Genomic analysis of an outbreak of Shiga toxin-producing Escherichia coli O183:H18 in the United Kingdom, 2023.

In June 2023, UKHSA surveillance systems detected an outbreak of severe gastrointestinal symptoms caused by a rare serotype of Shiga toxin-producing Escherichia coli, STEC O183:H18. There were 26 cases aged 6 months to 74 years (42 % cases were aged 0-9 years), distributed across the UK with onset dates range between 22 May 2023 and 4 July 2023. The epidemiological and food chain investigations were inconclusive, although meat products made from beef mince were implicated as a potential vehicle. The outbreak strain belonged to sequence type (ST) 657 and harboured a Shiga toxin (stx) subtype stx2a located on a prophage that was unique in the UKHSA stx-encoding bacteriophage database. Plasmid encoded, putative virulence genes subA, ehxA, saa, iha, lpfA and iss were detected, however, the established STEC virulence genes involved in attachment to the gut mucosa (eae and aggR) were absent. The acquisition of stx across the global population structure of ST657 appeared to correspond with the presence of subA, ehxA, saa, iha, lpfA and iss. During the outbreak investigation, we used long read sequencing to characterise the plasmid and prophage content of this atypical STEC, to look for evidence to explain its recent emergence. Although we were unable to determine source and transmission route of the outbreak strain, the genomic analysis revealed potential clues as to how novel strains for STEC evolve. With the implementation of PCR capable of detecting all STEC, and genome sequencing for typing and virulence profiling, we have the tools to enable us to monitor the changing landscape of STEC. Improvements in the standardised collection of epidemiological data and trace-back strategies within the food industry, will ensure we have a surveillance system capable of alerting us to emerging threats to public health.

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.

Survival of O157 and non-O157 shiga toxin-producing Escherichia coli in Korean style kimchi.

Korean style kimchi contaminated with Shiga toxin-producing Escherichia coli (STEC) O157:H7 was the cause of an outbreak in Canada from December 2021 to January 2022. To determine if this STEC O157:H7 has greater potential for survival in kimchi than other STEC, the outbreak strain and six other STEC strains (O26:H11, O91:H21, O103:H2, O121:H19, and two O157:H7) were inoculated individually at 6 to 6.5 log CFU/g into commercially sourced kimchi and incubation at 4 °C. At intervals of seven days inoculated and control kimchi was plated onto MacConkey agar to enumerate lactose utilising bacteria. The colony counts were interpreted as enumerating the inoculated STEC, since no colonies were observed on MacConkey agar plated with uninoculated kimchi. Over eight weeks of incubation the pH was stable at 4.10 to 4.05 and the STEC strains declined by 0.7-1.0 log, with a median reduction of 0.9 log. The linear rate of reduction of kimchi outbreak STEC O157:H7 was -0.4 log per 30 days (Slope Uncertainty 0.05), which was not significantly different from the other O157 and nonO157 STEC strains (P = 0.091). These results indicate that the outbreak was not due to the presence of strain better adapted to survival in kimchi than other STEC, and that STEC can persist in refrigerated Korean style kimchi with a minimal decline over the shelf-life of the product.

Prevalence and antibiogram of shigatoxinproducing E. coli in camel meat and offal.

Background: Camels are important animals in Egypt and other Arab countries on the basis of their economic value and ethnic culture. Escherichia coli is implicated in several gastrointestinal infections and outbreaks worldwide, especially in developing countries. It causes infections that might lead to death. Numerous biological activities, such as antioxidative, antibacterial, anti-diabetic, anti-mutagenic, anti-inflammatory, neuroprotective, and diuretic, are associated with coriander and coriander essential oils. Aim: This work targeted investigation of the prevalence, antibiogram, and occurrence of virulence genes of E. coli in camel meat liver and kidney. Besides, the anti-E. coli activity of coriander oil was further examined. Methods: Camel meat, liver, and kidneys were collected from local markets in Egypt. Isolation and identification of E. coli were performed. The antimicrobial susceptibility of the obtained E. coli isolates was screened using the disk diffusion assay. To detect the presence of virulence-associated genes (stx1, stx2, eaeA, and hylA gens), polymerase chain reaction was used. An experimental trial was done to investigate the anti-E. coli activity of coriander oil. Results: The obtained results revealed isolation of the following E. coli pathotypes: O17:H18, O128:H2, O119:H6, O103:H4, O145:H-, and O121:H7. The recovered E. coli isolates practiced multidrug resistance profiling with higher resistance toward Erythromycin, Nalidixic Acid, Clindamycin, and Ampicillin. However, the isolates were sensitive to Meropenem and cefoxitin. The recovered isolates had expressed different virulence attributes. Coriander oil of 2% could significantly reduce E. coli O128 count in camel meat by 65%. Conclusion: Therefore, strict hygienic measures are highly recommended during the processing of camel meat. The use of coriander oil during camel meat processing is highly recommended to reduce E. coli count.

