Enterohemorrhagic Research Articles

Cytoskeletal mechanisms regulating attaching/effacing bacteria interactions with host cells: It takes a village to build the pedestal.

The actin cytoskeleton is a key cellular structure subverted by pathogens to infect and survive in or on host cells. Several pathogenic strains of Escherichia coli, such as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), developed a unique mechanism to remodel the actin cytoskeleton that involves the assembly of actin filament-rich pedestals beneath the bacterial attachment sites. Actin pedestal assembly is driven by bacterial effectors injected into the host cells, and this structure is important for EPEC and EHEC colonization. While the interplay between bacterial effectors and the actin polymerization machinery of host cells is well-understood, how other mechanisms of actin filament remodelling regulate pedestal assembly and bacterial attachment are poorly investigated. This review discusses the gaps in our understanding of the complexity of the actin cytoskeletal remodelling during EPEC and EHEC infection. We describe possible roles of actin depolymerizing, crosslinking and motor proteins in pedestal dynamics, and bacterial interactions with the host cells. We also discuss the biological significance of pedestal assembly for bacterial infection.

Chlorine dioxide is a broad-spectrum disinfectant against Shiga toxin-producing Escherichia coli and Listeria monocytogenes in agricultural water

Agricultural water is commonly treated with chlorine-based disinfectants, which are impacted by water quality. Understanding how water quality influences disinfectants such as chlorine dioxide (ClO2) against pathogenic bacteria is important for creating efficacious sanitation regimens. In this study, the minimum inhibitory concentration (MIC) of ClO2 needed to achieve a 3-Log reduction against Shiga toxin-producing Escherichia coli (STEC) and Listeria monocytogenes was compared across agricultural water samples. Sterile ddH2O served as a control to compare with environmental samples from Salinas Valley, CA, and laboratory standards. To test different dosages and water qualities, stock ClO2 was diluted in 24-well plates with target concentrations of 10, 5, 2.5, and 1.25 mg/L. Well plates were inoculated with pathogens and treated with sanitizer for 5 min. Following treatment, surviving pathogens were enumerated using viable cell counts. The results demonstrate that groundwater samples had the highest water quality of the environmental samples and required the lowest concentration of disinfectant to achieve 3-Log reduction against both bacteria, with MIC between 1.4 and 2.0 mg/L. Open-source samples had lower water quality and required a higher concentration of ClO2 for 3-Log reduction, with MIC between 2.8 and 5.8 mg/L for both pathogens. There was no correlation between pH, turbidity, or conductivity/TDS and reduction for either STEC or L. monocytogenes, suggesting no individual water metric was driving reduction. A lower dosage was required to achieve 3-Log reduction against STEC, while L. monocytogenes required greater concentrations to achieve the same level of reduction. Overall, these results help guide growers in using ClO2 as a broad-spectrum disinfectant and demonstrate its efficacy in reaching 3-Log reduction across agricultural water samples.

Shiga toxin-producing Escherichia coli in the milk production chain: Evaluation of virulence genes and clonal diversity of O157:H7 strains

Microbial contamination of milk and dairy products is a significant issue due to sensory changes and the risk of foodborne diseases. Shiga toxin-producing Escherichia coli (STEC), particularly the O157:H7, are major pathogens transmitted by dairy products. This study aimed to evaluate the microbiological quality of bovine milk at various stages of the production chain in a dairy cooperative and its suppliers in Rio de Janeiro, Brazil. Additionally, the presence of STEC in milk and faeces from dairy cattle was investigated, along with the isolation of STEC O157:H7 strains and the evaluation of virulence genes and clonal diversity. These findings were compared with previously isolated STEC O157:H7 strains from the same region. A total of 100 raw milk samples (54 farm gallon (FG) samples, 25 community bulk tank milk (CBTM) samples, 14 isothermal car-tanker milk (ICTM) samples, and 7 industry tank milk (ITM) samples) and 10 pasteurized milk samples, along with 63 bovine faeces samples, were analysed. The stx gene, a marker for STEC, was detected in 65% of raw milk samples and 88.9% of faecal samples, indicating a high risk of contamination throughout the production chain. None of the pasteurized milk samples tested positive for the stx gene. The prevalence of stx in faeces and raw milk underscores the need for rigorous controls to prevent contamination. While pasteurization was effective, the microbiological quality of raw milk remained unsatisfactory, highlighting the importance of good hygiene practices and continuous monitoring to ensure dairy safety.

