Shiga Toxin-Producing Escherichia coli O157:H7 Illness Outbreak Associated with Untreated, Pressurized, Municipal Irrigation Water - Utah, 2023.

During July-September 2023, an outbreak of Shiga toxin-producing Escherichia coli O157:H7 illness among children in city A, Utah, caused 13 confirmed illnesses; seven patients were hospitalized, including two with hemolytic uremic syndrome. Local, state, and federal public health partners investigating the outbreak linked the illnesses to untreated, pressurized, municipal irrigation water (UPMIW) exposure in city A; 12 of 13 ill children reported playing in or drinking UPMIW. Clinical isolates were genetically highly related to one another and to environmental isolates from multiple locations within city A's UPMIW system. Microbial source tracking, a method to indicate possible contamination sources, identified birds and ruminants as potential sources of fecal contamination of UPMIW. Public health and city A officials issued multiple press releases regarding the outbreak reminding residents that UPMIW is not intended for drinking or recreation. Public education and UPMIW management and operations interventions, including assessing and mitigating potential contamination sources, covering UPMIW sources and reservoirs, indicating UPMIW lines and spigots with a designated color, and providing conspicuous signage to communicate risk and intended use might help prevent future UPMIW-associated illnesses.

Isolation of Shiga Toxin-Producing Escherichia coli from the Surfaces of Beef Carcasses in Slaughterhouses in Japan.

Shiga toxin-producing E. coli (STEC) is an important foodborne pathogen worldwide. It is necessary to control and prevent STEC contamination on beef carcasses in slaughterhouses because STEC infection is associated with beef consumption. However, the frequencies of STEC contamination of beef carcasses in various slaughterhouses in Japan are not well known. Herein, we investigated the contamination of beef carcasses with STEC in slaughterhouses to assess the potential risks of STEC. In total, 524 gauze samples were collected from the surfaces of beef carcasses at 12 domestic slaughterhouses from November 2020 to February 2023. The samples were measured for aerobic plate counts and tested for pathogenic genes (stx and eae) and major O-serogroups (O26, O45, O103, O111, O121, O145, and O157) by real-time PCR screening. Subsequently, immunomagnetic separation (IMS) was performed on samples positive for stx, eae, and at least one of the seven O-serogroups of STEC. Isolation process without IMS was performed on samples positive for stx, including those subjected to IMS. STEC O157:H7 and stx-positive E. coli other than serotype O157:H7 were isolated from 0.6% and 4.6% of beef carcass surfaces, respectively. Although the STEC O157:H7 isolation rate was low and stx-positive E. coli other than serotype O157:H7 belonged to minor O-serogroups, the results mean a risk of foodborne illness. Furthermore, a moderate correlation was observed between aerobic plate counts and detection rates of stx-positive samples by real-time PCR screening. The STEC O157:H7 isolated facilities showed higher values on aerobic plate counts and detection rates of stx-positive samples than the mean values of total samples. Therefore, these results suggest that it is important to evaluate hygiene treatments against beef carcasses for the reduction of STEC contamination risk, particularly in facilities with high aerobic plate counts.

Comparison of Two Shiga Toxin-producing Escherichia coli (STEC) Isolation Protocols in Raw Cow's Milk Cheese Enrichment Broths: Direct STEC Isolation Versus Techniques Based on Immuno-concentration.

The presence of Shiga toxin-producing Escherichia coli (STEC) in dairy products made with raw milk is a major concern for food safety authorities and industries. Two approaches have been proposed to isolate STEC from food. In the IC-Protocol (immuno-concentration protocol), specific serogroups are identified in the enrichment broth after the detection of the stx and eae genes. An immuno-concentration of the targeted serogroups is performed before isolating them on specific media. In the DI-Protocol (direct isolation protocol), a direct isolation of all STEC present in the enrichment broth is carried out after the detection of stx genes. We compared the ability of these two methods to isolate STEC O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7 after artificial inoculation in four different raw milk cheeses. Across all serogroups and cheese types, STEC were isolated in 83.3% of samples when using the IC-Protocol but only 53.3% of samples with the DI-Protocol. For two cheese types, the DI-Protocol failed to isolate STEC O157:H7 strains altogether. Our results suggest that IC-Protocol is a robust methodology to effectively isolate STEC across a range of cheese types.

