Shiga Toxin-producing Escherichia Coli Contamination Research Articles

Characterization of Shiga Toxin-producing Escherichia coli Isolated from Cattle Around Ulaanbaatar City, Mongolia

Shiga toxin-producing Escherichia coli (STEC) are associated with severe infections including hemorrhagic colitis and hemolytic uremic syndrome in humans. Ruminants are known as reservoirs of STEC; however, no data are available on STEC in ruminants in Mongolia, where more than 5 million cattle and 25 million sheep are raised. To disclose the existence and characteristics of STEC in Mongolia, in this study, we isolated and characterized STEC from cattle in Mongolia. We collected 350 rectal swabs of cattle from 30 farms near Ulaanbaatar city and isolated 45 STEC from 21 farms. Rectal swabs were precultured with modified Escherichia coli broth and then inoculated to Cefixime-Tellurite Sorbitol MacConkey agar plate and/or CHROMagar STEC agar plate for the isolation of STEC. The isolation ratios in each farm were from 0% to 40%. Multiplex PCR for the estimation of O- and H-serotypes identified 12 O-genotypes (Og-types) and 11 H-genotypes (Hg-types) from 45 isolates; however, Og-types of 19 isolates could not be determined. Stx gene subtyping by PCR identified 2 stx1 subtypes (1a and 1c) and 4 stx2 subtypes (2a, 2c, 2d, and 2g). Forty-five isolates were divided into 21 different groups based on the Og- and Hg-types, stx gene subtypes and the existence of virulence factors, ehxA, eae, and saa, which includes several major serotypes associated with human illness such as O26:H11 and O157:H7. The most dominant isolate, OgUT:H19 [stx1a (+), stx2a (+), ehxA (+) and saa (+)], was isolated from eight farms. This is the first report on the characterization of STEC in cattle in Mongolia, and the results suggest the importance of further monitoring of STEC contamination in the food chains as well as STEC infection in humans.

Development of Recombinase Aided Amplification (RAA)-Exo-Probe Assay for the Rapid Detection of Shiga Toxin-Producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) is a significant cause of foodborne illness causing various gastrointestinal diseases including hemolytic uremic syndrome (HUS), the most severe form, which can lead to kidney failure or even death. Here, we report the development of recombinase aided amplification (RAA)-exo-probe assays targeting the stx1 and stx2 genes for the rapid detection of STEC in food samples. Primers and exo-probes were designed and optimized for the detection of stx1 and stx2 using RAA technology. The optimal STEC RAA-exo-probe assays were then tested for specificity and sensitivity, and validated in both spiked and real food samples. These assays were found to be 100% specific to STEC strains and were also highly sensitive with a detection limit of 1.6 × 103 CFU/mL or 32 copies/reaction. Importantly, the assays were able to successfully detect STEC in spiked and real food samples (beef, mutton, and pork), with a detection limit as low as 0.35 CFU/25g in beef samples after an overnight enrichment step. Overall, the RAA assay reactions completed within ∼20 min and were less dependent on expensive equipment, suggesting they can be easily adopted for in-field testing requiring only a fluorescent reader. As such, we have developed two rapid, sensitive, and specific assays that can be used for the routine monitoring of STEC contamination in food samples, particularly in the field or in poorly equipped labs.

Characterization of Shiga toxin-producing Escherichia coli presence, serogroups and risk factors from private groundwater sources in western Ireland

