The rectal-anal junction (RAJ) is the major colonization site of Shiga toxin-producing Escherichia coli (STEC) O157 in beef cattle, leading to transmission of this foodborne pathogen from farms to food chains. To date, there is limited understanding regarding whether the mucosa-attached microbiome has a profound impact on host-STEC interactions. In this study, the active RAJ mucosa-attached microbiota and its potential role in host immunity-STEC commensal interactions were investigated using RAJ mucosal biopsies collected from calves orally challenged with two STEC O157 strains with or without functional stx2a (stx2a+ or stx2a-). The results revealed that shifts of microbial diversity, topology, and assembly patterns were subjected to stx2a production post-challenge and Paeniclostridium and Gallibacterium were the keystone taxa for both microbial interactions and assembly. Additional mucosal transcriptome profiling showed stx2a-dependent host immune responses (i.e. B- and T-cell signaling and antigen processing and presentation) post-challenge. Further integrated analysis revealed that mucosa-attached beneficial microbes (i.e. Provotella, Faecalibacterium, and Dorea) interacted with host immune genes pre-challenge to maintain host homeostasis; however, opportunistic pathogenic microbes (i.e. Paeniclostridium) could interact with host immune genes after the STEC O157 colonization and interactions were stx2a-dependent. Furthermore, predicted bacterial functions involved in pathogen (O157 and Paeniclostridium) colonization and metabolism were related to host immunity. These findings suggest that during pathogen colonization, host-microbe interactions could shift from beneficial to opportunistic pathogenic bacteria driven and be dependent on the production of particular virulence factors, highlighting the potential regulatory role of mucosa-attached microbiota in affecting pathogen-commensal host interactions in calves with STEC O157 infection.

1. Shiga toxin-producing Escherichia coli (STEC) strains are associated with disease outbreaks which cause a public health problem. The aim of this study was to determine the frequency of STEC strains, their virulence factors, phylogenetic groups and antimicrobial resistance profiles in broiler chickens.2. A total of 222 E.coli isolates were collected from the caecum of chickens intended to be slaughtered. Antibiotic susceptibility was tested against 21 antimicrobial agents and ESBL phenotype was assessed by double-disk synergy test. The presence of STEC virulence genes stx1, stx2,eaeA and ehxA was detected by PCR. The identification of STEC serogroups was realised by PCR amplification. Additive virulence genes, phylogenetic groups and integrons were examined among the STEC isolates.3. Out of 222 E.coli isolates, 72 (32%) were identified as STEC strains and the most predominant serogroups were O103, O145 and O157. Shiga toxin gene 1 (stx1) was found in 84.7% (61/72) of the STEC strains, and eae and stx2 were detected in 38.8% and 13.8%, respectively. The ESBL phenotype was documented in 48.6% (35/72) of isolates. Most of the isolates (90.3%) carried class 1 integron with the gene cassette encoding resistance to trimethoprim (dfrA) and streptomycin (aadA) in 31.9% of the isolates. Class 2 integron was identified in 36.1% of isolates.4. Broilers can be considered as a reservoir of STEC strains which have high virulence factors and integrons that might be transmitted to other chickens, environments and humans. It is important to undertake surveillance and efficient control measures in slaughterhouses and farms to control measures of STEC bacteria.

Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens causing severe diseases. The ability of STEC to produce disease is associated with Shiga toxin (Stx) production. We investigated the occurrence of STEC on bovine and pork carcasses and walls of trucks where they were transported, and we characterized virulence genes and serotypes of STEC strains. We compared the whole genomic sequencing of a STEC O157:H7 strain isolated from a bovine carcass in this work and a STEC O157:H7 strain isolated from a child with HUS, both isolated in 2019. We studied the relationship between these isolates and others collected in the database. The results show a 40% of STEC and two different serogroups were identified (O130 and O157). STEC O157:H7 were isolated from bovine carcasses and harbored stx2, eae, ehxA, katP, espP, stcE, ECSP_0242/1773/2687/2870/2872/3286/3620 and were classified as lineage I/II. In STEC non-O157 isolates, three isolates were isolated from bovine carcasses and harbored the serogroup O130 and one strain isolated from pork carcasses was O-non-typeable. All STEC non-O157 harbored sxt1 gene. The analysis from the whole genome showed that both STEC O157:H7 strains belonged to the hypervirulent clade 8, ST11, phylogroup E, carried the allele tir 255 T > A T, and they were not clonal. The analysis of information allows us to conclude that the STEC strains circulate in pork and bovine carcasses arriving in transport. This situation represents a risk for the consumers and the need to implement an integrated STEC control in the food chain.