First molecular detection of shiga toxin-producing Escherichia coli in dogs from serbia: a potential threat to human health?

Shiga toxin­producing Escherichia coli (STEC) are considered one of the most significant E. coli pathotypes transmitted by food, causing life­threatening conditions in children and elderly people. The aim of this study was to investigate the presence and determine the prevalence of STEC in dogs in Serbia by conventional PCR method, targeting three major virulence genes (stx1, stx2, and eae). The overall percentage of positive samples was 12.87% (13/101), with the stx2 gene, the more potent of the two toxins, found in all the positive samples. The finding of eae gene in combination with stx genes (8/13) within the same genetic pool implies the potential presence of enterohemorrhagic E. coli or the potential emergence of these strains, considering an efficient mechanism of horizontal transfer of three major virulence genes. Our results also highlight dogs' lifestyle as a risk factor for STEC colonisation. These E. coli strains, according to our results, are more likely to be found in dogs living outdoors than those kept in house. Due to significant prevalence of STEC in dogs determined in this research and due to close contact between dogs and humans, dogs could be considered a source of human infections.

Validation of the 3M™ Molecular Detection Assay 2-STEC Gene Screen (stx) for Detection of Shiga Toxin Gene (stx1 and/or stx2) in Dried Cannabis Flower and Dried Hemp Flower: AOAC Performance Tested MethodSM 071903.

BACKGROUND: The 3M™ Molecular Detection Assay 2-STEC Gene Screen (stx) method is based on gene amplification by the use of real-time loop-mediated isothermal amplification when used with the 3M Molecular Detection System for the rapid and specific detection of Shiga toxin gene (stx1 and/or stx2) from Shiga toxin-producing Escherichia coli (STEC) in enriched products. The 3M Molecular Detection Assay 2-STEC Gene Screen (stx) was approved as AOAC Performance Tested MethodSM Certificate No. 071903. OBJECTIVE: This matrix extension study evaluated the 3M Molecular Detection Assay 2-STEC Gene Screen (stx) method for detection of STECs in dried cannabis flower [>0.3% delta 9-tetrahydrocannabinol (THC)] and dried hemp flower (≤0.3% THC) at a 10 g test portion size. METHOD: Testing followed procedures outlined in 3M Molecular Detection Assay 2-STEC Gene Screen (stx) product instructions and Standard Method Performance Requirement (SMPR®) for Detection of Shiga Toxin-Producing Escherichia coli in Cannabis and Cannabis Products (AOAC SMPR 2020.012). The method was evaluated at low, high, and non-inoculated levels. RESULTS: Results showed no statistically significant difference between the presumptive positive 3M Molecular Detection Assay 2-STEC Gene Screen (stx) results and the SMPR 2020.012 recommended cultural confirmations. CONCLUSIONS: This study provides data that demonstrate the 3M Molecular Detection Assay 2-STEC Gene Screen (stx) is a reliable method for the rapid and specific detection of STECs in dried cannabis flower and dried hemp flower. HIGHLIGHTS: The 3M Molecular Detection Assay 2-STEC Gene Screen (stx) method is suitable for the rapid and specific detection of STECs in dried cannabis flower and dried hemp flower.

Validation of the 3M™ Molecular Detection Assay 2-STEC Gene Screen (stx and eae) for Detection of Shiga Toxin Gene (stx1 and/or stx2) and Intimin Gene (eae) in Dried Cannabis Flower and Dried Hemp Flower: AOAC Performance Tested MethodSM 071902.

