality and severe complications, such as chronic renal disease. Immunology, 1998 Jun, 94(2), 228 - 34 Prostaglandin and fatty acid modulation of Escherichia coli O157 phagocytosis by human monocytic cells; Davidson J et al.; Phagocytosis by human monocytes is an important primary survival mechanism particularly during bacterial infection . However, the processes that control the events and mediators involved in the activation of monocytes and their impact on the phagocytosis of bacteria are poorly understood . The effect of bacterial endotoxin, interleukin-1 beta (IL-1 beta), fatty acids and prostaglandin E2 (PGE2) on the phagocytosis of fluoroscein isothiocyanate (FITC)-labelled Escherichia coli (O157) by human blood monocytes and U937 cells was studied by flow cytometry . Endotoxin increased the phagocytosis of labelled bacteria by both monocytes and U937 cells . IL-1 beta and the polyunsaturated fatty acids; dihomo-gamma-linolenic and arachidonic acids also increased the phagocytic activity of both monocytes and U937 cells . In contrast, PGE2 suppressed phagocytosis in a concentration-dependent manner . The cyclo-oxygenase inhibitor, ketoprofen, further enhanced the increased phagocytic activity in the presence of endotoxin and interleukin-1 (IL-1) indicating suppression by endogenous prostaglandins . This was confirmed by the data which showed that lipopolysaccharide (LPS) and IL-1 increased PGE2 release and ketoprofen inhibited release . Endotoxin and fatty acids increased IL-1 beta release also, whereas PGE2 inhibited release . The data suggest that phagocytic activity may be linked to changes in IL-1 levels . The data presented in this study also suggest that monocyte phagocytosis in the course of bacterial infection would be altered during pathophysiological events which result in elevation of extracellular fatty acids. FEMS Microbiol Lett, 1998 Sep 1, 166(1), 43 - 8 Isolation of an Escherichia coli O157:H7 strain producing Shiga toxin 1 but not Shiga toxin 2 from a patient with hemolytic uremic syndrome in Korea; Kim YB et al.; Escherichia coli strains isolated from patients with diarrhea or hemolytic uremic syndrome (HUS) at Pusan University Hospital, South Korea, between 1990 and 1996 were examined for traits of the O157:H7 serogroup . One strain isolated from a patient with HUS belonged to the O157:H7 serotype, possessed a 60-MDa plasmid, the eae gene, and ability to produce Shiga toxin 1 but not Shiga toxin 2 . Arbitrarily primed PCR analysis suggested that this strain is genetically very close to a O157:H7 strain isolated in Japan. Appl Environ Microbiol, 1998 Sep, 64(9), 3389 - 96 PCR-based DNA amplification and presumptive detection of Escherichia coli O157:H7 with an internal fluorogenic probe and the 5' nuclease (TaqMan) assay; Oberst RD et al.; Presumptive identification of Escherichia coli O157:H7 is possible in an individual, nonmultiplexed PCR if the reaction targets the enterohemorrhagic E . coli (EHEC) eaeA gene . In this report, we describe the development and evaluation of the sensitivity and specificity of a PCR-based 5' nuclease assay for presumptively detecting E . coli O157:H7 DNA . The specificity of the eaeA-based 5' nuclease assay system was sufficient to correctly identify all E . coli O157:H7 strains evaluated, mirroring the previously described specificity of the PCR primers . The SZ-primed, eaeA-targeted 5' nuclease detection assay was capable of rapid, semiautomated, presumptive detection of E . coli O157:H7 when >/=10(3) CFU/ml was present in modified tryptic soy broth (mTSB) or modified E . coli broth and when >/=10(4) CFU/ml was present in ground beef-mTSB mixtures . Incorporating an immunomagnetic separation (IMS) step, followed by a secondary enrichment culturing step and DNA recovery with a QIAamp tissue kit (Qiagen), improved the detection threshold to >/=10(2) CFU/ml . Surprisingly, immediately after IMS, the sensitivity of culturing on sorbitol MacConkey agar containing cefeximine and tellurite (CT-SMAC) was such that identifiable colonies were demonstrated only when >/=10(4) CFU/ml was present in the sample . Several factors that might be involved in creating these false-negative CT-SMAC culture results are discussed . The SZ-primed, eaeA-targeted 5' nuclease detection system demonstrated that it can be integrated readily into standard culturing procedures and that the assay can be useful as a rapid, automatable process for the presumptive identification of E . coli O157:H7 in ground beef and potentially in other food and environmental samples. Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10449 - 52 Altering the anaerobic transcription factor FNR confers a hemolytic phenotype on Escherichia coli K12; Ralph ET et al.; The recent outbreaks of Escherichia coli O157-associated food poisoning have focused attention on the virulence determinants of E . coli . Here, it is reported that single base substitutions in the fnr gene encoding the oxygen-responsive transcription regulator FNR (fumarate and nitrate reduction regulator) are sufficient to confer a hemolytic phenotype on E . coli K12, the widely used laboratory strain . The mechanism involves enhancing the expression of a normally dormant hemolysin gene (hlyE) located in the E . coli chromosome . The mutations direct single amino acid substitutions in the activating regions (AR1 and AR3) of FNR that contact RNA polymerase . It is concluded that altering a resident transcription regulator, or acquisition of a competent heterologous regulator, could generate a pool of hemolytic, and therefore more virulent, strains of E . coli in nature. Commun Dis Public Health, 1998 Mar, 1(1), 60 - 1 Improved serological detection of infection with Vero cytotoxin producing Escherichia coli; Jenkins C et al.; Vero cytotoxin producing Escherichia coli (VTEC)--including all those of serogroup O157--and enteropathogenic E . coli produce attaching and effacing lesions in gut epithelium . Immunoblotting was used to detect antibodies to secreted proteins associated with the formation of these lesions . These tests should provide additional evidence of VTEC infection in conjunction with current assays for antibioties to E . coli O157 lipopolysaccharide. J Food Prot, 1998 Aug, 61(8), 948 - 52 Survival and recovery of Escherichia coli O157:H7 in inoculated bottled water; Warburton DW et al.; A methodology used to isolate Escherichia coli O157:H7 from water and survival of this pathogen in inoculated water is described . The methodology used in the isolation of E . coli O157:H7 included the use of selective plating on Sorbitol MacConkey agar (supplemented with potassium tellurite {2.5 mg/liter}, cefixime {0.05 mg/liter}, and cefsulodin {10 mg/liter}, and modified hemorrhagic colitis agar (also supplemented with potassium tellurite {2.5 mg/liter}) and cefsulodin {10 mg/liter}) . There were no significant differences (P < 0.05) between the recoveries of E . coli O157:H7 on these two selective media . Direct plating on these selective agars was used to determine the length of time that E . coli O157:H7 was able to grow, remain viable, and be resistant to the selective agents . E . coli O157:H7 survived in inoculated water for up to > 300 days, depending on the type of water . Observation by scanning electron microscopy indicated that E . coli O157:H7 cells attached to, and multiplied on, the container walls. J Food Prot, 1998 Aug, 61(8), 939 - 47 Survival of Escherichia coli O157:H7 in synthetic gastric fluid after cold and acid habituation in apple juice or trypticase soy broth acidified with hydrochloric acid or organic acids; Uljas HE et al.; Extreme acid tolerance of Escherichia coli O157:H7 has raised doubts about the safety of acidic foods . This study examined whether prior storage in acidic and/or cold conditions enhanced survival of E . coli O157:H7 in synthetic gastric fluid (SGF) . Three E . coli O157:H7 strains were stored in trypticase soy broth (TSB; acidified with HCl, malic acid, citric acid, or lactic acid) or pH 3.5 and 6.5 (nonacidic control) apple juice at 4 and 21 degrees C for < or = 7 days and then were incubated in pH 2.5 SGF at 37 degrees C for 4 h . Cells survived better in apple juice than in TSB containing organic acids, suggesting that juice constituents other than organic acids protect E . coli O157:H7 . Refrigeration combined with low pH best protected cells in apple juice and acidified TSB, but, compared to the nonacidic control, only acidified TSB enhanced subsequent survival in pH 2.5 SGF . Equal survival in SGF occurred after storage in pH 3.5 or 6.5 apple juice at 4 degrees C, suggesting that low temperature alone in apple juice enhanced acid tolerance . Two strains stored at 4 degrees C in TSB containing malic or citric acid subsequently survived better in SGF than cells stored in nonacidified TSB but poorer than cells stored in the presence of HCl . These differences reflect the higher pKa of these organic acids . However, subsequent survival of these strains in SGF was poorer after refrigerated storage in apple juice than in TSB containing citric or malic acids . Cells stored in lactic acid were most likely to be completely eliminated upon transfer to SGF . Differences in survival in storage media or SGF related to strain, storage conditions, or acidifier were consistent and often statistically significant (P < 0.05) . Although the survival of E . coli O157:H7 in refrigerated acidic beverages may not be affected by the type of acidifier used, the subsequent survival in SGF of this pathogen may be critically dependent on this factor. Infect Immun, 1998 Sep, 66(9), 4560 - 3 Escherichia coli O157:H7 requires intimin for enteropathogenicity in calves; Dean-Nystrom EA et al.; Enterohemorrhagic Escherichia coli (EHEC) strains require intimin to induce attaching and effacing (A/E) lesions in newborn piglets . Infection of newborn calves with intimin-positive or intimin-negative EHEC O157:H7 demonstrated that intimin is needed for colonization, A/E lesions, and disease in cattle . These results suggest that experiments to determine if intimin-based vaccines reduce O157:H7 levels in cattle are warranted. J Food Prot, 1998 Jun, 61(6), 662 - 7 Survival of enterohemorrhagic Escherichia coli O157:H7 in water; Wang G et al.; Several recent Escherichia coli O157:H7 outbreaks associated with both drinking and recreational water raise concerns about waterborne illness caused by this pathogen . The survival characteristics of a mixture of five nalidixic acid-resistant E . coli O157:H7 strains (10(3) CFU/ml) in filtered and autoclaved municipal water, in reservoir water, and in water from two recreational lakes were determined for a period of 91 days at 8, 15 or 25 degrees C . Greatest survival was in filtered autoclaved municipal water and least in lake water . Regardless of the water source, survival was greatest at 8 degrees C and least at 25 degrees C . E . coli O157:H7 populations decreased by 1 to 2 log10 by 91 days at 8 degrees C, whereas the pathogen was not detectable (> or 3 = log10 decrease) within 49 to 84 days at 25 degrees C in three of the four water sources . SDS-PAGE of surface antigens of surviving cells revealed that there was no major alteration in lipopolysaccharide pattern, but outer membrane protein composition did change . These studies indicate that E . coli O157:H7 is a hardy pathogen that can survive for long periods of time in water, especially at cold temperatures . However, direct viable counts of E . coli O157:H7 determined by acridine orange staining remained essentially the same for 12 weeks at 25 degrees C, whereas viable counts on tryptic soy agar plates decreased to undetectable levels within 12 weeks . Results suggest that E . coli O157:H7 can enter a viable but nonculturable (VBNC) state in water. J Food Prot, 1998 May, 61(5), 547 - 50 Decontaminating beef for Escherichia coli O157:H7; Delazari I et al.; Beef lean, fat, and connective tissues were inoculated with Escherichia coli O157:H7 before and after a prewashing procedure to compare the efficacy of prewashing and no prewashing on bacterial adherence and, consequently, on the removal of bacteria from the inoculated surfaces . Prewashing consisted of spraying tissues with tap water before inoculation . Final washing with disinfectant solutions compared the efficacy of several chemicals for the removal or destruction of E . coli O157:H7 . The results showed that prewashing was very effective in reducing the numbers of bacterial cells on beef tissues, mainly lean tissue, in the control samples which received final washing with water . An opposite effect of prewashing was observed when disinfectant solutions were used for final washing; this may be due to dilution by water carried on the tissues after prewashing . The efficacy of chemicals was dependent on the type of exposed tissue . Hydrogen peroxide (3%) was more efficient in the removal of E . coli O157:H7 from connective tissues, with reductions greater than 4 log CFU/cm2, compared to a normally washed control (P < 0.01) . Chlorhexidine (0.1%) was very efficient on fat and lean tissues, causing reductions over 5 log CFU/cm2 on not prewashed fat and lean tissues, compared to the control (P < 0.01) . Acetic acid (5%) was the least effective, decreasing the number of CFU by under 1 log/cm2 as compared to the control; and no statistically significant difference was found among tissues, even though the removal of bacteria seemed less in lean tissue compared to fat or connective tissues. J Food Prot, 1998 May, 61(5), 542 - 6 Influence of incubation conditions on survival and acid tolerance response of Escherichia coli O157:H7 and non-O157:H7 isolates exposed to acetic acid; Brudzinski L et al.; The increasing frequency of Escherichia coli O157:H7 outbreaks, especially in acidic foods, raises the concern of an acid tolerance response (ATR) . Organic acids can be present in processed and preserved foods: shifts in the acid levels of foods due to these acids may allow E . coli to adapt and later tolerate pH levels that would normally inactivate the organism . The effect of temperature and agitation on the ATRs of three E . coli O157:H7 and two non-O157:H7 isolates were determined . Triggered at pH 5.0, the adaptive system of the ATR allowed for up to nearly 1,000-fold enhanced survival of E . coli O157:H7 cells in some cases compared to survival of nonadapted cells at pH 4.0 . E . coli O157:H7 isolates revealed greater acid tolerance responses when incubated statically at 32 degrees C, whereas the non-O157:H7 E . coli isolates exhibited a greater acid tolerance response with orbital agitation at 25 degrees C . The magnitude of response changed over the incubation period. J Food Prot, 1998 Apr, 61(4), 490 - 4 Evaluating survival of Escherichia coli O157:H7 in frozen and thawed apple cider: potential use of a hydrophobic grid membrane filter-SD-39 agar method; Sage JR et al.; To determine the susceptibility of Escherichia coli O157:H7 to freezing and thawing in apple cider, methods that recover injured cells are needed for accurate enumeration . This study compared the ISO-GRID hydrophobic grid membrane filter (HGMF) SD-39 agar method to two other methods: a reference most probable number (MPN) method, and plating on sorbitol MacConkey agar (SMA) . To determine numbers of injured cells, SMA spread plating was also compared to Trypticase soy agar (TSA) spread plating . Two strains of E . coli O157:H7 QA 326 and ATCC 43895, were inoculated into presterilized apple cider (10 ml) which was then frozen (-20 degrees C for 24 h) . Samples were thawed at 4 degrees C for 4 h, or at 23 degrees C for 1.5 h, or in a microwave oven (700 W for 10 s) . Substantial cell death (0.69- to 6.33 log10 CFU/ml decreases) and injury (0.70- to 2.38-log10 CFU/ml decreases) occurred during freezing and thawing . The extent of death and injury varied with strain and thawing method . The TSA spread plating method recovered the most cells while the HGMF method always recovered more viable cells than the reference MPN method and also either recovered significantly more (P < 0.05) cells or a not significantly different number of cells than SMA spread plating . Some injured cells of both strains were not counted by the HGMF method . Significant numbers of cells injured by freezing and thawing at 4 degrees C in apple cider were enumerated in the cider was diluted 1:2 Trypticase soy broth immediately before plating . Two epifluorescent microscopic methods showed that injury was not associated with loss of cell membrane integrity. J Food Prot, 1998 Apr, 61(4), 395 - 401 Influence of growth temperature on inactivation and injury of Escherichia coli O157:H7 by heat, acid, and freezing; Semanchek JJ et al.; The influence of growth temperature on heat-, lactic acid-, and freeze-induced inactivation and injury of Escherichia coli O157:H7 in 0.1% peptone water was investigated . Three strains of E . coli O157:H7 isolated respectively from salami, apple cider, and ground beef were evaluated . Growth of strains at 10 degrees C compared with growth at 37 degrees C had a significant impact on reducing (P < 0.01) D values obtained for heating (DH value), acid exposure (DA value), with the exception of the cider strain stored in lactic acid solutions . When strains were cultivated at 10 and 37 degrees C and heated at 54 and 56 degrees C, the salami strain possessed the highest (P < 0.01) DH values (5.9 to 59.7 min) . When grown at 10 degrees C, the beef strain had the lowest (P < 0.01) DH values after heating at 52, 54, and 56 degrees C (11.2, 4.1 and 2.5 min, respectively) . The salami strain grown at 10 degrees C had the highest (P < 0.01) DA values in all concentrations of lactic acid . When grown at 37 degrees C, the salami strain had the highest (P < 0.01) DA values after storage in 0.1 and 0.25% lactic acid, while DA values for the salami and beef strains did not differ (P > 0.05) when stored in 0.5% lactic acid . Portions of strain populations were sublethally injured by heat and lactic acid treatments, as evidenced by the inability of injured organisms to form colonies on tryptone soy agar containing 2% NaCl . Strains cultured at 10 degrees C were more susceptible to sublethal heat injury than the strains cultured at 37 degrees C . Storage of test strains at -20 degrees C for 7 months resulted in a 4- to 6-log CFU/ml reduction in viable population, but induced only minimal sublethal injury . After 5 months at -20 degrees C, strains cultured at 10 degrees C were more sensitive to freeze inactivation than strains cultured at 37 degrees C . When grown at 10 and cultured at 37 degrees C . When grown at 10 and 37 degrees C and stored at -20 degrees C for 7 months, the cider strain possessed higher (P < 0.01) DF values than the beef and salami strains. J Food Prot, 1998 Apr, 61(4), 390 - 4 Prior storage conditions influence the destruction of Escherichia coli O157:H7 during heating of apple cider and juice; Ingham SC et al.; In apple beverage manufacture, cider and juice may be stored for a short time prior to pasteurization . Storage time and temperature may affect the subsequent thermotolerance of bacteria in these beverages . This study examined whether prior storage in pH 3.4 apple cider or apple juice affected the thermotolerance of two Escherichia coli O157:H7 strains in the same beverages at 61 degrees C . Both strains exhibited biphasic survivor curves . Strain ATCC 43894 was consistently more thermotolerant than strain ATCC 43889, with 33 to 153% greater D values derived from the linear portion of each survivor curve . Prior storage at 21 degrees C for 2 or 6 h hastened thermal destruction of both strains in apple cider, but not to a statistically significant extent . In apple juice, prior storage at 21 degrees C for 2 h significantly decreased thermotolerance of strain ATCC 43889, but not of strain ATCC 43894 . During 6 h of storage in 21 degrees C apple juice, populations of strains ATCC 43889 and 43894 decreased by 2.1 and 0.5 log10 CFU/ml, respectively, and died rapidly during subsequent heating . Prior storage in apple juice at 4 degrees C for 24 h significantly decreased thermotolerance of both strains, but this effect was not seen after 2 h of storage at 4 degrees C . Experiments with filtered apple cider showed that presence of filterable pulp enhanced the thermotolerance of both strains . These results show that short-term (< or = 6h) room temperature storage of pH 3.4 apple cider and apple juice may enhance the lethality of subsequent pasteurization. J Food Prot, 1998 Mar, 61(3), 285 - 9 Thermal inactivation of Escherichia coli O157:H7 isolated from ground beef and bovine feces, and suitability of media for enumeration; Clavero MR et al.; Rates of thermal inactivation of five strains of Escherichia coli O157:H7 isolated from ground beef implicated in outbreaks of hemorrhagic colitis and five strains isolated from bovine feces were determined . Ground beef (22% fat, 10 g), inoculated with individual test strains at populations ranging from 6.85 to 7.40 log10 CFU g-1 of beef, was formed into patties (0.3 cm thick and 8.0 cm in diameter) and sealed in polyethylene bags . For each strain and treatment temperature (54.4, 58.9, 62.8, 65.6, or 68.3 degrees C), 6 bags were simultaneously immersed into a recirculating water bath . Viable cells in patties heated for various lengths of time were enumerated by plating diluted samples on sorbitol MacConkey agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide (MSMA) and modified eosin methylene blue (MEMB) agar . Regardless of strain or treatment temperature, higher numbers of E . coli O157:H7 cells were generally recovered on MEMB agar than on MSMA, indicating the inferiority of MSMA as a recovery medium for quantitative determination of E . coli O157:H7 cells in heat-processed ground beef . Significantly (P < or = 0.05) higher D values when enumeration was done using MEMB agar compared with MSMA . Mean D values for combined strain data at 54.4, 58.9, 62.8, and 65.6 degrees C from cultures on MEMB agar were 123.90, 6.47, 0.62, and 0.20 min, respectively, whereas D values of 25.5, 5.21, and 0.18 min were obtained at the same temperatures from cultures on MSMA . Results suggest that cooking ground beef patties to an internal temperature of 68.3 degrees C for 40 s will inactivate at least 99.99% of E . coli O157:H7 cells; z values of 4.0 and 5.1 degrees C were calculated from mean D values obtained from MEMB agar and MSMA, respectively, as recovery media . Differences in D values existed among strains but rates of thermal inactivation do not appear to be correlated with the sources of the isolates. J Food Prot, 1998 Feb, 61(2), 158 - 61 Acid tolerance and acid shock response of Escherichia coli O157:H7 and non-O157:H7 isolates provide cross protection to sodium lactate and sodium chloride; Garren DM et al.; The survival of Escherichia coli O157:H7 and non-O157:H7 due to an enhanced acid tolerance response (ATR), and enhanced acid shock response (ASR), or the stationary phase protective system when exposed to lactic acid and the resulting cross protection against increased concentration of sodium chloride and sodium lactate was studied . Escherichia coli O157:H7 isolates (1932 and 009) and a non-O157:H7 strain (ATCC 23716) were grown to stationary phase at 32 degrees C and O157:H7 to one of two treatments in an attempt to either acid shock or acid adapt the survivors . Acid shocked cells were exposed to lactic acid at pH 4.0 . Acid-adapted cells were first exposed to a pH of 5.5 