Escherichia coli O157:H7

Escherichia coli O157 was first identified as a human pathogen in 1982. One of several Shiga toxin-producing serotypes known to cause human illness, the organism probably evolved through horizontal acquisition of genes for Shiga toxins and other virulence factors. E. coli O157 is found regularly in the faeces of healthy cattle, and is transmitted to humans through contaminated food, water, and direct contact with infected people or animals. Human infection is associated with a wide range of clinical illness, including asymptomatic shedding, non-bloody diarrhoea, haemorrhagic colitis, haemolytic uraemic syndrome, and death. Since laboratory practices vary, physicians need to know whether laboratories in their area routinely test for E. coli O157 in stool specimens. Treatment with antimicrobial agents remains controversial: some studies suggest that treatment may precipitate haemolytic uraemic syndrome, and other studies suggest no effect or even a protective effect. Physicians can help to prevent E. coli O157 infections by counselling patients about the hazards of consuming undercooked ground meat or unpasteurised milk products and juices, and about the importance of handwashing to prevent the spread of diarrhoeal illness, and by informing public-health authorities when they see unusual numbers of cases of bloody diarrhoea or haemolytic uraemic syndrome.     

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.     

Diversity of DNA sequences among enterohemorrhagic Escherichia coli O157:H7 detected by PCR-based DNA fingerprinting

A random amplified polymorphic DNA(RAPD) fingerprinting method has been developed to differentiate Enterohemorrhagic Escherichia coli(EHEC) O157:H7 isolates. This method uses an oligonucleotide of arbitrarily chosen sequence to prime DNA synthesis from pairs of sites to which it is matched or practically matched and results in strain-specific arrays of DNA products. By the RAPD analysis using A07(5'-TGCCTCGCACCA-3'), EHEC strains tested in this study were found to be divided into 5 groups. The RAPD arrays among 5EHEC clinical isolates from a single outbreak were identical from each other, and different from other origin. The data indicate that the RAPD method is feasible for investigating the source of an outbreak associated with EHEC O157:H7 infection.     

Enterohemorrhagic Escherichia coli O157:H7--an emerging pathogen

Enterohemorrhagic Escherichia coli O157:H7 has become an important public health problem in recent years, causing more than 20,000 cases of infection and up to 250 deaths per year in the United States. Transmission of infection is most commonly linked to consumption of undercooked ground beef, contaminated drinking water or unpasteurized milk. Patients with this infection most often present with an acute onset of diarrhea and abdominal cramping that progresses over days to bloody stools. The most serious complications of E. coli O157:H7 infection include hemolytic-uremic syndrome and thrombotic thrombocytopenic purpura. Hemolytic-uremic syndrome occurs most often in children less than five years of age and the elderly, while thrombotic thrombocytopenic purpura occurs only in adults. Detection of E. coli O157:H7 requires specific testing that is not performed in routine stool cultures. All patients with documented infection require close observation for the development of possible complications. Use of antibiotics and antimotility agents may worsen the course of the infection and should be avoided.     

Inactivation of Escherichia coli O157:H7 in apple juice by irradiation

Three strains (932, Ent-C9490, and SEA13B88) of Escherichia coli O157:H7 were used to determine the effectiveness of low-dose gamma irradiation for eliminating E. coli O157:H7 from apple juice or cider and to characterize the effect of inducing pH-dependent, stationary-phase acid resistance on radiation resistance. The strains were grown in tryptic soy broth with or without 1% dextrose for 18 h to produce cells that were or were not induced to pH-dependent stationary-phase acid resistance. The bacteria were then transferred to clarified apple juice and irradiated at 2 degrees C with a cesium-137 irradiator. Non-acid-adapted cells had radiation D values (radiation doses needed to decrease a microbial population by 90%) ranging from 0.12 to 0.21 kGy. D values increased to 0.22 to 0.31 kGy for acid-adapted cells. When acid-adapted SEA13B88 cells were tested in five apple juice brands having different levels of suspended solids (absorbances ranging from 0.04 to 2.01 at 550 nm), radiation resistance increased with increasing levels of suspended solids, with D values ranging from 0.26 to 0.35 kGy. Based on these results, a dose of 1.8 kGy should be sufficient to achieve the 5D inactivation of E. coli recommended by the National Advisory Committee for Microbiological Criteria for Foods.     