In November 2019, an outbreak of Shiga toxin-producing Escherichia coli O157:H7 was detected in South Yorkshire, England. Initial investigations established consumption of milk from a local dairy as a common exposure. A sample of pasteurised milk tested the next day failed the phosphatase test, indicating contamination of the pasteurised milk by unpasteurised (raw) milk. The dairy owner agreed to immediately cease production and initiate a recall. Inspection of the pasteuriser revealed a damaged seal on the flow divert valve. Ultimately, there were 21 confirmed cases linked to the outbreak, of which 11 (52%) were female, and 12/21 (57%) were either <15 or >65 years of age. Twelve (57%) patients were treated in hospital, and three cases developed haemolytic uraemic syndrome. Although the outbreak strain was not detected in the milk samples, it was detected in faecal samples from the cattle on the farm. Outbreaks of gastrointestinal disease caused by milk pasteurisation failures are rare in the UK. However, such outbreaks are a major public health concern as, unlike unpasteurised milk, pasteurised milk is marketed as \'safe to drink\' and sold to a larger, and more dispersed, population. The rapid, co-ordinated multi-agency investigation initiated in response to this outbreak undoubtedly prevented further cases.

Environmental survival time is important when evaluating adverse health outcomes from foodborne pathogens. Although outbreaks associated with manure-impacted irrigation or runoff water are relatively infrequent, their broad scope, regulatory importance, and severe health outcomes highlight the need to better understand the environmental survival of manure-borne pathogens. Shiga toxigenic Escherichia coli (STEC) are excreted in feces and persist in the environment until they die or recolonize a new host. Surface waters contaminated with manure-borne STEC can infect humans through drinking and recreational water use or irrigated crops that are minimally cooked. In this study, manure-impacted water microcosms mimicking beef cattle feedlot runoff were used to assess survival of STEC strains representing seven STEC serotypes (O26, O45, O103, O111, O121, O145, and O157) and persistence of target O antigen genes. Microcosms were sampled over the course of 1 year, and the entire experiment was repeated in a second year. Culture and polymerase chain reaction (PCR)-based techniques were used for detection and enumeration. Serotype-specific survival results were observed. Both STEC O26 and O45 declined slowly and remained culturable at 24 months. In contrast, STEC O121 and O145 decreased rapidly (-0.84 and -1.99 log10 abundance per month, respectively) and were unculturable by months 4 and 5, but detectable by PCR for a mean of 4.5 and 8.3 months, respectively. STEC O103, O111, and O157 remained culturable for a mean of 11.6, 5.5, and 15 months and detectable by PCR for a mean of 12, 13.8, and 18.6 months after inoculation, respectively. Results document that some STEC serotypes have the biological potential to survive in manure-impacted waters for extended periods of time when competing microflora are eliminated. Serotype-specific differences in survival of target bacteria and persistence of target genes were observed in this sample set, with STEC O26 and O45 strains appearing the most robust in these microcosm studies.

The role of environmental transmission of typically foodborne pathogens like Shiga toxin-producing Escherichia coli (STEC) O157 is increasingly recognized. To gain more insights into spatially restricted risk factors that play a role in this transmission, we assessed the spatial association between sporadic STEC O157 human infections and the exposure to livestock (i.e. small ruminants, cattle, poultry, and pigs) in a densely populated country: the Netherlands. This was done for the years 2007-2016, using a state-of-the-art spatial analysis method in which hexagonal areas with different sizes (90, 50, 25 and 10 km2) were used in combination with a novel probability of exposure metric: the population-weighted number of animals per hexagon. To identify risk factors for STEC O157 infections and their population attributable fraction (PAF), a spatial regression model was fitted using integrated nested Laplace approximation (INLA). Living in hexagonal areas of 25, 50 and 90 km2 with twice as much population-weighted small ruminants was associated with an increase of the incidence rate of human STEC O157 infections in summer (RR of 1.09 [95%CI;1.01-1.17], RR of 1.17 [95%CI;1.07-1.28] and RR of 1.13 [95%CI;1.01-1.26]), with a PAF of 49% (95%CI;8-72%). Results suggest exposure to small ruminants to be a risk factor, although no evidence on the mode of transmission is provided. Therefore, the underlying mechanisms warrant further investigation and could offer new targets for control. The newly proposed exposure metric has potential to improve existing spatial modeling studies on infectious diseases related to livestock exposure, especially in densely populated countries like the Netherlands.

