Phenotypic and genomic comparison of three human outbreak and one cattle-associated Shiga toxin-producing Escherichia coli O157:H7.

Escherichia coli O157:H7-adulterated food products are associated with disease outbreaks in humans. Although cattle feces are a source for E. coli O157:H7 contamination, it is unclear if human-associated outbreak isolates differentially colonize and shed in the feces of cattle from that of non-outbreak isolates. It is also unclear if phenotypes, such as biofilm formation, cell attachment, or toxin production, differentiate environmental E. coli O157:H7 isolates from those associated with human illness. The objective of this study was to compare the genotypes and phenotypes of a diverse set of E. coli O157:H7 isolates, with the intent of identifying differences that could inform cattle colonization and fecal shedding, along with virulence potential in humans. Isolates differed in attachment phenotypes on human Caco-2 cells and bovine-derived recto-anal junction squamous epithelial cells, with curli having a strong impact on attachment to the human-derived cell line. The prototypical E. coli O157 isolate EDL933 had the greatest expression of the adhesin gene iha, yet it had decreased expression of the virulence genes stx2, eae, and ehxA compared the lineage I/II isolates RM6067W and/or FRIK1989. Strong or weak biofilm production was not associated with significant differences in cattle colonization or shedding, suggesting biofilms may not play a major role in cattle colonization. No significant differences in cattle colonization and fecal shedding were detected, despite genomic and in vitro phenotypic differences. The outbreak isolate associated with the greatest incidence of hemolytic uremic syndrome, RM6067W, induced the greatest Vero cell cytotoxicity and had the greatest stx2 gene expression. IMPORTANCE: Foodborne illness has major impacts on global health and imposes financial hardships on food industries. Escherichia coli serotype O157:H7 is associated with foodborne illness. Cattle feces are a source of E. coli O157:H7, and routine surveillance has led to an abundance of E. coli O157:H7 genomic data. The relationship between E. coli O157:H7 genome and phenotype is not clearly discerned for cattle colonization/shedding and improved understanding could lead to additional strategies to limit E. coli O157:H7 in the food chain. The goal of the research was to evaluate genomic and phenotypic attributes of E. coli O157:H7 associated with cattle colonization and shedding, environmental persistence, and human illness. Our results indicate variations in biofilm formation and in vitro cellular adherence was not associated with differences in cattle colonization or shedding. Overall, processes involved in cattle colonization and various phenotypes in relation to genotype are complex and remain not well understood.

Evidence of on-going transmission of Shiga toxin-producing Escherichia coli O157:H7 following a foodborne outbreak.

In August 2019, public health surveillance systems in Scotland and England identified seven, geographically dispersed cases infected with the same strain (defined as isolates that fell within the same five single nucleotide polymorphism single linage cluster) of Shiga toxin-producing Escherichia coli O157:H7. Epidemiological analysis of enhanced surveillance questionnaire data identified handling raw beef and shopping from the same national retailer (retailer A) as the common exposure. Concurrently, a microbiological survey of minced beef at retail identified the same strain in a sample of minced beef sold by retailer A, providing microbiological evidence of the link. Between September and November 2019, a further four primary and two secondary cases infected with the same strain were identified; two cases developed haemolytic uraemic syndrome. None of the four primary cases reported consumption of beef from retailer A and the transmission route of these subsequent cases was not identified, although all four primary cases visited the same petting farm. Generally, outbreaks of STEC O157:H7 in the UK appear to be distinct, short-lived events; however, on-going transmission linked to contaminated food, animals or environmental exposures and person-to-person contact do occur. Although outbreaks of STEC caused by contaminated fresh produce are increasingly common, undercooked meat products remain a risk of infection.

Prevalence, characterization and antibiotic resistance of Shiga toxigenic Escherichia coli serogroups isolated from fresh beef and locally processed ready-to-eat meat products in Lagos, Nigeria.