and then an acid challenge of pH 4.0 . Sodium lactate (10%, 20%, or 30%) or sodium chloride (5%, 10%, or 15%) were added to a minimal glucose medium after the acidification treatment . When acid shocked and acid adapted isolate 932 and strain ATCC 23716 tolerated the elevated levels of sodium lactate, and the strain ATCC 23716 tolerated the elevated levels of sodium chloride . Acid adaption allowed isolate 932 to tolerate higher levels of sodium chloride; however, the acid shocking did not provide the same protection . Neither of the acid treatment provided increased tolerance to sodium chloride for isolate E009 . Evidence of cross protection against acid and sodium chloride or acid and sodium lactate in E . coli O157:H7 could point to a need for further evaluation of whether these combinations of preservation means are sufficient to control this pathogen. J Food Prot, 1998 Jan, 61(1), 110 - 2 Comparison of the Difco EZ Coil rapid detection system and Petrifilm test kit-HEC for detection of Escherichia coli O157:H7 in fresh and frozen ground beef; Woody JM et al.; The Difco EZ Coli Rapid Detection System was compared to the 3M Petrifilm method for detection of Escherichia coli O157:H7 in raw ground beef . Raw meatballs (25 g) were inoculated with 10 to 15 cells of Escherichia coli O157:H7, stored for various times and at different temperatures, and then stomached for 2 min in 225 ml of EZ Coli enrichment broth, which was then incubated at 42 degrees C for 18 to 24 h . A 1-ml sample of the enrichment broth was loaded into the top of the detector tips and the remaining EZ Coli broth held at 35 degrees C before streaking onto MacConkey sorbitol agar and tryptic soy agar with yeast extract . A duplicate set of meatballs were tested using the 3M Petrifilm Test Kit-HEC for hemorrhagic Escherichia coli O157:H7 . In this method raw meatballs (25 g) were enriched for 6 h in modified EC broth containing novobiocin at 37 degrees C prior to inoculation of the Petrifilm E . coli Count Plates, which were incubated at 42 degrees C for 18 h . The immunoblot ELISA was performed following this incubation . Presumptive positive isolates from both methods were confirmed using Oxoid E . coli Latex Agglutination and Difco Pasco ID Tripanels . Both methods permitted detection of 10 to 15 cells of E . coli O157:H7 per ml (i) immediately following inoculation, (ii) after 3 days of refrigerated storage at 8 degrees C, and (iii) after 30 days in frozen storage at -20 degrees C . The Difco EZ Coli Detection System proved to be a simpler and faster screening method with identification of negative and presumptive positive samples within 15 to 18 h. Int J Food Microbiol, 1998 Jun 16, 41(3), 213 - 21 Viability of Escherichia coli O157:H7 in ground and formed beef jerky prepared at levels of 5 and 20% fat and dried at 52, 57, 63 or 68 degrees C in a home-style dehydrator; Faith NG et al.; Beef jerky batter was prepared to fat contents of about 5 and 20% and inoculated with about 10(8) cfu g(-1) of a five-strain inoculum of Escherichia coli O157:H7 . Pathogen numbers were determined in the raw batter and in the strips formed from it after drying at 52, 57, 63, and 68 degrees C for times that ranged from 2 to 20 h . For both the high and low fat products, pathogen numbers were reduced by about 5 log10 cfu g(-1) within 4 h drying at 68 degrees C and within 8 h drying at 63 degrees C . At 57 degrees C, a 5-log10-unit reduction was achieved within 10h drying for the 5% fat product and within 16 h drying for the 20% fat product . At 52 degrees C, a 5-log10-unit reduction was achieved within 10 h drying for the 5% fat product and within 20 h drying for the 20% fat product . In at least one of the three trials for all four drying temperatures tested, the pathogen was present following enrichment of the samples in synthetic media . The calculated D values decreased from 2.59, 2.48, 1.23, and 1.17 as the temperature increased from 52, 57, 63, and 68 degrees C and as the fat content decreased from 20 to 5% . However, there was no direct correlation between the moisture-to-protein ratio and either the doneness of the strips or the viability of the pathogen . These data indicate that the fat content and the time and temperature at which strips are dried directly impact the viability of E . coli O157:H7 in ground and formed beef jerky. Int Arch Allergy Immunol, 1998 Aug, 116(4), 313 - 7 Induction of protection against oral infection with cytotoxin-producing Escherichia coli O157:H7 in mice by shiga-like toxin-liposome conjugate; Fukuda T et al.; We have previously reported that purified Shiga-like toxins (SLT), SLT-I and SLT-II coupled with liposomes induced a substantial amount of anti-SLT-I and anti-SLT-II IgG antibody production, respectively, in mice . The levels of anti-SLT antibody in the sera of SLT-liposome-immune mice correlated well with the protection against subsequent challenge with SLT . In this study, mice were immunized intraperitoneally with the mixture of SLT-I-liposome and SLT-II-liposome and protection against oral infection with cytotoxin-producing Escherichia coli O157:H7 was evaluated . All of the mice that received immunization with the mixture of SLT-I-liposome and SLT-II-liposome were protected against subsequent intravenous challenge with 10 LD50 of either SLT-I or SLT-II . Eight weeks after primary immunization, mice were inoculated intragastrically with 10(9) CFU of E . coli O157:H7 strain 96-60 . All SLT-liposome-immune mice tested survived without any apparent symptom while control mice died within 5 days . In addition, as shown by other antigen-liposome conjugates, SLT-liposome induced undetectable anti-SLT IgE antibody production while they induced substantial amounts of anti-SLT IgG antibodies . These results suggest that SLT-liposome conjugate may serve as a candidate vaccine that induces protection against cytotoxin-producing E . coli infection. FEMS Microbiol Lett, 1998 Jul 15, 164(2), 283 - 8 Inhibitory activity of gut bacteria against Escherichia coli O157 mediated by dietary plant metabolites; Duncan SH et al.; Under both aerobic and anaerobic conditions, the growth of Escherichia coli O157 strain NCTC 12,900 was inhibited by the coumarins esculetin, umbelliferone and scopoletin, but not by the coumarin glycoside esculin . Esculin-hydrolysing bacteria from the rumen, the pig gut and the human gut inhibited growth of E . coli in an overlay-plate assay in the presence of esculin . The combined effect of esculetin and volatile fatty acids was greater than the effect of either factor alone suggesting that coumarin glycosides in the diet might reduce the growth or survival of E . coli O157 in the gut . Adding esculin to incubations of mixed rumen contents significantly reduced the survival of E . coli O157. J Food Prot, 1998 Jul, 61(7), 917 - 20 Evaluation of the VIDAS methodology for detection of Escherichia coli O157 in food samples; Vernozy-Rozand C et al.; An automated enzyme-linked fluorescence immunoassay (ELFA), the VIDAS E . coli O157 method, was compared with immunomagnetic separation (IMS) followed by culture on cefixime tellurite sorbitol MacConkey agar (CT-SMAC) for detecting Escherichia coli O157 in artificially and naturally contaminated food samples including raw milk cheeses, poultry, raw sausages, and ground beef retail samples . Confirmation of the samples positive according to the ELFA was performed by use of an automated immunoconcentration system, VIDAS ICE, which allows selective capture and release of target organisms . A total of 496 retail food samples were examined . Seventeen food samples gave positive values with the ELFA method, and among them 9 food samples were confirmed by the ICE method . Eight were shown to contain sorbitol-positive, O157-positive, H7-negative, motile, non-verotoxin-producing E . coli . The ninth positive sample contained an O157-positive, H7-negative, sorbitol-negative, non-verotoxin-producing E . coli . The IMS technique only allowed confirmation of this sorbitol-negative, non-verotoxin-producing E . coli O157. J Food Prot, 1998 Jul, 61(7), 817 - 22 Attachment of Escherichia coli O157:H7 in ground beef to meat grinders and survival after sanitation with chlorine and peroxyacetic acid; Farrell BL et al.; The potential for transfer of Escherichia coli O157:H7 from contaminated ground beef to grinding equipment and the inactivation of attached cells during cleaning and sanitizing was examined . Chub-packed ground beef with lean:fat ratios of 75:25, 80:20 or 90:10 was inoculated with 6 log CFU/g or 2 log CFU/g E . coli O157:H7 strain FRIK 910 . Samples were consecutively ground in a Hobart meat grinder with stainless steel (SS) chips (1 cm2) glued to the auger housing . Chips were harvested after grinding, detergent washing with or without manual scrubbing and rinsing, sanitizing in a chlorine or peroxyacetic acid sanitizer, and overnight storage . Survival of E . coli O157:H7 was evaluated both by plate count and enrichment in trypticase soy broth . Approximately 3 to 4 log CFU/cm2 were attached to the SS after grinding with all three fat contents . After washing and sanitizing in a chlorine or peroxyacetic acid sanitizer, viable bacteria were infrequently recovered by plate count . Enrichment of chips resulted in a higher survival rate with both sanitizing treatments, indicating that cell numbers below the limit of detection (5 CFU/cm2) or potentially injured organisms remained on the surface . Manual scrubbing during the washing step reduced the recovery rate . The scrubbing step also increased the number of passing scores assigned using an ATP bioluminescence assay of total residual soil on the chips sanitized in chlorine . The overall results indicate that plate counts alone may not be a reliable indicator of sanitation efficacy and may be validated by enrichment assay. FEMS Microbiol Lett, 1998 Jul 1, 164(1), 133 - 9 Characterization of the locus of enterocyte effacement (LEE) in different enteropathogenic Escherichia coli (EPEC) and Shiga-toxin producing Escherichia coli (STEC) serotypes; Sperandio V et al.; All proteins involved in the attachment and effacement lesion produced by enteropathogenic Escherichia coli (EPEC) and Shiga-toxin producing E . coli (STEC) are encoded by the locus of enterocyte effacement (LEE) . We studied the presence and insertion site of the LEE in different EPEC and STEC strains . In serotypes O119:H6/H-, O55:H6, O55:H7, O142:H6, O111ac:H9/H-, O111ab:H9/H- LEE is inserted downstream of selC as previously described for EPEC O127:H6 and STEC O157:H7 . In serotypes O111ac:H8/H- and O26:H11/H- the LEE is inserted in pheU as previously described for STEC O26:H- . However in EPEC from serotype O111ab:H25 the LEE is not inserted in either site suggesting a third insertion site in the K12 chromosome . We also cloned fragments of 2.3 kb and 1.0 kb from the right and left hand sides of the LEE of a O111ac:H- strain and identified additional insertion sequences on these LEE fragments, suggesting that the LEE may be larger and may have undergone more recombination events in these serotypes. Infect Immun, 1998 Aug, 66(8), 3810 - 7 Molecular evolution of a pathogenicity island from enterohemorrhagic Escherichia coli O157:H7; Perna NT et al.; We report the complete 43,359-bp sequence of the locus of enterocyte effacement (LEE) from EDL933, an enterohemorrhagic Escherichia coli O157:H7 serovar originally isolated from contaminated hamburger implicated in an outbreak of hemorrhagic colitis . The locus was isolated from the EDL933 chromosome with a homologous-recombination-driven targeting vector . Recent completion of the LEE sequence from enteropathogenic E . coli (EPEC) E2348/69 afforded the opportunity for a comparative analysis of the entire pathogenicity