Environmental air pollution and DNA adducts in Copenhagen bus drivers--effect of GSTM1 and NAT2 genotypes on adduct levels

Fecal samples from 335 dairy farm residents and 1458 cattle on 80 farms were tested for Vero cytotoxin (VT)-producing Escherichia coli (VTEC). Residents were also tested for antibodies to VT1 and O157 lipopolysaccharide (LPS). Residents and cattle on farms with VTEC-positive persons or E. coli O157:H7-positive cattle were retested. Twenty-one persons (6.3%) on 16 farms (20.8%) and 46% of cattle on 100% of the farms had VTEC in fecal samples. Human VTEC isolates included E. coli O157:H7 and 8 other serotypes, 4 of which were present in cattle on the same farms. More persons had antibodies to VT1 (41%) than to O157 LPS (12.5%). Seropositivity to O157 LPS was associated with isolation of E. coli O157:H7 on the farm (P = .022). Human VTEC infection was negatively associated with age (P < .05) and was not associated with clinical illness. Many dairy farm residents experience subclinical immunizing VTEC infections at a young age, which frequently involve non-O157 VTEC found in cattle.     

Prevalence and clonal nature of Escherichia coli O157:H7 on dairy farms in Wisconsin

A survey was conducted between March and October of 1994 to determine the prevalence and identify the sources of serotype O157:H7 isolates of Escherichia coli in Wisconsin dairy herds. A stratified sample of 400 farms was identified, and 70 farms with weaned calves less than 4 months old were included in the study. During the prevalence study, 5 of the 70 farms (herd prevalence, 7.1 +/- 4.5%) and fecal samples from 10 of 560 calves (animal prevalence, 1.8%) tested positive for serotype O157:H7. In a follow-up study, the five O157:H7-positive farms and seven of the O157:H7-negative farms identified in the prevalence study were visited again. An additional 517 fecal samples from cattle of various ages were tested, and a total of 15 animals from four of the five herds that were previously positive and 4 animals from two of seven herds that were previously negative tested positive for E. coli O157:H7. Observations made during the follow-up study suggested that horizontal transmission was an important means of E. coli O157:H7 dissemination on the farms. A total of 302 environmental samples, were examined, and 2 animal drinking water samples from one previously negative farm and 1 animal drinking water sample from a previously positive farm contained E. coli O157:H7. Analyses by the pulsed-field gel electrophoresis technique of contour-clamped homogeneous electric field electrophoresis revealed that isolates from the same farm displayed identical or very similar XbaI restriction endonuclease digestion profiles (REDP), whereas isolates from different farms typically displayed different REDP. However, more than one REDP was usually observed for a given herd over the 8-month sampling period. Analyses of multiple isolates from an animal revealed that some animals harbored O157:H7 strains that had different REDP, although the REDP of isolates obtained from the same fecal sample were very similar. Collectively, 160 bovine isolates obtained from 29 different animals and three water isolates displayed 20 distinct XbaI REDP. Our data revealed that there are several clonal types of serotype O157:H7 isolates in Wisconsin and indicated that there is probably more than one source of this pathogen on the dairy farms studied. However, animal drinking water was identified as one source of E. coli O157:H7 on one farm.     

Polymorphism in tumor necrosis factor genes associated with myasthenia gravis

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.     

Evaluation of dietary influences on Escherichia coli O157:H7 shedding by sheep

The effect of diet, an abrupt diet change, and fasting on the shedding of Escherichia coli O157:H7 was investigated with experimentally inoculated sheep as a ruminant model. Sheep were fed a grass hay diet (G), which was low in protein and digestible energy and high in fiber, or a mixture of corn and pelleted alfalfa (C), which was high in protein and digestible energy and low in fiber. After a single oral inoculation of E. coli O157:H7, all the animals shed fecal E. coli O157:H7. However, sheep that were fed G shed the bacterium almost twice as long as, and in larger numbers than, did sheep that were fed C. The number of culture-positive animals increased after the diet was abruptly changed from C to G and decreased with the opposite change (G to C). A 24-h fast did not influence E. coli O157:H7 shedding. Horizontal transmission of infection between animals occurred. Recent shedding of E. coli O157:H7 did not affect recolonization with E. coli O157:H7. The findings presented in this study indicate that preharvest control of diet may reduce the risk of E. coli O157:H7-positive animals entering the food chain.     