Using CHROMagar™ STEC medium exclusively does not recover all clinically relevant Shiga toxin-producing Escherichia coli in Aotearoa, New Zealand.

Diagnostic laboratories in Aotearoa, New Zealand (NZ) refer cultures from faecal samples positive for Shiga toxin genes to the national Enteric Reference Laboratory for isolation of Shiga toxin-producing Escherichia coli (STEC) for epidemiological typing. As there was variation in the culture media being referred, a panel of 75 clinical isolates of STEC, representing 28 different serotypes, was used to assess six commercially available media and provide guidance to clinical laboratories. Recommendations were subsequently tested for a 3-month period, where STEC isolations and confirmations were assessed by whole genome sequencing analysis against the culture media referred. CHROMagar™ STEC (CH-STEC; CHROMagar Microbiology, Paris, France) or CH-STEC plus cefixime-tellurite sorbitol MacConkey agar was confirmed inferior to CH-STEC plus blood agar with vancomycin, cefsulodin, and cefixime (BVCC). The former resulted in fewer STEC types (n = 18) being confirmed compared to those from a combination of CH-STEC and BVCC (n = 42). A significant (P < .05) association with an STEC's ability to grow on CH-STEC and the presence of the ter gene cluster, and eae was observed. Culturing screen positive STEC samples onto both CH-STEC and BVCC ensures a consistently higher recovery of STEC from all clinical samples in NZ than CH-STEC alone.

Determining the prevalence of Escherichia coli, Salmonella, and shiga toxin-producing Escherichia coli in manure of dairy lagoons.

AIM: The aim of this study was to determine the prevalence of microbial pathogens in manure of dairy lagoons in California. METHODS AND RESULTS: To determine pathogens in dairy manure stored in anaerobic lagoons of dairy farm, an extensive field study was conducted across California to sample manure from 20 dairy farms. Samples were analyzed to determine the prevalence of indicator Escherichia coli, Shiga toxin producing E. coli (STEC), Salmonella, and E. coli O157: H7. To test the E. coli, STEC, and Salmonella, we used agar culture-based method followed by polymerase chain reaction (PCR) method. In addition, a real- time PCR based method was used to determine the presence of E coli O157: H7. Study demonstrated that the prevalence of Salmonella in manure sample is lower than E. coli. The presence of Salmonella was found in 2.26% of the samples, and both the culture-based and PCR methods yielded comparable outcomes in detecting Salmonella. Moreover, ∼11.30% of the total samples out of the 177 were identified as positive for STEC by qPCR. CONCLUSION: These findings demonstrate that indicator E. coli are abundantly present in anaerobic lagoons. However, the presence of STEC, and Salmonella is substantially low.

Shiga toxin-producing Escherichia coli isolated from hunted wild boar (Sus scrofa) in Switzerland.