Isolation and evaluation of the pathogenicity of a hybrid shiga toxin-producing and Enterotoxigenic Escherichia coli in pigs.

Porcine pathogenic Escherichia coli (E. coli), the globally recognized important pathogen, causes significant economic loss in the field. Enterotoxigenic E. coli (ETEC) causes porcine neonatal and post-weaning diarrhea (PWD), frequently carrying F4 adhesin, F18 adhesin, Heat-Stable toxin (ST), and Heat-Labile toxin (LT). Shiga Toxin-Producing E. coli (STEC) produces F18 adhesin and Shiga toxin type 2e (stx2e), majorly leading to systemic endothelial cell damage and edema disease. In this study, hemolytic pathogenic hybrid STEC/ETEC strains carrying ST and LT genes of ETEC and the Stx2e gene of STEC isolated from pigs with PWD in Taiwan were identified. The pathogenicity of a Taiwan hybrid STEC/ETEC strain was evaluated by oral inoculation in post-weaning pigs. Next generation sequencing and multilocus sequence typing of two hybrid Taiwan porcine STEC/ETEC isolates indicated that these two isolates were closely related to the ST88 porcine hybrid STEC/ETEC isolated from pigs with watery diarrhea. Furthermore, the two hybrid Taiwan porcine STEC/ETEC isolates also displayed combinations of multiple resistance genes encoding mechanisms for target modification and antibiotic inactivation. Animal experiments confirmed that the Taiwan hybrid STEC/ETEC could cause watery diarrhea in post-weaning pigs with no signs of edema disease and minimal histopathological lesions. To the best of the authors' knowledge, the present study is the first study demonstrating intestinal pathogenicity of the hybrid STEC/ETEC in pigs. The result suggests that the hybrid STEC/ETEC should be considered as a new emerging pathogen and a new target for vaccine development.

The inactivation of Shiga toxin-producing Escherichia coli (STEC) and Listeria monocytogenes using isochoric freezing in raw milk and carrot juice

Isochoric freezing is a novel food preservation method that may maintain the quality and safety of products by simultaneously applying low temperatures and high pressures. This work aims to determine whether isochoric freezing can improve food safety by inactivating Shiga toxin-producing Escherichia coli (STEC) and Listeria monocytogenes in raw milk and carrot juices. Raw milk and carrot juice samples were inoculated with cocktails of either STEC or L. monocytogenes and then subjected to isochoric freezing conditions in pressurized chambers set at −5, −10, and −15 °C and treated for 1, 5, 7, or 10 days. Following treatment, the product was sampled for surviving pathogens. The results demonstrated that a 5-log reduction of both STEC and L. monocytogenes can be achieved for raw milk and carrot juice without adversely affecting quality. For STEC inoculated in raw milk, both the linear and Weibull models suggest that 10 days is required to achieve 5-log reduction at −10 °C. Decreasing the temperature to −15 °C led to accelerated log reduction but induced phase separation in the milk. Listeria in raw milk demonstrated a biphasic reduction, indicating 1.3 days is required for a 5-log reduction at −10 °C. In comparison, pathogens demonstrated faster log reduction in carrot juice due to its intrinsic properties. The survival curves for STEC-inoculated carrot juice demonstrated that the 5-log reduction times were 6.9 days at −10 °C and 3.8 days at −15 °C. L. monocytogenes in carrot juice required 1.5 days at −10 °C for a 5-log reduction and was eliminated (7 log cfu/ml) within 24 h at −15 °C. Isochoric freezing could be an option for milk and carrot juice processors wishing to improve food safety without applying heat. This method is not a one-size-fits-all solution, and the time and temperature will need further optimization depending on the target pathogen and intended commodity.