Fresh beef and meat products have been implicated in outbreaks of Shiga toxin-producing Escherichia coli (STEC) worldwide. This study investigated the prevalence of E. coli O157: H7 and non-O157 STEC serogroups in fresh beef in the open market and street vended meat products (n = 180) in Lagos metropolis, Nigeria. A combination of culture media and immunomagnetic separation followed by typing for associated virulence factors and serotypes was performed. Antimicrobial susceptibility testing was performed on the isolated STEC serotypes using the disk diffusion method. A total of 72 STEC serogroup isolates were detected from 61 out of 180 samples. The O157 STEC serotypes were detected in fresh beef, suya, minced meat and tsire with prevalence of 20.8% while non-O157 STEC serogroups were detected in all the samples. Molecular typing revealed 25% (n = 18) of the STEC serogroups showed presence of all the stx1, stx2, eaeA, fliCH7 and rfbEO157 virulence factors while 54.2% (n = 39) possessed a combination of two virulence genes. Multidrug resistance was discovered in 23.6% (n = 17) of the total STEC serogroups. Locally processed ready-to-eat meat products in Lagos metropolis, Nigeria harbour potentially pathogenic multi-drug resistant STEC serogroups that can constitute public health hazard.

Analysis of a small outbreak of Shiga toxin-producing Escherichia coli O157:H7 using long-read sequencing.

Compared to short-read sequencing data, long-read sequencing facilitates single contiguous de novo assemblies and characterization of the prophage region of the genome. Here, we describe our methodological approach to using Oxford Nanopore Technology (ONT) sequencing data to quantify genetic relatedness and to look for microevolutionary events in the core and accessory genomes to assess the within-outbreak variation of four genetically and epidemiologically linked isolates. Analysis of both Illumina and ONT sequencing data detected one SNP between the four sequences of the outbreak isolates. The variant calling procedure highlighted the importance of masking homologous sequences in the reference genome regardless of the sequencing technology used. Variant calling also highlighted the systemic errors in ONT base-calling and ambiguous mapping of Illumina reads that results in variations in the genetic distance when comparing one technology to the other. The prophage component of the outbreak strain was analysed, and nine of the 16 prophages showed some similarity to the prophage in the Sakai reference genome, including the stx2a-encoding phage. Prophage comparison between the outbreak isolates identified minor genome rearrangements in one of the isolates, including an inversion and a deletion event. The ability to characterize the accessory genome in this way is the first step to understanding the significance of these microevolutionary events and their impact on the evolutionary history, virulence and potentially the likely source and transmission of this zoonotic, foodborne pathogen.

Precision long-read metagenomics sequencing for food safety by detection and assembly of Shiga toxin-producing Escherichia coli in irrigation water.

Shiga toxin-producing Escherichia coli (STEC) contamination of agricultural water might be an important factor to recent foodborne illness and outbreaks involving leafy greens. Closed bacterial genomes from whole genome sequencing play an important role in source tracking. We aimed to determine the limits of detection and classification of STECs by qPCR and nanopore sequencing using 24 hour enriched irrigation water artificially contaminated with E. coli O157:H7 (EDL933). We determined the limit of STEC detection by qPCR to be 30 CFU/reaction, which is equivalent to 105 CFU/ml in the enrichment. By using Oxford Nanopore's EPI2ME WIMP workflow and de novo assembly with Flye followed by taxon classification with a k-mer analysis software (Kraken2), E. coli O157:H7 could be detected at 103 CFU/ml (68 reads) and a complete fragmented E. coli O157:H7 metagenome-assembled genome (MAG) was obtained at 105-108 CFU/ml. Using a custom script to extract the E. coli reads, a completely closed MAG was obtained at 107-108 CFU/ml and a complete, fragmented MAG was obtained at 105-106 CFU/ml. In silico virulence detection for E. coli MAGs for 105-108 CFU/ml showed that the virulotype was indistinguishable from the spiked E. coli O157:H7 strain. We further identified the bacterial species in the un-spiked enrichment, including antimicrobial resistance genes, which could have important implications to food safety. We propose this workflow provides proof of concept for faster detection and complete genomic characterization of STECs from a complex microbial sample compared to current reporting protocols and could be applied to determine the limit of detection and assembly of other foodborne bacterial pathogens.