BACKGROUND: The 3M™ Molecular Detection Assay 2-STEC Gene Screen (stx and eae) method is based on gene amplification by the use of real time loop-mediated isothermal amplification when used with the 3M Molecular Detection System for the rapid and specific detection of Shiga toxin gene (stx1 and/or stx2) and intimin gene (eae) from Shiga toxin-producing Escherichia coli (STEC) in enriched products. The 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) was approved as AOAC Performance Tested MethodSM Certificate No. 071902. OBJECTIVE: This matrix extension study evaluated the 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) method for detection of STECs in dried cannabis flower [>0.3% delta 9-tetrahydrocannabinol (THC)] and dried hemp flower (≤0.3% THC) at a 10 g test portion size. METHOD: Testing followed procedures outlined in 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) product instructions and Standard Method Performance Requirements (SMPR®) for Detection of Shiga Toxin-Producing Escherichia coli in Cannabis and Cannabis Products (AOAC SMPR 2020.012). The method was evaluated at low, high, and non-inoculated levels. RESULTS: Results showed no statistically significant difference between the presumptive positive 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) results and the SMPR 2020.012 recommended cultural confirmations. CONCLUSIONS: This study provides data that demonstrate the 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) is a reliable method for the rapid and specific detection of STEC organisms in dried cannabis flower and dried hemp flower. HIGHLIGHTS: The 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) method is suitable for the rapid and specific detection of STEC organisms in dried cannabis flower and dried hemp flower.

Escherichia coli serogroups in slaughterhouses: Antibiotic susceptibility and molecular typing of isolates.

This study aimed to investigate the contamination of carcasses and slaughterhouse environment with Escherichia coli O157:H7 and non-O157 serogroups (O45:H2, O103:H2, O121:H19, O145:H28, O26:H11, O111:H8). For this purpose, a total of 150 samples (30 carcasses, 30 shredding units, 30 knives, 30 slaughterhouse waste water and 30 wall surfaces) were collected from 5 different slaughterhouses in Kayseri, Turkey. The conventional and molecular methods were performed in order to detect Escherichia coli and its serogroups. Of the 150 samples, 55 (36%) were found to be contaminated with E. coli. Among isolates, E. coli serogroup (O157:H7) were detected in 2 (11%) carcass and 2 (11%) wastewater samples. None of the E. coli isolates harbored tested genes (stx1, stx2, eaeA, and hylA). Effective infection control measures and antibiotic stewardship programs should be adopted to limit the spread of multidrug-resistant bacteria. It was also deduced that these isolates resistance to different antibiotics could be hazardous for public health.

Large-Scale Phylogenetic Analysis Reveals a New Genetic Clade among Escherichia coli O26 Strains.

Shiga toxin-producing Escherichia coli (STEC) O26 is the predominant non-O157 serogroup causing hemolytic uremic syndrome worldwide. Moreover, the serogroup is highly dynamic and harbors several pathogenic clones. Here, we investigated the phylogenetic relationship of STEC O26 at a global level based on 1,367 strains from 20 countries deposited in NCBI and Enterobase databases. The whole-genome-based analysis identified a new genetic clade, called ST29C4. The new clade was unique in terms of multilocus sequence type (ST29), CRISPR (group Ia), and dominant plasmid gene profile (ehxA+/katP-/espP-/etpD-). Moreover, the combination of multiple typing methods (core genome single nucleotide polymorphism [SNP] typing, CRISPR typing, and virulence genes analysis) demonstrated that this new lineage ST29C4 was in the intermediate phylogenetic position between ST29C3 and other non-ST29C3 strains. Besides, we observed that ST29C4 harbored extraintestinal pathogenic E. coli (ExPEC)-related virulence gene (VG), tsh, and STEC-associated VG, stx2a, suggesting the emergence of a hybrid pathogen. The ST29C4 strains also exhibited high similarity in stx2a-prophage and integrase with the O104:H4 strain, further demonstrating its potential risk to human health. Collectively, the large-scale phylogenetic analysis extends the understanding of the clonal structure of O26 strains and provides new insights for O26 strain microevolution. IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) O26 is the second prevalent STEC serogroup only to O157, which can cause a series of diseases ranging from mild diarrhea to life-threatening hemolytic uremic syndrome (HUS). The serogroup is highly diverse and multiple clones are characterized, including ST29C1-C3 and ST21C1-C2. However, the phylogenetic relationship of these clones remains fully unclear. In this study, we revealed a new genetic clade among O26 strains, ST29C4, which was unique in terms of CRISPR, multilocus sequence type (MLST), and plasmid gene profile (PGP). Moreover, the combination of multiple typing methods demonstrated that this new clone was located in the intermediate phylogenetic position between ST29C3 and other non-ST29C3 strains (i.e., ST29C1-C2 and ST21C1-C2). Overall, the large-scale phylogenetic analysis extends our current understanding of O26 microevolution.

