First Isolation of the Heteropathotype Shiga Toxin-Producing and Extra-Intestinal Pathogenic (STEC-ExPEC) E. coli O80:H2 in French Healthy Cattle: Genomic Characterization and Phylogenetic Position.

The emerging heteropathotype shigatoxigenic (STEC) and extra-intestinal pathogenic Escherichia coli (ExPEC) O80:H2 has been the second leading cause of pediatric HUS in France since the mid-2010s. In contrast with other highly pathogenic STEC serotypes, for which ruminants have clearly been identified as the main human infection source, this heteropathotype's reservoir remains unknown. In this context, we describe for the first time the isolation of seven STEC O80:H2 strains from healthy cattle on a single cattle farm in France. This study aimed at (i) characterizing the genome and (ii) investigating the phylogenetic positions of these O80:H2 STEC strains. The virulomes, resistomes, and phylogenetic positions of the seven bovine isolates were investigated using in silico typing tools, antimicrobial susceptibility testing and cgMLST analysis after short-read whole genome sequencing (WGS). One representative isolate (A13P112V1) was also subjected to long-read sequencing. The seven isolates possessed ExPEC-related virulence genes on a pR444_A-like mosaic plasmid, previously described in strain RDEx444 and known to confer multi-drug resistance. All isolates were clonally related and clustered with human clinical strains from France and Switzerland with a range of locus differences of only one to five. In conclusion, our findings suggest that healthy cattle in France could potentially act as a reservoir of the STEC-ExPEC O80:H2 pathotype.

Transcription levels of hes and their involvement in the biofilm formation of Shiga toxin-producing Escherichia coli O91.

Shiga toxin-producing Escherichia coli (STEC) are recognized as being responsible for many cases of foodborne diseases worldwide. Cattle are the main reservoir of STEC, shedding the microorganisms in their feces. The serogroup STEC O91 has been associated with hemorrhagic colitis and hemolytic uremic syndrome. Locus of Adhesion and Autoaggregation (LAA) and its hes gene are related to the pathogenicity of STEC and the ability to form biofilms. Considering the frequent isolation of STEC O91, the biofilm-forming ability, and the possible role of hes in the pathogenicity of STEC, we propose to evaluate the ability of STEC to form biofilms and to evaluate the expression of hes before and after of biofilm formation. All strains were classified as strong biofilm-forming. The hes expression showed variability between strains before and after biofilm formation, and this may be due to other genes carried by each strain. This study is the first to report the relationship between biofilm formation, and hes expression and proposes that the analysis and diagnosis of LAA, especially hes as STEC O91 virulence factors, could elucidate these unknown mechanisms. Considering that there is no specific treatment for HUS, only supportive care, it is necessary to know the survival and virulence mechanisms of STEC O91.

A prophage encoded ribosomal RNA methyltransferase regulates the virulence of Shiga-toxin-producing Escherichia coli (STEC).

Shiga toxin (Stx) released by Shiga toxin producing Escherichia coli (STEC) causes life-threatening illness. Its production and release require induction of Stx-encoding prophage resident within the STEC genome. We identified two different STEC strains, PA2 and PA8, bearing Stx-encoding prophage whose sequences primarily differ by the position of an IS629 insertion element, yet differ in their abilities to kill eukaryotic cells and whose prophages differ in their spontaneous induction frequencies. The IS629 element in ϕPA2, disrupts an ORF predicted to encode a DNA adenine methyltransferase, whereas in ϕPA8, this element lies in an intergenic region. Introducing a plasmid expressing the methyltransferase gene product into ϕPA2 bearing-strains increases both the prophage spontaneous induction frequency and virulence to those exhibited by ϕPA8 bearing-strains. However, a plasmid bearing mutations predicted to disrupt the putative active site of the methyltransferase does not complement either of these defects. When complexed with a second protein, the methyltransferase holoenzyme preferentially uses 16S rRNA as a substrate. The second subunit is responsible for directing the preferential methylation of rRNA. Together these findings reveal a previously unrecognized role for rRNA methylation in regulating induction of Stx-encoding prophage.

Presence and Virulence Characteristics of Shiga Toxin Escherichia coli and Non-Shiga Toxin-Producing Escherichia coli O157 in Products from Animal Protein Supply Chain Enterprises in South Africa.