A quasimetagenomics method for concerted detection and subtyping of Salmonella enterica and E. coli O157:H7 from romaine lettuce.

Quasimetagenomics refers to the sequencing of a modified food microbiome to facilitate combined detection and subtyping of targeted pathogens in a single workflow. Through quasimetagenomic sequencing, pathogens are detected and subtyped in a shortened time frame compared to traditional culture enrichment and whole genome sequencing-based analyses. While this method was previously used to detect and subtype Salmonella enterica from chicken, iceberg lettuce, and black pepper, it has not been applied to investigate multiple pathogens in one workflow. A quasimetagenomic method to concertedly detect and subtype Salmonella enterica and Escherichia coli O157:H7 from artificially contaminated romaine lettuce in a single workflow was developed. All quasimetagenomic samples with initial target pathogen inoculum levels of ~1 CFU/g were detected and serotyped after co-enrichment of the two pathogens for 12 h. Single nucleotide polymorphism typing was achievable for some initial pathogen inoculum levels as low as ~0.1 CFU/g. Our results suggest that this method can be used for concerted detection and subtyping of multiple bacterial pathogens from romaine lettuce even at low contamination levels.

Phylo-diversity of prevalent human E. coli O157:H7 with strains from retailed meat and fish in selected markets in Ibadan Nigeria.

Global prevalence of ESBL-biotypes poses a serious threat to public health as a result of severity and morbidity caused by beta-lactam encoded Escherichia coli. Therefore, the prevalent shiga toxigenic Escherichia coli of ESBL variant was investigated in various retailed food animals and cooking materials. A total of 823 samples consisting of raw meat (297) and fish (132) samples retailed at various major markets in Ibadan were collected and 394 swabs were taken from the butchers' processing tables and utensils used in retailing meat and fish. The samples were cultured and biotyped for Escherichia coli. Serological and PCR assay were used to identify O157:H7 variant and antibiotics resistant determinants. Genetic relatedness of characterized E. coli O157 from human and meat products was evaluated with phylogenetic analysis. Of all the isolates, 130 (15.8%) were E. coli and only 8 (1.0) were O157:H7 while 4 (50%) were resistant to one or more antibiotics with resistance index ranging from 0.1 to 0.5. More than 25% E. coli O157:H7 were resistant to Ampicillin, Pefloxacin and Gentamicin and blaSHV and blaCTX-M were detected in 1/8 (12.5%) of E. coli O157:H7 and blaTEM 3/8 (37.5%) respectively. Only 1 genotyped human Escherichia coli .0157:H7 clustered with beef strain There is evidence of blaTEM encoded E. coli O157:H7 causing infection in human from food animals retailed in many markets within various communities. Therefore, urgent surveillance with public health education, food, and environmental hygiene are highly needed to prevent its spread.

Reaction of Surrogate Escherichia coli Serotype O157:H7 and Non-O157 Strains to Nutrient Starvation: Variation in Phenotype and Transcription of Stress Response Genes and Behavior on Lettuce Plants in the Field.