A master regulator of central carbon metabolism directly activates virulence gene expression in attaching and effacing pathogens.

The ability of the attaching and effacing pathogens enterohaemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium to overcome colonisation resistance is reliant on a type 3 secretion system used to intimately attach to the colonic epithelium. This crucial virulence factor is encoded on a pathogenicity island known as the Locus of Enterocyte Effacement (LEE) but its expression is regulated by several core-genome encoded transcription factors. Here, we unveil that the core transcription factor PdhR, traditionally known as a regulator of central metabolism in response to cellular pyruvate levels, is a key activator of the LEE. Through genetic and molecular analyses, we demonstrate that PdhR directly binds to a specific motif within the LEE master regulatory region, thus activating type 3 secretion directly and enhancing host cell adhesion. Deletion of pdhR in EHEC significantly impacted the transcription of hundreds of genes, with pathogenesis and protein secretion emerging as the most affected functional categories. Furthermore, in vivo studies using C. rodentium, a murine model for EHEC infection, revealed that PdhR is essential for effective host colonization and maximal LEE expression within the host. Our findings provide new insights into the complex regulatory networks governing bacterial pathogenesis. This research highlights the intricate relationship between virulence and metabolic processes in attaching and effacing pathogens, demonstrating how core transcriptional regulators can be co-opted to control virulence factor expression in tandem with the cell's essential metabolic circuitry.

Investigation of Shiga toxin-producing Escherichia coli and evaluation of the microbiological quality of raw vegetable sal-ads

Foodborne diseases (FBD) are a significant public health concern worldwide, affecting millions of people annually. Symptoms of FBD range from mild gastrointestinal discomfort to more severe conditions, which in some cases can lead to death. Raw vegetables, especially those consumed in ready-to-eat (RTE) salads, are particularly susceptible to microbial contamination. This study aimed to investigate the presence of Shiga toxin-producing Escherichia coli (STEC) and to evaluate the microbiological quality of 77 RTE raw vegetable salads from 20 restaurants in Niterói, RJ, Brazil. None samples presented STEC, nor the safety indicators (SI) Salmonella spp. and Listeria monocytogenes. However, 89.6% of the samples were considered unfit for human consumption, since they presented at least one hygiene indicator (HI) outside the established standards. The majority of samples (95.5%) were unsatisfactory for Enterobacteriaceae (EB), followed by total aerobic bacteria (TAB) (76.6%), thermotolerant coliforms (C45) (32.5%) and Escherichia coli (EC) (1.3%). The difference between the occurrences of hygiene indicators was not significant. Given the high percentage of inadequate samples found in the study, it is clear that there is a need for stricter surveillance and adequate hygiene practices in restaurants. To reduce the risks of microbial contamination in RTE raw vegetable salads, several approaches can be implemented. These include continuous training of food handlers, implementation of food safety management systems such as Hazard Analysis and Critical Control Points (HACCP), and regular inspections. In addition, consumers also play an important role by demanding safe food and following safe food handling and consumption practices at home.

Prevalence and characteristics of 11 potentially diarrhoeagenic microbes in asymptomatic individuals in Norway, 2015-2020.