Over recent years, Ireland has reported the highest crude incidence rates of Shiga toxin-producing Escherichia coli (STEC) enteritis in Europe. Unregulated private groundwater sources have emerged as an important potential transmission route for STEC, with up to 750,000 Irish residents reliant on these sources for domestic waters. This study aimed to investigate the prevalence and serogroup profile of STEC contamination from domestic private wells in western Ireland. Fifty-two groundwater sources were analysed during two sampling campaigns in the autumn (September/October) of 2019 (n = 21) and 2021 (n = 31). Untreated groundwater samples (30 L) were collected and analysed using the “CapE” (capture, amplify, extract) method. Extracted DNA was tested using multiplex real-time PCR for Shiga toxin stx1 and/or stx2 and eae genes. STEC positive DNA samples were tested for clinically relevant serogroups by real-time PCR. Data relating to 27 potential groundwater contamination risk factors were geospatially linked to each well and assessed for association with E. coli, stx1 and/or stx2 and eae presence/absence. Overall, 20/52 wells (38.4 %) were positive for E. coli (median concentration 8.5 MPN/100 mL as assessed by Colilert-18 method). Stx1 and/or stx2 was detected in 10/52 (19.2 %) wells overall and 8/20 E. coli positive wells, equating to a STEC to “generic” E. coli detection ratio of 40 %. Six of these wells (30 %) were also positive for eae. One or more serogroup-specific gene targets were identified in all but one stx1 and/or stx2 positive sample, with O145 (n = 6), O157 (n = 5) and O103 (n = 4) most prevalent. STEC presence was significantly associated with decreasing well depth (U = −2.243; p = 0.024) and increasing 30-day mean antecedent rainfall (U = 2.126; p = 0.034). Serogroup O104 was associated with increased sheep density (U = 2.089; p = 0.044) and detection of stx1 and/or stx2 + eae with increased septic tank density (U = 2.246 p = 0.023). Findings indicate high detection rates of clinically relevant STEC in E. coli contaminated groundwater sources in Ireland.

The predominance of Shiga toxin-producing E. coli in the Southeast Coast of India

In this study, we reported Shiga toxin-producing Escherichia coli (STEC) in 847 samples, including those in coastal waters, sediments, and fish samples in the Southeast Coast of India. A total of 3742 E. coli strains were identified using conventional and molecular identification methods. Of these, 1518 isolates expressed virulent genes Stx1, Stx2, and Eae; effects on these genes on toxicity were examined. Furthermore, 2224 non-STEC isolates caused hemolytic uremic syndrome and played a key role in the persistence of STEC contamination. We conclude that toxin production is not adequate to cause disease, and the pathogenic mechanism of STEC remains poorly defined. Therefore, the present study indicates the status of pollution, highlighting the need for sanitation in public health.

Shiga Toxin–Producing Escherichia coli (STEC) and STEC-Associated Virulence Genes in Raw Ground Pork in Canada

Shiga toxin-producing Escherichia coli (STEC) O157:H7/nonmotile and some non-O157 STEC strains are foodborne pathogens. In response to pork-associated O157 STEC outbreaks in Canada, we investigated the occurrence of STEC in Canadian retail raw ground pork during the period of 1 November 2014 to 31 March 2016. Isolated STEC strains were characterized to determine the Shiga toxin gene (stx) subtype and the presence of virulence genes encoding intimin (eae) and enterohemorrhagic E. coli hemolysin (hlyA). O157 STEC and non-O157 STEC strains were isolated from 1 (0.11%) of 879 and 13 (2.24%) of 580 pork samples, respectively. STEC virulence gene profiles containing both eae and hlyA were found only in the O157 STEC (stx2a, eae, hlyA) isolate. The eae gene was absent from all non-O157 STEC isolates. Of the 13 non-O157 STEC isolates, two virulence genes of stx1a and hlyA were found in four (30.8%) O91:H14 STEC isolates, whereas one virulence gene of stx2e, stx1a, and stx2a was identified in five (38.5%), two (15.4%), and one (7.7%) STEC isolates, respectively, of various serotypes. The remaining non-O157 STEC isolate carried stx2, but the subtype is unknown because this isolate could not be recovered for sequencing. O91:H14 STEC (stx1a, hlyA) was previously reported in association with diarrheal illnesses, whereas the other non-O157 STEC isolates identified in this study are not known to be associated with severe human illnesses. Virulence gene profiles identified in this study indicate that the occurrence of non-O157 STEC capable of causing severe human illness is rare in Canadian retail pork. However, O157 STEC in ground pork can occasionally occur; therefore, education regarding the potential risks associated with STEC contamination of pork would be beneficial for the public and those in the food industry to help reduce foodborne illnesses.

