Detection Of Shiga Toxin-producing Escherichia Coli Research Articles

A rapid on-site loop-mediated isothermal amplification technology as an early warning system for the detection of Shiga toxin-producing Escherichia coli in water.

Shiga toxin-producing Escherichia coli (STEC) is an important waterborne pathogen capable of causing serious gastrointestinal infections with potentially fatal complications, including haemolytic-uremic syndrome. All STEC serogroups harbour genes that encode at least one Shiga toxin (stx1 and/or stx2), which constitute the primary virulence factors of STEC. Loop-mediated isothermal amplification (LAMP) enables rapid real-time pathogen detection with a high degree of specificity and sensitivity. The aim of this study was to develop and validate an on-site portable diagnostics workstation employing LAMP technology to permit rapid real-time STEC detection in environmental water samples. Water samples (n=28) were collected from groundwater wells (n=13), rivers (n=12), a turlough (n=2) and an agricultural drain (n=1) from the Corrib catchment in Galway. Water samples (100 ml) were passed through a 0.22 µm filter, and buffer was added to elute captured cells. Following filtration, eluates were tested directly using LAMP assays targeting stx1, stx2 and E. coli phoA genes. The portable diagnostics workstation was used in field studies to demonstrate the on-site testing capabilities of the instrument. Real-time PCR assays targeting stx1 and stx2 genes were used to confirm the results. The limit of detection for stx1, stx2 and phoA LAMP assays were 2, 2 and 6 copies, respectively. Overall, stx1, stx2 and phoA genes were detected by LAMP in 15/28 (53.6 %), 9/28 (32.2 %) and 24/28 (85.7 %) samples, respectively. For confirmation, the LAMP results for stx1 and stx2 correlated perfectly (100 %) with those obtained using PCR. The portable diagnostics workstation exhibited high sensitivity throughout the on-site operation, and the average time from sample collection to final result was 40 min. We describe a simple, transferable and efficient diagnostic technology for on-site molecular analysis of various water sources. This method allows on-site testing of drinking water, enabling evidence-based decision-making by public health and water management authorities.

A Health Threat from Farm to Fork: Shiga Toxin-Producing Escherichia coli Co-Harboring blaNDM-1 and mcr-1 in Various Sources of the Food Supply Chain.

The dissemination of resistant pathogens through food supply chains poses a significant public health risk, spanning from farm to fork. This study analyzed the distribution of Shiga toxin-producing Escherichia coli (STEC) across various sources within the animal-based food supply chain. A total of 500 samples were collected from livestock, poultry, the environment, fisheries, and dairy. Standard microbiological procedures were employed to isolate and identify E. coli isolates, which were further confirmed using MALDI-TOF and virulence-associated genes (VAGs) such as stx1, stx2, ompT, hylF, iutA, fimH, and iss. The phenotypic resistance patterns of the isolates were determined using the disc diffusion method, followed by molecular identification of antibiotic resistance genes (ARGs) through PCR. STEC were subjected to PCR-based O typing using specific primers for different O types. Overall, 154 (30.5%) samples were confirmed as E. coli, of which 77 (50%) were multidrug-resistant (MDR) E. coli. Among these, 52 (67.53%) isolates exhibited an array of VAGs, and 21 (40.38%) were confirmed as STEC based on the presence of stx1 and stx2. Additionally, 12 out of 52 (23.07%) isolates were identified as non-O157 STEC co-harbouring mcr-1 and blaNDM-1. O26 STEC was found to be the most prevalent among the non-O157 types. The results suggest that the detection of STEC in food supply chains may lead to serious health consequences, particularly in developing countries with limited healthcare resources.

Detection of Escherichia coli O157:H7 in Ground Beef Using Long-Read Sequencing.