We aimed to estimate the prevalence of potentially diarrhoeagenic microbes (PDMs) in faecal samples from asymptomatic individuals in a high-income country, identify risk factors for carriage and to identify microbial factors that differ between PDMs in asymptomatic versus symptomatic individuals. Samples from 1000 asymptomatic participants were collected, together with a questionnaire, between 2015 and 2020 and examined by PCR for 11 PDMs. Isolates were characterised and potential risk factors were registered. Atypical enteropathogenic Escherichia coli (aEPEC), Yersinia enterocolitica, Shiga toxin-producing E. coli (STEC), enterotoxigenic E. coli (ETEC) and Campylobacter spp. were found in 163 (16%), 20 (2.0%), 17 (1.7%), 12 (1.2%) and 11 (1.1%) asymptomatic individuals, respectively. Other PDMs were rare. Only low virulent STEC, with stx1c, stx2b or stx2f, was detected. Travels outside Europe was a significant risk factor for detecting Campylobacter spp. (odds ratio (OR) 6.99; 95% CI 1.12-43.6) and ETEC (OR 11.4; 95% CI 1.26-102). Individuals ≥65 years of age had lower odds of carrying STEC (OR 0.11; 95% CI 0.02-0.57) or EPEC (OR 0.09; 95% CI 0.05-0.16) than individuals ≤5 years of age. The common finding of PDMs in asymptomatic individuals could have implications for the interpretation of positive findings in clinical samples and infection control measures.

Commercial bacteriophage preparations for the control of Listeria monocytogenes and Shiga toxin-producing Escherichia coli in raw and pasteurized milk

Listeria monocytogenes was the etiologic agent in nearly all recent outbreaks in North America attributed to pasteurized dairy products, whereas Escherichia coli O157 infections were responsible for most of the rare, yet serious complications from outbreaks involving unpasteurized dairy. This study determined the susceptibility of selected strains of L. monocytogenes and Shiga toxin-producing E. coli (STEC) to commercial phage preparations and their ability to control these pathogens in pasteurized and raw milk during 7-day storage at 7 °C. Both phage products demonstrated high lytic efficiency against 17 strains of L. monocytogenes whereas the efficiency of E. coli phages was more variable against 11 strains of O157 and non-O157 STEC. Broth microdilution assays identified effective endpoint multiplicities of infection (MOI) ranging from log 2.53 to 5.13, which differed between strains of L. monocytogenes and phage products. Mean log MOIs of E. coli phages against STEC also varied within and between products from 0.43 to 7.05. Despite these observations, the change in counts over time of three L. monocytogenes strains exposed to phage in pasteurized milk (log MOI 6) was similar with counts ∼ 4 log CFU/mL lower than control at day 7. Results for STEC O157 varied by strain but counts were lower than control in all cases by 72 h thorough day 7. Titers on isolates of both pathogens isolated from pasteurized milk indicated that the surviving populations were less susceptible to phage. The addition of a phage preparation to raw milk did not reduce populations of either pathogen or affect the change in counts of any strain over time when compared to control. Reduced efficacy in raw milk may be attributed to reduced phage binding as titers in raw milk decreased steadily (∼2 log PFU/mL) during storage. Commercial phage products may be a promising pathogen control intervention for pasteurized dairy products, warranting further investigation.

Host-Pathogen Interactions during Shiga Toxin-Producing Escherichia coli Adherence and Colonization in the Bovine Gut: A Comprehensive Review.

Shiga toxin-producing Escherichia coli (STEC) is a significant public health threat due to its ability to cause severe gastrointestinal diseases in humans, ranging from diarrhea to life-threatening conditions such as hemorrhagic colitis and hemolytic uremic syndrome (HUS). As the primary reservoir of STEC, cattle play a crucial role in its transmission through contaminated food and water, posing a considerable risk to human health. This comprehensive review explores host-pathogen interactions during STEC colonization of the bovine gut, focusing on the role of gut microbiota in modulating these interactions and influencing disease outcomes. We integrated findings from published transcriptomics, proteomics, and genomics studies to provide a thorough understanding of how STEC adheres to and colonizes the bovine gastrointestinal tract. The insights from this review offer potential avenues for the development of novel preventative and therapeutic strategies aimed at controlling STEC colonization in cattle, thereby reducing the risk of zoonotic transmission.

