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Abstract: A molecular-based detection method was developed to detect Escherichia coli O26, O111 and O157 in minced (ground) beef samples. This method consists of an initial overnight enrichment in modified tryptone soya broth (mTSB) and novobiocin prior to DNA extraction and subsequent serogrouping using a triplex PCR. This method has a low limit of detection and results are available within 24 hours of receipt of samples. Once optimized, this rapid method was utilized to determine the prevalence of these E. coli serogroups in six hundred minced beef samples all of which were previously examined by immunomagnetic separation (IMS) and selective plating for E. coli O26 and O111. Using IMS, two E. coli O26 isolates were detected. No E. coli O111 were recovered. The multiplex PCR technique described here did not detect E. coli O111 nor O157 in any of the samples, however six minced beef samples were positive for E. coli O26 using our method, only two of these were previously detected by IMS and culture. Application of molecular methods are useful to support culture-based approaches thereby further contributing to risk reduction along the food chain. [Copyright &y& Elsevier]

The high level of human morbidity caused by E. coli O157:H7 necessitates an improved understanding of the infection dynamics of this bacterium within the bovine reservoir. Until recently, a degree of uncertainty surrounded the issue of whether these bacteria colonize the bovine gut and as yet, only incomplete in-vivo datasets are available. Such data typically consist of bacterial counts from fecal samples. The development of a deterministic model, which has been devised to make good use of such data, is presented. A partial differential equation, which includes advection, diffusion and growth terms, is used to model the (unobserved) passage of bacteria through the bovine gut. A set of experimentally-obtained fecal count data is used to parameterize the model. Between-animal variability is found to be greater than between-strain variability, with some results adding further weight to the hypothesis that E. coli O157:H7 can colonize the bovine gastrointestinal tract. [ABSTRACT FROM AUTHOR]

Abstract: Environmental and food contamination with Shiga toxin-producing Escherichia coli (STEC) pose a threat to public health worldwide, with notable geographic differences in incidence of human disease caused by these organisms. The prevalence of E. coli O157 and total stx-positive specimens collected from mature dairy cattle in Ohio and Norwegian dairy farms was compared using identical laboratory methods in a cross-sectional survey. E. coli O157 was isolated from 5/750 (0.66%) of Ohio dairy cows from 4/50 (8%) different herds, whereas E. coli O157 was not isolated from any (0/680) cattle present in 50 Norwegian dairy herds. In contrast, at least one stx-positive faecal sample was identified by PCR on all (50/50) Norwegian farms but only on 70% (35/50) of Ohio farms. Average animal stx prevalence on Ohio farms was also lower; 14% vs. 61% in Ohio and Norwegian herds, respectively. Livestock feed contamination with generic E. coli was uncommon in Norway, 1/50 feeds testing positive, whereas 19/50 (38%) of feeds collected from Ohio farms were contaminated, some as high as 105 CFU/g. Despite extreme differences in on-farm management practices between countries, stx appear to be widely disseminated in cattle in both countries, while the human pathogenic O157 serotype is less widely disseminated in Norway than it is in Ohio. Geographic distribution differences of human pathogenic STEC serogroups in the bovine reservoir, as opposed to specific farm management practices affecting on farm STEC prevalence, may be an important defining factor influencing the incidence of human illnesses associated in different areas of the world. [Copyright &y& Elsevier]

Escherichia coli O157, an etiological agent of hemorrhagic colitis and hemolytic uremic syndrome, is one of the leading worldwide public health threats. Genome sequencing of two O157 strains have revealed that the chromosome is comprised of a 4.1 Mb backbone shared by K-12 and a total of 1.4 Mb O157-specific sequences. Most of the large O157-specific sequences are prophages and prophage-like elements, which have carried many virulence genes into the O157 genome. This suggests that bacteriophages have played the key roles in the emergence of O157. The Whole Genome PCR Scanning (WGPScanning) analysis of O157 strains, on the other hand, revealed a high level of genomic diversity in O157. Variation of prophages has also been suggested as a major factor generating such diversity. In this study, we analyzed the gene content of O157 strains, by an oligoDNA microarray, using the same set of strains as examined by the WGPScanning method. Although most of the strains were typical O157 : H7, they differed remarkably in gene composition, particularly in those on prophages, and we identified more than 400 ‘variably absent or present’ genes which included virulence-related genes. This confirms the role of prophages in generating the genomic diversity, and raises a possibility that some level of variation in potential virulence is present among O157 strains. Fine comparison of the two datasets obtained by microarray and WGPScanning provided much further details on the O157 genome diversity than illustrated by each method alone, indicating the usefulness of this combinational approach in the genomic comparison of closely related strains. [ABSTRACT FROM PUBLISHER]

Diarrheagenic E. coli strains contribute to water related diseases in urban and rural environment in developing and developed world. E. coli pathotype and pathogenicity varies due to complex multifactorial mechanism involving a large number of virulence factors. Rapid assessment of the virulence pattern of E. coli isolates is possible by Real-Time PCR probes like TaqMan. For designing TaqMan probes and primers for multiplex PCR selected E. coli gene sequences: stx1, stx2, hlyA, chuA, eae, lacZ, lamB and fimA were retrieved from NCBI's GenBank database. The alignment of the multiple sequences and analysis of conserved sequences was carried out using ClustalW and BLAST programs. The primers and Taqmen probes were designed using Beacon Designer software version 2.1 for two multiplexed PCR assays. In silico PCR simulation of these assays showed PCR products for stx2 (248 bp) stx1 (102 bp), lacZ (228bp) and lamB (86 bp) in multiplex #1 and eae (200 bp), chuA (147 bp), hlyA (141bp) and fimA (79 bp) in multiplex #2, respectively. These multiplexed PCR amplification products and probes can be used to identify and confirm presence of O157:H7/H7-, O157:H43/45 and O26:H^{-}/H11 serotypes. In conclusion, multiplex Real-Time Polymerase Chain Reaction oligomers and TaqMan probes designed and validated in silico will be helpful in management of water quality and outbreaks, by improving specificity and minimizing time needed for in vitro verification work. [ABSTRACT FROM AUTHOR]