Prevalence of enterohemorrhagic Escherichia coli O157:H7 in a survey of dairy herds

The prevalence of Escherichia coli O157:H7 in dairy herds is poorly understood, even though young dairy animals have been reported to be a host. From February to May 1993, 662 fecal samples from 50 control herds in 14 states, and from June to August 1993, 303 fecal samples from 14 case herds in 11 states were collected for isolation of E. coli O157:H7. Case herds were those in which E. coli O157:H7 was isolated from preweaned calves in a previous U.S. Department of Agriculture study, whereas control herds from which E. coli O157:H7 had not been isolated previously were randomly selected from the same states as case herds. Among the control herds, E. coli O157:H7 was isolated from 6 of 399 calves (1.5%) that were between 24 h old and the age of weaning and from 13 of 263 calves (4.9%) that were between the ages of weaning and 4 months. Eleven of 50 control herds (22%) were positive. Among the case herds, E. coli O157:H7 was isolated from 5 of 171 calves (2.9%) that were between 24 h old and the age of weaning and from 7 of 132 calves (5.3%) that were between the ages of weaning and 4 months. Seven of 14 case herds (50%) were positive. Sixteen of 31 isolates were obtained by direct plating, with populations ranging from 10(3) to 10(5) CFU/g. Fifteen of 31 isolates were isolated by enrichment only. Nineteen of the isolates produced both verocytotoxin 1 (VT-1) and VT-2, whereas 12 produced VT-2 only.     

Survival and recovery of Escherichia coli O157:H7 in inoculated bottled water

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.     

Jurisdictional trends in opioid overdose deaths, 1988-96

Management factors in 36 Pacific Northwest dairy herds were evaluated for their association with the prevalence of Shiga toxin-positive Escherichia coli O157 (E. coli O157) in dairy cattle. The within-herd prevalence of E. coli O157 was estimated by bacteriological culture of fecal pat samples, collected monthly for 6 months (approximately 60 per visit), from heifer cattle. During the first visit to each farm, a management questionnaire was administered that covered a broad range of animal husbandry practices. On each subsequent visit, a brief questionnaire was administered to detect changes in management practices. A significantly higher prevalence of E. coli O157 was noted in herds that fed corn silage to heifers compared to herds that did not feed corn silage. More tentative associations of E. coli O157 prevalence were observed for weaning method, protein level of calf starter, feeding of ionophores in heifer rations, feeding of grain screens to heifers, and feeding of animal by-products to cows.     

A cluster of Escherichia coli O157:H7 infections with the hemolytic-uremic syndrome and death in California. A mandate for improved surveillance

In mid-January 1993, an outbreak of Escherichia coli O157:H7 infections associated with eating hamburger patties at a fast-food restaurant chain (chain A) was reported in Washington State. From mid-December to mid-January, 9 cases of E coli O157:H7-associated bloody diarrhea and the hemolytic-uremic syndrome had been reported in San Diego County, California. A total of 34 persons had bloody diarrhea, the hemolytic-uremic syndrome, or E coli O157:H7 organisms isolated from stool during the period November 15, 1992, through January 31, 1993. Organisms of E coli O157:H7 identified from 6 persons were indistinguishable from those of the Washington outbreak strain. Illness was associated with eating at chain A restaurants in San Diego (odds ratio, 13; 95% confidence interval, 1.7, 99) and with eating regular-sized hamburgers (odds ratio, undefined; lower-limit 95% confidence interval, 1.3). Improved surveillance by mandating laboratory- and physician-based reporting of cases of E coli O157:H7 infection and the hemolytic-uremic syndrome might have alerted health officials to this outbreak sooner, which could have resulted in earlier investigation and the institution of measures to prevent more cases.     

Use of the flagellar H7 gene as a target in multiplex PCR assays and improved specificity in identification of enterohemorrhagic Escherichia coli strains