Free-ranging red deer (Cervus elaphus) as carriers of potentially zoonotic Shiga toxin-producing Escherichia coli.

Shiga toxin-producing E. coli (STEC) are zoonotic foodborne pathogens of outmost importance and interest has been raised in recent years to define the potential zoonotic role of wildlife in STEC infection. This study aimed to estimate prevalence of STEC in free-ranging red deer (Cervus elaphus) living in areas with different anthropisation levels and describe the characteristics of strains in order to evaluate the potential risk posed to humans. Two-hundred one deer faecal samples collected in 2016-2018 from animals of Central Italian Alps were examined by bacteriological analysis and PCR screening of E. coli colonies for stx1, stx2 and eae genes. STEC strains were detected in 40 (19.9%) deer, with significantly higher prevalence in offspring than in yearlings. Whole genome analysis was performed to characterise a subset of 31 STEC strains. The most frequently detected serotype was O146:H28 (n = 10, 32.3%). Virulotyping showed different stx subtypes combinations, with stx2b-only (n = 15, 48.4%) being the most prevalent. All STEC lacked the eae gene but harbored additional virulence genes, particularly adhesins, toxins and/or other colonisation factors also described in STEC isolated from disease in humans. The most frequently detected genes were astA (n = 22, 71%), subAB (n = 21, 68%), iha (n = 26, 83.9%) and lpfA (n = 24, 77%). Four hybrid STEC/Enterotoxigenic E. coli strains were also identified. According to the most recent paradigm for pathogenicity assessment of STEC issued by the European Food Safety Authority, our results suggest that red deer are carriers of STEC strains that may have zoonotic potential, regardless of the anthropisation levels. Particular attention should be drawn to these findings while handling and preparing game meat. Furthermore, deer may release STEC in the environment, possibly leading to the contamination of soil and water sources.

Multiplex PCR method for the detection of human norovirus, Salmonella spp., Shigella spp., and shiga toxin producing Escherichia coli in blackberry, coriander, lettuce and strawberry.

A multiplex PCR method was developed for the simultaneous detection of murine norovirus (MNV-1) as a surrogate for human norovirus (HuNoV) GI and GII, Salmonella spp., Shigella spp., and Shiga toxin producing Escherichia coli (STEC) in fresh produce. The toxicity of the glycine buffer on bacterial pathogens viability was evaluated. The growth of each of the three pathogens (previously stressed) was evaluated at 35 and 41.5 °C in modified buffered peptone water (mBPW) and trypticase soy broth (TSB), supplemented with vancomycin, novobiocin and brilliant green at two concentration levels. The selected conditions for simultaneous enrichment were: 41.5 °C/mBPW/supplemented with 8 ppm vancomycin, 0.6 ppm novobiocin and 0.2 ppm brilliant green. The pathogens and aerobic plate count (APC) growth was evaluated in the enrichment of lettuce, coriander, strawberry and blackberry under the best enrichment conditions. Starting from 1 to 10 CFU/mL, Salmonella reached from 7.63 to 8.91, Shigella 6.81 to 7.76 and STEC 7.43 to 9.27 log CFU/mL. The population reached for the APC was 5.11-6.56 log CFU/mL. Simultaneous detection by PCR was done using designed primers targeting invA, ipaH, stx1 and stx2 genes, and MNV-1. The detection sensitivity was 10-100 PFU for the MNV-1 and 1-10 CFU for each pathogenic bacteria. This protocol takes 6 h for MNV-1 and 24 h for Salmonella spp., Shigella spp., and STEC detection from the same food portion. In total, 200 samples were analyzed from retail markets from Queretaro, Mexico. Two strawberry samples were positive for HuNoV GI and one lettuce sample was positive for STEC. In conclusion, the method developed in this study is capable of detecting HuNoV GI and GII, Salmonella spp., Shigella spp and STEC from the same fresh produce sample.