Preharvest contamination with bacteria borne by irrigation water may result in leafy vegetables serving as vehicles for transmission of Shiga toxin-producing Escherichia coli (STEC) to humans. The influence of starvation-associated stress on the behavior of non-toxin-producing strains of E. coli serotype O157:H7 and serotypes O26, O103, O111, and O145 was examined subsequent to their introduction to the phyllosphere of field-grown romaine lettuce as inocula simulating starved (96 h in sterile deionized water) and nutrient-depleted (24 h broth culture) cells. As with E. coli O157:H7, leaf populations of the non-O157 strains declined rapidly during the first 72 h postinoculation, displaying the biphasic decay curve typical of serotype O157:H7 isolates. Preinoculation treatment appeared not to influence decay rates greatly (P > 0.5), but strain-specific differences (persistence period and attachment proficiency) indicated that serotype O103:H2 strain PARC445 was a better survivor. Also assessed was the impact of preinoculation treatment on phenotypes key to leaf colonization and survival and the expression of starvation stress-associated genes. The 96-h starvation period enhanced biofilm formation in one strain but reduced motility and autoinducer 2 formation in all five study strains relative to those characteristics in stationary-phase cells. Transcription of rpoS, dps, uspA, and gapA was reduced significantly (P < 0.05) in starvation-stressed cells relative to that for exponential- and stationary-phase cultures. Strain-specific differences were observed; serotype O103:H2 PARC445 had greater downturns than did serotype O157:H7 and other non-O157 strains. Within this particular cohort, the behavior of the representative serotype O157:H7 strain, PARC443 (ATCC 700728), was not predictive of behavior of non-O157 members of this STEC group.

High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution.

Targeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.

Prevalence of Shiga toxin-producing Escherichia coli in pasture-based dairy herds.

Shiga toxin-producing Escherichia coli strains (STEC) are food-borne pathogens. While E. coli O157:H7 is commonly associated with cattle, less is known about the prevalence of non-O157 STEC serogroups in bovines. This study evaluated the prevalence and virulence status of O157:H7 and six E. coli O-serogroups (O26, O103, O45, O145, O121, O111) in New Zealand dairy farms using molecular as well as culture-based methods. Fresh farm dairy effluent (FDE) (n = 36) and composite calf faeces (n = 12) were collected over three samplings from 12 dairy farms. All seven target serogroups were detected through molecular techniques. Of the 202 isolates which were serologically confirmed following traditional culturing and immunomagnetic separation (IMS), O103, O26, O45 and O121 were the most common serogroups, being found in 81, 47, 42 and 32% of the FDE and in 17, 33, 25 and 9% of the calf faeces respectively. The majority (157/202) of the isolates were negative for stx and eae virulence genes. The prevalence of the seven target STEC was low, and only nine O26 isolates (4%) were recovered from four of the farms. The study has highlighted the need for improving the isolation of Top 7 STEC from the stx-negative populations present in fresh dairy effluent and calf faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens that can cause severe illness in humans. Cattle are asymptomatic reservoirs for STEC, and transmission to humans can be by consumption of food products or water contaminated with cattle faeces. Our study investigated the prevalence of O157:H7 and six E. coli serogroups of STEC (O26, O103, O45, O145, O121, O111) over time in the dairy reservoir and increases the knowledge and understanding of these pathogens on pasture-based farms. Such information is required to develop risk-assessment models aiming at limiting transmission of these STEC to human.

Highly Pathogenic Clone of Shiga Toxin-Producing Escherichia coli O157:H7, England and Wales.

We used whole-genome sequencing to investigate the evolutionary context of an emerging highly pathogenic strain of Shiga toxin-producing Escherichia coli (STEC) O157:H7 in England and Wales. A timed phylogeny of sublineage IIb revealed that the emerging clone evolved from a STEC O157:H7 stx-negative ancestor ≈10 years ago after acquisition of a bacteriophage encoding Shiga toxin (stx) 2a, which in turn had evolved from a stx2c progenitor ≈20 years ago. Infection with the stx2a clone was a significant risk factor for bloody diarrhea (OR 4.61, 95% CI 2.24-9.48; p<0.001), compared with infection with other strains within sublineage IIb. Clinical symptoms of cases infected with sublineage IIb stx2c and stx-negative clones were comparable, despite the loss of stx2c. Our analysis highlighted the highly dynamic nature of STEC O157:H7 Stx-encoding bacteriophages and revealed the evolutionary history of a highly pathogenic clone emerging within sublineage IIb, a sublineage not previously associated with severe clinical symptoms.

Genomic Characterization of ß-Glucuronidase-Positive Escherichia coli O157:H7 Producing Stx2a.