Foodborne pathogens are a significant cause of illness, and infection with Shiga toxin-producing Escherichia coli (STEC) may lead to life-threatening complications. The current methods to identify STEC in meat involve culture-based, molecular, and proteomic assays and take at least four days to complete. This time could be reduced by using long-read whole-genome sequencing to identify foodborne pathogens. Therefore, the goal of this project was to evaluate the use of long-read sequencing to detect STEC in ground beef. The objectives of the project included establishing optimal sequencing parameters, determining the limit of detection of all STEC virulence genes of interest in pure cultures and spiked ground beef, and evaluating selective sequencing to enhance STEC detection in ground beef. Sequencing libraries were run on the Oxford Nanopore Technologies' MinION sequencer. Optimal sequencing output was obtained using the default parameters in MinKNOW, except for setting the minimum read length to 1 kb. All genes of interest (eae, stx1, stx2, fliC, wzx, wzy, and rrsC) were detected in DNA extracted from STEC pure cultures within 1 h of sequencing, and 30× coverage was obtained within 2 h. All virulence genes were confidently detected in STEC DNA quantities as low as 12.5 ng. In STEC-inoculated ground beef, software-controlled selective sequencing improved virulence gene detection; however, several virulence genes were not detected due to high bovine DNA concentrations in the samples. The growth enrichment of inoculated meat samples in mTSB resulted in a 100-fold increase in virulence gene detection as compared to the unenriched samples. The results of this project suggest that further development of long-read sequencing protocols may result in a faster, less labor-intensive method to detect STEC in ground beef.

Shiga Toxin-Producing Escherichia coli Testing in New York 2011-2022 Reveals Increase in Non-O157 Identifications.

Shiga toxin-producing Escherichia coli (STEC) are an important cause of bacterial enteric infection. STEC strains cause serious human gastrointestinal disease, which may result in life-threatening complications such as hemolytic uremic syndrome. They have the potential to impact public health due to diagnostic challenges of identifying non-O157 strains in the clinical laboratory. The Wadsworth Center (WC), the public health laboratory of the New York State Department of Health, has isolated and identified non-O157 STEC for decades. A shift from initially available enzyme immunoassay testing to culture-independent diagnostic tests (CIDTs) has increased the uptake of testing at clinical microbiology laboratories. This testing change has resulted in an increased number of specimen submissions to WC. During a 12-year period between 2011 and 2022, WC received 5037 broths and/or stool specimens for STEC confirmation from clinical microbiology laboratories. Of these, 3992 were positive for Shiga toxin genes (stx1 and/or stx2) by real-time PCR. Furthermore, culture methods were utilized to isolate, identify, and characterize 2925 STEC from these primary specimens. Notably, WC observed a >200% increase in the number of STEC specimens received in 2021-2022 compared with 2011-2012 and an 18% increase in the number of non-O157 STEC identified using the same methodologies. During the past decade, the WC testing algorithm has been updated to manage the increase in specimens received, while also navigating the novel COVID-19 pandemic, which took priority over other testing for a period of time. This report summarizes updated methods for confirmation, surveillance, and outbreak detection of STEC and describes findings that may be related to our algorithm updates and the increased use of CIDTs, which is starting to elucidate the true incidence of non-O157 STEC.

Phage Tail Fiber Protein as a Specific Probe for Recognition of Shiga Toxin-Producing Escherichia coli O91, O103, and O111.

The ability to quickly identify specific serotypes of Shiga toxin-producing Escherichia coli (STEC) could facilitate the monitoring and control of STEC pathogens. In this study, we identified the receptors and receptor-binding proteins (RBPs) of three novel phages (pO91, pO103, and pO111) isolated from hospital wastewater. Recombinant versions of these RBPs (pO91-ORF43, pO103-ORF42, and pO111-ORF8) fused to a fluorescent reporter protein were then constructed. Both fluorescence microscopy and transmission electron microscopy showed that all three recombinant RBPs were bound to the bacterial surface. Indirect enzyme-linked immunosorbent assay was used to verify that each recombinant RBP bound specifically to E. coli O91, O103, or O111, but not to any of the 83 strains of E. coli with different O-antigens, nor to 10 other bacterial species that were tested. The recombinant RBPs adsorbed to their respective host bacteria within 10 min of incubation. The minimum concentration of bacteria required for detection by the recombinant RBPs was 33 colony-forming units (CFU)/mL (range: 3.3 × 10 to 3.3 × 108 CFU/mL). Furthermore, each recombinant RBP was also able to detect bacteria in lettuce, chicken breast meat, and infected mice, indicating that their usage will facilitate the detection of STEC and may help to reduce the spread of STEC-related infections and diseases.