PCR products of 1.8 kb were generated with DNAs from all Escherichia coli H7 strains tested by using oligonucleotide primers which flank the fliC gene. Three RsaI digestion profiles of these PCR products were evident on agarose gels; the first occurred with serotype O55:H7, O157:H7, or nonmotile (NM) strains, the second occurred with serotype O1:H7 and O18:H7 strains, and the third occurred with serotype O?:H7, O19:H7, O121:H7, O88:H7, and O156:H7 strains. Despite these differences, the nucleotide sequences of the E. coli E32511 (O157:NM) and U5-41 (O1:H7) fliC genes were 97% homologous. Two PCR primer pairs synthesized on the basis of the E32511 H7 fliC sequence amplified specific DNA fragments from all E. coli H7 strains, but did not amplify DNA fragments from the other bacterial strains. The H7-specific primers were used in combination with other primers which target the Verotoxin 1(VT1) and VT2 genes and the E. coli O157:H7 eaeA gene in multiplex PCR assays. In these assays, vt and eaeA PCR products were observed with DNAs from the majority of EHEC strains and vt, eaeA, and fliC PCR products were observed with DNAs from E. coli O157:H7 or NM strains. Only eaeA PCR products were present with DNA from enteropathogenic E. coli, and only vt PCR products occurred with VT-producing E. coli which are not EHEC. The multiplex PCR assays described allow for the specific identification of E. coli O157:H7 or NM and other EHEC strains.     

Fate of Escherichia coli O157:H7 in ground apples used in cider production

Survival of Escherichia coli O157:H7 in ground Golden Delicious, Red Delicious, Rome, and Winesap apples stored at 4, 10, and 25 degrees C was determined. E. coli O157:H7 populations were monitored for up to 18 days (4 degrees C), 12 days (10 degrees C), and 5 days (25 degrees C), when mold contamination became visible. At 25 degrees C, Red Delicious apples supported survival of E. coli O157:H7 better (P < 0.05) than the other cultivars, followed by Golden Delicious and Rome apples, which were not statistically different (P > 0.05). Winesap apples were the least favorable (P < 0.05) for survival of E. coli O157:H7 at 25 degrees C. E. coli O157:H7 was recovered at similar rates from Golden Delicious and Red Delicious apples, (P > 0.05), but pathogen populations increased in both cultivars (P < 0.05) during storage at 25 degrees C. At 10 degrees C, survival of E. coli O157:H7 was poorest (P < 0.05) in ground Red Delicious apples, while there was no significant difference in survival of E. coli O157:H7 among ground Golden Delicious, Rome, or Winesap cultivars (P > 0.05). When stored at 4 degrees C, Golden Delicious and Rome apples were not statistically different in supporting survival of the pathogen (P > 0.05). In general, apple pH increased during storage and was associated with mold growth. Results of this investigation indicate that there is no trend toward a particular apple cultivar supporting survival of E. coli O157:H7. However, variation in apple pH during storage can negatively or positively influence E. coli O157:H7 survival at 25 degrees C.     

Arthroscopic treatment of partial rotator cuff tears in young athletes. A preliminary report

A cluster of four cases of haemolytic uraemic syndrome in children occurred in Northern Bohemia, Czech Republic, between 15 June and 7 July, 1995. All the cases had significantly elevated titres of anti-O157 lipopolysaccharide (LPS) antibodies as detected by the indirect haemagglutination assay. All but one of them had drunk unpasteurized goat's milk from the same farm within the week before the disease. Evidence of E. coli O157 infection was subsequently found in 5 of 15 regular drinkers of the farm's raw goat's milk; four of them were asymptomatic, 1 had mild diarrhoea at the end of June. Verocytotoxin 2-producing E. coli O157:H7 strains of phage type 2 and of identical pulsed-field gel electrophoresis patterns were isolated from 1 of 2 farm goats and from 1 of the asymptomatic goat's milk drinkers. The frequency of anti-O157 LPS antibodies found among regular drinkers of the farm's raw goat's milk (33%; 5 of 15) was significantly higher than that found in control population (0%; none of 45) (P = 0.0005; Fisher's exact test). Our findings indicate that goats may be a reservoir of E. coli O157:H7 and a source of the infection for humans; raw goat's milk may serve as a vehicle of the pathogen transmission.     