Consumption of food that is contaminated with Shiga toxin-producing Escherichia coli (STEC) has been linked to serious foodborne disease outbreaks. Our aim was to provide a descriptive study on the presence and virulence factors of STEC and non-STEC O157 isolates recovered from 2017 diverse meat and meat product samples from all provinces of South Africa (n = 1758) and imported meat from South Africa's major ports of entry (n = 259). A cross-sectional study was undertaken to analyze raw intact meat, raw processed (nonintact) meat, and ready-to-eat (RTE) meat from cattle, game, sheep, pork, and poultry. Isolation was performed using International Organization for Standardization-based microbiological techniques, while detection and characterization were performed using real-time PCR (RT-PCR) and conventional PCR targeting the stx1, stx2, eae, and ehxA genes. A total of 28 of 1758 (1.59%; confidence interval [CI] 1.1-2) samples from the domestic market tested positive (n = 10 Escherichia coli O157:H7; n = 14 Escherichia coli O157: non-H7; and n = 4 non-O157 STEC), while 4/259 (1.54%; CI 0.4-4) samples from ports of entry tested positive for Escherichia coli O157:H7 based on RT-PCR. On average, diverse samples from domestic meat and meat products from cattle showed the highest number of positive samples (22/1758; 1.3%; CI 0.8-2). RT-PCR detected more positive samples (n = 32) compared with culture (n = 17). Sixteen different virulence factor combinations were observed. Our findings demonstrate a relatively low presence of diverse STEC strains along the meat value chain. To our knowledge, this is the first extensive report in South Africa to analyze STEC and non-STEC O157 from local and imported samples from many animal species. This is important as it reveals virulence factors in STEC strains circulating in meat and meat products in South Africa, which contribute to the risk of infection.

The global population structure and evolutionary history of the acquisition of major virulence factor-encoding genetic elements in Shiga toxin-producing Escherichia coli O121:H19.

Shiga toxin (Stx)-producing Escherichia coli (STEC) are foodborne pathogens causing serious diseases, such as haemorrhagic colitis and haemolytic uraemic syndrome. Although O157:H7 STEC strains have been the most prevalent, incidences of STEC infections by several other serotypes have recently increased. O121:H19 STEC is one of these major non-O157 STECs, but systematic whole genome sequence (WGS) analyses have not yet been conducted on this STEC. Here, we performed a global WGS analysis of 638 O121:H19 strains, including 143 sequenced in this study, and a detailed comparison of 11 complete genomes, including four obtained in this study. By serotype-wide WGS analysis, we found that O121:H19 strains were divided into four lineages, including major and second major lineages (named L1 and L3, respectively), and that the locus of enterocyte effacement (LEE) encoding a type III secretion system (T3SS) was acquired by the common ancestor of O121:H19. Analyses of 11 complete genomes belonging to L1 or L3 revealed remarkable interlineage differences in the prophage pool and prophage-encoded T3SS effector repertoire, independent acquisition of virulence plasmids by the two lineages, and high conservation in the prophage repertoire, including that for Stx2a phages in lineage L1. Further sequence determination of complete Stx2a phage genomes of 49 strains confirmed that Stx2a phages in lineage L1 are highly conserved short-tailed phages, while those in lineage L3 are long-tailed lambda-like phages with notable genomic diversity, suggesting that an Stx2a phage was acquired by the common ancestor of L1 and has been stably maintained. Consistent with these genomic features of Stx2a phages, most lineage L1 strains produced much higher levels of Stx2a than lineage L3 strains. Altogether, this study provides a global phylogenetic overview of O121:H19 STEC and shows the interlineage genomic differences and the highly conserved genomic features of the major lineage within this serotype of STEC.

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.

Emergence of New ST301 Shiga Toxin-Producing Escherichia coli Clones Harboring Extra-Intestinal Virulence Traits in Europe.

O80:H2 enterohemorrhagic Escherichia coli (EHEC) of sequence type ST301 is one of the main serotypes causing European hemolytic and uremic syndrome, but also invasive infections, due to extra-intestinal virulence factors (VFs). Here, we determined whether other such heteropathotypes exist among ST301. EnteroBase was screened for ST301 strains that were included in a general SNP-phylogeny. French strains belonging to a new heteropathotype clone were sequenced. ST, hierarchical clusters (HC), serotype, resistome, and virulome were determined using EnteroBase, the CGE website, and local BLAST. The ST301 general phylogeny shows two groups. Group A (n = 25) is mainly composed of enteropathogenic E. coli, whereas group B (n = 55) includes mostly EHEC. Three serotypes, O186:H2, O45:H2 and O55:H9, share the same virulome as one of the O80:H2 sub-clones from which they derive subsequent O-antigen switches. The O55:H9 clone, mainly present in France (n = 29), as well as in the UK (n = 5) and Germany (n = 1), has a low background of genetic diversity (four HC20), although it has three Stx subtypes, an H-antigen switch, and genes encoding the major extra-intestinal VF yersiniabactin, and extended-spectrum beta-lactamases. Diverse heteropathotype clones genetically close to the O80:H2 clone are present among the ST301, requiring close European monitoring, especially the virulent O55:H9 clone.