A differential agar for the further detection of presumptive Escherichia coli O157 strains among typical sorbitol negative and cefixime-tellurite resistant strains was investigated. For this purpose, in parallel to colonies of 8 reference strains, 1130 suspect colonies picked from the surface of cefixime-tellurite (CT)-sorbitol MacConkey (SMAC) (CT-SMAC) agar plates of food samples (white cheese, raw milk and ground beef) were transferred to differential agar plates. After a 6-h incubation period at 42 ºC, only 47 (4.15%) of the 1130 colonies appeared as typical E. coli O157 on the differential agar plates (100 x 15 mm) gridded into 25 numbered sections. Salicin (S), rhamnose (R) and cellobiose (C) were tested separately and together in combination with MUG. The best results were obtained from the combined use of the 3 in SMAC medium (SRC-MUG-SMAC). When violet red bile lactose agar (VL) was used in the same manner as SRC-MUG-SMAC, only 16 (1.41%) of the 1130 colonies remained typical. In this procedure, no biochemical test was required for further identification. The use of an SRC-MUG-SMAC plate immediately after the colony selection step appeared to be more discriminative than the addition of these sugars and MUG to the primary isolation media. As a result, the testing of presumptive E. coli O157 colonies using a differential agar plate including MUG and 3 (or more) sugars that are not fermented by this organism is a rapid, easy, precise and relatively inexpensive isolation procedure, and it is particularly ideal for use in routine screening laboratories. [ABSTRACT FROM AUTHOR]

Abstract: Escherichia coli O157 is a major etiological agent of food-borne illness. Bovine animals are recognized reservoirs for this organism and represent a significant source from where these pathogens can enter the food chain. Food products derived from these animals are convenient vehicles, and are often the focal point(s) of infection. As a useful strategy to provide herd-level surveillance and to investigate for the presence of this pathogen in a population of Irish dairy cattle, milk filters from 97 farms were analysed by conventional culture and other methods. Five hundred and thirty-six milk filters were evaluated over a 2-year period. Filters from 12 of the 97 farms (12%) were found to contain E. coli O157, based on culture methods. Sixteen verocytotoxigenic E. coli O157 organisms were recovered and characterized in detail. The farm families in each case were consuming raw milk from their respective herds. The potential risk to public health associated with the detection of E. coli O157 and the local consumption of raw milk are discussed. [Copyright &y& Elsevier]

Abstract: Escherichia coli O157 is an important cause of food-borne illness. The primary reservoir for this organism is cattle and at present the major site of control is within abattoirs. Recent data have highlighted the importance of the pathogen load entering abattoirs on harvest-ready feedlot cattle. The likelihood for in-plant intervention failure increases as the proportion of cattle carrying E. coli O157 within a pen increases. Pre-harvest reduction of E. coli O157 colonization will require targeted intervention strategies and should reduce contamination of carcasses thereby enhancing public health. Several pre-harvest interventions show substantial promise, such as specific strains of direct-fed microbials, vaccine technology, sodium chlorate, and neomycin sulfate, whereas others such as Brown Seaweed or chlorination of water have little or no detectable benefit. Selection of validated interventions strategies will be important as efforts to control pre-harvest carriage of E. coli O157 increase. [Copyright &y& Elsevier]

Abstract: A human dose response model for Escherichia coli O157 would enable prediction of risk of infection to humans following exposure from either foodborne or environmental pathways. However, due to the severe nature of the disease, volunteer human dose response studies cannot be carried out. Surrogate models from Shigella fed to humans and E. coli O157 to rabbits have been utilised but are significantly different to one another. In addition data obtained by animal exposure may not be representative for human beings. An alternative approach to generating and validating a dose response model is to use quantitative data obtained from actual human outbreaks. This work collates outbreak data obtained from global sources and these are fitted using exponential and beta-Poisson models. The best fitting model was found to be the beta-Poisson model using a beta-binomial likelihood and the authors favour the exact version of this model. The confidence levels in this model encompass a previously published Shigella dose response model. The potential incorporation of this model into QMRAs is discussed together with applications of the model to help explain foodborne outbreaks. [Copyright &y& Elsevier]

A total of 107 coastal environment samples collected, during the period from February to December 2002, from different sites along the seashore of Alexandria City were examined for the presence of enterohaemorrhagic Escherichia coli O157 and total colifrom bacteria (TC). Of the total examined samples, 51 (48%) were found to harbour the pathogen. The bacterium was recovered from 23 (48%) of 48 seawater, 15 (37%) of 41 fish and 13 (72%) of 18 invertebrate samples. The pathogen was detected in the environmental samples during all the sampling months. Some stations proved to be contaminated over the entire period of study; however, others were not. As a general trend, a positive correlation between TC counts and the presence of the pathogen was observed. Also, numbers of TC and the incidence of the bacterium were relatively high during the warmest months. Measures that prevent the risk of infection by such pathogen are discussed. [ABSTRACT FROM AUTHOR]

Abstract: The standard method of immunomagnetic separation of pathogenic bacteria from food and environmental matrices processes 1ml volumes. Pathogens present at low levels (<1 pathogenic bacteria per ml) will not be consistently detected by this method. Here a flow through immunomagnetic separator (FTIMS) has been designed and tested to process large volume samples (>50ml). Preliminary results show that between 70 and 113 times more Escherchia coli O157 are recovered compared with the standard 1ml method. [Copyright &y& Elsevier]