island . We have identified a total of 54 open reading frames in the EDL933 LEE . Of these, 13 fall within a putative P4 family prophage designated 933L . The prophage is not present in E2348/69 but is found in a closely related EPEC O55:H7 serovar and other O157:H7 isolates . The remaining 41 genes are shared by the two complete LEEs, and we describe the nature and extent of variation among the two strains for each gene . The rate of divergence is heterogeneous along the locus . Most genes show greater than 95% identity between the two strains, but other genes vary more than expected for clonal divergence among E . coli strains . Several of these highly divergent genes encode proteins that are known to be involved in interactions with the host cell . This pattern suggests recombinational divergence coupled with natural selection and has implications for our understanding of the interaction of both pathogens with their host, for the emergence of O157:H7, and for the evolutionary history of pathogens in general. Nucleic Acids Res, 1998 Aug 1, 26(15), 3614 - 5 Quantitative analysis of polymerase chain reaction using anisotropy ratio and relative hydrodynamic volume of fluorescence polarization method; Ye BC et al.; The method based on the combination of polymerase chain reaction (PCR) and fluorescence polarization is presented . A targeted DNA was amplified with a 5'-fluorescein labeled primer, using a 256 bp DNA fragment of stx2 gene in Escherichia coli O157:H7 (188-443 bp) as a template . The fluorescence anisotropy of the 5'-fluorescein labeled primer increased upon the polymerization through Taq polymerase . The conversion of primer to PCR product was quantitatively monitored by anisotropy ratio and relative hydrodynamic volume . This system was also applied to the determination of E.coli O157:H7. J Clin Microbiol, 1998 Aug, 36(8), 2339 - 41 Identification of a rough strain of Escherichia coli O157:H7 that produces no detectable O157 antigen; Feng P et al.; MA6, an O157:H7-like strain, did not react with most anti-O157 kits examined; however, it had the rfbE gene that is essential for O157 expression and carried O157:H7 virulence factors . Lipopolysaccharide analysis showed that MA6 is a rough strain that does not produce the O157 antigen, but genetically, it belongs in the O157:H7 clonal group. Vet Microbiol, 1998 Mar 15, 61(1-2), 137 - 43 Prevalence of verotoxin-producing Escherichia coli harbored in the intestine of cattle in Japan; Miyao Y et al.; The production of verotoxin was examined in 2152 Escherichia coli isolates from 387 cattle in Japan from 1992 to 1994 . The toxin was detected in 263 isolates from 94 cattle (detection rate: 24.3%) . Verotoxin-producing E . coli (VTEC) was isolated from the cattle in 15 out of 17 prefectures, and the strains were divided into 33 serotypes . E . coli O157:H7 was isolated from 7 out of 387 cattle (detection rate: 1.8%) in four prefectures . These results suggest that VTEC is widely distributed in Japan and include a wide variety of serotypes. Acta Paediatr, 1998 May, 87(5), 593 - 4 Bacterial genotype and neurological complications of Escherichia coli O157:H7-associated haemolytic uraemic syndrome; Cimolai N et al.; We examined the possibility of an association between the bacterial genotype of Escherichia coli O157:H7 and the likelihood of progression to neurological complications in childhood gastroenteritis-associated haemolytic uraemic syndrome (D+HUS) . Bacterial stool isolates were available from 51 patients with HUS; 11 of these patients suffered a neurological complication . Bacteria were assessed for plasmid content, verotoxin (Shiga-like toxin) profile, verotoxin 2 subtype, and presence of the eaeA (effacement and attachment) marker . No association of bacterial genotype with central nervous system (CNS) manifestations was observed . Whilst the cause of CNS manifestations may be multifactorial, there is no evidence at present to implicate specific bacterial traits. Lett Appl Microbiol, 1998 Apr, 26(4), 325 - 30 Factors affecting the heat resistance of Escherichia coli O157:H7; Kaur J et al.; Escherichia coli O157:H7 has been reported as being not particularly heat resistant . However, several factors which might increase its heat resistance have been investigated in this study using five strains . Increase in growth temperature to 40 degrees C, as found in the cow gut, heat-shock at sub-lethal temperatures of 42, 45, 48 and 50 degrees C, and variable heating rate (1 degree C min-1 to 23 degrees C min-1) had no dramatic effect on heat resistance . Growth phase had a marked impact on heat resistance; late stationary phase cells were more heat-resistant than were log phase cells . The difference in heat resistance between the two phases of growth became more pronounced when cells were resuspended in fresh nutrient broth; heat resistance of late stationary phase cells increased dramatically whereas no such effect was observed with log phase cells . The addition of polyphosphates to the heating medium did not increase heat resistance . A reduction in water activity of the heating medium from 0.995 to levels between 0.980 and 0.960 also resulted in a marked increase in heat resistance . This effect was more pronounced under conditions of extremely low water activity created by resuspending late stationary phase cells in sunflower oil . Survivors were detected even after a heat treatment at 60 degrees C for 1 h or 70 degrees C for 5 min . It can be confirmed that this serotype has no unusual heat resistance and that the heating environment markedly affects resistance. Microbiol Immunol, 1998, 42(4), 265 - 9 Studies of Escherichia coli cultured on Rainbow Agar O157 with particular reference to enterohaemorrhagic Escherichia coli (EHEC); Bettelheim KA; Rainbow Agar O157 is designed for the rapid isolation and identification of enterohaemorrhagic Escherichia coli (EHEC), particularly O157, characterised by black colonies . Five-hundred-eighty-five E . coli strains, including O157, O111 and O113 serogroups from many sources were examined on Rainbow Agar O157 . EHEC O157 could readily be isolated and recognized uniquely by typical black colonies . Some other EHEC also stand out as blue-black, whereas O113 and some other EHEC strains were mauve, red or pink and indistinguishable from SLT-negative strains of E . coli. J Clin Microbiol, 1998 Jun, 36(6), 1801 - 4 A PCR specific for Escherichia coli O157 based on the rfb locus encoding O157 lipopolysaccharide; Desmarchelier PM et al.; A PCR was developed for the detection of Escherichia coli O157 based on the rfbE O-antigen synthesis genes . A 479-bp PCR product was amplified specifically from E . coli O157 in cell lysates containing 200 or 2 CFU following crude DNA extraction . The PCR detected < 1 CFU of E . coli O157 per ml in raw milk following enrichment. J Clin Microbiol, 1998 Jun, 36(6), 1777 - 80 Non-O157:H7 Stx2-producing Escherichia coli strains associated with sporadic cases of hemolytic-uremic syndrome in adults; Bonnet R et al.; From August 1996 to May 1997, six verotoxin-producing Escherichia coli (VTEC) strains were isolated from stool specimens of adults suffering from hemolytic-uremic syndrome (HUS) . All the isolates were stx2 positive and belonged to different serotypes: O6:H4, O91:H10, O91:H21, O rough:H16, OX3:H-, and O nontypeable: H- . The enterohemolysin (Ehly)-encoding genes were detected in two isolates, and none of the isolates harbors the intimin (Eae)-encoding gene . These findings suggest that stx2-positive non-O157:H7 VTEC is a major cause of HUS in adults and that several sources of pathogens are responsible for local endemic infections. J Immunol Methods, 1998 Feb 1, 211(1-2), 1 - 8 Filtration capture and immunoelectrochemical detection for rapid assay of Escherichia coli O157:H7; Brewster JD et al.; A new approach for rapid assay of bacteria in liquid samples is described . Cells were labeled by incubation with an enzyme-antibody conjugate and captured by filtration of the sample/conjugate mixture through a 0.2 microm filter . The enzyme-labeled cells were detected by placing the filter on the surface of an electrode, incubating with enzyme substrate, and measuring the current produced by oxidation of the electroactive enzyme product . Assay time was 25 min and a detection limit of approximately 5000 cells/ml was obtained for E . coli O157:H7 . Background current due to non-specific binding of conjugate to the filter was the primary factor controlling the detection limit, and fewer than 50 cells could be detected when very small sample volumes (10 microl) were used to minimize background current. J Bacteriol, 1998 May, 180(10), 2670 - 5 The wzz (cld) protein in Escherichia coli: amino acid sequence variation determines O-antigen chain length specificity; Franco AV et al.; The O antigen is a polymer with a repeated unit . The chain length in most Escherichia coli strains has a modal value of 10 to 18 O units, but other strains have higher or lower modal values . wzz (cld/rol) mutants have a random chain length distribution, showing that the modal distribution is determined by the Wzz protein . Cloned wzz genes from E . coli strains with short (7 to 16), intermediate (10 to 18), and long (16 to 25) modal chain lengths were transferred to a model system, and their effects on O111 antigen were studied . The O111 chain length closely resembled that of the parent strains . We present data based on the construction of chimeric wzz genes and site-directed mutagenesis of the wzz gene to show that the modal value of O-antigen chain length of E . coli O1, O2, O7, and O157 strains can be changed by specific amino acid substitutions in wzz . It is concluded that the O-antigen chain length heterogeneity in E . coli strains is the result of amino acid sequence variation of the Wzz protein. J Infect Dis, 1998 Jun, 177(6), 1588 - 93 An outbreak of Escherichia coli O157:H7 infections associated with leaf lettuce consumption; Ackers ML et al.; In July 1995, 40 Montana residents were identified with laboratory-confirmed Escherichia coli O157:H7 infection; 52 residents had bloody diarrhea without laboratory confirmation . The median age of those with laboratory-confirmed cases was 42 years (range, 4- 86); 58% were female . Thirteen patients were hospitalized, and 1 developed hemolytic-uremic syndrome . A case-control study showed that 19 (70%) of 27 patients but only 8 (17%) of 46 controls reported eating purchased (not home-grown) leaf lettuce before illness (matched odds ratio, 25.3; 95% confidence interval, 3.9-1065.6) . Pulsed-field gel electrophoresis identified a common strain among 22 of 23 isolates tested . Implicated lettuce was traced to two sources: a local Montana farm and six farms in Washington State that shipped under the same label . This outbreak highlights the increasing importance of fresh produce as a vehicle in foodborne illness . Sanitary growing and handling procedures are necessary to prevent these infections. J Pediatr, 1998 May, 132(5), 777 - 82 Risk of hemolytic uremic syndrome after sporadic Escherichia coli O157:H7 infection: results of a Canadian collaborative study . Investigators of the Canadian Pediatric Kidney Disease Research Center; Rowe PC et al.; OBJECTIVES: The objectives of this study were to better estimate the age-specific risks of hemolytic uremic syndrome (HUS) and hemolytic anemia after Escherichia coli O157:H7 infection among a representative cohort of both referred and nonreferred children with documented illness from the province of Alberta and to compare this with the rates in children evaluated at referral centers in the rest of Canada . STUDY DESIGN: Children with HUS or E . coli O157:H7 gastroenteritis were eligible if they were < 15 years of age . Hemoglobin, blood smear, urinalysis, and serum creatinine were obtained 8 to 10 days after the onset of diarrhea to ascertain for hemolysis, anemia, thrombocytopenia, and renal injury . Subjects were monitored for 1 month . RESULTS: From June 1991 to March 1994, HUS was diagnosed in 205 children . Of these 77% had evidence of E . coli O157:H7 infection . A further 582 children had E . coli O157:H7 gastroenteritis, of whom 18 had hemolytic anemia . The risk of HUS after E . coli O157:H7 infection in Alberta was 8.1% (95% confidence interval, 5.3 to 11.6) compared with 31.4% in referral centers in the rest of Canada . In Alberta the highest age-specific risk of HUS/hemolytic anemia was 12.9% in those < 5 years of age . CONCLUSIONS: These data will help guide clinical care and provide a basis for estimating the sample sizes required in future treatment trials for the secondary prevention of HUS. Epidemiol Infect, 1998 Mar, 120(2), 187 - 92 Studies of the presence of verocytotoxic Escherichia coli O157 in bovine faeces submitted for diagnostic purposes in England and Wales and on beef carcases in abattoirs in the United Kingdom; Richards MS et al.; A survey of beef carcases in abattoirs in the UK was carried out in order to estimate the prevalence of contamination with verocytotoxin-producing Escherichia coli (VTEC) serogroup O157 . Contamination with verocytotoxin-producing E . coli (VTEC) O157 was confirmed in 0.47% of the 4067 (95% confidence limits 0.22-1.00%) of neck muscle samples . A significant tendency for carcases present in the same abattoir on the same day to have similar results was found, thus suggesting cross contamination . VTEC O157 was found in 0.83% of 6495 bovine faeces samples routinely submitted for diagnostic purposes to Veterinary Investigation Centres in England and Wales . Of the samples from cattle less than 6 months old, 3.7% of 68 samples from animals without gastrointestinal disease were positive for E . coli O157, in contrast to 0.75% of 2321 samples from cases of gastrointestinal disease . No association with season or herd type (beef or dairy) was found. J Clin Microbiol, 1998 May, 36(5), 1180 - 4 Insertion element IS3-based PCR method for subtyping Escherichia coli O157:H7; Thompson CJ et al.; An Escherichia coli O157:H7 subtyping method based on PCR amplification of variable DNA sequences between the repetitive element IS3 was developed . Template DNA was prepared by boiling cells in Chelex . Two separate IS3 PCR amplifications were performed for each isolate: one with a single primer (primer IS3A) and one with two primers (primers IS3A and IS3B) . The IS3 PCR subtyping method was applied to 35 epidemiologically related and unrelated E . coli O157:H7 isolates that had been previously characterized by pulsed-field gel electrophoresis (PFGE) . PFGE identified 25 different subtypes (difference of one or more bands) . PCR with single primer IS3A and primer pair IS3A-IS3B identified 6 and 14 different subtypes, respectively . By combining the results of the two PCR amplifications, 15 different IS3 PCR subtypes were identified . While not as sensitive as PFGE, IS3 PCR subtyping grouped all outbreak-related isolates . IS3 PCR banding patterns were reproducible between amplifications and between subcultures . IS3 PCR could serve as a simple, rapid screening method for the identification of unrelated E . coli O157:H7 isolates. Microbiol Immunol, 1998, 42(2), 125 - 8 Protective effect of Japanese green tea extract on gnotobiotic mice infected with an Escherichia coli O157:H7 strain; Isogai E et al.; We examined the effect of Japanese green tea extract (JGTE) on enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection in a gnotobiotic mouse model . Gnotobiotic mice inoculated with an EHEC strain developed neurologic and systemic symptoms, usually culminating in death . In contrast, none of mice receiving dietary JGTE showed clinical signs or death . This report describes the effect of JGTE, which includes the inhibition of bacterial growth in vivo . The Shiga-like toxin (SLT) level in the feces of the JGTE diet group was significantly lower than that of the control group. Anal Biochem, 1998 May 1, 258(2), 293 - 8 Use of a light-addressable potentiometric sensor for the detection of Escherichia coli O157:H7; Gehring AG et al.; We describe the development of an immunoligand assay (ILA) in conjunction with a light-addressable potentiometric sensor (LAPS) for the rapid detection of Escherichia coli O157:H7 cells in buffered saline . The ILA protocol consists of "sandwiching" bacterial analyte between biotinylated and fluoresceinated antibodies, indirect enzyme labeling of the bacteria with urease-labeled anti-fluorescein antibody, and active capture of the immune complex at a biotinylated bovine serum albumin-blocked nitrocellulose filter membrane with streptavidin . Using live E . coli O157:H7, the efficiency of the ILA was compared using various ratios of the biotinylated and fluoresceinated antibodies . Simultaneous addition of equimolar biotinylated and fluoresceinated antibodies effected optimal urease labeling and subsequent active capture of the bacteria in the ILA . Equimolar concentrations of the antibodies were varied to achieve optimal LAPS detection response for the live bacteria . Using ILA with LAPS, a minimum detectable level of ca . 7.1 x 10(2) cells/ml of heat-killed or ca . 2.5 x 10(4) cells/ml of live E . coli O157:H7 bacteria was achieved in Tris-buffered saline in an assay time of ca . 45 or ca . 30 min, respectively. Lett Appl Microbiol, 1998 Feb, 26(2), 93 - 7 Evaluation of DNA preparation techniques for detection of the SLT-1 gene of Escherichia coli O157: H7 in bovine faeces using the polymerase chain reaction; Stewart DS et al.; The polymerase chain reaction (PCR) has the potential to detect low levels of the human pathogen Escherichia coli O157: H7 in bovine faeces . To improve the utility of PCR for this application, several methods for preparing template DNA from bovine faeces, both directly and after non-selective enrichment, were tested . These were boiling, enzyme treatment, enzyme treatment plus phenol-chloroform extraction, and enzyme treatment plus phenol-chloroform extraction plus Geneclean purification . Of these, the boiling method was the most consistent and had a sensitivity of approximately 3 cfu g-1 faeces, with an assay time of less than 32 h . The boiling method was also combined with immunomagnetic separation (IMS) to detect E . coli O157: H7 in less than 8 h, but with a sensitivity of approximately 10(3) cfu g-1 faeces . These methods can be used to prepare template for PCR screening of bovine faeces using any appropriate PCR primers. J Burn Care Rehabil, 1998 Mar-Apr, 19(2), 135 - 7 Hemolytic uremic syndrome in a child with burn injuries; Emil S et al.; We report the first case of hemolytic uremic syndrome in a patient with burns . An unusual case of the syndrome developed in a 1-year-old black girl hospitalized after second-degree burns to 33% of her total body surface area . Acute abdominal distention, hemolytic anemia, hematuria, and oliguric renal failure developed 1 week after admission to the burn unit . Blood cultures grew Escherichia coli O157:H7 . She received supportive care and antibiotics, in addition to low-dose dopamine, which promptly reversed the oliguria . Dialysis was not required, and the child made a complete recovery. J AOAC Int, 1998 Mar-Apr, 81(2), 403 - 18 Direct 24-hour presumptive enumeration of Escherichia coli O157:H7 in foods using hydrophobic grid membrane filter followed by serological confirmation: collaborative study; Entis P; Fifteen laboratories took part in a collaborative study to validate a method for enumerating Escherichia coli O157:H7 . The method is based on use of a hydrophobic grid membrane filter and consists of 24 h presumptive enumeration on SD-39 Agar and serological confirmation to yield a confirmed E . coli O157:H7 count . Six food products were analyzed: pasteurized apple cider, pasteurized 2% milk, cottage cheese, cooked ground pork, raw ground beef, and frozen whole egg . The test method produced significantly higher confirmed count results than did the reference method for milk, pork, and beef . Test method results were numerically higher than but statistically equivalent to reference method results for cheese, cider, and egg . The test method produced lower repeatability and reproducibility values than did the reference method for most food/inoculation level combinations and values very similar to those of the reference method for the remaining combinations . Overall, 94% of presumptive positive isolates from the test method were confirmed serologically as E coli O157:H7, and 98% of these were also biochemically typical of E . coli O157:H7 (completed test) . Corresponding rates for the reference method were 69 and 98%, respectively . On the basis of the results of this collaborative study and the precollaborative study that preceded it, it is recommended that this method be adopted official first action for enumeration of E . coli O157:H7 in meats, poultry, dairy foods, infant formula, liquid eggs, mayonnaise, and apple cider. Lancet, 1998 Apr 4, 351(9108), 1019 - 22 Risk factors for and prevention of sporadic infections with vero cytotoxin (shiga toxin) producing Escherichia coli O157; Parry SM et al.; BACKGROUND: Recent outbreaks of vero cytotoxin (shiga toxin) producing Escherichia coli O157 (VTEC O157) infection have stimulated debate on food safety . However, 90% of cases in England and Wales are sporadic . We report a case-control study of sporadic VTEC O157 infection . METHODS: We compared 85 sporadic cases of VTEC O157 infection, identified through population surveillance, with 142 controls, randomly selected from general practitioners' lists . We matched cases and controls for age, sex, and family doctor's practice . Exposures to foods, water, animals, farms, and environmental factors were recorded . We visited the premises concerned when cases had eaten beefburgers or cooked sliced meats from caterers or had had contact with a farm . FINDINGS: Consumption of a beefburger from a catering premises other than from a fast-food chain A (a national chain) and consumption of cold cooked sliced meat (eg, in a salad or sandwich) from caterers, but not butchers, was associated with VTEC O157 infection (odds ratios 4.63 {95% CI 1.33-30.14} and 3.36 {1.04-12.74}, respectively) . Policies for ensuring thorough cooking of burgers by one national fast-food chain differed from the other catering premises we visited . There was evidence of person-to-person spread and transmission of VTEC O157 infection from animals . INTERPRETATION: Local inspection of catering establishments that serve cooked meats together with public education to prevent spread on farms and in houses would reduce the burden of VTEC O157 infection by about 10% for each risk factor. Appl Environ Microbiol, 1998 Apr, 64(4), 1390 - 9 Longitudinal study of Escherichia coli O157:H7 dissemination on four dairy farms in Wisconsin; Shere JA et al.; A 14-month longitudinal study was conducted on four dairy farms (C, H, R, and X) in Wisconsin to ascertain the source(s) and dissemination of Escherichia coli O157:H7 . A cohort of 15 heifer calves from each farm were sampled weekly by digital rectal retrieval from birth to a minimum of 7 months of age (range, 7 to 13 months) . Over the 14 months of the study, the cohort heifers and other randomly selected cattle from farms C and H tested negative . Farm R had two separate periods of E . coli O157:H7 shedding lasting 4 months (November 1995 to February 1996) and 1 month (July to August 1996), while farm X had at least one positive cohort animal for a 5-month period (May to October 1996) . Heifers