Genetic and antimicrobial resistance profiles of non-O157 Shiga toxin-producing Escherichia coli from different sources in Egypt.

BACKGROUND: The Shiga toxin-producing Escherichia coli (STEC) represented a great risk to public health. In this study, 60 STEC strains recovered from broiler and duck fecal samples, cow's milk, cattle beef, human urine, and ear discharge were screened for 12 virulence genes, phenotypic and genotypic antimicrobial resistance, and multiple-locus variable-number tandem-repeat analysis (MLVA). RESULTS: The majority of strains harbored Shiga toxin 1 (stx1) and stx1d, stx2 and stx2e, and ehxA genes, while a minority harbored stx2c subtype and eaeA. We identified 10 stx gene combinations; most of strains 31/60 (51.7%) exhibited four copies of stx genes, namely the stx1, stx1d, stx2, and stx2e, and the strains exhibited a high range of multiple antimicrobial resistance indices. The resistance genes blaCTX-M-1 and blaTEM were detected. For the oxytetracycline resistance genes, most of strains contained tetA, tetB, tetE, and tetG while the tetC was present at low frequency. MLVA genotyping resolved 26 unique genotypes; genotype 21 was highly prevalent. The six highly discriminatory loci DI = 0.9138 are suitable for the preliminary genotyping of STEC from animals and humans. CONCLUSIONS: The STEC isolated from animals are virulent, resistant to antimicrobials, and genetically diverse, thus demands greater attention for the potential risk to human.

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.

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.

Whole-genome sequencing analysis of uncommon Shiga toxin-producing Escherichia coli from cattle: Virulence gene profiles, antimicrobial resistance predictions, and identification of novel O-serogroups.

Shiga toxin-producing E. coli (STEC) are major foodborne pathogens. While many studies have focused on the "top-7 STEC", little is known for minor serogroups. A total of 284 non-top-7 STEC strains isolated from cattle feces were subjected to whole-genome sequencing (WGS) to determine the serotypes, the presence of virulence genes and antimicrobial resistance (AMR) determinants. Nineteen typeable and three non-typeable serotypes with novel O-antigen loci were identified. Twenty-one AMR genes and point mutations in another six genes that conferred resistance to 10 antimicrobial classes were detected, as well as 46 virulence genes. The distribution of 33 virulence genes and 15 AMR determinants exhibited significant differences among serotypes (p < 0.05). Among all strains, 81.7% (n = 232) and 14.1% (n = 40) carried stx2 and stx1 only, respectively; only 4.2% (n = 12) carried both. Subtypes stx1a, stx1c, stx2a, stx2c, stx2d, and stx2g were identified. Forty-six strains carried eae and stx2a and therefore had the potential cause severe diseases; 47 strains were genetically related to human clinical strains inferred from a pan-genome phylogenetic tree. We were able to demonstrate the utility of WGS as a surveillance tool to characterize the novel serotypes, as well as AMR and virulence profiles of uncommon STEC that could potentially cause human illness.

Hemoconcentration and predictors in Shiga toxin-producing E. coli-hemolytic uremic syndrome (STEC-HUS).

BACKGROUND: Hemoconcentration has been identified as a risk factor for a complicated course in Shiga toxin-producing E. coli-hemolytic uremic syndrome (STEC-HUS). This single-center study assesses hemoconcentration and predictors at presentation in STEC-HUS treated from 2009-2017. METHODS: Data of 107 pediatric patients with STEC-HUS were analyzed retrospectively. Patients with mild HUS (mHUS, definition: max. serum creatinine < 1.5 mg/dL and no major neurological symptoms) were compared to patients with severe HUS (sHUS, definition: max. serum creatinine ≥ 1.5 mg/dL ± major neurological symptoms). Additionally, predictors of complicated HUS (dialysis ± major neurological symptoms) were analyzed. RESULTS: Sixteen of one hundred seven (15%) patients had mHUS. Admission of patients with sHUS occurred median 2 days earlier after the onset of symptoms than in patients with mHUS. On admission, patients with subsequent sHUS had significantly higher median hemoglobin (9.5 g/dL (3.6-15.7) vs. 8.5 g/dL (4.2-11.5), p = 0.016) than patients with mHUS. The product of hemoglobin (g/dL) and LDH (U/L) (cutoff value 13,302, sensitivity 78.0%, specificity of 87.5%) was a predictor of severe vs. mild HUS. Creatinine (AUC 0.86, 95% CI 0.79-0.93) and the previously published score hemoglobin (g/dL) + 2 × creatinine (mg/dL) showed a good prediction for development of complicated HUS (AUC 0.87, 95% CI 0.80-0.93). CONCLUSIONS: At presentation, patients with subsequent severe STEC-HUS had a higher degree of hemoconcentration. This underlines that fluid loss or reduced fluid intake/administration may be a risk factor for severe HUS. The good predictive value of the score hemoglobin (g/dL) + 2 × creatinine (mg/dL) for complicated HUS could be validated in our cohort. A higher resolution version of the Graphical abstract is available as Supplementary Information.