INTRODUCTION: Shiga toxin-producing Escherichia (E.) coli (STEC) are zoonotic foodborne pathogens of significant public health importance. While ruminants are considered the main reservoir, wild animals are increasingly acknowledged as carriers and potential reservoirs of STEC. The aim of this study was to determine the occurrence of STEC in a total of 59 faecal samples of hunted wild boars (Sus scrofa) from two different regions in Switzerland (canton Thurgau in northern Switzerland and canton Ticino in southern Switzerland), and to characterise the isolates using a whole genome sequencing approach. After an enrichment step, Shiga-toxin encoding genes (stx) were detected by real-time PCR in 41 % (95 % confidence interval (95 %CI) 0,29 - 0,53) of the samples, and STEC were subsequently recovered from 22 % (95 %CI 0,13 - 0,34) of the same samples. Seven different serotypes and six different sequence types (STs) were found, with O146:H28 ST738 (n = 4) and O100:H20 ST2514 (n = 4) predominating. Subtyping of stx identified isolates with stx1c/stx2b (n = 1), stx2a (n = 1), stx2b (n = 6), and stx2e (n = 6). No isolate contained the eae gene, but all harboured additional virulence genes, most commonly astA (n = 10), hlyE (n = 9), and hra (n = 9). STEC O11:H5, O21:H21, and O146:H28 harboured virulence factors associated with extra-intestinal pathogenic E. coli (ExPEC), and STEC O100:H20 and O155:H26 possessed sta1 and/or stb and were STEC/enterotoxigenic E. coli (ETEC) hybrid pathotypes. Our results show that wild boars are carriers of STEC which may be distributed in the environment, possibly leading to the contamination of agricultural crops and water sources. The serogroups included STEC O146 which belongs to the most common non-O157 serogroups associated with human illness in Europe, with implications for public health. Since Stx2e-producing STEC have frequently been reported in swine and pork, STEC O100:H20 harbouring stx2e in faeces of wild boars may be relevant to free-range systems of pig farming because of the potential risk of transmission events at the wildlife-livestock interface.

INTRODUCTION: Les Escherichia (E.) coli producteurs de shiga-toxine (STEC) sont des agents pathogènes zoonotiques d'origine alimentaire qui revêtent une grande importance pour la santé publique. Alors que les ruminants sont considérés comme le principal réservoir, les animaux sauvages sont de plus en plus souvent reconnus comme porteurs et réservoirs potentiels de STEC. L'objectif de cette étude était de déterminer la présence de STEC dans un total de 59 échantillons fécaux de sangliers (Sus scrofa) chassés provenant de deux régions différentes de Suisse (canton de Thurgovie dans le nord de la Suisse et canton du Tessin dans le sud de la Suisse) et de caractériser les isolats en utilisant une approche de séquençage du génome entier. Après une étape d'enrichissement, les gènes codant pour la Shiga-toxine (stx) ont été détectés par PCR en temps réel dans 41% (intervalle de confiance à 95% (95%CI) 0,29 - 0,53) des échantillons, et les STEC ont ensuite été récupérés dans 22% (95%CI 0,13 - 0,34) des mêmes échantillons. Sept sérotypes différents et six types de séquence (ST) différents ont été trouvés, avec une prédominance de O146:H28 ST738 (n = 4) et O100:H20 ST2514 (n = 4). Le sous-typage des stx a permis d'identifier des isolats avec stx1c/stx2b (n = 1), stx2a (n = 1), stx2b (n = 6) et stx2e (n = 6). Aucun isolat ne contenait le gène eae, mais tous hébergeaient d'autres gènes de virulence, le plus souvent astA (n = 10), hlyE (n = 9) et hra (n = 9). Les STEC O11:H5, O21:H21 et O146:H28 présentaient des facteurs de virulence associés à des E. coli pathogènes extra-intestinaux (ExPEC), et les STEC O100:H20 et O155:H26 possédaient sta1 et/ou stb et étaient des pathotypes hybrides STEC/E. coli entérotoxinogène (ETEC).

Molecular characterization of enteropathogenic Escherichia coli isolated from patients with gastroenteritis in a tertiary referral hospital of northeast India.