shed O157:H7 strains in feces for 1 to 16 weeks at levels ranging from 2.0 x 10(2) to 8.7 x 10(4) CFU per g . E . coli O157:H7 was also isolated from other noncohort cattle, feed, flies, a pigeon, and water associated with the cohort heifers on farms R and/or X . When present in animal drinking water, E . coli O157:H7 disseminated through the cohort cattle and other cattle that used the water source . E . coli O157:H7 was found in water at < 1 to 23 CFU/ml . Genomic subtyping by pulsed-field gel electrophoresis demonstrated that a single O157:H7 strain comprised a majority of the isolates from cohort and noncohort cattle, water, and other positive samples (i.e., from feed, flies, and a pigeon, etc.) on a farm . The isolates from farm R displayed two predominant XbaI restriction endonuclease digestion profiles (REDP), REDP 3 and REDP 7, during the first and second periods of shedding, respectively . Six additional REDP that were > or = 89% similar to REDP 3 or REDP 7 were identified among the farm R isolates . Additionally, the REDP of an O157:H7 isolate from a heifer on farm R in 1994 was indistinguishable from REDP 3 . Farm X had one O157:H7 strain that predominated (96% of positive samples had strains with REDP 9), and the REDP of an isolate from a heifer in 1994 was indistinguishable from REDP 9 . These results suggest that E . coli O157:H7 is disseminated from a common source on farms and that strains can persist in a herd for a 2-year period. FEMS Immunol Med Microbiol, 1997 Dec, 19(4), 285 - 8 Culture supernatant of Shiga toxin-producing Escherichia coli strains provoke fluid accumulation in rabbit ileal loops; Ferreira AJ et al.; Production of Shiga toxin (Stx) in Escherichia coli strains belonging to serogroups O26, O111, and O157 was evaluated in the rabbit ileal loop assay and results were compared to those using tissue culture assays and DNA hybridization with specific probes for Stx1 and Stx2 . All 14 Shiga toxin-producing E . coli strains tested provoked fluid accumulation in the rabbit intestinal loop . Eleven strains hybridized with Stx1 probe, one strain with Stx2 and two strains with both probes . Filtered culture supernatants of all E . coli strains presented cytotoxic effects in both HeLa and Vero cells . In this study, we found a strong association between the production of Stx and its effect in an animal model . This is the first description of high-level Stx-producing E . coli O111ac isolated in Brazil. J Infect Dis, 1998 Apr, 177(4), 962 - 6 A nationwide case-control study of Escherichia coli O157:H7 infection in the United States; Slutsker L et al.; Risk factors for Escherichia coli O157:H7 infection were investigated in a case-control study at 10 medical centers throughout the United States . Among 73 case-patients and 142 matched controls, exposures in the 7 days before illness associated with E . coli O157:H7 infection in univariate analysis included consumption of hamburger (matched odds ratio {MOR}, 3.8; 95% confidence interval {CI}, 1.9-7.9), undercooked hamburger (MOR, 4.5; 95% CI, 1.6-12.2), or hot dogs (MOR, 2.2; 95% CI, 1.1-4.4); eating at a fast-food restaurant (MOR, 2.3; 95% CI, 1.1-4.6); drinking unchlorinated well water (MOR, 2.4; 95% CI, 1.1-5.7); swimming in a pond (MOR, 5.4; 95% CI, 1.1-26.0); and having a household member with diarrhea (MOR, 11.9; 95% CI, 2.7-53.5) . In multivariate analysis, only eating undercooked hamburger remained associated with infection . Seven (8%) of 93 patients developed hemolytic uremic syndrome and 1 died . Prevention strategies aimed at modifying risk factors may help to reduce the risk of infection with E . coli O157:H7. Anal Chem, 1998 Mar 15, 70(6), 1108 - 11 Diffraction-based cell detection using a microcontact printed antibody grating; St John PM et al.; An optical detector has been fabricated that is specific for targeted bacterial cells, by stamping an antibody grating pattern on a silicon surface . The antibody grating alone produces insignificant optical diffraction, but upon immunocapture of cells, the optical phase change produces a diffraction pattern . This technique eliminates much of the surface modifications and the secondary immunochemical or enzyme-linked steps that are common in immunoassays . Microcontact printing provides an alternative to previously reported photolithographic-mediated antibody patterning processes and uses a photolithographic process simply to produce the elastomeric stamp . We have stamped antibodies directly onto clean native oxide silicon substrates with no other chemical surface treatments . Direct binding of the antibodies to the silicon occurs in a way that still allows them to function and selectively bind antigen . The performance of the sensor was evaluated by capturing Escherichia coli O157:H7 cells on the antibody-stamped lines and measuring the intensity of the first-order diffraction beam resulting from the attachment of cells . The diffraction intensity increases in proportion to the cell density bound on the surface. Infect Immun, 1998 Apr, 66(4), 1726 - 34 Enhancement of susceptibility to Shiga toxin-producing Escherichia coli O157:H7 by protein calorie malnutrition in mice; Kurioka T et al.; Infection with Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli is increasing among children . In this study, 5-week-old C57BL/6 mice with protein calorie malnutrition (PCM) that had been fed a 5% protein diet for 2 weeks since ablactation were inoculated intragastrically with 2 x 106 CFU of Stx-producing E . coli O157:H7 . More than 75% of infected mice with PCM died by 10 days postinfection . Infected mice with PCM developed neurologic symptoms 5 days after infection, while well-nourished control mice receiving a 25% protein diet did not . In the intestinal tracts of infected mice with PCM, inoculated E . coli O157:H7 multiplied between days 2 and 4 of infection, with a peak of growth at day 4 . Although the pathogens were not culturable from the stool after day 7, 0157 lipopolysaccharide was detectable in the stool by enzyme-linked immunosorbent assay even after day 8 . Stx was detectable in the stool after day 2 of infection and increased in proportion to the growth of inoculated organisms . The maximal production of Stx occurred at 4 days postchallenge, and Stx was detectable in the blood on days 3 to 5 . In contrast, well-nourished control mice survived the infection, and all of them remained well even after 3 weeks of infection . In these control mice, inoculated E . coli O157:H7 disappeared from the stool before day 3 . Stx was not detectable in the stool and blood of infected control mice at any time from day 1 through day 8 . Histologically, cerebral hemorrhages seemed to be the cause of acute death of infected mice with PCM . Immunocytochemical staining demonstrated the positive immunoreaction to Stx at the alveus and stratum pyramidale of the hippocampus and in renal tubules of infected malnourished mice . Such immunoreactions were not found in tissues from infected control mice . Histological study of the intestinal epithelium before infection showed that PCM severely affected the development of intestinal epithelia . These findings strongly indicate that PCM-induced nondevelopment of intestinal physical barrier is one of the predisposing factors for infection with Stx-producing E . coli O157:H7 in mice and suggest that our mouse model may explain the high incidence of infection with Stx-producing E . coli O157:H7 in the children whose intestinal epithelia have not yet completely developed. Infect Immun, 1998 Apr, 66(4), 1688 - 96 Divergent signal transduction responses to infection with attaching and effacing Escherichia coli; Ismaili A et al.; Shiga toxin-producing Escherichia coli (STEC) O157:H7 is an attaching and effacing pathogen that causes hemorrhagic colitis and the hemolytic-uremic syndrome . Although this organism causes adhesion pedestals, the cellular signals responsible for the formation of these lesions have not been clearly defined . We have shown previously that STEC O157:H7 does not induce detectable tyrosine phosphorylation of host cell proteins upon binding to eukaryotic cells and is not internalized into nonphagocytic epithelial cells . In the present study, tyrosine-phosphorylated proteins were detected under adherent STEC O157:H7 when coincubated with the non-intimately adhering, intimin-deficient, enteropathogenic E . coli (EPEC) strain CVD206 . The ability to be internalized into epithelial cells was also conferred on STEC O157:H7 when coincubated with CVD206 ({158 +/- 21} % of control) . Neither the ability to rearrange phosphotyrosine proteins nor that to be internalized into epithelial cells was evident following coincubation with another STEC O157:H7 strain or with the nonsignaling espB mutant of EPEC . E . coli JM101(pMH34/pSSS1C), which overproduces surface-localized O157 intimin, also rearranged tyrosine-phosphorylated and cytoskeletal proteins when coincubated with CVD206 . In contrast, JM101 (pMH34/pSSS1C) demonstrated rearrangement of cytoskeletal proteins, but not tyrosine-phosphorylated proteins, when coincubated with intimin-deficient STEC (strains CL8KO1 and CL15) . These findings indicate that STEC O157:H7 forms adhesion pedestals by mechanisms that are distinct from those in attaching and effacing EPEC . Taken together, these findings point to diverging signal transduction responses to infection with attaching and effacing bacterial enteropathogens. Infect Immun, 1998 Apr, 66(4), 1680 - 7 Signal transduction pathways involved in enterohemorrhagic Escherichia coli-induced alterations in T84 epithelial permeability; Philpott DJ et al.; Enterohemorrhagic Escherichia coli (EHEC) infection is associated with watery diarrhea and can lead to complications, including hemorrhagic colitis and the hemolytic-uremic syndrome . The mechanisms by which these organisms produce diarrheal disease remain to be elucidated . Changes in T84 epithelial cell electrophysiology were examined following EHEC infection . T84 cell monolayers infected with EHEC O157:H7 displayed a time-dependent decrease in transepithelial resistance . Increases in the transepithelial flux of both {3H}mannitol and 51Cr-EDTA accompanied the EHEC-induced decreases in T84 resistance . Altered barrier function induced by EHEC occurred at the level of the tight junction since immunofluorescent staining of the tight-junction-associated protein ZO-1 was disrupted when examined by confocal microscopy . Decreased resistance induced by EHEC involved a protein kinase C (PKC)-dependent pathway as the highly specific PKC inhibitor, CGP41251, abrogated the EHEC-induced drop in resistance . PKC activity was also increased in T84 cells infected with EHEC . Calmodulin and myosin light chain kinase played a role in EHEC-induced resistance changes as inhibition of these effector molecules partially reversed the effects of EHEC on barrier function . These studies demonstrate that intracellular signal transduction pathways activated following EHEC infection link the increases in T84 epithelial permeability induced by this pathogen. Epidemiol Infect, 1998 Feb, 120(1), 17 - 20 Escherichia coli O157:H7 diarrhoea associated with well water and infected cattle on an Ontario farm; Jackson SG et al.; A 16-month old female child living on an Ontario dairy farm was taken to hospital suffering from bloody diarrhoea . Escherichia coli O157:H7 was isolated from her stool . Initial tests of well water samples were negative for E . coli by standard methods but culture of selected coliform colonies on sorbitol-MacConkey agar led to isolation of E . coli O157:H7 . E . coli O157:H7 was also isolated from 63% of cattle on the farm . The E . coli O157:H7 isolates from the child, the water and the cattle were phage type 14, produced verotoxins 1 and 2, and were highly related on analysis by pulsed field gel electrophoresis . The child did not have known direct contact with the cattle and did not consume unpasteurized milk . Hydrogeological investigation revealed the design and location of the well would allow manure-contaminated surface water to flow into the well . This investigation demonstrates that cattle farm well water is a potential source of E . coli O157:H7 which may not be identified by standard screening for E . coli in water. Pediatrics . 