Abstract: Optimised immunomagnetic separation methods to detect Cryptosporidium parvum and Escherichia coli O157 in UK shellfish are described. Whole tissue homogenates gave the best recoveries for C. parvum oocysts compared with gill or haemolymph extracts. The sensitivity of recovery from spiked samples was comparable to that achieved when processing water and varied from 12–34% in mussels, 48–69.5% in oysters and 30–65% in scallops. Maximum recovery of E. coli O157 was achieved by enriching in buffered peptone water supplemented with vancomycin at 42 °C. Increasing enrichment temperatures from 37 to 42 °C gave a significant increase in target number recovery. Implementation of these methods into monitoring programmes and end-product testing will enable shellfish producers to better assess product safety. [Copyright &y& Elsevier]

Abstract: Our objective was to generate hypotheses about associations between management, climate, and the presence of Escherichia coli O157 in feedlot–cattle water tanks and in feedlot–cattle feed. Water samples from 710 tanks on 73 feedlots, and feed-samples from a subset of 504 pens on 54 feedlots, in four US states were tested for E. coli O157. Management and climate factors were ascertained by survey and observation. Escherichia coli O157 were isolated from 13% of the water tanks and at least one water tank was positive on 60% of the feedlots. The factors significantly associated with E. coli O157 in water were greater percentage of cattle shedding E. coli O157 in faeces within the same pen, higher concentration of total E. coli in the water, lack of the clarity of the water, the use of fly traps, the reported frequency of rodent sightings in the pen or alley area, and the weather at the time of sampling. Escherichia coli O157 were isolated from 14.9% of the feed samples obtained from the feedbunks. Factors positively associated with E. coli O157 in feed were higher heat index at the time of sampling, the presence of cottonseed meal in the ration, and the feedlot location (state). Coliform counts in feed, presence of E. coli O157 in water tanks and faecal prevalence of E. coli O157 were not associated with the presence of E. coli O157 in feed. [Copyright &y& Elsevier]

Abstract: Our objective was to generate hypotheses for potential on-farm control strategies for Escherichia coli O157 by identifying associations between management practices and climate, and the presence of E. coli O157 in feedlot cattle. Faeces were obtained from 10,622 cattle in 711 pens on 73 feedlots between May and August 2001. Management and climate information was obtained by questionnaire and observation at the time of sampling. The prevalence of E. coli O157 was 10.2% at the sample level, 52.0% at the pen-level, and 95.9% at the feedlot-level. The factors associated with the presence of E. coli O157 in cattle faeces were the frequency of observing cats in the pens or alleys (most common when observed daily), the presence of E. coli O157 in the water tanks (positive association), the historical use of injectable mass medication (positive association), the use of antibiotics in the ration or water (negative association), the wetness of the pen, number of cattle in the pen (negative association), wind velocity (positive association), and height of the feed bunk (positive association). [Copyright &y& Elsevier]

Abstract: We examined 476 faecal samples from subjects aged from 0 to >60 years, 283 with diarrhoea and 193 with illnesses involving other sites or clinically healthy, and 154 samples of faeces of healthy cattle, in order to define the diffusion of E. coli O157 in the Urbino area. The samples were seeded by both direct streaking onto cefixime tellurite sorbitol Mac Conkey agar (CT-SMAC) and previous enrichment in cefixime tellurite tryptone soya broth for human specimens and in cefixime vancomicin tryptone soya broth for bovine samples. The strains of E. coliO157 were characterized by verocytotoxin and adhesin eae genes detection. We isolated one strain of E. coli O157 (0.2%) from a man 68 year old who had bloody diarrhoea, and one strain (0.64%) from a weaned calf. Both isolates carried the adhesin eae gene, but only the bovine strain was VT2+. The study shows a low diffusion of E. coli O157 in the Urbino area, confirming the epidemiological data on the national territory. [ABSTRACT FROM AUTHOR]

Shiga toxigenic Escherichia coli (STEC) isolated from raw beef samples in northern Palestine during a 1-year period were characterized for virulence genes by a polymerase chain reaction (PCR) assay and screened for their antibiotic resistance. STEC was identified in 44 (14.7%) of 300 raw beef samples. Twelve (27.3%) of the STEC isolates were serotype O157. Nine of those were isolated during summer. The majority of STEC isolates (70.5%) harbored both stx1 and stx2 genes, while the others harbored either stx1 or stx2. High levels of resistance against different antimicrobial agents were detected. Resistance to at least three drugs was found in 55% of the isolates. [Copyright &y& Elsevier]

Ruminants harbour both O157:H7 and non-O157 Attaching Effacing Escherichia coli (AEEC) strains but to date only non-O157 AEEC have been shown to induce attaching effacing lesions in naturally infected animals. However, O157 may induce lesions in deliberate oral inoculation studies and persistence is considered dependent upon the bacterially encoded locus for enterocyte effacement. In concurrent infections in ruminants it is unclear whether non-O157 AEEC contribute either positively or negatively to the persistence of E. coli O157:H7. To investigate this, and prior to animal studies, E. coli O157:H7 NCTC 12900, a non-toxigenic strain that persists in conventionally reared sheep, and non-toxigenic AEEC O26:K60 isolates of sheep origin were tested for adherence to HEp-2 tissue culture alone and in competition one with another. Applied together, both strains adhered in similar numbers but lower than when either was applied separately. Pre-incubation of tissue culture with either one strain reduced significantly (P < 0.05) the extent of adherence of the strain that was applied second. It was particularly noticeable that AEEC O26 when applied first reduced adherence and inhibited microcolony formation, as demonstrated by confocal microscopy, of E. coli O157:H7. The possibility that prior colonisation of a ruminant by non-O157 AEEC such as O26 may antagonise O157 colonisation and persistence in ruminants is discussed. [Copyright &y& Elsevier]