Pathogenic potential assessment of the Shiga toxin-producing Escherichia coli by a source attribution-considered machine learning model.

Instead of conventional serotyping and virulence gene combination methods, methods have been developed to evaluate the pathogenic potential of newly emerging pathogens. Among them, the machine learning (ML)-based method using whole-genome sequencing (WGS) data are getting attention because of the recent advances in ML algorithms and sequencing technologies. Here, we developed various ML models to predict the pathogenicity of Shiga toxin-producing Escherichia coli (STEC) isolates using their WGS data. The input dataset for the ML models was generated using distinct gene repertoires from positive (pathogenic) and negative (nonpathogenic) control groups in which each STEC isolate was designated based on the source attribution, the relative risk potential of the isolation sources. Among the various ML models examined, a model using the support vector machine (SVM) algorithm, the SVM model, discriminated between the two control groups most accurately. The SVM model successfully predicted the pathogenicity of the isolates from the major sources of STEC outbreaks, the isolates with the history of outbreaks, and the isolates that cannot be assessed by conventional methods. Furthermore, the SVM model effectively differentiated the pathogenic potentials of the isolates at a finer resolution. Permutation importance analyses of the input dataset further revealed the genes important for the estimation, proposing the genes potentially essential for the pathogenicity of STEC. Altogether, these results suggest that the SVM model is a more reliable and broadly applicable method to evaluate the pathogenic potential of STEC isolates compared with conventional methods.

Epidemiological investigation of recurrent outbreaks of haemolytic uraemic syndrome caused by Shiga toxin-producing Escherichia coli serotype O55:H7 in England, 2014-2018.

Recurrent outbreaks of haemolytic uraemic syndrome (HUS) caused by Shiga toxin-producing Escherichia coli (STEC) serotype O55:H7 occurred in England between 2014 and 2018. We reviewed the epidemiological evidence to identify potential source(s) and transmission routes of the pathogen, and to assess the on-going risk to public health. Over the 5-year period, there were 43 confirmed and three probable cases of STEC O55:H7. The median age of cases was 4 years old (range 6 months to 69 years old) and over half of all cases were female (28/46, 61%). There were 36/46 (78.3%) symptomatic cases, and over half of all cases developed HUS (25/46, 54%), including two fatal cases. No common food or environmental exposures were identified, although the majority of cases lived in rural or semi-rural environments and reported contact with both wild and domestic animals. This investigation informed policy on the clinical and public health management of HUS caused by STEC other than serotype O157:H7 (non-O157 STEC) in England, including comprehensive testing of all household contacts and household pets and more widespread use of polymerase chain reaction assays for the rapid diagnosis of STEC-HUS.

Choice of library preparation affects sequence quality, genome assembly, and precise in silico prediction of virulence genes in shiga toxin-producing Escherichia coli.

Whole genome sequencing (WGS) provides essential public health information and is used worldwide for pathogen surveillance, epidemiology, and source tracking. Foodborne pathogens are often sequenced using rapid library preparation chemistries based on transposon technology; however, this method may miss random segments of genomes that can be important for accurate downstream analyses. As new technologies become available, it may become possible to achieve better overall coverage. Here we compare the sequence quality obtained using libraries prepared from the Nextera XT and Nextera DNA Prep (Illumina, San Diego, CA) chemistries for 31 Shiga toxin-producing Escherichia coli (STEC) O121:H19 strains, which had been isolated from flour during a 2016 outbreak. The Nextera DNA Prep gave superior performance metrics including sequence quality, assembly quality, uniformity of genome coverage, and virulence gene identification, among other metrics. Comprehensive detection of virulence genes is essential for making educated assessments of STECs virulence potential. The phylogenetic SNP analysis did not show any differences in the variants detected by either library preparation method which allows isolates prepared from either library method to be analysed together. Our comprehensive comparison of these chemistries should assist researchers wishing to improve their sequencing workflow for STECs and other genomic risk assessments.