Precision metagenomics sequencing for food safety: hybrid assembly of Shiga toxin-producing Escherichia coli in enriched agricultural water.

Culture-independent metagenomic sequencing of enriched agricultural water could expedite the detection and virulotyping of Shiga toxin-producing Escherichia coli (STEC). We previously determined the limits of a complete, closed metagenome-assembled genome (MAG) assembly and of a complete, fragmented MAG assembly for O157:H7 in enriched agricultural water using long reads (Oxford Nanopore Technologies, Oxford), which were 107 and 105 CFU/ml, respectively. However, the nanopore assemblies did not have enough accuracy to be used in Single Nucleotide Polymorphism (SNP) phylogenies and cannot be used for the precise identification of an outbreak STEC strain. The present study aimed to determine the limits of detection and assembly for STECs in enriched agricultural water by Illumina MiSeq sequencing technology alone, followed by establishing the limit of hybrid assembly with nanopore long-read sequencing using three different hybrid assemblers (SPAdes, Unicycler, and OPERA-MS). We also aimed to generate a genome with enough accuracy to be used in a SNP phylogeny. The classification of MiSeq and nanopore sequencing identified the same highly abundant species. Using the totality of the MiSeq output and a precision metagenomics approach in which the E. coli reads are binned before assembly, the limit of detection and assembly of STECs by MiSeq were determined to be 105 and 107 CFU/ml, respectively. While a complete, closed MAG could not be generated at any concentration, a complete, fragmented MAG was produced using the SPAdes assembler with an STEC concentration of at least 107 CFU/ml. At this concentration, hybrid assembled contigs aligned to the nanopore-assembled genome could be accurately placed in a neighbor-joining tree. The MiSeq limit of detection and assembly was less sensitive than nanopore sequencing, which was likely due to factors including the small starting material (50 vs. 1 μg) and the dilution of the library loaded on the cartridge. This pilot study demonstrates that MiSeq sequencing requires higher coverage in precision metagenomic samples; however, with sufficient concentration, STECs can be characterized and phylogeny can be accurately determined.

Detection and isolation of Shiga toxin-producing Escherichia coli and microbial population counts on Argentinean Kosher beef for export to Israel

The aim of this study was to provide data on the frequency of Top 7 Shiga toxin-producing Escherichia coli (STEC) and microbial population counts on processed beef from Argentinean Kosher cattle abattoirs authorized to export to Israel. A total of 480 samples (forequarters, primal cuts, trimmings) were taken and analyzed for Top 7 STEC detection and isolation and for mesophilic aerobic organism, coliform and E. coli enumeration. Differences in stx detection and microbial population counts on forequarter samples before and after salting were not statistically significant (P >0.05). All samples were negative for Top 7 STEC. Differences were significant for all microbial counts in primal cuts (P <0.001). Neck samples showed a higher level of contamination with the three groups of microorganisms than fore shank and brisket samples. The prevalence of stx was lower than that reported worldwide and in Argentinean export abattoirs. Salting did not significantly reduce the microbial load on forequarters.

Evaluation of DNA extraction methods for the detection of Shiga toxin producing Escherichia coli in food by polymerase chain reaction