s.e. robinson, e.j. wright, c.a. hart, m. bennett and n.p. french. 2004.We conducted two short-term studies of cohorts of naturally infected calves to determine the prevalence and concentrations ofEscherichia coliO157 shed in faeces.Two cohorts of calves were sampled; in the first study 14 calves were sampled up to five times a day for 5 days; in the second study a group of 16 separate calves were sampled once or twice a day for 15 days. All cattle within the two cohorts shedE. coliO157 at some point during the respective studies. In 18% of samples,E. coliO157 could only be isolated using immunomagnetic separation after an enrichment period, suggesting concentrations<250 CFU g−1. The highest concentrations recorded were 6·7 × 105 and 1·6 × 106 CFU g−1 for studies 1 and 2 respectively.Persistent, high shedders (shedding>103 CFU g−1) were evident in both studies but, in the majority of calves, the pathogen was isolated intermittently.The variable patterns of shedding have important implications for the design of appropriate sampling protocols and for gaining meaningful estimates of parameters used in mathematical models of transmission. [ABSTRACT FROM AUTHOR]

Escherichia coli O157:H7/NM (E. coli O157) is now recognized as an important cause of diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome worldwide. There have been several cases of human E. coli O157 infection in Korea since it was first isolated from a patient with hemolytic-uremic syndrome in 1998. Meat, other foods, and recreational and drinking water contaminated with animal feces are probably the major sources of the E. coli O157 infection. In this study, we investigated the prevalence of E. coli O157 in fecal and meat samples of cattle, pigs and chicken in Korea from April 2000 to July 2002. Eighty-six (3.03%) of 2843 samples were positive for E. coli O157. Most of the E. coli O157 strains were isolated from fecal samples of beef and dairy cattle from May to October of each year. Of 86 E. coli O157 isolates, 73 were serotype O157:H7 and 13 were serotype O157:NM. Polymerase chain reaction (PCR) analysis of E. coli O157 virulence markers revealed that all O157:H7/NM isolates were positive for EhlyA, eaeA and rfbO157, and 77 isolates were positive for stx1 and/or stx2. Cytotoxicity analysis revealed that many of the E. coli O157 isolates showed high cytotoxicity on Vero cells. Our data suggest that the majority of Korean E. coli O157 isolates from food animals can cause serious diseases in humans. [Copyright &y& Elsevier]

n. fegan, p. vanderlinde, g. higgs and p. desmarchelier. 2004. To determine the prevalence and concentration of Escherichia coli O157 shed in faeces at slaughter, by beef cattle from different production systems. Faecal samples were collected from grass-fed (pasture) and lot-fed (feedlot) cattle at slaughter and tested for the presence of E. coli O157 using automated immunomagnetic separation (AIMS). Escherichia coli O157 was enumerated in positive samples using the most probable number (MPN) technique and AIMS and total E. coli were enumerated using Petrifilm. A total of 310 faecal samples were tested (155 from each group). The geometric mean count of total E. coli was 5 × 105 and 2·5 × 105 CFU g−1 for lot- and grass-fed cattle, respectively. Escherichia coli O157 was isolated from 13% of faeces with no significant difference between grass-fed (10%) and lot-fed cattle (15%). The numbers of E. coli O157 in cattle faeces varied from undetectable (<3 MPN g−1) to 1·1 × 105 MPN g−1. Twenty-six (67%) of 39 O157 positive faeces had <10 MPN g−1 and three (8%) had counts between 103–105 MPN g−1. There was no significant difference between concentrations of E. coli O157 in the faeces of grass-fed or lot-fed cattle. The prevalence and numbers of E. coli O157 in the faeces of cattle at slaughter were not affected by the production systems evaluated in this study. Information on the prevalence and numbers of E. coli O157 can be used for formulating intervention strategies and in quantitative risk assessments. [ABSTRACT FROM AUTHOR]

The use of a minimal lactose enrichment broth (MLB) in conjunction with immunomagnetic electrochemiluminescence detection (IM-ECL) was evaluated for the estimation of viable Escherichia coli O157 populations in surface water samples. In principle, E. coli O157 populations (Cinitial E. coli O157) can be derived from enrichment data according to the equation: Cinitial E. coli O157=Cinitial coliforms×(Cfinal E. coli O157/Cfinal coliforms), assuming that the growth rates and lag times of water-borne E. coli O157 and collective coliforms are sufficiently comparable, or at least consistent. We have previously described a protocol for determining Cfinal E. coli O157 in MLB-enriched water samples. In the present study, 80% of coliforms (red/pink colonies on MacConkey Agar) grew in MLB, indicating that this provides reasonably accurate estimates of Cinitial coliforms. Estimates of Cfinal coliforms were determined from turbidity data. Initial E. coli O157 populations (Cinitial E. coli O157) were calculated for 33 Baltimore watershed samples giving a positive IM-ECL response. The majority of samples contained E. coli O157 concentrations of <1 cell per 100 ml. These data indicate that E. coli O157 are present in surface water samples but at very low levels. Growth rates for MLB-enriched coliforms were highly variable (k=0.47±0.13 h-1, n=72). There was no correlation between growth rates and any measured water parameter, suggesting that coliform populations in water samples are spatially and temporally unique. Although variability in growth rates was expected to yield some low values, the fact that most E. coli O157 concentrations were <1 suggests that other factor(s) were also responsible. Studies with E. coli O157:H7 and wild-type E. coli suggest that increased lag times due to starvation were at least partially responsible for the observed data. Based on estimates of Cinitial coliforms and kcoliforms, MLB was evaluated for sensitivity and quantitativeness. Simulated populations of E. coli O157:H7 at stationary phase varied from ca. 103 to 108 cells ml-1 enrichment culture. Although not suitable for quantitation, MLB enrichment in conjunction with IM-ECL can detect as few as one viable water-borne E. coli O157 cell per 100 ml surface water. Experiments are in progress to evaluate alternative media for sensitivity and quantitative detection of enterohemorrhagic E. coli. [Copyright &y& Elsevier]