PURPOSE: Diarrhoeal illness accounts for a high morbidity and mortality both in paediatric as well as adult groups and diarrhoeagenic Escherichia coli occupies a top position as a causative agent of infectious diarrhoeal illness worldwide. The aim of the current investigation was to determine the virulence and pattern of antibiotic resistance of enteropathogenic, enterotoxigenic, and shiga toxigenic Escherichia coli that are linked to diarrhoea in patients of both adult and paediatric age groups. METHODS: A total of 50 consecutive, nonduplicate Escherichia coli isolates were collected from patients with gastro-enteritis who were admitted to different clinical wards Silchar Medical College and Hospital, Silchar, India. PCR was used to identify the virulence genes of EPEC (eaeA and bfpA), STEC (stx1, stx2, and eae) and ETEC (eltA, eltB, estA1 and estA2) in the isolates of E. coli. The antibiotic susceptibility pattern of virulent E. coli isolates were checked using disc diffusion method. Molecular typing of the virulent E. coli detected in the study based on enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) was also done. RESULT: Out of 50 E. coli isolates, 13 (26%) were found to carry atleast one virulence gene. 11 isolates harboured eae gene and were characterized as EPEC and two isolates carried stx1 gene of STEC. These virulent isolates showed different antibiotic susceptibility pattern and harboured single or multiple antibiotic resistance genes. ERIC PCR established 12 different clonal patterns of the virulent study isolates of E. coli harbouring. CONCLUSION: EPEC pathotypes were found to be the most detected pathotype in the stool samples. Majority of the virulent isolates were also resistant to multiple antibiotics which is a serious public health concern and therefore requires a proper surveillance and studies to track their reservoirs to contain their spread.

A 6-year retrospective study of clinical features, microbiology, and genomics of Shiga toxin-producing Escherichia coli in children who presented to a tertiary referral hospital in Costa Rica.

Shiga-toxin-producing Escherichia coli (STEC) is associated with diarrhea and hemolytic uremic syndrome (HUS). STEC infections in Costa Rica are rarely reported in children. We gathered all the records of STEC infections in children documented at the National Children's Hospital, a tertiary referral hospital, from 2015 to 2020. Clinical, microbiological, and genomic information were analyzed and summarized. A total of 3,768 diarrheal episodes were reviewed. Among them, 31 STEC were characterized (29 fecal, 1 urine, and 1 bloodstream infection). The prevalence of diarrheal disease due to STEC was estimated at 0.8% (n = 29/3,768), and HUS development was 6.4% (n = 2/31). The stx1 gene was found in 77% (n = 24/31) of STEC strains. In silico genomic predictions revealed a predominant prevalence of serotype O118/O152:H2, accompanied by a cluster exhibiting allele differences ranging from 33 to 8, using a core-genome multilocus sequence typing (cgMLST) approach. This is the first study using a genomic approach for STEC infections in Costa Rica.IMPORTANCEThis study provides a comprehensive description of clinical, microbiological, genomic, and demographic data from patients who attended the only pediatric hospital in Costa Rica with Shiga-toxin-producing Escherichia coli (STEC) infections. Despite the low prevalence of STEC infections, we found a predominant serotype O118/O152:H2, highlighting the pivotal role of genomics in understanding the epidemiology of public health threats such as STEC. Employing a genomic approach for this pathogen for the first time in Costa Rica, we identified a higher prevalence of STEC in children under 2 years old, especially those with gastrointestinal comorbidities, residing in densely populated regions. Limitations such as potential geographic bias and lack of strains due to direct molecular diagnostics are acknowledged, emphasizing the need for continued surveillance to uncover the true extent of circulating serotypes and potential outbreaks in Costa Rica.

Prevalence of Salmonella spp. and Escherichia coli in the feces of free-roaming wildlife throughout South Korea.

Wildlife can carry pathogenic organisms, including viruses, bacteria, parasites, and fungi, which can spread to humans and cause mild to serious illnesses and even death. Spreading through animal feces, these pathogens significantly contributes to the global burden of human diseases. Therefore, the present study investigated the prevalence of zoonotic bacterial pathogens, such as Salmonella spp., Escherichia coli, and Shiga toxin-producing E. coli (STEC), in animal feces. Between September 2015 and August 2017, 699 wildlife fecal samples were collected from various agricultural production regions and mountainous areas in South Korea. Fecal samples were collected from wild mammals (85.26%, 596/699) and birds (14.73%, 103/699). Salmonella spp. and E. coli were present in 3% (21/699) and 45.63% (319/699) of the samples, respectively. Moreover, virulence genes stx1 and both stx1 and stx2 were detected in 13.30% (93/699) and 0.72% (5/699) of the samples, respectively. The 21 Salmonella spp. were detected in badgers (n = 5), leopard cats (n = 7), wild boars (n = 2), and magpies (n = 7); STEC was detected in roe deer, water deer, mice, and wild boars. Through phylogenetic and gene-network analyses, the Salmonella spp. isolates (n = 21 laboratory isolates, at least one isolate from each Salmonella-positive animal fecal sample, and n = 6 widely prevalent reference Salmonella serovars) were grouped into two major lineages: S. enterica subsp. enterica and S. enterica subsp. diarizonae. Similarly, 93 E. coli isolates belonged to stx1, including three major lineages (groups 1-3), and stx1 and stx2 detected groups. To the best of our knowledge, this is the first report of a wild leopard cat serving as a reservoir for Salmonella spp. in South Korea. The research findings can help manage the potential risk of wildlife contamination and improve precautionary measures to protect public health.