Occurrence of verocytotoxin-producing Escherichia coli O157 on Dutch dairy farms

During the period from September 1996 through November 1996, 10 Dutch dairy farms were visited to collect fecal samples from all cattle present. The samples were examined for the presence of verocytotoxin (VT)-producing Escherichia coli (VTEC) of serogroup O157 (O157 VTEC) by immunomagnetic separation following selective enrichment. Cattle on 7 of the 10 dairy farms tested positive for O157 VTEC, with the proportion of cattle infected varying from 0.8 to 22.4%. On the seven farms positive for O157 VTEC, the excretion rate was highest in calves ages 4 to 12 months (21.2%). In a follow-up study, two O157 VTEC-positive farms and two O157 VTEC-negative farms identified in the prevalence study were revisited five times at intervals of approximately 3 months. Cattle on each farm tested positive at least once. The proportion of cattle infected varied from 0 to 61.0%. Excretion rates peaked in summer and were lowest in winter. Again, the highest prevalence was observed in calves ages 4 to 12 months (11.8%). O157 VTEC strains were also isolated from fecal samples from horses, ponies, and sheep and from milk filters and stable flies. O157 VTEC isolates were characterized by VT production and type, the presence of the E. coli attaching-and-effacing gene, phage type, and pulsed-field gel electrophoretic genotype. No overlapping strain types were identified among isolates from different farms except one. The predominance of a single type at each sampling suggests that horizontal transmission is an important factor in dissemination of O157 VTEC within a farm. The presence of more than one strain type, both simultaneously and over time, suggests that there was more than one source of O157 VTEC on the farms. Furthermore, this study demonstrated that the O157 VTEC status of a farm cannot be ascertained from a single visit testing a small number of cattle.     

The immunohistological diagnosis of E. coli O157:H7 colitis: possible association with colonic ischemia

OBJECTIVE: E. coli O157:H7 may cause hemorrhagic colitis resembling ischemic colitis. Diagnosis is usually made by finding sorbitol-negative colonies on MacConkey agar that react with O157 and H7 antisera. Most ischemic colitis is idiopathic, but some may be caused by E. coli O157:H7, inasmuch as this organism can produce fibrin thrombi in colon vasculature. The objectives of this study were to determine whether E. coli O157:H7 infection can be diagnosed retrospectively from paraffin blocks of colon sections and whether an association exists between E. coli O157:H7 infection and colonic ischemia. METHODS: Paraffin-embedded sections of normal colon (n = 2) and various colitides [ischemic (n = 11), E. coli O157:H7 (n = 2), IBD (n = 8) and pseudomembranous (n = 3)] were used. Sections were deparaffinized, rehydrated, incubated with 3% peroxide in methanol, rinsed, and incubated with peroxidase-labeled antibody isolated from goats immunized with whole E. coli O157:H7. Sections were stained with peroxidase chromagen reagent and counterstained with hematoxylin. Coarse, granular, orange-brown staining was considered positive. To determine the localization of the chromagen deposits, three cases that stained positive, including one of the culture-proved E. coli O157:H7 colitis and two of colonic ischemia, were processed for electron microscopy. RESULTS: Both cases (100%) of E. coli O157:H7 colitis and three of 11 (27.3%) cases of ischemic colitis stained positive by light microscopy. In one culture-proved case, electron microscopy demonstrated staining of bacillary structures; in two cases of colonic ischemia, extensive deposits of chromagen material were present that were associated neither with inflammatory cells nor with bacterial forms. CONCLUSIONS: Immunoperoxidase staining of archival sections may be used to diagnose E. coli O157:H7 infection. An etiological role for this organism is possible in some cases of colonic ischemia.     

Sporadic cases of hemorrhagic colitis associated with Escherichia coli O157:H7 in rural Wisconsin

The epidemiology and clinical aspects of Escherichia coli O157:H7 (E. coli O157:H7) infections in rural Wisconsin have rarely been reported. In the last six years, 66 cases of E. coli O157:H7 infection were encountered at our institution. Bloody diarrhea was the universal presentation and all cases represented apparent sporadic infection as institutional or community-wide outbreaks were not found in our study. The mean age was 31 (range 7 months to 86 years), 25% less than 10 years old and 60% were female. Most cases were seen in summer and early autumn (88%). Two patients (3%) developed hemolytic-uremic syndrome. Case-fatality rate in this study was 1.5%. Antibiotic treatment and hospitalization did not change the course and outcome of the infection. Routine screening of E. coli O157:H7 during winter time (December and January) may not be necessary in our rural area. The understanding gained from our study might foster better infection control.     

Enterohaemorrhagic Escherichia coli O157:H7 infection

Escherichia coli O157:H7 differs from previously described diarrheagenic E. coli classes (enteropathogenic, enteroinvasive, enterotoxigenic) by distinct clinical symptoms, production of verotoxin (VT) and a specific plasmid. Cattle are the primary reservoirs of E. coli O157:H7. The organism may be transmitted through the consumption of contaminated foods (mainly of bovine origin) and by person-to-person contact. The most typical clinical manifestations of E. coli O157:H7 infection are hemorrhagic colitis and hemolytic-uremic syndrome. Since the 1982 many outbreaks of E. coli O157:H7 infections as well as sporadic cases have been documented. Diagnosis of E. coli O157:H7 is based on a positive stool culture, presence of VT and elevated serum antibodies. The best currently available and inexpensive method for diagnosing E. coli O157:H7 is culture of stool on sorbitol-Mac Conkey agar medium.     