Antibiotic Resistance and Genomic Analysis of Shiga Toxin-Producing Escherichia coli from Dairy Cattle, Raw Milk, and Farm Environment in Shaanxi Province, China.

To investigate the epidemiology of Shiga toxin-producing Escherichia coli (STEC) in dairy cattle, 975 samples (185 feces, 34 silage, 36 cattle drinking water, 360 raw milk, and 360 teat skin swabs) were collected from two dairy farms in Baoji and Yangling, Shaanxi Province, China, and were screened for STEC. Whole-genome sequencing was used to analyze the genomic characteristics and potential transmission of STEC isolates. A total of 32 samples were contaminated with STEC, including 4.0% (19/479) in Farm A and 2.6% (13/496) in Farm B. Compared with adult cows (4.5%), nonadult cows had a higher rate (21.3%) of STEC colonization. A total of 14 serotypes and 11 multilocus sequence typing were identified in 32 STEC isolates, among which O55:H12 (25.0%) and ST101 (31.3%) were the most predominant, respectively. Six stx subtypes/combinations were identified, including stx1a (53.1%), stx2g (15.6%), stx2d, stx2a+stx2d, stx1a+stx2a (6.3%, for each), and stx2a (3.1%). Of 32 STEC isolates, 159 virulence genes and 27 antibiotic resistance genes were detected. Overall, STEC isolates showed low levels of resistance to the 16 antibiotics tested (0-40.6%), with most common resistance to ampicillin (40.6%). The phylogenetic analysis confirmed that STEC in the gut of cattle can be transmitted through feces. The results of this study help to improve our understanding of the epidemiological aspects of STEC in dairy cattle and provide early warning and control of the prevalence and spread of the bacterium.

Shiga toxin-producing Escherichia coli infection as a precipitating factor for atypical hemolytic-uremic syndrome.

Hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy characterized by intravascular hemolysis. It can be classified as either typical, primarily caused by Shiga toxin-producing Escherichia coli (STEC) infection, or as atypical HUS (aHUS), which results from uncontrolled complement activation. We report the case of a 9-year-old boy with aHUS due to compound heterozygous complement factor H-related genes (CFHR) 1/3 and CFHR1-CFHR4 deletions, leading to the development of anti-complement factor H (CFH) autoantibodies. The patient presented nephrological and neurological thrombotic microangiopathy with STEC positivity. Additionally, we provide an extensive literature review of aHUS cases initially classified as typical. A total of 11 patients were included, 73% of whom were pediatric. Kidney replacement therapy was required in 73% of patients. The recurrence rate was 55%. All cases were found positive for pathological variants of the complement system genes. The most commonly implicated gene was CFH, while the CFHR genes were involved in 36% of cases, although none exhibited anti-CFH autoantibodies. Anti-complement therapy was administered in 54% of cases, and none of the patients who received it early progressed to kidney failure. STEC infection does not exclude aHUS diagnosis, and early use of anti-complement therapy might be reasonable in life-threatening conditions. Genetic testing can be helpful in patients with atypical presentations and can confirm the necessity of prolonged anti-complement therapy.

Characteristics of Enterohaemorrhagic Escherichia coli in Jeju Province, Korea: a study of registry routes from 2017 to 2023

Enterohemorrhagic Escherichia coli (EHEC) produces Shiga toxin, which causes diarrhea, hemorrhagic colitis, and hemorrhagic uremic syndrome. The Korea Center for Disease Control and Prevention (KCDC) has designated EHEC as a legal infectious disease and has conducted laboratory surveillance. This study aimed to compare the demographic, epidemiological, clinical, and laboratory characteristics of EHEC cases in Jeju Province between August 2017 and November 2023, notified by two registry routes. The data were sourced from the integrated disease health and health management system managed by the KCDC. Cases reported due to experiencing gastrointestinal symptoms, or contacts of each case were defined as the “symptomatic” group. In contrast, cases with positive findings during a health checkup or laboratory surveillance were defined as the “screening” group. The symptomatic and screening groups had 35 and eight patients, respectively. There were significant differences in the age and seasonal distribution. The selection group was 20-59 years old in the main age distribution and registered evenly throughout the four seasons. In comparison, the symptomatic group was younger or older and not registered in the winter. These findings may be used to establish a more appropriate surveillance system for diarrheal outbreaks.