Shiga Toxin (Stx)-Binding Glycosphingolipids of Primary Human Renal Cortical Epithelial Cells (pHRCEpiCs) and Stx-Mediated Cytotoxicity.

Human kidney epithelial cells are supposed to be directly involved in the pathogenesis of the hemolytic-uremic syndrome (HUS) caused by Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC). The characterization of the major and minor Stx-binding glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), respectively, of primary human renal cortical epithelial cells (pHRCEpiCs) revealed GSLs with Cer (d18:1, C16:0), Cer (d18:1, C22:0), and Cer (d18:1, C24:1/C24:0) as the dominant lipoforms. Using detergent-resistant membranes (DRMs) and non-DRMs, Gb3Cer and Gb4Cer prevailed in the DRM fractions, suggesting their association with microdomains in the liquid-ordered membrane phase. A preference of Gb3Cer and Gb4Cer endowed with C24:0 fatty acid accompanied by minor monounsaturated C24:1-harboring counterparts was observed in DRMs, whereas the C24:1 fatty acid increased in relation to the saturated equivalents in non-DRMs. A shift of the dominant phospholipid phosphatidylcholine with saturated fatty acids in the DRM to unsaturated species in the non-DRM fractions correlated with the GSL distribution. Cytotoxicity assays gave a moderate susceptibility of pHRCEpiCs to the Stx1a and Stx2a subtypes when compared to highly sensitive Vero-B4 cells. The results indicate that presence of Stx-binding GSLs per se and preferred occurrence in microdomains do not necessarily lead to a high cellular susceptibility towards Stx.

Shiga Toxin-Producing E. coli in Animals: Detection, Characterization, and Virulence Assessment.

Cattle and other ruminants are primary reservoirs for Shiga toxin-producing Escherichia coli (STEC) strains which have a highly variable, but unpredictable, pathogenic potential for humans. Domestic swine can carry and shed STEC, but only STEC strains producing the Shiga toxin (Stx) 2e variant and causing edema disease in piglets are considered pathogens of veterinary medical interest. In this chapter, we present general diagnostic workflows for sampling livestock animals to assess STEC prevalence, magnitude, and duration of host colonization. This is followed by detailed method protocols for STEC detection and typing at genetic and phenotypic levels to assess the relative virulence exerted by the strains.

Use of the Dynamic TIM-1 Model for an In-Depth Understanding of the Survival and Virulence Gene Expression of Shiga Toxin-Producing Escherichia coli in the Human Stomach and Small Intestine.

Due to obvious ethical and technical reasons, it remains very difficult to evaluate the survival and expression of virulence genes of food-borne pathogens, such as Shiga toxin-producing Escherichia coli (STEC) in the human gastrointestinal tract. Here, we describe the use of the dynamic TNO (Toegepast Natuurwetenschappelijk Onderzoek) gastrointestinal model (TIM-1) as a powerful in vitro tool to obtain the kinetics of STEC survival by plate counting, the regulation of major virulence genes by RT-qPCR, and the production of Shiga toxins by ELISA, in the human stomach and small intestine. The gut model was adapted in order that in vitro digestions were performed both under adult and child digestive conditions, specific at risk populations for STEC infections.

Measuring Effector-Mediated Modulation of Inflammatory Responses to Infection with Enteropathogenic and Shiga Toxin-Producing E. coli.

Shiga toxin-producing Escherichia coli (STEC) and the related pathogen enteropathogenic Escherichia coli (EPEC) use a type III secretion system to translocate effector proteins into host cells to modulate inflammatory signaling pathways during infection. Here we describe the procedures to investigate effector-driven modulation of host inflammatory signaling pathways in mammalian cells where bacterial effectors are ectopically expressed or in cell lines infected with STEC or EPEC. We focus on the TNF-induced NF-κB response by examining IκBα degradation by immunoblot and p65 nuclear localization in addition to using an NF-κB-dependent luciferase reporter and cytokine secretion assays. These methods can be adapted for examining effector-mediated modulation of other inflammatory stimuli and host signaling pathways.

Interaction of Bovine Lymphocytes with Products of Shiga Toxin-Producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) produce a number of virulence factors that interfere with lymphocyte functions, including mitogen- and antigen-activated proliferation and pro-inflammatory cytokine synthesis. Here we describe how to isolate lymphocyte subsets from bovine peripheral blood as well as methods that we have used to study the effects of STEC products on lymphocyte proliferation and cytokine production. We also describe an assay that allows for the detection of association of a given protein with lymphocytes.