1997 Jul;100(1):E12. Predictors of hemolytic uremic syndrome in children during a large outbreak of Escherichia coli O157:H7 infections; Bell BP et al.; OBJECTIVE: To evaluate risk factors for progression of Escherichia coli O157:H7 infection to the hemolytic uremic syndrome (HUS) . STUDY DESIGN: We conducted a retrospective cohort study among 278 Washington State children <16 years old who developed symptomatic culture-confirmed E coli O157:H7 infection during a large 1993 outbreak . The purpose of the study was to determine the relative risk (RR) of developing HUS according to demographic characteristics, symptoms, laboratory test results, and medication use in the first 3 days of illness . RESULTS: Thirty-seven (14%) children developed HUS . In univariate analysis, no associations were observed between HUS risk and any demographic characteristic, the presence of bloody diarrhea or of fever, or medication use . In multivariate analysis, HUS risk was associated with, in the first 3 days of illness, use of antimotility agents (odds ratio {OR} = 2.9; 95% confidence interval {CI} 1.2-7.5) and, among children <5.5 years old, vomiting (OR = 4 . 2; 95% CI 1.4-12.7) . Among the 128 children tested, those whose white blood cell (WBC) count was >/=13 000/microL in the first 3 days of illness had a 7-fold increased risk of developing HUS (RR 7 . 2; 95% CI 2.8-18.5) . Thirteen (38%) of the 34 patients with a WBC count >/=13 000/microL developed HUS, but only 5 (5%) of the 94 children whose initial WBC count was <13 000/microL progressed to HUS . Among children who did not develop HUS, use of antimotility agents in the first 3 days of illness was associated with longer duration of bloody diarrhea . CONCLUSIONS: Prospective studies are needed to further evaluate measures to prevent the progression of E coli O157:H7 infection to HUS and to assess further clinical and laboratory risk factors . These data argue against the use of antimotility agents in acute childhood diarrhea . Our finding that no intervention decreased HUS risk underscores the importance of preventing E coli O157:H7 infections. J Clin Microbiol, 1998 Mar, 36(3), 727 - 33 Genomic comparisons and Shiga toxin production among Escherichia coli O157:H7 isolates from a day care center outbreak and sporadic cases in southeastern Wisconsin; Gouveia S et al.; Contour-clamped homogeneous electric field pulsed-field gel electrophoresis (CHEF-PFGE) was used to compare Wisconsin isolates of Escherichia coli O157:H7, including 39 isolates from a 1994 day care center outbreak, 28 isolates from 18 individuals from the surrounding geographic area with sporadic cases occurring during the 3 months before the outbreak, and 3 isolates, collected in 1995, from patients with hemolytic-uremic syndrome (HUS) who were from eastern Wisconsin counties other than those inhabited by the day care center and sporadic-case individuals . The technique of CHEF-PFGE using XbaI identified seven highly related restriction endonuclease digestion profiles (REDPs) (93 to 98% similarity) among the 39 day care center isolates and nine XbaI REDPs (63 to 93% similarity) among the 28 isolates from sporadic-case individuals, including REDP 33, which was exhibited by both day care and sporadic-case isolates . PFGE analyses of sequential E . coli O157:H7 isolates from symptomatic day care center attendees revealed that the REDPs of 25 isolates from eight patients were indistinguishable whereas the REDPs of 2 of 6 isolates from two patients differed slightly (93 to 95% similarity) . The REDPs of the three isolates from 1995 HUS patients were 78 to 83% similar, with REDP 26 being exhibited by one HUS-associated isolate and an isolate from one day care attendee who did not develop HUS . The genes for both Shiga toxins I and II (stx1 and stx2, respectively) were detected in all but one isolate (sporadic case), and Shiga toxin production by the day care center isolates was not significantly different from that of the other isolates, including the three HUS-associated isolates . Analyses of E . coli O157:H7 isolates from both the day care center outbreak and sporadic cases by CHEF-PFGE permitted us to define the REDP variability of an outbreak and geographic region and demonstrated that the day care center outbreak and a HUS case in 1995 were caused by E . coli O157:H7 strains endemic to eastern Wisconsin. Kansenshogaku Zasshi, 1998 Jan, 72(1), 40 - 4 {Evaluation of commercial kits for a rapid detection of Escherichia coli O157 in stool}; Tsukamoto T et al.; To introduce rapid detection of E . coli O157 in the stool without culturing, nine different commercial kits for detection of E . coli O157 were compared . VIP, Reveal, Path Stik, Quix, Now EH and EZ coli were found to detect E . coli O157 greater than 10(6) CFU/ml . TECRA and Novapath could detect more than 10(5) CFU/ml . VIDAS reacted positive with more than 10(4) CFU/ml . TECRA, Novapath, Quix could detect 1:4, 1:4, 1:9 dilution of stool specimen spiked with E . coli O157 respectively, but others could not detect with 1:9 dilution of stool specimen . When stool specimens spiked with E . coli O157 were diluted ten times, centrifuged slightly and supernatants were tested, Path Stik, Quix and EZ coli could detect 10(7) CFU/ml . TECRA and Novapath could detect 10(6) CFU/ml, whereas VIDAS could detect 10(6) CFU/ml . Quix is therefore one of the most suitable kit for detection of E . coli O157 in the stool at the bedside, although some non-specific reactions were observed . Path Stik is also convenient kit at the bedside . VIDAS, although it requires special equipment, is the most sensitive kit for detection of E . coli O157 and is suitable for detection of low levels of E . coli O157 in the clinical laboratory. Appl Environ Microbiol, 1998 Mar, 64(3), 1153 - 6 Detection of Escherichia coli O157:H7 in the beef marketed in Malaysia; Radu S et al.; Twelve strains of Escherichia coli O157:H7 were isolated from 9 of 25 beef samples purchased from retail stores in Malaysia . These strains produced Shiga toxin 2 with or without Shiga toxin 1 and had the eae gene and a 60-MDa plasmid . The antibiograms and the profiles of the arbitrarily primed PCR of the strains were diverse, suggesting that the strains may have originated from diverse sources. Mol Cell Probes, 1997 Dec, 11(6), 397 - 406 Detection and characterization of the fimA gene of Escherichia coli O157:H7; Li B et al.; An expected 850-bp DNA fragment containing fimA, the structural gene for type 1 fimbriae, and flanking sequences was amplified from 39 (of 46) pathogenic and commensal strains of Escherichia coli using the polymerase chain reaction (PCR) . Restriction fragment length polymorphism (RFLP) analysis of the amplified products showed 13 HinP1 and four Sau961 restriction profiles among these 39 E . coli strains, revealing the polymorphic nature of this allele . A unique RFLP pattern was shared by E . coli O157:H7, O157:H- and a few O55 serotype strains . DNA sequence analysis of the fimA region demonstrated that E . coli O157:H7 strain 933 and O157:H- strain E32511 contained identical DNA sequences that were distinct from other E . coli strains, especially a 16-bp sequence 5' to fimA that was conspicuously absent only in E . coli O157 strains . Exploiting these differences, a PCR assay was developed that amplifies a 936-bp fragment from all E . coli O157:H7 strains examined to date . This PCR assay offers a simple, rapid, and reliable means to detect E . coli strains of the O157:H7 serotype. Lett Appl Microbiol, 1998 Jan, 26(1), 31 - 4 Heat shock response enhances acid tolerance of Escherichia coli O157:H7; Wang G et al.; Escherichia coli O157:H7 (O157) has unusual acid tolerance . The influence of heat shock on acid tolerance of O157 was studied . Seven strains of O157 and E . coli K-12 were tested for their ability to survive in minimum glucose medium (pH 2.5) at 37 degrees C . The survival of heat-shocked (10 min at 48 degrees C) cells was about 10-100 times greater compared with untreated cells depending on the strain . No significant difference (P > 0.05) for O157 strain 932 was observed between heat shock-induced and acid adaptation-induced (pH 5.0) acid tolerance . Chloramphenicol prevented heat shock-induced acid tolerance, indicating the requirement of newly synthesized protein(s) . Two outer membrane proteins (OMP) (22 and 15 kDa) were synthesized within 10 min of heat shock and were expressed for at least 6 h by cells held at 37 degrees C . N-terminal amino acid sequence analysis suggested that the 22 kDa OMP is a component of an alkyl hydroperoxide reductase . This protein contains a redox active disulphide, which is probably involved in H+ transport . Results indicate that sublethal heat treatment of O157 cells substantially increases their tolerance to acidic conditions . This could have practical implications for foods that receive a mild heat treatment and rely on acid as a preservative. Kansenshogaku Zasshi, 1997 Dec, 71(12), 1232 - 7 {Genotype analysis of enterohemorrhagic Escherichia coli O157:H7 strains isolated in Iwate Prefecture from 1996 to February 1997}; Ohara-Nemoto Y et al.; Genotypes of 38 isolates of enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolated from 11 sporadic cases and one outbreak in Iwate Prefecture from 1996 to February 1997 were studied by pulsed-field gel electrophoresis (PFGE), in comparison with a strain of EHEC O157:H7 isolated in 1992 in Ohazama-Cho, Iwate Prefecture, and two isolates of EHEC O157:NM . In order to substantiate the genotypes classified by PFGE, Southern blotting was performed to investigate integration sites of the Shiga toxin genes (stx) in the XbaI-digested genome DNA fragments . The stx1 gene existed on an approximately 130 kb fragment in all isolates except two ones . On the other hand, the stx2 gene was observed on 11 DNA fragments in different length from 600 kb to 155 kb, indicating that the stx2 gene integrates into more heterogeneous sites of genome DNA than stx1 does . From these analyses, EHEC O157:H7 isolates examined were classified into 7 genotypes . Since half of the isolates were the same genotype as that of the isolate in 1992, it is suggested that this type of EHEC O157:H7 strain is expanding from Ohazama-Cho and Morioka City in Iwate Prefecture. Acta Microbiol Immunol Hung, 1997, 44(3), 257 - 69 Detection of VTEC using specific DNA probes and complex typing of Escherichia coli O157; Milch H et al.; The incidence of E . coli causing hemorrhagic colitis (HC) or non-bloody enteritis in Hungary was studied using SLT-I and SLT-II gene-probes as well as Vero-cell toxicity and Verotox-F tests . Out of 41 E . coli O157 strains isolated in Hungary between 1987 and 1996 15 strains (O157:HNM 4, O157:H77 8, O157:HNT 3) derived from hemorrhagic colitis (HC) . Hybridization was observed with SLT-I and/or SLT-II in 19 strains . Verocytotoxin production of E . coli of 23 other serotypes was proven by hybridization of DNA probes . SLT production were demonstrated in 24 strains . Complex typing (sero-, phage-, colicin-typing and plasmid profile analysis) was carried out in E . coli serogroup O157 strains isolated from different geographical areas . Using the Hungarian phages the E . coli