Recovery of thermally-stressed Escherichia coli O157:H7 by media supplemented with pyruvate

Unheated and heat-stressed (57 degrees C, 50 min and 60 min) cells of Escherichia coli O157:H7, were enumerated using three media supplemented with 1% sodium pyruvate (NaPyr): plate count agar (PCA), tryptic soy agar (TSA) and phenol red sorbitol agar (PhRSA) using the spread plate method. The medium recovering the greatest numbers of severely heated E. coli O157:H7 was PCA with 1% NaPyr. Recovery of heat stressed E. coli O157:H7 on this medium was significantly higher (P < 0.05) than the two other media with pyruvate: 16.3% (50 min heating) and 0.55% (60 min heating) of the total population was recovered with TSA + 1% NaPyr when compared to those numbers found on PCA + 1% NaPyr. The ability of PhRSA + 1% NaPyr to recover heat-stressed E. coli O157:H7 was similar to that of TSA + 1% NaPyr. Using PhRSA + 1% NaPyr media. 12.9% (50 min heating) and 0.61% (60 min heating) of the total population were recovered when compared with the cells enumerated on PCA + 1% NaPyr. Recovery of the heat-stressed cells using the spread plate method was greater than using pour plate method. Recovery was significantly higher (P < 0.05) on the spread plates for highly stressed E. coli O157:H7(1.2 log) heated for 60 min than on the pour plates. Overall, the populations on the TSA spread and pour plates were low compared with the same heat-stressed cells recovered on media containing pyruvate. The     

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.     

Molecular evolution of a pathogenicity island from enterohemorrhagic Escherichia coli O157:H7

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.     

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.     

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.     

Mutators and long-term molecular evolution of pathogenic Escherichia coli O157:H7

We report the MRI features and correlative pathologic findings of a lung cancer in a patient with progressive massive fibrosis (PMF). In this case, MRI was able to distinguish the lung cancer as a high signal intensity area, and the fibrotic mass as a low signal intensity area, on both T1-weighted and T2-weighted images when compared with muscle. MRI is potentially useful in distinguishing cancer tissue from PMF in patients with pneumoconiosis.     

A solid phase fluorescent capillary immunoassay for the detection of Escherichia coli O157:H7 in ground beef and apple cider

A solid phase fluorescence-based immunoassay was developed for the detection of Escherichia coli O157:H7 using an antigen down competition format. A soft glass capillary tube served as the solid support, to which heat-killed E. coli O157:H7 were adsorbed. Polyclonal anti-E. coli O157:H7 antibody, conjugated with biotin, was used and the bound antigen-antibody complex was detected using avidin molecules labelled with Cy5, a fluorescent cyanine dye. Any E. coli O157:H7 in the sample would compete with the formation of this complex, reducing fluorescence. This assay was tested for sensitivity with spiked ground beef and apple cider samples. The minimum detectable number of cells present in the initial inoculum was calculated to be approximately 1 colony-forming unit (cfu) per 10 g of ground beef when samples were enriched in modified EC broth for 7 h at 37 degrees C. The minimum detectable number of cells for the apple cider samples was calculated to be approximately 0.5 cfu ml-1. The E. coli cells in the cider samples were captured with immunomagnetic beads, incubated for 7 h in the enrichment broth, and detected with the solid phase fluorescence immunoassay.     

The isolation and detection of Escherichia coli O157 by use of immunomagnetic separation and immunoassay procedures

Pelvic inflammatory disease (PID) has been associated with the use of intrauterine devices (IUDs) ever since they were introduced. In several mostly retrospective studies the incidence of PID was suggested to be as high as three- to ninefold in IUD users compared to non-users. Later epidemiological prospective studies showed a considerably lower risk comparing different types of contraception. Compared to non-contraceptive users the relative risk ranged from 1.8 in patients with copper-containing IUDs to 3.3 in patients using the older insert IUDs like the Dalkon Shield. More recent studies indicate that PID among IUD users is strongly related to the insertion process and to background risk of sexually transmissible disease. The incidence rate of PID decreases from 9.66 per 1000 woman years during the first 20 days following insertion to 1.38 per 1000 woman years beyond the first 20 days. This incidence is similar to the incidence in non-IUD users. PID is more prevalent in younger single women with different sexual partners. Nulliparous women with stable monogamous relationships are not at higher risk of PID than parous women. In conclusion, IUD users, selected for low risk of sexually transmissible disease, do not have excess PID. Proper counselling and selection is of the utmost importance. As there is a higher risk shortly after insertion, limiting IUD replacements will help diminish PID incidence.     

Polysaccharide side chains are not required for attaching and effacing adhesion of Escherichia coli O157:H7

Escherichia coli of the serotype O157:H7 is an enterohemorrhagic human pathogen which demonstrates attaching and effacing adhesion to colonocytes in vivo and to epithelial cells grown in tissue culture. Transposon TnphoA mutants of E. coli O157:H7 strain CL-8 were produced. Two of 300 alkaline phosphatase positive mutants, designated JB6 and JB27, did not express O157 side chains as assessed by agglutination with specific polyclonal O157 antiserum, silver staining of lipopolysaccharide extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western immunoblots with polyclonal O157-specific antiserum. Both O157-negative mutants and the parent strain demonstrated localized adherence to HEp-2 cells when examined by Giemsa staining and bright-field microscopy. Furthermore, both O157-negative mutants showed enhanced adherence to HEp-2 cells compared with the parent strain when assessed by quantification of adherent bacterial CFUs. The parent strain, CL-8, and both of the mutants produced fluorescent foci when adherent bacteria and HEp-2 cells were stained with fluorescein isothiocyanate-labelled phalloidin. Transmission electron microscopy confirmed attaching and effacing adherence of strain CL-8 and the OO7-negative mutants to HEp-2 cells. These findings indicate that mutants deficient in O157 polysaccharide repeats exhibit adherence to tissue culture cells in vitro and that O157 polysaccharide repeats are not required to produce the attaching and effacing lesion.     