In June 2017, an outbreak of Shiga toxin-producing Escherichia coli (STEC) O157 infection with phage type 21/28 and identical genotypic profiles involving three children from Staffordshire was reported. Two cases developed haemolytic uraemic syndrome (HUS). Person-to-person transmission via a shared inflatable home paddling pool was the most likely route of infection, following contamination by the first case. The source of infection in the first case was not identified. We recommend that individuals experiencing gastroenteritis should not bathe in paddling pools and that water should be changed at frequent intervals throughout the day to minimise the spread of infection.

In October 2014, Public Health England (PHE) identified cases of Shiga toxin-producing Escherichia coli (STEC) serogroup O157 sharing a multiple locus variable-number tandem repeat analysis (MLVA) profile. We conducted a case-control study using multivariable logistic regression to calculate adjusted odds ratios (aOR) and 95% confidence intervals (CI) testing a range of exposures. Cases were defined as laboratory-confirmed STEC O157 with the implicated MLVA profile, were UK residents aged ⩾18 years with symptom onset between 25 September and 30 October 2014, and had no history of travel abroad within 5 days of symptom onset. One hundred and two cases were identified. Cases were mostly female (65%; median age 49, range 2-92 years). It was the second largest outbreak seen in England, to date, and a case-control study was conducted using market research panel controls and online survey methods. These methods were instrumental in the rapid data collection and analysis necessary to allow traceback investigations for short shelf-life products. This is a new method of control recruitment and this is the first in which it was a standalone recruitment method. The case-control study suggested a strong association between consumption of a ready-to-eat food and disease (aOR 28, 95% CI 5·0-157) from one retailer. No reactive microbiological testing of food items during the outbreak was possible due to the short shelf-life of the product. Collaboration with industrial bodies is needed to ensure timely traceback exercises to identify contamination events and initiate appropriate and focused microbiological testing and implement control measures.

High-pressure treatments (400 and 600 MPa) combined with the addition of sodium lactate (1 and 3%) were tested to reduce Escherichia coli O157:H7 (STEC O157) and spoilage microbiota contamination in a manufactured cured beef carpaccio in fresh or frozen conditions. Counts of spoilage microorganisms and STEC O157 were also examined during the curing step to prepare the carpaccio. STEC O157 counts remained almost unchanged through the curing process performed at 1 ± 1 °C for 12 days, with a small decrease in samples with 3% of sodium lactate. High-pressure treatments at 600 MPa for 5 min achieved an immediate reduction of up to 2 logarithmic units of STEC O157 in frozen carpaccio, and up to 1.19 log in fresh condition. Counts of spoilage bacteria diminished below detection limits in fresh or frozen carpaccio added with sodium lactate by the application of 400 and 600 MPa. Maximum injury on STEC O157 cells was observed at 600 MPa in carpaccio in fresh condition without added sodium lactate. Lethality of high-pressure treatments on STEC O157 was enhanced in frozen carpaccio, while the addition of sodium lactate at 3% reduced the lethality on STEC O157 in frozen samples, and the degree of injury in fresh carpaccio.

An appropriate approach of high throughput multi-screening was verified for Shiga toxin-producing Escherichia coli (STEC) and Salmonella spp. in strawberries, lettuce and basil. Sample replicates were inoculated with STEC O157 or O26 and Salmonella Thompson (ca. 10-70, 100-700 and 1000-7000 cfu/25 g) and analysed after 1 and 5 days of storage (strawberries and lettuce at 7 °C and basil at 10 °C). After 18-24 h of enrichment at 37 °C in buffered peptone water, detection was performed using the GeneDisc multiplex PCR (stx1, stx2, eae and iroB genes) and selective culture media for isolation of STEC (with immunomagnetic separation (IMS)) and Salmonella spp. in parallel. After 1 day, the pathogenic strains were recovered from all samples for all inoculum levels, whereas reduced detection rates of STEC O157 and S. Thompson were observed after 5 days of storage in case of strawberries, in particular for the lowest inoculums level, suggesting superior survival potential for STEC O26. Overall, this study indicates the ability of PCR based screening methods for reproducible multi-detection of low numbers (10-70 cfu/25 g) of STEC and Salmonella in this type of foods. However, for the basil samples, PCR needed twofold dilution of the DNA extract to overcome inhibition. It was noted that on several occasions growth of competitive microbiota obstructed finding presumptive colonies on the selective agar media, whereas the use of an additional agar medium such as CHROMagar STEC (without IMS) improved recovery rate of STEC.