m.l. hutchison, l.d. walters, s.m. avery, b.a. synge and a. moore. 2004. To determine the prevalence and levels of zoonotic agents in livestock wastes. A proportionally weighted survey was undertaken and livestock waste samples analysed quantitatively for Escherichia coli O157, pathogenic Listeria, Salmonella, Campylobacter, Giardia and Cryptosporidium. A significant proportion of wastes contained at least one zoonotic agent. Relationships were found between dry matter content and the presence and levels of some zoonotic agents. British livestock wastes contain measurable levels of the zoonotic agents that cause most cases of gastroenteritis in the UK. Animal wastes are disposed of by spreading to agricultural land used for the production of crops and livestock grazing. As British wastes are contaminated with significant levels of zoonotic agents, the practice may represent a way for pathogens to travel further up the food chain. [ABSTRACT FROM AUTHOR]

The prevalence of Escherichia coli O157 in Scottish beef cattle at abattoir was found to be greater during the cooler months [11.2% (95% CI, 8.4–13.9%)] compared to the warmer months [7.5% (95% CI, 5.4–9.6%)]; the reverse of seasonality of human infections. However, high shedding beef cattle (excreting >104 g−1) appear to shed greater concentrations of E. coli O157 in the warmer months which may partly explain increased human infection seasonality at this time. [Copyright &y& Elsevier]

E. coli O157 is a foodborne pathogen responsible for serious human illnesses, such as hemorrhagic colitic and hemolytic uremic syndrome. Ground beef products are among the foods that are more commonly contaminated, and the strains isolated have been frequently found to carry virulence factors of this pathotype. This paper reports the results of serotyping assays and of investigations performed to screen for virulence factors of 10 E. coli O157 strains isolated from fresh sausages purchased at retail meat outlets in various provinces of Apulia (southern Italy). The presence of verocytotoxins was assessed on VERO cells and ELISA tests. Multiplex PCR assays were performed for the eae, stx1, stx2 and hlyA genes. Six of the 10 strains examined presented the H7 antigen and all of them proved to be potentially pathogenic due to the presence of individual or multiple virulence factors. [Copyright &y& Elsevier]

1 In this study, we examined the role of mitopen-activated protein (MAP) kinases in the effects of verotoxins (VTs), from Escherichia co/i 0157:H7. upon both apoptosis and the release of tumour necrosis factor alpha (TNF-α) and granulocyte-macrophag colony-stinulated factor (GM-CSF) from human monocytes. 2 Both VTl and VT2 stimuilated a weak, transient increase in c-Jun-N-terminal kinase (JNK) activity and a strong activation of both p[sup38] mitogen-activated protcin kinase (MAP kinase) and extracellular-regulated kinase ( EKK) activity in human monocytes, which was sustained in the case of p[sup38] MAP kinase. 3 Stimulition of human monocytes with VT2 (l00ngml[sup-1]) did not result in an increase in apoptosis: however, the toxin stimulated the release of both TNF-α and GM-CSF. 4 Pretrcatment of human monocytes with the p[sup38] MAP kinase inhibitor SB203580. at concentrations from l00nM to 10μM. significantly decreased the VT1- and VT2-iiiducecl TNF-α and GM-CSF release from monocytes. In contrast, inhibition of MEK1 with PD98059 only significantly decreased GM-CSF release. 5 Pretreatment of monocytes with SP600125 inhibited both GM-CSF and TNF-α production: however, significant effects upon p[sup38] MAP kinase and ERK activation were observed. 6 Taken together, these results suggest a role for p[sup38] MMP kinase and ERK in cytokine generation in response to the verotoxins. A role for JNK remains undetermined. [ABSTRACT FROM AUTHOR]

We report here the use of immunomagnetic (IM) electrochemiluminescence (ECL) for quantitative detection of Esherichia coli O157:H7 in water samples following enrichment in minimal lactose broth (MLB). IM beads prepared in-house with four commercial anti-O157 monoclonal antibodies were compared for efficiency of cell capture. IM-ECL responses for E. coli O157:H7 (strain SEA13B88) were similar for all four commercial anti-O157 LPS monoclonal antibodies. The ECL signal was linearly correlated with E. coli O157:H7 cell concentration, indicating a constant ECL response per cell. Twenty-two strains of E. coli O157:H7 or O157:NM gave comparable ECL signals using IM beads prepared in-house. To assess the potential for interference from background bacteria in MLB-enriched water samples, 104 cells of E. coli O157:H7 (strain SEA13B88) were added to enriched samples prior to analysis. There was considerable variability in recovery of E. coli O157:H7 cells; net ECL signals ranged from 1% to 100% of expected values (i.e., percent inhibition from 0% to 99%). Cultures of Klebsiella pneumoniae, Klebsiella oxytoca, and Enterobacter cloacae, subsequently isolated from MLB-enriched water samples via IM separation (IMS), were observed to interfere with the binding of E. coli O157:H7 cells to IM beads. Recoveries of 104 E. coli O157:H7 cells were ≤10% in the presence of ca. 108 K. pneumoniae, K. oxytoca, or E. cloacae cells. None of these strains gave a positive IM-ECL signal. Although competitive binding decreased sensitivity, there still was a linear correlation between ECL signal and higher E. coli O157:H7 cell concentrations. These studies indicate that IM-ECL in conjunction with MLB enrichment is capable of quantitatively detecting as few as 103 to 105 E. coli O157:H7 cells ml−1, depending on percent recoveries, in enriched samples that contain ca. 109 total lactose-fermenting bacteria ml−1. Assuming comparable growth rates for E. coli O157:H7 and other lactose-fermenting bacteria in MLB, it may be possible to detect as few as one E. coli O157:H7 in 100 ml of raw water containing as many as 104 to 106 lactose-fermenting bacteria (i.e., total coliforms). [Copyright &y& Elsevier]