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.

Validation of the 3M™ Molecular Detection Assay 2 - STEC Gene Screen (stx) for the Detection of Shiga Toxin Gene (stx1 and/or stx2) in Fresh Raw Ground Beef and Fresh Spinach: AOAC Performance Tested MethodSM 071903.

BACKGROUND: The 3M™ Molecular Detection Assay 2 - STEC Gene Screen (stx) method is based on gene amplification by the use of real time loop-mediated isothermal amplification when used with the 3M Molecular Detection System for the rapid and specific detection of Shiga toxin gene (stx1 and/or stx2) from Shiga toxin-producing Escherichia coli (STEC) in enriched foods. The stx assay does not differentiate between stx1 and stx2 but detects the presence of stx1 and/or stx2. OBJECTIVE: The 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) method was evaluated for AOAC®  Performance Tested MethodsSM certification. METHODS: Matrix studies, inclusivity/exclusivity, robustness testing, product stability, and lot-to-lot variability testing were conducted to assess the method's performance. RESULTS: The 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) demonstrated equivalent results to the United States Department of Agriculture/Food Safety and Inspection Service Microbiology Laboratory Guidebook Chapter 5C.00 reference method for fresh raw ground beef, and the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 4A reference method for fresh spinach. The 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) detected all STEC E. coli strains (E. coli strains with stx1 and/or stx2 genes) and did not detect any of the 45 strains from the exclusivity panel. Robustness testing indicated that small variations in critical test parameters did not adversely affect the assay's performance. Product consistency and stability testing demonstrated no differences between the lots evaluated. CONCLUSION: The data collected in these studies demonstrate that the 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) is a reliable method for the rapid and specific detection of Shiga toxin-producing E. coli in raw ground beef and spinach. HIGHLIGHTS: The 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) method is suitable for the rapid and specific detection of Shiga toxin-producing E. coli in fresh raw ground beef, and spinach.

A step forward for Shiga toxin-producing Escherichia coli identification and characterization in raw milk using long-read metagenomics.

Shiga toxin-producing Escherichia coli (STEC) are a cause of severe human illness and are frequently associated with haemolytic uraemic syndrome (HUS) in children. It remains difficult to identify virulence factors for STEC that absolutely predict the potential to cause human disease. In addition to the Shiga-toxin (stx genes), many additional factors have been reported, such as intimin (eae gene), which is clearly an aggravating factor for developing HUS. Current STEC detection methods classically rely on real-time PCR (qPCR) to detect the presence of the key virulence markers (stx and eae). Although qPCR gives an insight into the presence of these virulence markers, it is not appropriate for confirming their presence in the same strain. Therefore, isolation steps are necessary to confirm STEC viability and characterize STEC genomes. While STEC isolation is laborious and time-consuming, metagenomics has the potential to accelerate the STEC characterization process in an isolation-free manner. Recently, short-read sequencing metagenomics have been applied for this purpose, but assembly quality and contiguity suffer from the high proportion of mobile genetic elements occurring in STEC strains. To circumvent this problem, we used long-read sequencing metagenomics for identifying eae-positive STEC strains using raw cow's milk as a causative matrix for STEC food-borne outbreaks. By comparing enrichment conditions, optimizing library preparation for MinION sequencing and generating an easy-to-use STEC characterization pipeline, the direct identification of an eae-positive STEC strain was successful after enrichment of artificially contaminated raw cow's milk samples at a contamination level as low as 5 c.f.u. ml-1. Our newly developed method combines optimized enrichment conditions of STEC in raw milk in combination with a complete STEC analysis pipeline from long-read sequencing metagenomics data. This study shows the potential of the innovative methodology for characterizing STEC strains from complex matrices. Further developments will nonetheless be necessary for this method to be applied in STEC surveillance.