Among Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H7 strains, those producing Stx2a cause more severe diseases. Atypical STEC O157:H7 strains showing a ß-glucuronidase-positive phenotype (GP STEC O157:H7) have rarely been isolated from humans, mostly from persons with asymptomatic or mild infections; Stx2a-producing strains have not been reported. We isolated, from a patient with bloody diarrhea, a GP STEC O157:H7 strain (PV15-279) that produces Stx2a in addition to Stx1a and Stx2c. Genomic comparison with other STEC O157 strains revealed that PV15-279 recently emerged from the stx1a/stx2c-positive GP STEC O157:H7 clone circulating in Japan. Major virulence genes are shared between typical (ß-glucuronidase-negative) and GP STEC O157:H7 strains, and the Stx2-producing ability of PV15-279 is comparable to that of typical STEC O157:H7 strains; therefore, PV15-279 presents a virulence potential similar to that of typical STEC O157:H7. This study reveals the importance of GP O157:H7 as a source of highly pathogenic STEC clones.

A spatial and temporal analysis of risk factors associated with sporadic Shiga toxin-producing Escherichia coli O157 infection in England between 2009 and 2015.

Infection with STEC O157 is relatively rare but has potentially serious sequelae, particularly for children. Large outbreaks have prompted considerable efforts designed to reduce transmission primarily from food and direct animal contact. Despite these interventions, numbers of infections have remained constant for many years and the mechanisms leading to many sporadic infections remain unclear.Here, we show that two-thirds of all cases reported in England between 2009 and 2015 were sporadic. Crude rates of infection differed geographically and were highest in rural areas during the summer months. Living in rural areas with high densities of cattle, sheep or pigs and those served by private water supplies were associated with increased risk. Living in an area of lower deprivation contributed to increased risk but this appeared to be associated with reported travel abroad. Fresh water coverage and residential proximity to the coast were not risk factors.To reduce the overall burden of infection in England, interventions designed to reduce the number of sporadic infections with STEC should focus on the residents of rural areas with high densities of livestock and the effective management of non-municipal water supplies. The role of sheep as a reservoir and potential source of infection in humans should not be overlooked.

Whole genome shotgun sequencing revealed highly polymorphic genome regions and genes in Escherichia coli O157:H7 isolates collected from a single feedlot.

Escherichia coli serotype O157:H7 continues to pose a serious health threat to human beings. Cattle, a major reservoir of the pathogen, harbor E. coli O157:H7 in their gastrointestinal tract and shed variable concentrations of E. coli O157:H7 into the environment. Genetic characterization of cattle-shed E. coli O157 strains is of interest to the livestock industry, food business, and public health community. The present study applied whole genome shotgun sequencing (WGS) and single nucleotide variant (SNV) calling to characterize 279 cattle-shed E. coli O157:H7 strains isolated from a single feedlot located in southwestern region of the US. More than 4,000 SNVs were identified among the strains and the resultant phylogenomic tree revealed three major groups. Using the Sakai strain genome as reference, more than 2,000 SNVs were annotated and a detailed SNV map generated. Results clearly revealed highly polymorphic loci along the E. coli O157:H7 genome that aligned with the prophage regions and highly variant genes involved in processing bacterial genetic information. The WGS data were further profiled against a comprehensive virulence factor database (VFDB) for virulence gene identification. Among the total 285 virulence genes identified, only 132 were present in all the strains. There were six virulence genes unique to single isolates. Our findings suggested that the genome variations of the E. coli O157:H7 were mainly attributable to dynamics of certain phages, and the bacterial strains have variable virulence gene profiles, even though they came from a single cattle population, which may explain the differences in pathogenicity, host prevalence, and transmissibility by E. coli O157:H7.

Outbreak of Enterohemorrhagic Escherichia coli O157:H7 Infection Associated with Minced Meat Cutlets Consumption in Kanagawa, Japan.