A Validation System for Selection of Bacteriophages against Shiga Toxin-Producing Escherichia coli Contamination

Shiga toxin-producing Escherichia coli (STEC) can cause severe infections in humans, leading to serious diseases and dangerous complications, such as hemolytic-uremic syndrome. Although cattle are a major reservoir of STEC, the most commonly occurring source of human infections are food products (e.g., vegetables) contaminated with cow feces (often due to the use of natural fertilizers in agriculture). Since the use of antibiotics against STEC is controversial, other methods for protection of food against contaminations by these bacteria are required. Here, we propose a validation system for selection of bacteriophages against STEC contamination. As a model system, we have employed a STEC-specific bacteriophage vB_Eco4M-7 and the E. coli O157:H7 strain no. 86-24, bearing Shiga toxin-converting prophage ST2-8624 (Δstx2::cat gfp). When these bacteria were administered on the surface of sliced cucumber (as a model vegetable), significant decrease in number viable E. coli cells was observed after 6 h of incubation. No toxicity of vB_Eco4M-7 against mammalian cells (using the Balb/3T3 cell line as a model) was detected. A rapid decrease of optical density of STEC culture was demonstrated following addition of a vB_Eco4M-7 lysate. However, longer incubation of susceptible bacteria with this bacteriophage resulted in the appearance of phage-resistant cells which predominated in the culture after 24 h incubation. Interestingly, efficiency of selection of bacteria resistant to vB_Eco4M-7 was higher at higher multiplicity of infection (MOI); the highest efficiency was evident at MOI 10, while the lowest occurred at MOI 0.001. A similar phenomenon of selection of the phage-resistant bacteria was also observed in the experiment with the STEC-contaminated cucumber after 24 h incubation with phage lysate. On the other hand, bacteriophage vB_Eco4M-7 could efficiently develop in host bacterial cells, giving plaques at similar efficiency of plating at 37, 25 and 12 °C, indicating that it can destroy STEC cells at the range of temperatures commonly used for vegetable short-term storage. These results indicate that bacteriophage vB_Eco4M-7 may be considered for its use in food protection against STEC contamination; however, caution should be taken due to the phenomenon of the appearance of phage-resistant bacteria.

The interactions of bacteriophage Ace and Shiga toxin-producing Escherichia coli during biocontrol.

Strictly lytic phages are considered powerful tools for biocontrol of foodborne pathogens. Safety issues needed to be addressed for the biocontrol of Shiga toxin-producing Escherichia coli (STEC) include: lysogenic conversion, Shiga toxin production through phage induction, and emergence/proliferation of bacteriophage insensitive mutants (BIMs). To address these issues, two new lytic phages, vB_EcoS_Ace (Ace) and vB_EcoM_Shy (Shy), were isolated and characterized for life cycle, genome sequence and annotation, pH stability and efficacy at controlling STEC growth. Ace was efficient in controlling host planktonic cells and did not stimulate the production of the Stx prophage or Shiga toxin. A single dose of phage did not lead to the selection of BIMs. However, when reintroduced, BIMs were detected after 24 h of incubation. The gain of resistance was associated with lower virulence, as a subset of BIMs failed to agglutinate with O157-specific antibody and were more sensitive to human serum complement. BIM's biofilm formation capacity and susceptibility to disinfectants was equal to that of the wild-type strain. Overall, this work demonstrated that phage Ace is a safe biocontrol agent against STEC contamination and that the burden of BIM emergence did not represent a greater risk in environmental persistence and human pathogenicity.

Transmission Dynamics of Shiga Toxin-Producing Escherichia coli in New Zealand Cattle from Farm to Slaughter.