Reported food-borne outbreaks of Shiga toxin producing Escherichia coli (STEC) have involved a very diverse range of foods. Contemporary analytical methods for the detection of STEC in foods typically include PCR screening of enrichment media. However, PCR inhibitors present in food enrichments can produce false negative results when screening for DNA sequences associated with the pathogen. To avoid false negative results in enrichment screening, it is advantageous to have DNA extraction methods that are effective at removing PCR inhibitors from a wide range of foods. The standard Canadian STEC method MFLP-52 uses Bio-Rad Instagene Matrix for DNA extraction. In this study, three DNA extraction protocols using commercial kits (Instagene Matrix with Beckman Coulter Ampure XP Beads; Qiagen Gentra Puregene Yeast/Bact. Kit; Qiagen DNeasy Blood & Tissue) were assessed as alternative DNA extraction methods for the detection of the Shiga toxin gene by PCR in enrichments from sixteen different foods inoculated with STEC O157. The inoculated foods were bean sprouts, blackberries, blue cheese, cilantro, cocoa powder, coleslaw, cream of mushroom dried soup mix, cream of vegetable dried soup mix, flaxseed, guacamole, peanut butter, soft cheese, soy butter, spinach, walnut, and wheat flour. Two of the protocols, Instagene Matrix with Ampure XP Beads, and Gentra Puregene Yeast/Bact, produced no false-negative or false positive results in the analysis of triplicate enrichment samples from sixteen inoculated foods.

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.

Detection and analysis of Shiga toxin producing and enteropathogenic Escherichia coli in cattle from Tierra del Fuego, Argentina.

Shiga toxin producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) are pathovars that affect mainly infants' health. Cattle are the main reservoir of STEC. Uremic hemolytic syndrome and diarrheas can be found at high rates in Tierra del Fuego (TDF). This study aimed to establish the prevalence of STEC and EPEC in cattle at slaughterhouses in TDF and to analyze the isolated strains. Out of 194 samples from two slaughterhouses, STEC prevalence was 15%, and EPEC prevalence was 5%. Twenty-seven STEC strains and one EPEC were isolated. The most prevalent STEC serotypes were O185:H19 (7), O185:H7 (6), and O178:H19 (5). There were no STEC eae + strains (AE-STEC) or serogroup O157 detected in this study. The prevalent genotype was stx2c (10/27) followed by stx1a/stx2hb (4/27). Fourteen percent of the strains presented at least one stx non-typeable subtype (4/27). Shiga toxin production was detected in 25/27 STEC strains. The prevalent module for the Locus of Adhesion and Autoaggregation (LAA) island was module III (7/27). EPEC strain was categorized as atypical and with the ability to cause A/E lesion. The ehxA gene was present in 16/28 strains, 12 of which were capable of producing hemolysis. No hybrid strains were detected in this work. Antimicrobial susceptibility tests showed that all strains were resistant to ampicillin and 20/28 were resistant to aminoglycosides. No statistical differences could be seen in the detection of STEC or EPEC either by slaughterhouse location or by production system (extensive grass or feedlot). The rate of STEC detection was lower than the one reported for the rest of Argentina. STEC/EPEC relation was 3 to 1. This is the first study on cattle from TDF as reservoir for strains that are potentially pathogenic to humans.

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.

Antimicrobial Sensitivity of Shiga toxin-producing Escherichia coli (STEC) and Virulence Genes of Representative Isolates in Port Harcourt, Nigeria