An outbreak of enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection occurred in October 2016 in Kanagawa, Japan. A total of 61 patients and 17 asymptomatic cases of EHEC O157:H7 infection were confirmed by laboratory testing. Among them, 24 patients were hospitalized and 4 developed hemolytic-uremic syndrome. An epidemiological investigation revealed that this outbreak of EHEC O157:H7 infection was associated with the consumption of uncooked minced meat cutlets that were sold frozen at branches of a supermarket chain. The implicated uncooked meat cutlets were made of a mixture of minced beef, pork, onions, and eggs. All 40 meat cutlets tested from one particular batch were positive for EHEC O157:H7. The patterns observed on pulsed-field gel electrophoresis of strains isolated from the affected patients and meat cutlets were identical. The bacterial counts of EHEC O157:H7 and E. coli in meat cutlets ranged from 2.3 to 110 most-probable-number (MPN)/g and from 240 to 4,600 MPN/g, respectively. There are currently no national regulatory standards to ensure the safety of these types of meat products in Japan. Consumers should ensure that such products are cooked thoroughly and that safe food handling procedures are used to prevent infection.

Molecular Typing of Escherichia coli O157 Isolates from Romanian Human Cases.

Verocytotoxin-producing Escherichia coli (VTEC) of serogroup O157 are among the most important causes of severe cases of foodborne disease and outbreaks worldwide. As little is known about the characteristic of these strains in Romania, we aimed to provide reference information on the virulence gene content, phylogenetic background, and genetic diversity of 7 autochthonous O157 strains collected during 2016 and 2017 from epidemiologically non-related cases. These strains were typed by a combination of phenotypic and molecular methods routinely used by the national reference laboratory. Additionally, 4 of them were subjected to whole-genome sequencing (WGS), and public web-based tools were used to extract information on virulence gene profiles, multilocus sequence types (MLST), and single nucleotide polymorphism (SNP)-based phylogenetic relatedness. Molecular typing provided evidence of the circulation of a polyclonal population while distinguishing a cluster of non-sorbitol-fermenting, glucuronidase-negative, phylogenetic group E, MLST 1804 strains, representing lineage II and clade 7, which harbored vtx2c, eae-gamma, and ehxA genes. A good correlation between the routine typing methods and WGS data was observed. However, SNP-based genotyping provided a higher resolution in depicting the relationships between the O157:H7 strains than that provided by Pulse-field gel electrophoresis. This study should be a catalyst for improved laboratory-based surveillance of autochthonous VTEC.

Outbreak of Escherichia coli O157 Phage Type 32 linked to the consumption of venison products.

In September 2015, an outbreak of Escherichia coli Phage Type 32 with an indistinguishable multi locus variable number tandem repeat analysis profile was identified in Scotland. Twelve cases were identified; nine primary cases, two secondary and one asymptomatic case. Extensive food history investigations identified venison products containing wild venison produced by a single food business operator as the most likely source of the outbreak. Of the nine primary cases, eight had consumed venison products, and one case had not eaten venison themselves but had handled and cooked raw venison in the household. This was the first reported outbreak of Shiga toxin-producing Escherichia coli (STEC) linked to venison products in the UK, and was also notable due to the implicated products being commercially produced and widely distributed. In contrast, previous venison outbreaks reported from other countries have tended to be smaller and related to individually prepared carcases. The outbreak has highlighted some important knowledge gaps in relation to STEC in venison that are currently been investigated via a number of research studies.

Rapid and simultaneous detection of Salmonella spp., Escherichia coli O157, and Listeria monocytogenes by magnetic capture hybridization and multiplex real-time PCR.