The use of serodiagnosis in the retrospective investigation of a nursery outbreak associated with Escherichia coli O157:H7

AIMS: To use serology to investigate an outbreak of verocytotoxin (VT) producing Escherichia coli O157 in a hospital nursery, following the detection of faecal E coli O157 (phage type 49) producing VT type 2. METHODS: ELISA and immunoblotting techniques, based on lipopolysaccharide (LPS) purified from E coli O157; diagnostic bacteriology; serotyping and phage typing; DNA probes for VT. RESULTS: 29 of 126 sera contained antibodies to the LPS of E coli O157: 10 were from children, three were from staff, and 11 were from hospital kitchen staff. Five parents of children attending the nursery were antibody positive. Sixty four sera from other hospital staff and controls did not contain antibodies to the LPS of E coli O157. CONCLUSIONS: Serology detected evidence of infection with E coli O157 in 23% of sera examined. By bacteriology alone, only a single case of infection with E coli O157 would have been detected. Serology is valuable in providing evidence of infection with E coli O157.     

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.     

Abundance in sewage of bacteriophages that infect Escherichia coli O157:H7 and that carry the Shiga toxin 2 gene

Shiga toxin-converting bacteriophages are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7, but data on the occurrence and distribution of such phages as free particles in nature were not available. An experimental approach has been developed to detect the presence of the Shiga toxin 2 (Stx 2)-encoding bacteriophages in sewage. The Stx 2 gene was amplified by PCR from phages concentrated from 10-ml samples of sewage. Moreover, the phages carrying the Stx 2 gene were detected in supernatants from bacteriophage enrichment cultures by using an Stx 2-negative E. coli O157:H7 strain infected with phages purified from volumes of sewage as small as 0.02 ml. Additionally, the A subunit of Stx 2 was detected in the supernatants of the bacteriophage enrichment cultures, which also showed cytotoxic activity for Vero cells. By enrichment of phages concentrated from different volumes of sewage and applying the most-probable-number technique, it was estimated that the number of phages infectious for E. coli O157:H7 and carrying the Stx 2 gene was in the range of 1 to 10 per ml of sewage from two different origins. These values were approximately 1% of all phages infecting E. coli O157:H7.     

Detection of Escherichia coli O157:H7 in the beef marketed in Malaysia

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.     

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.     

Quantitative risk assessment for Escherichia coli O157:H7 in ground beef hamburgers

Quantitative Risk Assessment (QRA) is a methodology used to organize and analyze scientific information to estimate the probability and severity of an adverse event. Applied to microbial food safety, the methodology can also help to identify those stages in the manufacture, distribution, handling, and consumption of foods that contribute to an increased risk of foodborne illness, and help focus resources and efforts to most effectively reduce the risk of foodborne pathogens. The term Process Risk Model (PRM) is introduced in this paper to describe the integration and application of QRA methodology with scenario analysis and predictive microbiology to provide an objective assessment of the hygienic characteristics of a manufacturing process. The methodology was applied to model the human health risk associated with Escherichia coli O157:H7 in ground beef hamburgers. The PRM incorporated two mathematical submodels; the first was intended to described the behaviour of the pathogen from the production of the food through processing, handling, and consumption to predict human exposure. The exposure estimate was then used as input to a dose-response model to estimate the health risk associated with consuming food from the process. Monte Carlo simulation was used to assess the effect of the uncertainty and variability in the model parameters on the predicted human health risk. The model predicted a probability of Hemolytic Uremic Syndrome of 3.7 x 10(-6) and a probability of mortality of 1.9 x 10(-7) per meal for the very young. These estimates are likely high for all hamburger meals, but may be reasonable for the home-prepared hamburgers described by this model. The efficacy of three risk mitigation strategies were evaluated by modifying the values of the predictive factors and comparing the new predicted risk. The average probability of illness was predicted to be reduced by 80% under a hypothetical mitigation strategy directed at reducing microbial growth during retail storage through a reduction in storage temperature. This strategy was predicted to be more effective than a hypothetical intervention which estimated a plausible reduction in the concentration of E. coli O157:H7 in the feces of cattle shedding the pathogen and one aimed at convincing consumers to cook hamburgers more thoroughly. The conclusions of this approach are only accurate to the extent that the model accurately represents the process. Currently, uncertainty and ignorance about the hygienic effects of the individual operations during production, processing, and handling limit the applicability of a PRM to specify HACCP criteria in a quantitative manner. However, with continuous improvement through stimulated research, a PRM should encompass all available information about the process, food, and pathogen and should be the most appropriate decision-support tool since it represents current knowledge.