Aim: To assess the antimicrobial susceptibility, Shiga toxin-producing Escherichia coli (STEC) and virulence genes of representative isolates in some selected sites of Port Harcourt, Nigeria.
 Study Design: Case-controlled study.
 Place and Duration of Study: Selected places in Port Harcourt, Rivers State, Nigeria, between November, 2020 to November, 2021.
 Methodology: Three hundred and forty-nine (349) samples were analyzed, 80 meat and 63 waste waters from five abattoirs cited in the city, 46 meat samples from five selected roadside butchers sites in the city, 109 patients stool samples, 30 stool samples from food sellers, 20 stool samples from healthy subjects and 1 commercial bottled water which served as control samples. A combination of methods was employed: conventional culture using Tryptone soya broth as an enrichment media, chromagar STEC and serology with O157 latex reagents. Multiplex polymerase chain reaction analysis was used to screen for the presence of specific virulence genes in representative isolates and 16SrRNA sequence data was used to confirm the identity of isolates. GraphPad Prism 2.01 was used to perform the statistical analysis and p values < 0.05 were considered statistically significant.
 Results: The results showed that 20 (100%) of the selected isolates profiled for virulence gene harbored a combination of stx1 + stx2, while 45% carried the eaeA gene. The culture and molecular methods used in STEC detection showed a good level of agreement comparatively. The result of isolates sequenced for identification by targeting the 16S gene and compared with publicly available sequences on NCBI for phylogenetic analysis showed 100% identities to the ribosomal RNA gene of seven uncultured Escherichia coli strains with Gene Accession numbers (CP049290, MN208208, KY780343, CP058233 MK641327, CP082357, CP082129, MH346270). Isolates were resistant to Ceftazidime 98.9%, Cefuroxime 100%, Cefixime 100%, Augmentin 100% and sensitive to the quinolones and nitrofuratoin. The virulence gene combination pattern, the antibiogram pattern and the phylogenetic analysis result indicated genetic relatedness of isolates. 
 Conclusion: The representative isolates harbored stx1 and stx2 combination, only 45% harbored stx1, stx2 and eaeA genes combination. Phylogenetic analysis of the representative isolates confirmed the genetic similarity of isolates.

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.

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. 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. 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 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. 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. 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.

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.

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. 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. 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 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. 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. 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.

A systematized review and qualitative synthesis of potential risk factors associated with the occurrence of non-O157 Shiga toxin-producing Escherichia coli (STEC) in the primary production of cattle.

Human infection with Shiga toxin-producing Escherichia coli (STEC) causes an estimated 2.8 million cases of acute illness worldwide each year. Serogroup O157 is the most commonly diagnosed STEC in humans, but cases linked to non-O157 STEC serogroups have increased recently due to increased surveillance and improvements to detection methods. Cattle are an important reservoir for STEC O157 and the same may be true for non-O157 STEC; therefore, reducing the occurrence of these pathogens in cattle could mitigate human infection risk. A systematized literature review of articles published within the Scopus database since 2010 (employing a partially systematic approach) was therefore conducted followed by qualitative synthesis of evidence to provide a structured overview of potential risk factors for non-O157 STEC in primary cattle production. Overall, few relevant studies were identified (n=22), highlighting that more studies are needed. Consistently significant associations were only identified with respect to cattle age (broadly higher rate of isolation from young animals compared to adults) and season of sampling (generally increased isolation of non-O157 STEC in summer). However, wide variation in study designs, including notable differences in laboratory detection methods, means drawing more general conclusions is currently not possible based on the results of this review. However, it is likely that the development of more sensitive methods for non-O157 STEC detection in potential livestock reservoirs and increased standardization across statistically sound epidemiological investigations are required to identify pertinent risk factors.

Ruminant-Related Risk Factors are Associated with Shiga Toxin-Producing Escherichia coli Infection in Children in Southern Ghana.

Livestock can provide benefits to low-income households, yet may expose children to zoonotic enteropathogens that cause illness and negative long-term health outcomes. The aim of this cross-sectional study was to determine whether livestock-related risk factors, including animal ownership, exposure to animal feces, and consumption of animal-source foods, were associated with bacterial zoonotic enteropathogen infections in children 6-59 months old in Greater Accra, Ghana. Stool samples from 259 children and 156 household chickens were analyzed for atypical enteropathogenic Escherichia coli (aEPEC), Campylobacter jejuni/coli (C. jejuni/coli), Salmonella, and Shiga toxin-producing Escherichia coli (STEC) using quantitative polymerase chain reaction (qPCR). aEPEC, C. jejuni/coli, STEC, and Salmonella were detected in 45.6%, 11.6%, 4.3%, and 0.8% of children's stool samples, respectively. In adjusted logistic regression models, household ownership of goats or sheep was associated with STEC detection in children (odds ratio [95% confidence interval]: 4.30 [1.32, 14.08]), as were positive detection of STEC in chicken feces (7.85 [2.54, 24.30]) and frequent consumption of fresh cow's milk (3.03 [1.75, 5.24]). No livestock-related risk factors were associated with aEPEC or C. jejuni/coli infection in children. Our findings suggest that ruminant ownership in southern Ghana may expose children to STEC through household fecal contamination and foodborne routes. The lack of association between livestock risk factors and the more commonly detected pathogens, aEPEC and C. jejuni/coli, warrants further research, particularly to help explain how animal-keeping and sanitation practices affect transmission of fecal pathogens that were highly prevalent in chicken feces.