An outbreak of infection with Escherichia coli 0157 Phage Type 21/28 occurred between the 23rd November 2001 and the 7th December 2001 in Eccleston, Lancashire. There were 30 confirmed cases (23 with positive faecal isolates and seven serologically positive). Eccleston is a village of approximately 5,000 inhabitants with a single medical practice where many of the cases were patients. Initial investigations identified the suspected source as a butcher's counter, operated as a franchise, in a supermarket in Eccleston. The butcher closed voluntarily on the 24th November. The median age of cases was 60 with a mean of 56 and a range of 2-91 years. Of the 30 confirmed cases, 22 were admitted to hospital. Two patients developed serious complications but all 30 made a full recovery. Microbiological investigations confirmed the butcher's counter as the source of the outbreak. The epidemiological evidence implicated cooked meats and microbiological evidence confirmed that contamination had occurred between raw and cooked meats. The deficiencies in meat hygiene practice that were identified could have led to the cross contamination. This outbreak illustrates the risk associated with the handling of raw and cooked meats in the same shop. Complete physical separation of raw and cooked meat operations reduces the risk of such outbreaks. [ABSTRACT FROM AUTHOR]

The prevalence of Escherichia coli O157 was determined in 10,662 fecal samples, 2130 water and 1132 water tank-sediment samples collected during the summer months in 2001 from 711 pens in 73 feedlots located in Kansas, Nebraska, Texas, or Oklahoma, USA. Overall, 10.2% of fecal samples were positive for E. coli O157, with 52% of the pens and 95.9% of the feedlots having at least one positive fecal sample. There were no differences among states or months in the fecal prevalences. Water or water tank-sediment was positive in 13.1% of the water tanks, and 60.3% of feedlots had at least one positive tank. Cattle were more likely to be shedding E. coli O157 in pens with positive water tanks, and water was more likely to be positive when E. coli O157 was detected in the sediment. [Copyright &y& Elsevier]

SUMMARY Mannan-binding lectin (MBL) triggers complement activation upon binding to microbial surfaces. MBL deficiency has been associated with increased susceptibility to severe bacterial infections. We hypothesized that MBL deficiency may predispose children to Shiga toxin-producing Escherichia coli (STEC) O157:H7 infections and the associated haemolytic uraemic syndrome (HUS). We compared circulating levels of MBL among children with uncomplicated O157:H7 haemorrhagic colitis (HC), patients with O157:H7 HUS, normal and diseases control groups. Circulating MBL concentrations on admission were as follows: 3·22 ± 2·43 µ g/ml among normal controls (n = 23); 2·90 ± 2·44 µ g/ml in patients with rotavirus enteritis (n = 10); 2·78 ± 1·65 µ g/ml in children with HC due to non-STEC bacterial pathogen (n = 15); 2·67 ± 2·44 µ g/ml in patients with uncomplicated O157:H7 HC (n = 27); 2·80 ± 2·97 µ g/ml in children with O157:H7 HUS (n = 15); 6·70 ± 4·49 µ g/ml in patients with chronic renal failure unrelated to O157:H7 infection (n = 6). Higher MBL levels were found in patients with chronic renal failure compared to O157:H7 HC (P < 0·047). However, MBL concentrations <0·5 µ g/ml, which have been associated with MBL deficiency in relation to increased susceptibility to infections, were noted at comparable rates between the different groups (P = NS). Our data does not support that MBL deficiency may predispose to O157:H7 infections nor than the development of diarrhoea associated HUS. [ABSTRACT FROM AUTHOR]

From December 1999 to December 2000, 150 pigs were randomly selected in two large abattoirs of northern Italy. Caecal material and carcass swabs were collected and examined for Salmonella, Yersinia enterocolitica, and Escherichia coli O157. Tonsils were examined for Salmonella and Y. enterocolitica. Salmonella was isolated from the intestinal content of 55 (36.7%) specimens, from 8 (5.3%) tonsils, and from 9 (6.0%) carcasses. Ten different serotypes were detected; the more common were Salmonella derby (37.8%), Salmonella bredeney (21.6%), and Salmonella typhimurium (14.8%). S. typhimurium isolates that belonged to phage-types DT104 and DT208 were 45% and 27.3%, respectively; 18.2% belonged to U302 and 9.1% were non-typeable. Y. enterocolitica was detected in the intestinal matter of 6 (4.0%) slaughtered pigs and in 22 (14.7%) tonsils; however, this pathogen was not found on carcasses. The majority of Y. enterocolitica isolates (82.1%) belonged to serotype O:3 biotype 4, one (3.6%) belonged to serotype O:9, and 13% did not belong to any known biotype. Verocytotoxin-producing E. coli (VTEC) O157 was isolated from the intestinal content of one (0.7%) slaughtered pig and from one (0.7%) carcass; four (2.7%) faecal samples contained E. coli O157 strains negative for the presence of both eae and VT genes. [Copyright &y& Elsevier]