Simultaneous detection of six major virulent genes of Escherichia coli O157:H7 from seafood by multiplex PCR and its validation through spiked study

Enterohaemorrhagic Escherichia coli (EHEC), mainly the serotype O157:H7 is a global concern in foodborne outbreaks,. The incidence of E. coli O157:H7 in seafood sold in India has been reported as 8.4%. The conventional method for the detection of E. coli O157:H7 from seafood is cumbersome and time consuming making this strain particularly challenging to detect. In this study a multiplex PCR was developed targeting six major virulence factors of the pathogen viz. shigatoxin 1 and 2, attachment and effacement factor, flagellar antigen, hemolysin gene and O157 antigen. The sensitivity of the multiplex PCR developed in the study was found to be 120 pg μl-1 of DNA and that of E. coli 0157:H7 spiked samples was 2 cfu ml-1. The multiplex PCR developed in this study showed 100% specificity in identifying E. coli O157:H7, effectively distinguishing it from non-target bacteria. This highly sensitive and specific multiplex PCR is the first of its kind to detect E. coli O157:H7 in seafood by targeting six major virulence genes. Keywords: E. coli O157:H7, EHEC, Foodborne pathogen, Seafood, Sensitivity, Shigatoxin, Specificity

Detection of Shiga toxin-producing Escherichia coli in dairy cows: genetic characterization and inhibition of adherence by cattle anti-STEC antibodies to HEp-2 cell.

Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen associated with severe disease. Cattle are recognized as the primary animal reservoir of STEC. This study reports the occurrence and characterization of STEC from dairy cows and evaluates the inhibition of adherence by cattle anti-STEC antibodies to the HEp-2 cell. From 151 samples, 30% (n = 45) were positive for stx by PCR screening (25.21% of dairy cows and 46.8% of growing calves). From these positive samples, 17 STEC isolates were characterized. In dairy cows, 3 out of 17 samples carried stx2, 3 out of 17 possessed stx1, and 2 out of 17 carried stx1/stx2. In growing cows, 8 out of 17 samples carried stx1 and 1 out of 17 carried stx1/stx2. Other virulence factors such as ehxA, saa, iha, cah, and eae were detected. The strains were typed into 3 E. coli O groups (O26, O91, and O130). The analysis of the HEp-2 adherence assays indicated that all serum from both cattle categories presented high levels of inhibition of adherence. Considering the severity of the symptoms caused by STEC in humans, searching for factors influencing the colonization of STEC in cattle would help identify strategies to avoid transmission and STEC infection.

Ruminant-dense environments increase risk of Shiga toxin-producing Escherichia coli independently of ruminant contact.

Cattle and other domestic ruminants are the primary reservoirs of O157 and non-O157 Shiga toxin-producing Escherichia coli (STEC). Living in areas with high ruminant density has been associated with excess risk of infection, which could be due to both direct ruminant contact and residual environmental risk, but the role of each is unclear. We investigated whether there is any meaningful risk to individuals living in ruminant-dense areas if they do not have direct contact with ruminants. Using a Bayesian spatial framework, we investigated the association between the density of ruminants on feedlots and STEC incidence in Minnesota from 2010 to 2019, stratified by serogroup and season, and adjusting for direct ruminant contact. For every additional head of cattle or sheep per 10 acres, the incidence of O157 STEC infection increased by 30% (IRR 1.30; 95% CrI 1.18, 1.42) or 135% (IRR 2.35; 95% CrI 1.14, 4.20), respectively, during the summer months. Sheep density was also associated with O157 STEC risk during winter (IRR 4.28; 95% CrI 1.40, 8.92). The risk of non-O157 STEC infection was only elevated in areas with goat operations during summer (IRR 19.6; 95% CrI 1.69, 78.8). STEC risk associated with ruminant density was independent of direct ruminant contact across serogroups and seasons. Our findings demonstrate that living in a ruminant-dense area increases an individual's risk of O157 and non-O157 STEC infection even without direct ruminant contact, indicating that prevention efforts need to extend to community strategies for averting indirect transmission from local ruminant populations.