Prevalence of Shiga toxin-producing Escherichia coli in Yezo sika deer (Cervus nippon yesoensis) in the Tokachi sub-prefecture of Hokkaido, Japan.

In food hygiene, the surveillance of foodborne pathogens in wild animals is indispensable because we cannot control hygienic status of them. Yezo sika deer (Cervus nippon yesoensis), which are found only on the island of Hokkaido, Japan, are the most common game animal in the country. In this study, we analyzed the incidence of Shiga toxin-producing Escherichia coli (STEC) in Yezo sika deer hunted in the Tokachi sub-prefecture, which is one of the densest zones for the sub-species. Real-time polymerase chain reaction testing detected STEC in 18.3% of fecal samples (59/323) collected from deer hunted between 2016 and 2017, whereas no Shigella and Salmonella markers were detected. No correlation was found between STEC detection from fecal samples and characteristics of carcasses, such as hunting area, age, and fascioliasis. From 59 STEC-positive fecal samples, we isolated 37 STEC strains, including 34 O- and H-genotyped strains, in which 16 different serogroups were detected. Genetic analysis revealed that our isolates included various stx gene types (stx1+/stx2−, stx1+/stx2+, and stx1−/stx2+) and carried eae. This study demonstrated that STEC strains with various features colonized the Yezo sika deer, similar to other subspecies of sika deer. We conclude that continuous surveillance activity is important to monitor the suitability of game animals as a food source and to assess the validity of the food safety management system for game meat production.

Shiga-toxin producing Escherichia coli detection in samples from Abruzzo and Molise regions, Italy

Abstract Background Top five serogroups (O157, O111, O26, O103 and O145) of Shiga-toxin producing Escherichia coli (STEC) represent the third most important increasing zoonosis reported in European Union. The ISO methods of analysis, used in foods and environmental samples, quite often does not confirm the molecular detection of STEC genes (stx1 and stx2) in enrichment broth. The aim of this study was to summarize the results of the tests carried out at Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise in the last six years (2016-2021), comparing genes detection in enrichment broth and in colonies grown on agar plates. Methods Along the 2016-2021, a total of 1716 foods and environmental samples from Abruzzo and Molise regions were tested, according to ISO/TS 13136:2012. Results Out of 1716 samples, 16 (0.9%) shown presumptive positive presence of STEC (stx1 and/or stx2) in broth. In spite of that, the presence of STEC (positive colonies for stx1 and/or stx2) was confirmed only in 4 out of 16 samples (one sample of cheese and 3 of minced meat). Then, eae gene was found in 54 samples in broth and only in 3 samples from colonies, during a foodborne outbreak investigation. Serogroup specific targets were detected in 6 broth samples (O26, O145 and O157) and confirmed in colonies only in 2 minced meat samples (O157). Conclusions STEC are a heavy hazard for the consumers and the number of cases is slightly increasing since 2015. Our results highlight a low number of presumptive positive samples and even lower number of STEC detection obtained with the ISO classical method. Furthermore, detection of the target genes in broth was rarely confirmed after plating, highlighting a weak point of the analytical method that needs to be investigated. Efforts are necessary to improve the method and the STEC confirmation from colonies. Immunomagnetic beads targeting the top five serogroups and digital PCR could help in increasing the recovery rate of cultivable STEC. Key messages The presumptive positive samples for STEC genes in foodstuffs from the Abruzzo and Molise regions are quite rare, even lower is the number of confirmed colonies by agar plate isolation. Specific immunomagnetic beads for the top five STEC serogroups could improve the method and the STEC colonies detection, increasing the recovery rate of cultivable strains.

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.

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.