Cattle are asymptomatic carriers of Shiga toxin-producing Escherichiacoli (STEC) strains that can cause serious illness or death in humans. In New Zealand, contact with cattle feces and living near cattle populations are known risk factors for human STEC infection. Contamination of fresh meat with STEC strains also leads to the potential for rejection of consignments by importing countries. We used a combination of PCR/matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) and whole-genome sequencing (WGS) to evaluate the presence and transmission of STEC on farms and in processing plants to better understand the potential pathways for human exposure and thus mitigate risk. Animal and environmental samples (n = 2,580) were collected from six farms and three meat processing plants in New Zealand during multiple sampling sessions in spring of 2015 and 2016. PCR/MALDI-TOF analysis revealed that 6.2% were positive for "Top 7" STEC. Top 7 STEC strains were identified in all sample sources (n = 17) tested. A marked increase in Top 7 STEC prevalence was observed between calf hides on farm (6.3% prevalence) and calf hides at processing plants (25.1% prevalence). Whole-genome sequencing was performed on Top 7 STEC bacterial isolates (n = 40). Analysis of STEC O26 (n = 25 isolates) revealed relatively low genetic diversity on individual farms, consistent with the presence of a resident strain disseminated within the farm environment. Public health efforts should focus on minimizing human contact with fecal material on farms and during handling, transport, and slaughter of calves. Meat processing plants should focus on minimizing cross-contamination between the hides of calves in a cohort during transport, lairage, and slaughter.IMPORTANCE Cattle are asymptomatic carriers of Shiga toxin-producing E. coli (STEC) strains, which can cause serious illness or death in humans. Contact with cattle feces and living near cattle are known risk factors for human STEC infection. This study evaluated STEC carriage in young calves and the farm environment with an in-depth evaluation of six farms and three meat processing plants over 2 years. An advanced molecular detection method and whole-genome sequencing were used to provide a detailed evaluation of the transmission of STEC both within and between farms. The study revealed widespread STEC contamination within the farm environment, but no evidence of recent spread between farms. Contamination of young dairy calf hides increased following transport and holding at meat processing plants. The elimination of STEC in farm environments may be very difficult given the multiple transmission routes; interventions should be targeted at decreasing fecal contamination of calf hides during transport, lairage, and processing.

Genetic diversity and pathogenic potential of Shiga toxin-producing Escherichia coli (STEC) derived from German flour

Shiga toxin-producing Escherichia coli (STEC) can cause severe human illness, which are frequently linked to the consumption of contaminated beef or dairy products. However, recent outbreaks associated with contaminated flour and undercooked dough in the United States and Canada, highlight the potential of plant based food as transmission routes for STEC. In Germany STEC has been isolated from flour, but no cases of illness have been linked to flour.In this study, we characterized 123 STEC strains isolated from flour and flour products collected between 2015 and 2019 across Germany. In addition to determination of serotype and Shiga toxin subtype, whole genome sequencing (WGS) was used for isolates collected in 2018 to determine phylogenetic relationships, sequence type (ST), and virulence-associated genes (VAGs).We found a high diversity of serotypes including those frequently associated with human illness and outbreaks, such as O157:H7 (stx2c/d, eae), O145:H28 (stx2a, eae), O146:H28 (stx2b), and O103:H2 (stx1a, eae). Serotypes O187:H28 (ST200, stx2g) and O154:H31 (ST1892, stx1d) were most prevalent, but are rarely linked to human cases. However, WGS analysis revealed that these strains, as well as, O156:H25 (ST300, stx1a) harbour high numbers of VAGs, including eae, nleB and est1a/sta1.Although STEC-contaminated flour products have yet not been epidemiologically linked to human clinical cases in Germany, this study revealed that flour can serve as a vector for STEC strains with a high pathogenic potential. Further investigation is needed to determine the sources of STEC contamination in flour and flour products particularly in regards to these rare serotypes.

An Overview of Shiga-Toxin Producing Escherichia coli Carriage and Prevalence in the Ovine Meat Production Chain.

Shiga-toxin producing Escherichia coli (STEC) are zoonotic foodborne pathogens that are capable of causing serious human illness. Ovine ruminants are recognized as an important source of STEC and a notable contributor to contamination within the food industry. This review examined the prevalence of STEC in the ovine food production chain from farm-to-fork, reporting carriage in sheep herds, during abattoir processing, and in raw and ready-to-eat meats and meat products. Factors affecting the prevalence of STEC, including seasonality and animal age, were also examined. A relative prevalence can be obtained by calculating the mean prevalence observed over multiple surveys, weighted by sample number. A relative mean prevalence was obtained for STEC O157 and all STEC serogroups at multiple points along the ovine production chain by using suitable published surveys. A relative mean prevalence (and range) for STEC O157 was calculated: for feces 4.4% (0.2-28.1%), fleece 7.6% (0.8-12.8%), carcass 2.1% (0.2-9.8%), and raw ovine meat 1.9% (0.2-6.3%). For all STEC independent of serotype, a relative mean prevalence was calculated: for feces 33.3% (0.9-90.0%), carcass 58.7% (2.0-81.6%), and raw ovine meat 15.4% (2.7-35.5%). The prevalence of STEC in ovine fleece was reported in only one earlier survey, which recorded a prevalence of 86.2%. Animal age was reported to affect shedding in many surveys, with younger animals typically reported as having a higher prevalence of the pathogen. The prevalence of STEC decreases significantly along the ovine production chain after the application of postharvest interventions. Ovine products pose a small risk of potential STEC contamination to the food supply chain.