Epidemiological investigations identified an outbreak of Shiga toxin-producing Escherichia coli serotype O26:H11 associated with pre-packed sandwiches.

In October 2019, public health surveillance systems in Scotland identified an increase in the number of reported infections of Shiga toxin-producing Escherichia coli (STEC) O26:H11 involving bloody diarrhoea. Ultimately, across the United Kingdom (UK) 32 cases of STEC O26:H11 stx1a were identified, with the median age of 27 years and 64% were male; six cases were hospitalised. Among food exposures there was an association with consuming pre-packed sandwiches purchased at outlets belonging to a national food chain franchise (food outlet A) [odds ratio (OR) = 183.89, P < 0.001]. The common ingredient identified as a component of the majority of the sandwiches sold at food outlet A was a mixed salad of Apollo and Iceberg lettuce and spinach leaves. Microbiological testing of food and environmental samples were negative for STEC O26:H11, although STEC O36:H19 was isolated from a mixed salad sample taken from premises owned by food outlet A. Contamination of fresh produce is often due to a transient event and detection of the aetiological agent in food that has a short-shelf life is challenging. Robust, statistically significant epidemiological analysis should be sufficient evidence to direct timely and targeted on-farm investigations. A shift in focus from testing the microbiological quality of the produce to investigating the processes and practices through the supply chain and sampling the farm environment is recommended.

Prevalence and Whole-Genome Sequence-Based Analysis of Shiga Toxin-Producing Escherichia coli Isolates from the Recto-Anal Junction of Slaughter-Age Irish Sheep.

Shiga toxin-producing Escherichia coli (STEC) organisms are a diverse group of pathogenic bacteria capable of causing serious human illness, and serogroups O157 and O26 are frequently implicated in human disease. Ruminant hosts are the primary STEC reservoir, and small ruminants are important contributors to STEC transmission. This study investigated the prevalence, serotypes, and shedding dynamics of STEC, including the supershedding of serogroups O157 and O26, in Irish sheep. Recto-anal mucosal swab samples (n = 840) were collected over 24 months from two ovine slaughtering facilities. Samples were plated on selective agars and were quantitatively and qualitatively assessed via real-time PCR (RT-PCR) for Shiga toxin prevalence and serogroup. A subset of STEC isolates (n = 199) were selected for whole-genome sequencing and analyzed in silico. In total, 704/840 (83.8%) swab samples were Shiga toxin positive following RT-PCR screening, and 363/704 (51.6%) animals were subsequently culture positive for STEC. Five animals were shedding STEC O157, and three of these were identified as supershedders. No STEC O26 was isolated. Post hoc statistical analysis showed that younger animals are more likely to harbor STEC and that STEC carriage is most prevalent during the summer months. Following sequencing, 178/199 genomes were confirmed as STEC. Thirty-five different serotypes were identified, 15 of which were not yet reported for sheep. Serotype O91:H14 was the most frequently reported. Eight Shiga toxin gene variants were reported, two stx1 and six stx2, and three novel Shiga-toxin subunit combinations were observed. Variant stx1c was the most prevalent, while many strains also harbored stx2b. IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) bacteria are foodborne, zoonotic pathogens of significant public health concern. All STEC organisms harbor stx, a critical virulence determinant, but it is not expressed in most serotypes. Sheep shed the pathogen via fecal excretion and are increasingly recognized as important contributors to the dissemination of STEC. In this study, we have found that there is high prevalence of STEC circulating within sheep and that prevalence is related to animal age and seasonality. Further, sheep harbor a variety of non-O157 STEC, whose prevalence and contribution to human disease have been underinvestigated for many years. A variety of Stx variants were also observed, some of which are of high clinical importance.

Comparison of four enzymatic library preparation kits for sequencing Shiga toxin-producing Escherichia coli for surveillance and outbreak detection.