The application of rapid, specific, and sensitive methods for pathogen detection and quantification is very advantageous in diagnosis of human pathogens in several applications, including food analysis. The aim of this study was the evaluation of a method for the multiplexed detection and quantification of three significant foodborne pathogenic species (Escherichia coli O157, Salmonella spp., and Listeria monocytogenes). The assay combines specific DNA extraction by multiplex magnetic capture hybridization (mMCH) with multiplex real-time PCR. The amplification assay showed linearity in the range 106-10 genomic units (GU)/PCR for each co-amplified species. The sensitivity corresponded to 1 GU/PCR for E. coli O157 and L. monocytogenes, and 10 GU/PCR for Salmonella spp. The immobilization process and the hybrid capture of the MCH showed good efficiency and reproducibility for all targets, allowing the combination in equal amounts of the different nanoparticle types in mMCH. MCH and mMCH efficiencies were similar. The detection limit of the method was 10 CFU in samples with individual pathogens and 102 CFU in samples with combination of the three pathogens in unequal amounts (amount's differences of 2 or 3 log). In conclusion, this multiplex molecular platform can be applied to determine the presence of target species in food samples after culture enrichment. In this way, this method could be a time-saving and sensitive tool to be used in routine diagnosis.

Quantitative surveillance of shiga toxins 1 and 2, Escherichia coli O178 and O157 in feces of western-Canadian slaughter cattle enumerated by droplet digital PCR with a focus on seasonality and slaughterhouse location.

Often Escherichia coli are harmless and/or beneficial bacteria inhabiting the gastrointestinal tract of livestock and humans. However, Shiga toxin-producing E. coli (STEC) have been linked to human disease. Cattle are the primary reservoir for STEC and STEC "super-shedders" are considered to be a major contributor in animal to animal transmission. Among STEC, O157:H7 is the most recognized serotype, but in recent years, non-O157 STEC have been increasingly linked to human disease. In Argentina and Germany, O178 is considered an emerging pathogen. Our objective was to compare populations of E. coli O178, O157, shiga toxin 1 and 2 in western Canadian cattle feces from a sampling pool of ~80,000 beef cattle collected at two slaughterhouses. Conventional PCR was utilized to screen 1,773 samples for presence/absence of E. coli O178. A subset of samples (n = 168) was enumerated using droplet digital PCR (ddPCR) and proportions of O178, O157 and shiga toxins 1 & 2 specific-fragments were calculated as a proportion of generic E. coli (GEC) specific-fragments. Distribution of stx1 and stx2 was determined by comparing stx1, stx2 and O157 enumerations. Conventional PCR detected the presence of O178 in 873 of 1,773 samples and ddPCR found the average proportion of O178, O157, stx1 and stx2 in the samples 2.8%, 0.6%, 1.4% and 0.5%, respectively. Quantification of stx1 and stx2 revealed more virulence genes than could be exclusively attributed to O157. Our results confirmed the presence of E. coli O178 in western Canadian cattle and ddPCR revealed O178 as a greater proportion of GEC than was O157. Our results suggests: I) O178 may be an emerging subgroup in Canada and II) monitoring virulence genes may be a more relevant target for food-safety STEC surveillance compared to current serogroup screening.

Quantification of enterohemorrhagic Escherichia coli O157:H7 protein abundance by high-throughput proteome.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a human pathogen responsible for diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). To promote a comprehensive insight into the molecular basis of EHEC O157:H7 physiology and pathogenesis, the combined proteome of EHEC O157:H7 strains, Clade 8 and Clade 6 isolated from cattle in Argentina, and the standard EDL933 (clade 3) strain has been analyzed. From shotgun proteomic analysis a total of 2,644 non-redundant proteins of EHEC O157:H7 were identified, which correspond approximately 47% of the predicted proteome of this pathogen. Normalized spectrum abundance factor analysis was performed to estimate the protein abundance. According this analysis, 50 proteins were detected as the most abundant of EHEC O157:H7 proteome. COG analysis showed that the majority of the most abundant proteins are associated with translation processes. A KEGG enrichment analysis revealed that Glycolysis / Gluconeogenesis was the most significant pathway. On the other hand, the less abundant detected proteins are those related to DNA processes, cell respiration and prophage. Among the proteins that composed the Type III Secretion System, the most abundant protein was EspA. Altogether, the results show a subset of important proteins that contribute to physiology and pathogenicity of EHEC O157:H7.