Investigation of Shiga Toxin-Producing Escherichia coli in Groundwater, River Water, and Fecal Sources in the Kathmandu Valley, Nepal

Shiga toxin-producing Escherichia coli (STEC) is one of the most commonly heard sources of foodborne outbreaks but presently lacks studies of in the Kathmandu Valley. This study explored the presence of STEC in river water (n = 17), groundwater (n = 83), feces (n = 50), and manure (n = 20) in the Kathmandu Valley, Nepal. Samples that were confirmed to be E. coli–positive using the Colilert assay in previous studies were selected to examine the presence of the sfmD, stx1, and stx2 genes using quantitative polymerase chain reaction. Based on Colilert/sfmD gene ratios, the percentage of viable cells of E. coli in the fecal (2.0% ± 2.5%), manure (0.1% ± 0.1%), river water (3.4% ± 3.1%), and groundwater samples (3.9% ± 5.8%) were determined. The presence of the stx1 gene was observed in the fecal (6%), river water (53%), and groundwater (2%) samples, whereas the stx2 gene was detected in the fecal (8%), manure (5%), and river water (71%) samples. Interestingly, the stx/sfmD gene ratios in the groundwater samples were greater than 100%, suggesting the presence of other stx1/stx2-harboring microorganisms. These findings indicated the importance of continuing investigation into waterborne sources of STEC contamination within the Kathmandu Valley.

Prevalence, risk factors and molecular characteristics of Shiga toxin-producing Escherichia coli in beef abattoirs in Gauteng, South Africa

A cross-sectional study was conducted to determine the prevalence, risk factors, and molecular characteristics of Shiga toxin-producing Escherichia coli (STEC) in 12 beef abattoirs in Gauteng province, South Africa. The relationship between STEC contamination and aerobic bacterial counts on carcasses at various stages of processing was investigated. Multiplex PCR was used to detect virulence genes in broth-enriched samples, to determine O-serogroups in all samples positive for Shiga toxin-encoding genes (stx), and to characterize isolates of STEC. The overall prevalence of STEC determined by PCR in 419 selective enrichment broth samples was 35.1% (147/419), and was significantly higher (P = 0.037) in perineum hide swabs (50%) than in 24 h chilled carcass swabs (20%). The maximum total aerobic plate count (TAPC) was 3.8 log10 CFU/100 cm2 for carcass swabs, but was below the South African microbiological standard for meat export at all stages of carcass processing. There was no significant association between TAPC and STEC contamination. Serogroup O113 was the most prevalent serogroup (13.6%; 20/147) detected. Only 33 isolates, all non-O157 STEC, were recovered, amongst which six different genotype combinations were observed. Additionally, the clinically important serogroups O117, O8, and O2 were isolated. Multivariable logistic regression revealed that the odds of STEC contamination was lower in post-wash (OR = 0.42; 95% CI: 0.18–0.98; P = 0.045) and 24 h chilled (OR = 0.33; 95% CI: 0.12–0.91; P = 0.033) carcass swabs compared to pre- and post-evisceration swabs. It was concluded that non-O157 STEC serogroups more frequently colonize beef cattle slaughtered at abattoirs in our study area than O157 STEC, and therefore have a higher potential to enter the food chain during carcass processing, with food safety implications.

Lessons Learned from a Decade of Investigations of Shiga Toxin-Producing Escherichia coli Outbreaks Linked to Leafy Greens, United States and Canada.