Clinical Outcomes and Virulence Factors of Shiga Toxin-Producing Escherichia coli (STEC) from Southern Alberta, Canada, from 2020 to 2022.

Shiga toxin-producing Escherichia coli (STEC) can cause severe clinical disease in humans, particularly in young children. Recent advances have led to greater availability of sequencing technologies. We sought to use whole genome sequencing data to identify the presence or absence of known virulence factors in all clinical isolates submitted to our laboratory from Southern Alberta dated 2020-2022 and correlate these virulence factors with clinical outcomes obtained through chart review. Overall, the majority of HUS and hospitalizations were seen in patients with O157:H7 serotypes, and HUS cases were primarily in young children. The frequency of virulence factors differed between O157:H7 and non-O157 serotypes. Within the O157:H7 cases, certain virulence factors, including espP, espX1, and katP, were more frequent in HUS cases. The number of samples was too low to determine statistical significance.

Hydrolysis probe assays for the detection of pathogenic Enterohemorrhagic Escherichia coli: Multi-Country validation study

Enterohemorrhagic Escherichia coli (EHEC) strains are foodborne pathogens frequently associated with outbreaks linked to red meat, posing significant global food safety concerns. While several commercial assays exist for detecting EHEC strains belonging to serogroups O26, O103, O111, and O121, they often fail to distinguish pathogenic strains from non-pathogenic strains lacking virulence genes. These non-pathogenic E. coli interfere with the accuracy of existing assays, resulting in false-positives rates between 81 – 100 % for beef products. This study aimed to standardize and validate four hydrolysis probe assays for the specific detection of pathogenic strains of E. coli O26, O103, O111, and O121. Primers and probes were designed to target single nucleotide polymorphisms conserved among pathogenic strains of each target serogroup. The standardized assays were validated using a comprehensive collection of pure culture and DNA samples (n = 248) from Canada, France, Switzerland, and the United States; laboratory-inoculated beef and spinach (n = 132), fractionally inoculated ground beef (n = 390), and samples from the federal red meat surveillance program (n = 166). The results demonstrated an overall accuracy of 94.8 % in predicting the virulence profile of pure culture strains. These four assays are a single-step PCR screening tool that can determine the presence of virulent strains. These assays provide food testing laboratories worldwide with a set of reliable methods for identifying pathogenic O26, O103, O111, and O121 strains. Thus, this assay will help to reduce product waste and financial losses caused by misleading positive results caused by presence of non-pathogenic strains.

PSV-5 Characterizing the activity of bovine Lactobacillus strains against the top 7 Shiga-toxin Producing Escherichia coli strains