Four enzymatic DNA library preparation kits were compared for sequencing Shiga toxin-producing E. coli. All kits produced high quality sequence data which performed equally well in the downstream analyses for surveillance and outbreak detection. Important differences were noted in the workflow user-friendliness and per sample cost.

Inconsistent PCR detection of Shiga toxin-producing Escherichia coli: Insights from whole genome sequence analyses.

Shiga toxin-producing Escherichia coli (STEC) have been linked to food-borne disease outbreaks. As PCR is routinely used to screen foods for STEC, it is important that factors leading to inconsistent detection of STEC by PCR are understood. This study used whole genome sequencing (WGS) to investigate causes of inconsistent PCR detection of stx1, stx2, and serogroup-specific genes. Fifty strains isolated from Alberta feedlot cattle from three different studies were selected with inconsistent or consistent detection of stx and serogroup by PCR. All isolates were initially classified as STEC by PCR. Sequencing was performed using Illumina MiSeq® with sample library by Nextera XT. Virtual PCRs were performed using Geneious and bacteriophage content was determined using PHASTER. Sequencing coverage ranged from 47 to 102x, averaging 74x, with sequences deposited in the NCBI database. Eleven strains were confirmed by WGS as STEC having complete stxA and stxB subunits. However, truncated stx fragments occurred in twenty-two other isolates, some having multiple stx fragments in the genome. Isolates with complete stx by WGS had consistent stx1 and stx2 detection by PCR, although one also having a stx2 fragment had inconsistent stx2 PCR. For all STEC and 18/39 non-STEC, serogroups determined by PCR agreed with those determined by WGS. An additional three WGS serotypes were inconclusive and two isolates were Citrobacter spp. Results demonstrate that stx fragments associated with stx-carrying bacteriophages in the E. coli genome may contribute to inconsistent detection of stx1 and stx2 by PCR. Fourteen isolates had integrated stx bacteriophage but lacked complete or fragmentary stx possibly due to partial bacteriophage excision after sub-cultivation or other unclear mechanisms. The majority of STEC isolates (7/11) did not have identifiable bacteriophage DNA in the contig(s) where stx was located, likely increasing the stability of stx in the bacterial genome and its detection by PCR.

Molecular detection of Shiga toxin-producing Escherichia coli (STEC) O157 in sheep, goats, cows and buffaloes.

BACKGROUND: Shiga toxin-producing E. coli (STEC) are important foodborne pathogens that causing serious public health consequences worldwide. The present study aimed to estimate the prevalence ratio and to identify the zoonotic potential of E. coli O157 isolates in slaughtered adult sheep, goats, cows and buffaloes. MATERIALS AND METHODS: A total of 400 Recto-anal samples were collected from two targeted sites Rawalpindi and Islamabad. Among them, 200 samples were collected from the slaughterhouse of Rawalpindi included sheep (n = 75) and goats (n = 125). While, 200 samples were collected from the slaughterhouse of Islamabad included cows (n = 120) and buffalos (n = 80). All samples were initially processed in buffered peptone water and then amplified by conventional PCR. Samples positive for E. coli O157 were then streaked onto SMAC media plates. From each positive sample, six different Sorbitol fermented pink-colored colonies were isolated and analyzed again via conventional PCR to confirm the presence of rfbE O157 gene. Isolates positive for rfbE O157 gene were then further analyzed by multiplex PCR for the presence of STEC other virulent genes (sxt1, stx2, eae and ehlyA) simultaneously. RESULTS: Of 400 RAJ samples only 2 (0.5%) showed positive results for E. coli O157 gene, included sheep 1/75 (1.33%) and buffalo 1/80 (1.25%). However, goats (n = 125) and cows (n = 120) found negative for E. coli O157. Only 2 isolates from each positive sample of sheep (1/6) and buffalo (1/6) harbored rfbE O157 genes, while five isolates could not. The rfbE O157 isolate (01) of sheep sample did not carry any of STEC genes, while the rfbE O157 isolate (01) of buffalo sample carried sxt1, stx2, eae and ehlyA genes simultaneously. CONCLUSION: It was concluded that healthy adult sheep and buffalo are possibly essential carriers of STEC O157. However, rfbE O157 isolate of buffalo RAJ sample carried 4 STEC virulent genes, hence considered an important source of STEC infection to humans and environment which should need to devise proper control systems.