Shiga toxin-producing Escherichia coli (STEC) cause substantial and costly illnesses. Leafy greens are the second most common source of foodborne STEC O157 outbreaks. We examined STEC outbreaks linked to leafy greens during 2009-2018 in the United States and Canada. We identified 40 outbreaks, 1,212 illnesses, 77 cases of hemolytic uremic syndrome, and 8 deaths. More outbreaks were linked to romaine lettuce (54%) than to any other type of leafy green. More outbreaks occurred in the fall (45%) and spring (28%) than in other seasons. Barriers in epidemiologic and traceback investigations complicated identification of the ultimate outbreak source. Research on the seasonality of leafy green outbreaks and vulnerability to STEC contamination and bacterial survival dynamics by leafy green type are warranted. Improvements in traceability of leafy greens are also needed. Federal and state health partners, researchers, the leafy green industry, and retailers can work together on interventions to reduce STEC contamination.

Phage Biocontrol Improves Food Safety by Significantly Reducing the Level and Prevalence of Escherichia coli O157:H7 in Various Foods

Management of Shiga toxin-producing Escherichia coli (STEC), including E. coli O157:H7, in food products is a major challenge for the food industry. Several interventions, such as irradiation, chemical disinfection, and pasteurization, have had variable success controlling STEC contamination. However, these interventions also indiscriminately kill beneficial bacteria in foods, may impact organoleptic properties of foods, and are not always environmentally friendly. Biocontrol using bacteriophage-based products to reduce or eliminate specific foodborne pathogens in food products has been gaining attention due to the specificity, safety, and environmentally friendly properties of lytic bacteriophages. We developed EcoShield PX, a cocktail of lytic bacteriophages, that specifically targets STEC. This study was conducted to examine the efficacy of this bacteriophage cocktail for reducing the levels of E. coli O157:H7 in eight food products: beef chuck roast, ground beef, chicken breast, cooked chicken, salmon, cheese, cantaloupe, and romaine lettuce. The food products were challenged with E. coli O157:H7 at ca. 3.0 log CFU/g and treated with the bacteriophage preparation at ca. 1 × 106, 5 × 106, or 1 × 107 PFU/g. Application of 5 × 106 and 1 × 107 PFU/g resulted in significant reductions (P < 0.05) in E. coli O157:H7 levels of up to 97% in all foods. When bacteriophages (ca. 1 × 106 PFU/g) were used to treat lower levels of E. coli O157:H7 (ca. 1 to 10 CFU/10 g) on beef chuck roast samples, mimicking the levels of STEC found under real-life conditions in food processing plants, the prevalence of STEC in the samples was significantly reduced (P < 0.05) by ≥80%. Our results suggest that this STEC-targeting bacteriophage preparation can result in significant reduction of both the levels and prevalence of STEC in various foods and, therefore, may help improve the safety and reduce the risk of recalls of foods at high risk for STEC contamination.

Shiga Toxin-Producing Escherichia coli Infections Associated With Romaine Lettuce-United States, 2018.

Produce-associated outbreaks of Shiga toxin-producing Escherichia coli (STEC) were first identified in 1991. In April 2018, New Jersey and Pennsylvania officials reported a cluster of STEC O157 infections associated with multiple locations of a restaurant chain. The Centers for Disease Control and Prevention (CDC) queried PulseNet, the national laboratory network for foodborne disease surveillance, for additional cases and began a national investigation. A case was defined as an infection between 13 March and 22 August 2018 with 1 of the 22 identified outbreak-associated E. coli O157:H7 or E. coli O61 pulsed-field gel electrophoresis pattern combinations, or with a strain STEC O157 that was closely related to the main outbreak strain by whole-genome sequencing. We conducted epidemiologic and traceback investigations to identify illness subclusters and common sources. A US Food and Drug Administration-led environmental assessment, which tested water, soil, manure, compost, and scat samples, was conducted to evaluate potential sources of STEC contamination. We identified 240 case-patients from 37 states; 104 were hospitalized, 28 developed hemolytic uremic syndrome, and 5 died. Of 179 people who were interviewed, 152 (85%) reported consuming romaine lettuce in the week before illness onset. Twenty subclusters were identified. Product traceback from subcluster restaurants identified numerous romaine lettuce distributors and growers; all lettuce originated from the Yuma growing region. Water samples collected from an irrigation canal in the region yielded the outbreak strain of STEC O157. We report on the largest multistate leafy greens-linked STEC O157 outbreak in several decades. The investigation highlights the complexities associated with investigating outbreaks involving widespread environmental contamination.