Abstract Shiga toxigenic Escherichia coli (STEC), specifically serogroups O26, O45, O111, O103, O121, O145, and O157 (Top 7), pose significant health risks. Cattle are the primary reservoir of STEC, and effective intervention remains elusive. The identification of Lactobacillus spp. with antimicrobial activity as direct-fed microbials (DFMs) presents a potential strategy for pathogen reduction. These strains are not only capable of favorably modulating the gut microbiota but also possess the innate ability to produce bacteriocins that can target specific pathogens including STEC. Selection of viable anti-STEC Lactobacillus strains requires assessment of the presence or absence of prophage that may revert from the lysogenic to lytic phase and affect their efficacy. This study aimed to characterize anti-STEC potential of 14 Lactobacillus strains isolated from cattle and to identify their associated bacteriocins and prophages. Antimicrobial activity of 14 Lactobacillus strains against the top 7 STEC strains was determined using live culture spot assays and cell-free supernatants (CFS) well-diffusion assays. To further assess their anti-STEC efficacy, Lactobacillus strains with the strongest activity were selected and further evaluated in MRS broth culture and rumen fluid. The ability of Lactobacillus strains to inhibit STEC attachment to Caco-2 epithelial cells was also assessed. Prokka 1.14.6, and Bagel5 were utilized to identify genes encoding bacteriocins. Additionally, PHASTEST 3.0 was utilized to identify prophages. All 14 strains demonstrated antimicrobial activity against the top 7 STEC serogroups. Notably, L. agilis strains L3 and L6, with genome sizes of 2,063,576 bp and 2,071,091 bp respectively, and L. amylovorus L7 (883,016 bp) exhibited the greatest anti-STEC activities, targeting O157, O26, O45, and O103 for L3 and L6, and O157, O45, and O103 for L7. The CFS from these strains showed anti-STEC activity at pH 4.4-4.6, but not at 7, suggesting that organic acids played a role in inhibition. In MRS, DFM reduced STEC O157:H7 by 5 to 7.4 logs after 24 h. However, in rumen fluid, no notable reduction in STEC O157:H7 was observed. Bioinformatic analysis revealed that the L7 genome encoded Helveticin-J, a Class III bacteriocin. Prophage identified included 1 intact (51,818 bp) and 1 non-intact (17,267 bp) in L3, and 2 intact (46,916 bp, 20,295 bp) and 1 non-intact (13,939 bp) in L6, while no prophage was identified in L7. This study identified novel DFMs with activity against STEC, but future studies with cattle are required to confirm their ability to inhibit STEC.

Molecular and in silico analyses for detection of Shiga toxin-producing Escherichia coli (STEC) and highly pathogenic enterohemorrhagic Escherichia coli (EHEC) using genetic markers located on plasmid, O Island 57 and O Island 71

BackgroundDue to the diversity of Shiga toxin-producing Escherichia coli (STEC) isolates, detecting highly pathogenic strains in foodstuffs is challenging. Currently, reference protocols for STEC rely on the molecular detection of eae and the stx1 and/or stx2 genes, followed by the detection of serogroup-specific wzx or wzy genes related to the top 7 serogroups. However, these screening methods do not distinguish between samples in which a STEC possessing both determinants are present and those containing two or more organisms, each containing one of these genes. This study aimed to evaluate ecf1, Z2098, Z2099, and nleA genes as single markers and their combinations (ecf1/Z2098, ecf1/Z2099, ecf1/nleA, Z2098/Z2099, Z2098/nleA, and Z2099/nleA) as genetic markers to detect potentially pathogenic STEC by the polymerase chain reaction (PCR) in 96 animal samples, as well as in 52 whole genome sequences of human samples via in silico PCR analyses.ResultsIn animal isolates, Z2098 and Z2098/Z2099 showed a strong association with the detected top 7 isolates, with 100% and 69.2% of them testing positive, respectively. In human isolates, Z2099 was detected in 95% of the top 7 HUS isolates, while Z2098/Z2099 and ecf1/Z2099 were detected in 87.5% of the top 7 HUS isolates.ConclusionsOverall, using a single gene marker, Z2098, Z2099, and ecf1 are sensitive targets for screening the top 7 STEC isolates, and the combination of Z2098/Z2099 offers a more targeted initial screening method to detect the top 7 STEC isolates. Detecting non-top 7 STEC in both animal and human samples proved challenging due to inconsistent characteristics associated with the genetic markers studied.