Escherichia coli O157:H7 requires intimin for enteropathogenicity in calves

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.     

Detection and characterization of the fimA gene of Escherichia coli O157:H7

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.     

eaeA genes in Escherichia coli derived from Japanese patients with sporadic diarrhea

To investigate the prevalence of attaching and effacing Escherichia coli, we examined 364 strains isolated from the feces of 9,684 patients with diarrhea at the Anjo Kosei Hospital in Japan for the presence of eaeA. Twenty-nine (8%) of the strains were eaeA positive. Of enteropathogenic E. coli (EPEC), 11 of the 87 (13%) strains were for the positive eaeA gene. The serotypes and the numbers of eaeA-positive strains among the strains tested were as follows: O26:H-(2/3), O55:H7 (4/4), O55:H-(2/ 2) and O128:H2(3/3). Two enterohemorrhagic E. coli (EHEC) strains (Verotoxin positive O157:H7) were also eaeA positive. Among 260 non-EPEC strains that were not categorized as diarrheagenic E. coli, 16 (6%) were eaeA positive. Those serotypes were as follows: O15:H2, O20:H6, O28:H28, O63:H6. O153:H7, O28:H6, O153:H19 and O157:H45. EPEC strains including O18:H7 and six other serotypes, enteroinvasive E. coli (EIEC), and enterotoxigenic E. coli (ETEC) were all eaeA negative.     

Evaluation of the premier EHEC assay for detection of Shiga toxin-producing Escherichia coli

An enzyme-linked immunosorbent assay for the detection of Shiga toxins (Premier EHEC assay; Meridian Diagnostics, Inc.) was compared to conventional sorbitol-MacConkey culture for the recovery of enterohemorrhagic Escherichia coli. A total of 74 enteric pathogens, including 8 E. coli O157:H7 isolates, were recovered from 974 stool specimens. Two of these specimens were not tested by Premier assaying due to insufficient sample and are not considered in the data analysis. The Premier EHEC assay detected the 6 evaluable specimens which were culture positive for E. coli O157:H7 and identified an additional 10 specimens as containing Shiga toxin. Seven isolates were recovered from these 10 specimens by an immunoblot assay and were confirmed as toxin producers by a cytotoxin assay. Of these seven, four isolates were serotype O157:H7, one was O26:NM, one was O6:H-, and one was O untypeable:H untypeable. Three specimens contained Shiga toxin by both EHEC immunoassaying and cytotoxin testing; however, no cytotoxin-producing E. coli could be recovered. The sorbitol-MacConkey method had a sensitivity and a specificity of 60 and 100%, respectively, while the Premier EHEC assay had a sensitivity and a specificity of 100 and 99.7%, respectively, for E. coli O157:H7 only. The Premier EHEC assay also detected an additional 20% Shiga toxin-producing E. coli (STEC) that were non-O157:H7. Thus, the Premier EHEC assay is a sensitive and specific method for the detection of all STEC isolates. Routine use would improve the detection of E. coli O157:H7 and allow for determination of the true incidence of STEC other than O157:H7. The presence of blood in the stool and/or the ages of the patients were poor predictors of the presence of STEC. Criteria need to be determined which would allow for the cost-effective incorporation of this assay into the routine screen for enteric pathogens in high-risk individuals, especially children.     

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An assay was developed for the specific detection of Escherichia coli O157 using PCR, because O serological cross-reactivities have been reported between E. coli O157 and some E. coli, other bacterial species. PCR amplification of E. coli O157 rfbE (Ec O157:H7) gene that is necessary for the expression of the O157 antigen, was performed for the identification of E. coli O157. All Shiga toxin-producing Escherichia coli (STEC) O157:H7 and O157:H, non-STEC O157 strains were positive, and other non-O157 E. coli strains were negative by PCR. All tested strains of other bacterial species, like Salmonella O30 and Citrobacter freundii which gave positive results with O157 detection kits, were negative by PCR. It is recommended that PCR amplification of O157 rfbE gene is one of the most specific method for E. coli O157 identification.