Immunomagnetic Capture of Big Six Shiga Toxin–Producing Escherichia coli Strains in Apple Juice with Detection by Multiplex Real-Time PCR Eliminates Interference from the Food Matrix

Having reliable methods for detecting Shiga toxin-producing Escherichia coli (STEC) in foods is an important food safety goal. The majority of STEC outbreaks have involved either the O157:H7 serotype or one of six non-O157 serogroups, O26, O45, O103, O111, O121, and O145, termed "The Big Six." We have compared detection by PCR of the Shiga toxin genes stx1a and stx2a from STEC bacteria isolated from unclarified apple juice by simple centrifugation with the use of an immunocapture technique to minimize contaminants (such as pectin and polyphenols that may copurify with DNA) that may interfere with DNA amplification efficiencies and limit sensitivity. An internal control for successful immunocapture, DNA extraction, and PCR amplification was generated by introducing the pmRaspberry plasmid into an stx null strain, yielding an E. coli O45 pmRaspberry derivative that can be added to food samples directly. Using serial dilutions of a representative Big Six STEC in apple juice, our immunocapture method resulted in a 50% probability of detection value of 3.34, 2.25, and 4.25 CFU for detection by multiplex real-time PCR, growth on solid agar, and multiplex endpoint PCR, respectively. The time to result was 6.5 h, 9.5 h, and 1.5 days for immunocapture of Big Six STECs and detection by multiplex real-time PCR, endpoint PCR, and growth on solid agar, respectively. A set of 52 Big Six STEC isolates and 30 non-Big Six STEC strains was used to establish the inclusivity and exclusivity of the method. Finally, the ability to detect Big Six STEC contamination reliably was confirmed at 4.5 and 45 CFU/25-mL portions of refrigerated apple juice.

Global and regional source attribution of Shiga toxin-producing Escherichia coli infections using analysis of outbreak surveillance data.

Shiga toxin-producing Escherichia coli (STEC) infections pose a substantial health and economic burden worldwide. To target interventions to prevent foodborne infections, it is important to determine the types of foods leading to illness. Our objective was to determine the food sources of STEC globally and for the six World Health Organization regions. We used data from STEC outbreaks that have occurred globally to estimate source attribution fractions. We categorised foods according to their ingredients and applied a probabilistic model that used information on implicated foods for source attribution. Data were received from 27 countries covering the period between 1998 and 2017 and three regions: the Americas (AMR), Europe (EUR) and Western-Pacific (WPR). Results showed that the top foods varied across regions. The most important sources in AMR were beef (40%; 95% Uncertainty Interval 39-41%) and produce (35%; 95% UI 34-36%). In EUR, the ranking was similar though with less marked differences between sources (beef 31%; 95% UI 28-34% and produce 30%; 95% UI 27-33%). In contrast, the most common source of STEC in WPR was produce (43%; 95% UI 36-46%), followed by dairy (27%; 95% UI 27-27%). Possible explanations for regional variability include differences in food consumption and preparation, frequency of STEC contamination, the potential of regionally predominant STEC strains to cause severe illness and differences in outbreak investigation and reporting. Despite data gaps, these results provide important information to inform the development of strategies for lowering the global burden of STEC infections.

Escherichia coli O26 and O113:H21 on Carcasses and Beef from a Slaughterhouse Located in Mato Grosso, Brazil.