O157:HNM, O157-H7 strains could be distributed into 6 phage groups each and these phage groups could be further divided according to colicin production and plasmid profile . The Hungarian phage typing method for E . coli strains used since 1978 was compared to the method elaborated in Canada in 1990 . Out of the most frequent Canadian phage types (1, 4, 8, 31, 14) phage types 8, 31 and 14 were observed in Hungary. Microbios, 1997, 91(366), 37 - 46 Development of a spectrophotometric immuno-agglutination assay for quantitation of IgG for Escherichia coli O157; Abolmaaty A et al.; A direct spectrophotometric immuno-agglutination assay for quantitation of specific Escherichia coli O157 IgG was developed . Initial linear rates of increase in absorbance at 550 nm as a result of agglutination were found to increase with both cell and antiserum concentrations . Optimum conditions consisted of 1 x 10(8) cells/ml, 40 degrees C, and 0.005 M phosphate buffer (PB) containing 0.05% NaCl and 0.02% sodium azide at pH 7.4 . A completely linear increase in absorbance was obtained with affinity purified IgG under optimum conditions of the assay . The useful range of the assay was between 13 and 104 micrograms of O157 specific IgG per ml of reaction mixture. J Clin Microbiol, 1998 Jan, 36(1), 24 - 9 Structure and function of plasmid pColD157 of enterohemorrhagic Escherichia coli O157 and its distribution among strains from patients with diarrhea and hemolytic-uremic syndrome; Hofinger C et al.; In this study, pColD157, a 6.7-kb colicinogenic plasmid of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain CL40cu, was characterized by restriction mapping and determination of its complete nucleotide sequence . The sequence consists of 6,675 bp and shows a high degree of similarity to the nucleotide sequence of colicinogenic plasmids pColD-CA23 and pColK . Seven potential genes were located on pColD157, three of which were closely related (>97.9%) to the colicin D structural gene and the corresponding immunity and lysis genes of plasmid pColD-CA23, and these were therefore designated cda, cdi, and cdl, respectively, using the reference extension -CL40 for differentiation . The adjacent 3' region is related to the origin of replication of pColD-CA23 . In contrast, the remaining part of the plasmid harbors a cluster of genes, closely related to the mobilization genes of pColK, which is followed by a 0.3-kb stretch homologous to the pColK resolution function . These determinants were designated mbdA, mbdB, mbdC, and mbdD and cdr, respectively . Southern blot analysis was performed with a probe specific for the cda gene of pColD157 and two groups of EHEC O157:H7 isolates from patients with diarrhea or hemolytic-uremic syndrome resident in Germany . Whereas 16 of 46 E . coli O157 strains isolated between 1987 and 1991 harbored plasmid pColD157, only 1 of 50 strains isolated during 1996 carried this plasmid . In addition, all strains harboring plasmid pColD157 were shown to have colicinogenic activity. Wien Klin Wochenschr, 1997 Sep 19, 109(17), 669 - 77 {Enterohemorrhagic Escherichia coli and hemolytic-uremic syndrome}; Allerberger F et al.; Enterohemorrhagic Escherichia coli (EHEC) are increasingly identified as the cause of diarrhea and hemorrhagic colitis in countries with highly developed livestock . In 5-10% of patients, full-blown hemolytic uremic syndrome (HUS) occurs as a postinfectious life-threatening complication . Up to 1996, 5 out of 39 patients (12.8%) with EHEC O157 infections in Austria developed HUS . Acute complications of HUS such as brain edema may also lead to death; one fatal outcome has been observed so far in Austrian patients . Aside from the cytotoxic Shiga toxins, other different pathogenic factors are often found in clinical EHEC isolates . These include a cytolysin termed EHEC-hemolysin and a low molecular heat-stabile enterotoxin . Furthermore, most EHEC strains express an important surface protein, intimin, which is important for adherence to intestinal epithelial cells . EHEC are heterogeneous in their antigenic structure (O-, H-antigens) . In Austria O157:H7 and O157:H- are the dominating serogroups; in 1997 the first Austrian case of HUS due to EHEC O26:H11 was documented . Because there are no known reliable phenotypical markers for EHEC, diagnostic strategies should focus on the demonstration of Shiga toxins or Shiga toxin genes . For epidemiological purposes it is also important to attempt to isolate the causative agent . Cows and other ruminants are reservoirs for EHEC . In the Tyrol 3% of unpasteurised milk samples, up to 10% of minced beef samples, and 6% of calves yield EHEC O157 . Aside from transmission via contaminated food, direct transmission from person to person also plays a major role in the chain of EHEC infection . In contrast to Italy and Bavaria, Austria has not experienced a major outbreak due to this organism so far . A nationwide surveillance system of HUS has shown an incidence of 0.37 HUS cases per 100,000 residents in the age group 0-14 years for 1995 (Italy: 0.2 cases per 100,000; Bavaria: approx . 1.5 cases per 100,000). Kansenshogaku Zasshi, 1997 Nov, 71(11), 1131 - 6 {Enterohemorrhagic Escherichia coli O157 outbreak in Obihiro-city--biological and molecular characteristics among O157 isolates}; Makino S et al.; The outbreak of enterohaemorrhagic Escherichia coli (EHEC) O157:H7 infection in Obihiro City, Japan, occurred in late October 1996 . The infection affected a total of 169 kindergarten pupils and school staff members in a private kindergarten . Twenty-one children (12.4%) progressed into hemolytic uremic syndrome (HUS) . Moreover, the person-to-person infections in 9 families and the duration of excretion of EHEC in 13 patients were observed . The contaminated food was identified as the potato-salad served at lunch . Analysis of biological characteristics, the ability of toxin production, and the DNA analysis by PCR-based fingerprinting, the RAPD tests, among all clinical isolates, clarified a homologous origin of contamination. Infect Immun, 1998 Feb, 66(2), 636 - 44 Apoptosis of renal cortical cells in the hemolytic-uremic syndrome: in vivo and in vitro studies; Karpman D et al.; This study examined apoptotic cell death associated with Shiga-like toxin (Stx)-producing Escherichia coli . Renal cortices from three children with postenteropathic hemolytic-uremic syndrome (HUS) and from mice infected with E . coli O157:H7 and pediatric renal tubular epithelial cells stimulated with Stx and E . coli O157:H7 extracts were examined for apoptotic changes . Apoptotic cells were detected by terminal dUTP nick end labeling of tubuli and glomeruli from HUS patients and from mice inoculated with Stx-2-positive and Stx-negative strains . Apoptosis was more extensive and severe ultramorphological nuclear and cytoplasmic changes were seen in the Stx-2-positive group . Stx caused DNA fragmentation and ultramorphological changes indicating apoptosis in cultured pediatric tubular cells . DNA fragmentation increased when cells were pre-stimulated with tumor necrosis factor alpha . Polymyxin extracts from Stx-2-positive and Stx-negative strains induced DNA fragmentation, but only extracts from Stx-2-positive strains caused ultramorphological changes and extensive DNA fragmentation . The results indicate that HUS is accompanied by increased apoptosis of kidney cells and that bacterial factors, possibly together with host cytokines in vivo, may activate apoptotic tissue injury. J Clin Invest, 1998 Jan 15, 101(2), 372 - 82 Verotoxin and ricin have novel effects on preproendothelin-1 expression but fail to modify nitric oxide synthase (ecNOS) expression and NO production in vascular endothelium; Bitzan MM et al.; Interaction of bipartite Escherichia coli O157-derived verotoxins (VTs) 1 and 2 (Shiga toxin 1 and 2) with vascular endothelium is believed to play a central role in the pathogenesis of the thrombotic microangiopathy and ischemic lesions characteristic of hemolytic uremic syndrome and of E . coli O157-associated hemorrhagic colitis . We defined the effects of VTs on the expression of potent endothelial cell-derived regulators of vascular wall function, namely endothelin-1 (ET-1) and nitric oxide (NO) . In quiescent bovine aortic endothelial cells, both VT1 and VT2, but not receptor-binding VT B-subunit which lacks N-glycosidase activity, induced concentration-dependent (0.1-10 nM) increases in steady state preproET-1 mRNA transcript levels, an effect that was maximal at 12-24 h . Metabolic-labeling experiments indicated that VTs increased preproET-1 mRNA transcript levels at concentrations that had trivial effects on nascent DNA, RNA, and protein synthesis . In contrast to preproET-1, endothelin converting enzyme-1 and endothelial constitutive NO synthase mRNA transcript levels remained unchanged . Consistent with these findings, VTs failed to modulate immunoreactive endothelial constitutive NO synthase expression and basal and calcium-dependent L-{14C}arginine to L-{14C}citrulline conversion or the NO chemiluminescence signal . The plant-derived toxin ricin, which shows a similar molecular mechanism of enzymatic ribosomal modification to VTs, caused comparable effects on these endothelial vasomediators and metabolite incorporation, at 3 log orders lower concentrations . Nuclear transcription and actinomycin D chase experiments indicated that VTs stabilize labile preproET-1 mRNA transcripts in endothelial cells . Therefore, VTs potently increase select mRNA transcript levels in endothelial cells at concentrations of toxins that have minimal effects on protein synthesis . Perturbed expression of endothelial-derived vasomediators may play a pathophysiologic role in the microvascular dysfunction that is the hallmark of hemolytic uremic syndrome and hemorrhagic colitis. Commun Dis Rep CDR Rev, 1997 Dec 12, 7(13), R206 - 11 A community outbreak of Vero cytotoxin producing Escherichia coli O157 infection linked to a small farm dairy; Clark A et al.; A community outbreak of infection with Vero cytotoxin producing Escherichia coli O157 (VTEC 0157) occurred in a small area of north west England in 1996 . An outbreak control team was established to investigate the outbreak and implement control measures . Nine people developed symptomatic infections with VTEC O157, and a further three were found to be excreting the bacteria . All were infected with the same genotype of VTEC O157 . Three children under 5 years of age and one adult were admitted to hospital . One child developed haemolytic uraemic syndrome . All cases recovered . All primary cases had consumed milk from a particular farm dairy . No other common foods were identified . The farm dairy had a faulty pasteuriser and the potential for post pasteurisation contamination existed . VTEC O157 was isolated from a milk sock specimen and from two cows, but these strains differed from that infecting the cases . All local doctors and the public were alerted and advised about preventative measures . Distribution of unpasteurised milk from the farm was discontinued as was the sale of pasteurised milk when the faulty pasteuriser was discovered . A replacement pasteuriser was installed and checked before milk was released for human consumption . No conclusive evidence of the origin of this outbreak was found, but the farm was the most probable source . The investigations raised concerns about the distribution of VTEC O157 colonised dairy cattle, the natural history of such colonisation, the effectiveness of pasteurisation with respect to the elimination of VTEC O157, and the effectiveness of current legislation for the prevention and control of milkborne infection. Epidemiol Infect, 1997 Dec, 119(3), 299 - 305 Human Escherichia coli O157:H7 infect