From 1999 until 2001, 3625 food samples were examined for the presence of Escherichia coli O157. Samples were from bovine origin (ground beef, n=549; carcasses, n=2452), calves (carcasses, n=147), chicken (breast, n=203; carcasses, n=71) and pigs (carcasses, n=85; trimmings, n=118). Vidas ECO detected 451 (12%) samples positive, but from only 27 (0.74%) samples was E. coli O157 isolated. One strain was isolated from bovine ground beef (0.18%), one from a pig carcass (1.17%) and all others were isolated from bovine carcasses (1.02%). All strains possessed the attaching-and-effacing gene, the enterohemorrhagic plasmid and verotoxin (VT) genes, except the strain isolated from the pig carcass that was therefore eliminated. Six of the strains were urease-positive. Strains were typed by two DNA fingerprinting methods: random amplification of polymorphic DNA (RAPD) and pulsed field gel electrophoresis (PFGE). PFGE revealed a similarity of 71.05%, while RAPD was 77.36% similar. None of the typing methods were able to classify all urease-positive strains to one pattern. Strains in the same PFGE cluster did not belong to one RAPD cluster. This paper highlights that Belgian fresh meat at retail level can be contaminated with E. coli O157 and that two different typing methods divide strains into different types. [Copyright &y& Elsevier]

Abstract Aims: To compare the sensitivity of two pre-enrichment broth media prior to immunomagnetic separation for the isolation of Escherichia coli O157 from cattle faeces. Methods and Results: One-gram portions of 721 cattle faeces collected from 43 farms were pre-enriched in buffered peptone water containing vancomycin, cefixime and cefsulodin (BPW-VCC) and buffered peptone water without additives (BPW-WOA), respectively. A total of 137 samples were positive for E. coli O157: 127 pre-enriched with BPW-WOA and 89 pre-enriched in BPW-VCC. Representative isolates were tested for phage type, verotoxin and eae (E. coli attaching and effacing) gene sequences, resulting in the recognition of eight different types. All the E. coli O157 types recognized were isolated by both methods except for three different strains, each of which were isolated only on a single occasion: two by BPW-WOA and another by BPW-VCC. Conclusions: The results clearly demonstrate, under the conditions of this study, that BPW without antibiotics was the superior pre-enrichment medium for the isolation of E. coli O157 from cattle faeces. Significance and Impact of the Study: The use of BPW-WOA in preference to BPW-VCC for the isolation of E. coli O157 from cattle faeces in future research and outbreak studies should lead to a higher number of positive isolates. [ABSTRACT FROM AUTHOR]

Verocytotoxin-producing Escherichia coli (VTEC) O157 is an important emerging human pathogen. Cattle are considered to be the main reservoir for VTEC O157. The objectives of this study were to determine the prevalence of VTEC O157 in Danish dairy herds and to investigate the relationship between shedding of VTEC O157 and a number of animal and herd characteristics. Sixty dairy farms were visited once in August–October, and from each herd faecal samples from up to 50 animals were analysed for VTEC O157 by enrichment, immunomagnetic separation (IMS), and plating on selective agar. In total, 2419 animals were sampled, and 3.6% of these excreted VTEC O157. These animals were located on 10 farms (17%). On average, 21% of the sampled animals in the positive herds excreted VTEC O157. Register data, including age, sex, breed, housing conditions and herd composition, were extracted from a database. No influence of herd size or housing conditions was found. A strong effect of age was seen with 2–6-month-old calves as the high-risk age group (8.6% positive) in contrast to calves <2 months (0.7%) and cows (2.4%). There was a non-significant tendency of bull calves to have a higher prevalence than heifers within the age group of 2–6 months. Significantly, more of the herds characterised by having relatively many bull calves or many animals bought into the herd were positive for VTEC O157. Despite the low incidence of human VTEC O157 infections in Denmark, the prevalence in Danish dairy herds was found to be at a similar level as in many other countries. [Copyright &y& Elsevier]

Verocytotoxin-producing Escherichia coli O157 is a serious pathogen in man that is carried by ruminants and has been isolated from some other animal species. Except in the very young of certain species and in greyhounds, the organism is not associated with disease in animals. Humans may be infected by ingestion of the organism through direct animal contact, from contaminated food or water or from the environment. Great efforts have been made to improve hygienic food production and handling, to protect water supplies and to give adequate advice to people handling animals. It is also essential to try to reduce the numbers of organisms shed by animals and, to do this, a clear understanding of the ecology of the organism is required. [ABSTRACT FROM AUTHOR]

Background. The aim of the study was to assess the impact of an outbreak of Escherichia coli O157:H7 that occurred in 1994 in a rural community, with a population of approximately 107 000, to the west of Edinburgh. [ABSTRACT FROM PUBLISHER]