Escherichia coli and their potential transmission of carbapenem and colistin-resistant genes in camels.

BACKGROUND: Camels harbouring multidrug-resistant Gram-negative bacteria are capable of transmitting various microorganisms to humans. This study aimed to determine the distribution of anti-microbial resistance among Escherichia coli (E. coli) isolated from the feces of apparently healthy camels in Egyptian abattoirs. Additionally, we sought to characterize Shiga toxin-producing E. coli (STEC) strains, assess their virulence potential, and investigate the possibility of camels spreading carbapenem- and colistin-resistant E. coli. METHODS: 121 fecal swaps were collected from camels in different abattoirs in Egypt. Isolation and identification of E. coli were performed using conventional culture techniques and biochemical identification. All isolates obtained from the examined samples underwent genotyping through polymerase chain reaction (PCR) of the Shiga toxin-encoding genes (Stx1 and Stx2), the carbapenemase-encoding genes (blaKPC, blaOXA-48, blaNDM, and blaVIM), and the mcr genes for mcr-1 to mcr-5. RESULT: Bacteriological examination revealed 75 E. coli isolates. PCR results revealed that one strain (1.3%) tested positive for Stx1, and five (6.6%) were positive for Stx2. Among the total 75 strains of E. coli, the overall prevalence of carbapenemase-producing E. coli was 27, with 7 carrying blaOXA48, 14 carrying blaNDM, and 6 carrying blaVIM. Notably, no strains were positive for blaKPC but a high prevalence rate of mcr genes were detected. mcr-1, mcr-2, mcr-3, and mcr-4 genes were detected among 3, 2, 21, and 3 strains, respectively. CONCLUSION: The results indicate that camels in Egypt may be a primary source of anti-microbial resistance (AMR) E. coli, which could potentially be transmitted directly to humans or through the food chain.

Gastrointestinal involvement in STEC-associated hemolytic uremic syndrome: 10 years in a pediatric center.

BACKGROUND: The gastrointestinal (GI) tract represents one of the main targets of typical hemolytic uremic syndrome (HUS) in children. In this observational study, we tried to establish (1) the main features of GI complications during STEC-HUS and (2) the relationship between Escherichia coli serotypes and Shiga toxin (Stx) variants with hepatopancreatic involvement. METHODS: A total of 79 STEC-HUS patients were admitted to our pediatric nephrology department between January 2012 and June 2021. Evidence of intestinal, hepatobiliary, and pancreatic involvements was reported for each patient, alongside demographic, clinical, and laboratory features. Frequency of gastrointestinal complications across groups of patients infected by specific E. coli serotypes and Stx gene variants was evaluated. RESULTS: Six patients developed a bowel complication: two developed rectal prolapse, and four developed bowel perforation which resulted in death for three of them and in bowel stenosis in one patient. Acute pancreatitis was diagnosed in 13 patients. An isolated increase in pancreatic enzymes and/or liver transaminases was observed in 41 and 15 patients, respectively. Biliary sludge was detected in three, cholelithiasis in one. Forty-seven patients developed direct hyperbilirubinemia. Neither E. coli serotypes nor Shiga toxin variants correlated with hepatic or pancreatic involvement. CONCLUSIONS: During STEC-HUS, GI complications are common, ranging from self-limited elevation of laboratory markers to bowel perforation, a severe complication with a relevant impact on morbidity and mortality. Hepatopancreatic involvement is frequent, but usually short-lasting and self-limiting.

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.

RNA-Seq analysis of long non-coding RNA in human intestinal epithelial cells infected by Shiga toxin-producing Escherichia coli.