Isolation and Characterization of Shiga Toxin-Producing Escherichia coli from Retail Beef Samples from Eight Provinces in China.

While Shiga toxin-producing Escherichia coli (STEC) is a major foodborne pathogen worldwide, data on the molecular and phylogenetic properties of STEC isolates from retail beef samples in China remain scant. Fresh retail beef samples (n = 1062) were collected from eight provinces, and STEC isolates were recovered and characterized. PCR data showed that more than 50% of the samples were stx positive, and 82 STEC isolates were recovered from 14.8% (79/535) stx-positive enriched broths. In contrast, all ciprofloxacin resistant isolates (n = 19) and 13 cefotaxime (CTX) resistant isolates were eae positive and belonged to three serotypes: O111:H8, O26:H11, or O157:H7. Point mutations in quinolone resistance-determining regions and plasmid-mediated quinolone resistance determinants were identified in 16 and 20 isolates, respectively. BlaCTX-M and a point mutation (C-42T) in ampC promoter were detected in 15 and 8 of the CTX resistant isolates, respectively. In addition, macrolide resistance gene mphA was identified in eight azithromycin resistant O111:H8 isolates and one O26:H11 isolate. Single nucleotide polymorphism analysis demonstrated that the O26 and O157 isolates had multiple origins, but the O111 isolates were closely related. Taken together, our data demonstrated that several sequence types associated with hemolytic uremic syndrome from the retail beef samples in China had developed into dangerous multidrug resistant pathogens. The resistant phenotype can facilitate their transmission among the farm animals and human beings when there is an antimicrobial selective pressure.

The emerging importance of Shiga toxin-producing Escherichia coli other than serogroup O157 in England.

Introduction. Shiga toxin-producing Escherichia coli (STEC) can cause severe disease and large outbreaks. In England, the incidence and clinical significance of STEC serogroups other than O157 (non-O157) is unknown due to a testing bias for detection of STEC O157. Since 2013, the implementation of PCR to detect all STEC serogroups by an increasing number of diagnostic laboratories has led to an increase in the detection of non-O157 STEC.Hypothesis/Gap statement. Due to a bias in testing methodologies to select for STEC serogroup O157 in frontline diagnostic laboratories in most countries, very little surveillance data have been previously generated on non-O157 STEC.Aim. Five years (2014-2018) of STEC national surveillance data were extracted and descriptive analysis undertaken to assess disease severity of non-O157 STEC strains.Methods. Data from 1 January 2014 to 31 December 2018 were extracted from the National Enhanced Surveillance System for STEC and analysed.Results. The implementation of Gastrointestinal Polymerase Chain Reaction (GI-PCR) has resulted in a four-fold increase in the detection of non-O157 STEC cases between 2014 and 2018. There were 2579 cases infected with 97 different non-O157 serogroups. The gender distribution was similar amongst STEC O157 and non-O157 STEC cases with 57 and 56 % of cases being female respectively, but a significantly higher proportion of cases (P <0.001) under 5 years of age was observed among STEC O157 (22 %) cases compared to non-O157 STEC (14 %). The most common non-O157 serogroups were O26 (16 %), O146 (11 %), O91 (10 %), O128 (7 %), O103 (5 %) and O117 (3 %). Overall, rates of bloody diarrhoea were highest in O26 (44 %) and O103 (48 %) cases and lowest in STEC O117 cases (17 %). Strains harbouring Shiga toxin stx1a caused the highest proportion of diarrhoea (93 %) and caused the same level of bloody diarrhoea as stx2a (39 %). However, stx2a caused the highest proportion of vomiting (46 %), hospitalisation (49 %) and considerably more HUS (29 %) than other stx profiles.Conclusion. The implementation of PCR targeting stx at diagnostic laboratories has shown that non-O157 STEC, most notably STEC O26, are an emerging risk to public health.