Shiga toxin-producing Escherichia coli (STEC) is a group of emerging pathogens that can cause human diseases, including hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC). Monitoring slaughtering stages and checking contamination points are crucial for the production of safe food. In this context, the aim of this study was to verify contamination by STEC strains, to determine the contamination points and evaluate the resistance profile to 12 antimicrobials used in both veterinary and human medicine. A total of 80 samples were obtained from eight collection points (pen floor, rectum, hide, carcass swabs and esophagus, diaphragm, masseter, and retail beef tissue samples). The isolates were collected by dilution plating on MacConkey agar with sorbitol, cefixime, and tellurite and analyzed by multiplex polymerase chain reaction for virulence genes. Serotyping of non-O157 was performed, and testing for 12 antibiotics by disk diffusion was carried out. A total of 18 STEC strains were isolated, presenting different virulence profiles. Contamination by STEC was observed in the rectum (5/18), carcass surface (5/18), hide (3/18), diaphragm (2/18), retail beef (2/18), and masseter muscle (1/18). Pen floor swabs and esophagus tissues showed no STEC contamination. Moreover, three strains were identified as O26 and three as O113:H21 strains, which have been linked to HUS and HC outbreak cases in Brazil. All STEC isolates were susceptible to all evaluated antimicrobials, except streptomycin. The presence of STEC strains is a direct risk to the consumer, especially when isolated from retail beef, and contamination can occur during different slaughter stages. However, antimicrobial resistance profiles did not identify multidrug-resistant strains, limiting potential antimicrobial resistance transmission to other pathogens.

Evaluation of decontamination efficacy of commonly used antimicrobial interventions for beef carcasses against Shiga toxin-producing Escherichia coli

In Argentina, Shiga toxin producing Escherichia coli (STEC) serogroups O157, O26, O103, O111, O145 and O121 are adulterant in ground beef. In other countries, the zero-tolerance approach to all STEC is implemented for chilled beef. Argentinean abattoirs are interested in implementing effective interventions against STEC on carcasses. Pre-rigor beef carcasses were used to determine whether nine antimicrobial strategies effectively reduced aerobic plate, coliform and E. coli counts and stx and eae gene prevalence. These strategies were: citric acid (2%; automated), acetic acid (2%; manual and automated), lactic acid (LA 2%; manual and automated), LA (3%; automated), electrolytically-generated hypochlorous acid (400 ppm; manual), hot water (82 °C; automated) and INSPEXX (0.2%; automated). Automated application of 2% LA after 30–60-min aeration and 3% LA at 55 °C were the most effective interventions. Automated application was more effective than manual application. Decontamination of beef carcasses through automated application of lactic acid and hot water would reduce public health risks associated with STEC contamination.

Prevalence, virulence potential, and pulsed-field gel electrophoresis profiling of Shiga toxin-producing Escherichia coli strains from cattle

BackgroundAs a primary source of Shiga-toxin-producing Escherichia coli (STEC) infection, cattle are often targeted to develop strategies for reducing STEC contamination. Monitoring the virulence potentials of STEC isolates from cattle is important for tracing contamination sources, managing outbreaks or sporadic cases, and reducing the risks for human infection. This study aimed to investigate the prevalence of STEC in cattle farm samples in South Korea and to assess their virulence potentials.ResultsIn total, 63 STEC were isolated from 496 cattle farm samples, and temperature and rainfall affected STEC prevalence (p < 0.001). The O157 serogroup was most prevalent, followed by O108, O8, O84, O15, and O119. In the stx variant test, high prevalence of stx2a and stx2c (known to be associated with high STEC virulence) were observed, and stx2g, a bovine STEC variant, was detected in STEC O15 and O109. Additionally, stx1c was detected in eae-positive STEC, suggesting genetic dynamics among the virulence genes in the STEC isolates. STEC non-O157 strains were resistant to tetracycline (17.9%), ampicillin (14.3%), and cefotaxime (3.6%), while STEC O157 was susceptible to all tested antimicrobials, except cefotaxime. The antimicrobial resistance genes, blaTEM (17.5%), tetB (6.3%), and tetC (4.8%), were only detected in STEC non-O157, whereas tetE (54.0%) was detected in STEC O157. AmpC was detected in all STEC isolates. Clustering was performed based on the virulence gene profiles, which grouped STEC O84, O108, O111, and O157 together as potentially pathogenic STEC strains. Finally, PFGE suggested the presence of a prototype STEC that continues to evolve by genetic mutation and causes within- and between-farm transmission within the Gyeonggi province.ConclusionsConsiderable numbers of STEC non-O157 were isolated from cattle farms, and the virulence and antimicrobial resistance features were different between the STEC O157 and non-O157 strains. STEC from cattle with virulence or antimicrobial resistance genes might represent a threat to public health and therefore, continual surveillance of both STEC O157 and non-O157 would be beneficial for controlling and preventing STEC-related illness.