BACKGROUND: The Shiga toxin-producing Escherichia coli (STEC) infects animals and induces acute intestinal inflammation. Long non-coding RNAs (lncRNAs) are known to play crucial roles in modulating inflammation response. However, it is not clear whether lncRNAs are involved in STEC-induced inflammation. METHODS AND RESULTS: To understand the association of lncRNAs with STEC infection, we used RNA-seq technology to analyze the profiles of lncRNAs in Mock-infected and STEC-infected human intestinal epithelial cells (HIECs). We detected a total of 702 lncRNAs differentially expressed by STEC infection. 583 differentially expressed lncRNAs acted as competitive microRNAs (miRNAs) binding elements in regulating the gene expression involved in TNF signaling pathway, IL-17 signaling pathway, PI3K-Akt signaling pathway, and apoptosis pathways. We analyzed 3 targeted genes, TRADD, TRAF1 and TGFB2, which were differentially regulated by mRNA-miRNA-lncRNA interaction network, potentially involved in the inflammatory and apoptotic response to STEC infection. Functional analysis of up/downstream genes associated with differentially expressed lncRNAs revealed their role in adheres junction and endocytosis. We also used the qRT-PCR technique to validate 8 randomly selected differentially expressed lncRNAs and mRNAs in STEC-infected HIECs. CONCLUSION: Our results, for the first time, revealed differentially expressed lncRNAs induced by STEC infection of HIECs. The results will help investigate the molecular mechanisms for the inflammatory responses induced by STEC.

Unusual modifications of protein biomarkers expressed by plasmid, prophage, and bacterial host of pathogenic Escherichia coli identified using top-down proteomic analysis.

RATIONALE: Pathogenic bacteria often carry prophage (bacterial viruses) and plasmids (small circular pieces of DNA) that may harbor toxin, antibacterial, and antibiotic resistance genes. Proteomic characterization of pathogenic bacteria should include the identification of host proteins and proteins produced by prophage and plasmid genomes. METHODS: Protein biomarkers of two strains of Shiga toxin-producing Escherichia coli (STEC) were identified using antibiotic induction, matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF-TOF) tandem mass spectrometry (MS/MS) with post-source decay (PSD), top-down proteomic (TDP) analysis, and plasmid sequencing. Alphafold2 was also used to compare predicted in silico structures of the identified proteins to prominent fragment ions generated using MS/MS-PSD. Strain samples were also analyzed with and without chemical reduction treatment to detect the attachment of pendant groups bound by thioester or disulfide bonds. RESULTS: Shiga toxin was detected and/or identified in both STEC strains. For the first time, we also identified the osmotically inducible protein (OsmY) whose sequence unexpectedly had two forms: a full and a truncated sequence. The truncated OsmY terminates in the middle of an α-helix as determined by Alphafold2. A plasmid-encoded colicin immunity protein was also identified with and without attachment of an unidentified cysteine-bound pendant group (~307 Da). Plasmid sequencing confirmed top-down analysis and the identification of a promoter upstream of the immunity gene that is activated by antibiotic induction, that is, SOS box. CONCLUSIONS: TDP analysis, coupled with other techniques (e.g., antibiotic induction, chemical reduction, plasmid sequencing, and in silico protein modeling), is a powerful tool to identify proteins (and their modifications), including prophage- and plasmid-encoded proteins, produced by pathogenic microorganisms.

Shiga Toxin‒Producing Escherichia coli Diagnoses from Health Practitioners, Queensland, Australia.

In Queensland, Australia, 31 of 96 Shiga toxin‒producing Escherichia coli cases during 2020-2022 were reported by a specialty pathology laboratory servicing alternative health practitioners. Those new cases were more likely to be asymptomatic or paucisymptomatic, prompting a review of the standard public health response.

Study of the transfer of Shiga toxin-producing Escherichia coli during the slaughter of cattle using molecular typing combined with epidemiologic data.