For food quality and safety issues, the emergence of foodborne pathogenic bacteria has further accelerated the spread of antibiotic residues and drug resistance genes. To alleviate the harm caused by bacterial infections, it is necessary to seek novel antimicrobial agents as biopreservatives to prevent microbial spoilage. Nanoantimicrobials have been widely used in the direct treatment of bacterial infections. CNMs, formed by chitosan nanoparticles and peptides, are promising antibiotic alternatives for use as excellent new antibacterial drugs against pathogenic bacteria. Herein, the current study evaluated the function of CNMs in the protection of foodborne pathogen Escherichia coli (E. coli) O157 infection using an intestinal epithelial cell model. Antibacterial activity assays indicated that CNMs exerted excellent bactericidal activity against E. coli O157. Assessment of the cytotoxicity risks toward cells demonstrated that 0.0125–0.02% of CNMs did not cause toxicity, but 0.4% of CNMs caused cytotoxicity. Additionally, CNMs did not induced genotoxicity either. CNMs protected against E. coli O157-induced barrier dysfunction by increasing transepithelial electrical resistance, decreasing lactate dehydrogenase and promoting the protein expression of occludin. CNMs were further found to ameliorate inflammation via modulation of tumor factor α, toll-like receptor 4 and nuclear factor κB (NF-κB) expression via inhibition of mitogen-activated protein kinase and NF-κB activation and improved antioxidant activity. Taken together, CNMs could protect the host against E. coli O157-induced intestinal barrier damage and inflammation, showing that CNMs have great advantages and potential application as novel antimicrobial polymers in the food industry as food biopreservatives, bringing new hope for the treatment of bacterial infections. [ABSTRACT FROM AUTHOR]

Foodborne pathogens significantly impact public health globally. Excessive antimicrobial use plays a significant role in the development of the public health crisis of antibiotic resistance. Here, we determined the prevalence and antimicrobial resistance profiles of E. coli O157, Salmonella, L. monocytogenes, and Campylobacter isolated between 2016 and 2020 from small scale agricultural settings that were amended with dairy cattle or poultry manure in Northeastern Ohio. The total prevalence of the foodborne pathogens was 19.3%: Campylobacter 8%, Listeria monocytogenes 7.9%, Escherichia coli O157 1.8%, and Salmonella 1.5%. The prevalence was significantly higher in dairy cattle (87.7%) compared to poultry (12.2%) manure amended farms. Furthermore, the prevalence was higher in manure samples (84%) compared to soil samples (15.9%; p < 0.05). Multiple drug resistance was observed in 73%, 77%, 100%, and 57.3% of E. coli O157, Salmonella, L. monocytogenes, and Campylobacter isolates recovered, respectively. The most frequently observed resistance genes were mphA, aadA, and aphA1 in E. coli O157; blaTEM, tet(B), and strA in Salmonella; penA, ampC, lde, ermB, tet(O), and aadB in L. monocytogenes and blaOXA-61, tet(O), and aadE in Campylobacter. Our results highlight the critical need to address the dissemination of foodborne pathogens and antibiotic resistance in agricultural settings. [ABSTRACT FROM AUTHOR]

Infectious Escherichia coli O157:H7 threatens the health of millions people each year. Thus, it is important to establish a simple and sensitive method for bacterial detection and eradication. Herein, a DNA-programming strategy is explored to synthesize anisotropic dumbbell-like Au-Pt nanoparticles with excellent catalytic and anti-bacterial activities, which were applied in the simultaneous detection and eradication of pathogenic bacteria. The DNA sequence-dependent growth of bimetallic nanoparticles is firstly studied and polyT20 has the tendency to form dumbbell-like Au-Pt bimetallic structures based on gold nanorods seeds. PolyA20 and polyC20 can also form similar structures but only at much lower DNA concentrations, which can be explained by their much higher affinity to the metal surfaces than T20. The as-prepared nanoparticles exhibit high nanozyme catalytic activity resulting from the synergistic effect of Au and Pt. Under light irradiation, the Au-Pt nanoparticles show high photothermal conversion efficiency and enhanced catalytic activity, which can be applied for the eradication and detection of E. coli O157:H7 with a robust efficacy (95%) in 5 min and provides excellent linear detection (10–107 CFU/mL), with a detection limit of 2 CFU/mL. This study offered new insights into DNA-directed synthesis of nanomaterials with excellent biosensing and antibiotic applications. [Display omitted] • DNA-encoded seed-growth method was firstly used to synthesis of bimetallic dumbbell-like Au-Pt nanoparticles. • Au-Pt nanoparticles have enhanced photothermal conversion efficiency and nanozyme catalytic performance. • Au-Pt nanoparticles were used for high-performance detection and eradication of pathogenic bacteria. [ABSTRACT FROM AUTHOR]

Increased speed and sensitivity of testing are always desired for the detection of pathogens in foods. Presented a test sample with low microbial analyte concentration, it is an advantage to analyze as much volume as possible of the sample to attain the best limit of detection (LOD). Therefore, a rapid screening method using a novel flow-through immunoelectrochemical biosensor was developed for the detection of pathogenic bacteria (E. coli O157:H7 and Salmonella) in food (ground beef). As the working electrode employed was comprised of a porous, antibody-coated graphite felt electrode that served as both a biorecognition-element coated solid support for capture of targeted pathogens as well as a signal transducer, high volumes of aqueous sample could be rapidly exposed to the solid support via gravity flow. Flow rates as high as 16.7 mL/min and 12.3 mL/min could be achieved for bacterial samples in buffer and 1:4 ground meat (beef) homogenate, respectively, with no significant effect on LOD. Fastest flow rates for beef homogenate, without clogging of the porous electrode as well as reduction in apparent electrochemical interference, was realized with a tandem combination of sample pretreatment strategies that included filtration with glass wool and graphite felt as well as continuous flow centrifugation. The LOD for 10,000 E. coli O157 cells in 5, 60, and 1000 mL of buffer was 2000, 170, and 10 cells/mL, respectively in a total assay time of 3 h whereas the LOD for E. coli O157 was 400 cells/mL in 1:4 beef homogenate. • Rapid, field portable biosensor for detecting microbial pathogen in foods. • Flow-through system enables screening of large sample volumes without subsampling. • The working electrode serves as selective capture surface and as signal transducer. [ABSTRACT FROM AUTHOR]