Investigation on the distribution and biological characteristics of Shiga-toxin producing Escherichia coli (STEC) during beef processing is essential for in-plant critical control points and food safety risk assessment. Serogroups and subtypes of stx genes of STEC strains isolated from beef processing lines were first investigated. Identification to cross-contamination among different sampling sites was further conducted by combining multilocus sequence typing (MLST) with the previous distribution and characterization data. The PCR-positive rate for STEC in 435 samples from two slaughter plants in China was 14.3% and the isolation rate for the 62 PCR positive and the entire set of 435 samples were 26% and 3.68% respectively. The existence of serotype O157:H7 (33%) and serogroups O121 (42%) and O26 (21%) as well as the high detection rate of high pathogenic gene stx2a (68%) in these serogroups indicated potential risk to the safety of beef. Traceability analysis showed that hide plays a critical role in cross-contamination between feces, lairage pens and post-washing carcasses from a molecular perspective. Intervening measures revolves around de-hiding should be involved in the in-plant safety control policy according to the tracing analysis.

Genetic Characterization of Intimin Gene (eae) in Clinical Shiga Toxin-Producing Escherichia coli Strains from Pediatric Patients in Finland.

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections cause outbreaks of severe disease in children ranging from bloody diarrhea to hemolytic uremic syndrome (HUS). The adherent factor intimin, encoded by eae, can facilitate the colonization process of strains and is frequently associated with severe disease. The purpose of this study was to examine and analyze the prevalence and polymorphisms of eae in clinical STEC strains from pediatric patients under 17 years old with and without HUS, and to assess the pathogenic risk of different eae subtypes. We studied 240 STEC strains isolated from pediatric patients in Finland with whole genome sequencing. The gene eae was present in 209 (87.1%) strains, among which 49 (23.4%) were from patients with HUS, and 160 (76.6%) were from patients without HUS. O157:H7 (126, 60.3%) was the most predominant serotype among eae-positive STEC strains. Twenty-three different eae genotypes were identified, which were categorized into five eae subtypes, i.e., γ1, ß3, ε1, θ and ζ3. The subtype eae-γ1 was significantly overrepresented in strains from patients aged 5-17 years, while ß3 and ε1 were more commonly found in strains from patients under 5 years. All O157:H7 strains carried eae-γ1; among non-O157 strains, strains of each serotype harbored one eae subtype. No association was observed between the presence of eae/its subtypes and HUS. However, the combination of eae-γ1+stx2a was significantly associated with HUS. In conclusion, this study demonstrated a high occurrence and genetic variety of eae in clinical STEC from pediatric patients under 17 years old in Finland, and that eae is not essential for STEC-associated HUS. However, the combination of certain eae subtypes with stx subtypes, i.e., eae-γ1+stx2a, may be used as risk predictors for the development of severe disease in children.

Oral Immunization with Attenuated Salmonella Choleraesuis Expressing the FedF Antigens Protects Mice against the Shiga-Toxin-Producing Escherichia coli Challenge.

Edema disease (ED) is a severe and lethal infectious ailment in swine, stemming from Shiga-toxin-producing Escherichia coli (STEC). An efficient, user-friendly, and safe vaccine against ED is urgently required to improve animal welfare and decrease antibiotic consumption. Recombinant attenuated Salmonella vaccines (RASV) administered orally induce both humoral and mucosal immune responses to the immunizing antigen. Their potential for inducing protective immunity against ED is significant through the delivery of STEC antigens. rSC0016 represents an enhanced recombinant attenuated vaccine vector designed for Salmonella enterica serotype Choleraesuis. It combines sopB mutations with a regulated delay system to strike a well-balanced equilibrium between host safety and immunogenicity. We generated recombinant vaccine strains, namely rSC0016 (pS-FedF) and rSC0016 (pS-rStx2eA), and assessed their safety and immunogenicity in vivo. The findings demonstrated that the mouse models immunized with rSC0016 (pS-FedF) and rSC0016 (pS-rStx2eA) generated substantial IgG antibody responses to FedF and rStx2eA, while also provoking robust mucosal and cellular immune responses against both FedF and rStx2eA. The protective impact of rSC0016 (pS-FedF) against Shiga-toxin-producing Escherichia coli surpassed that of rSC0016 (pS-rStx2eA), with percentages of 83.3%. These findings underscore that FedF has greater suitability for vaccine delivery via recombinant attenuated Salmonella vaccines (RASVs). Overall, this study provides a promising candidate vaccine for infection with STEC.