O26 Polysaccharides as Key Players in Enteropathogenic E. coli Immune Evasion and Vaccine Development.

Enteropathogenic Escherichia coli (EPEC) produce a capsule of polysaccharides identical to those composing the O-antigen polysaccharide of its LPS (lipopolysaccharide) molecules. In light of this, the impact of O26 polysaccharides on the immune evasion mechanisms of capsulated O26 EPEC compared to non-capsulated enterohemorrhagic Escherichia coli (EHEC) was investigated. Our findings reveal that there was no significant difference between the levels in EPEC and EHEC of rhamnose (2.8:2.5), a molecule considered to be a PAMP (Pathogen Associated Molecular Patterns). However, the levels of glucose (10:1.69), heptose (3.6:0.89) and N-acetylglucosamine (4.5:2.10), were significantly higher in EPEC than EHEC, respectively. It was also observed that the presence of a capsule in EPEC inhibited the deposition of C3b on the bacterial surface and protected the pathogen against lysis by the complement system. In addition, the presence of a capsule also protected EPEC against phagocytosis by macrophages. However, the immune evasion provided by the capsule was overcome in the presence of anti-O26 polysaccharide antibodies, and additionally, these antibodies were able to inhibit O26 EPEC adhesion to human epithelial cells. Finally, the results indicate that O26 polysaccharides can generate an effective humoral immune response, making them promising antigens for the development of a vaccine against capsulated O26 E. coli.

O26 Polysaccharides as key players in enteropathogenic E. coli immune evasion and vaccine development

Enteropathogenic Escherichia coli (EPEC) produce a capsule of polysaccharides identical to those composing the O-antigen polysaccharide of its LPS (lipopolysaccharide) molecules. In light of this, the impact of O26 polysaccharides on the immune evasion mechanisms of capsulated O26 EPEC compared to non-capsulated enterohemorrhagic Escherichia coli (EHEC) was investigated. Our findings reveal that there was no significant difference between the levels in EPEC and EHEC of rhamnose (2.8:2.5), a molecule considered to be a PAMP (Pathogen Associated Molecular Patterns). However, the levels of glucose (10:1.69), heptose (3.6:0.89) and N-acetylglucosamine (4.5:2.10), were significantly higher in EPEC than EHEC, respectively. It was also observed that the presence of a capsule in EPEC inhibited the deposition of C3b on the bacterial surface and protected the pathogen against lysis by the complement system. In addition, the presence of a capsule also protected EPEC against phagocytosis by macrophages. However, the immune evasion provided by the capsule was overcome in the presence of anti-O26 polysaccharide antibodies, and additionally, these antibodies were able to inhibit O26 EPEC adhesion to human epithelial cells. Finally, the results indicate that O26 polysaccharides can generate an effective humoral immune response, making them promising antigens for the development of a vaccine against capsulated O26 E. coli.

Phylogenetic relationship and virulence composition of Escherichia coli O26:H11 cattle and human strain collections in Scotland; 2002-2020.

O26 is the commonest non-O157 Shiga toxin (stx)-producing Escherichia coli serogroup reported in human infections worldwide. Ruminants, particularly cattle, are the primary reservoir source for human infection. In this study, we compared the whole genomes and virulence profiles of O26:H11 strains (n = 99) isolated from Scottish cattle with strains from human infections (n = 96) held by the Scottish Escherichia coli O157/STEC Reference Laboratory, isolated between 2002 and 2020. Bovine strains were from two national cross-sectional cattle surveys conducted between 2002-2004 and 2014-2015. A maximum likelihood phylogeny was constructed from a core-genome alignment with the O26:H11 strain 11368 reference genome. Genomes were screened against a panel of 2,710 virulence genes using the Virulence Finder Database. All stx-positive bovine O26:H11 strains belonged to the ST21 lineage and were grouped into three main clades. Bovine and human source strains were interspersed, and the stx subtype was relatively clade-specific. Highly pathogenic stx2a-only ST21 strains were identified in two herds sampled in the second cattle survey and in human clinical infections from 2010 onwards. The closest pairwise distance was 9 single-nucleotide polymorphisms (SNPs) between Scottish bovine and human strains and 69 SNPs between the two cattle surveys. Bovine O26:H11 was compared to public EnteroBase ST29 complex genomes and found to have the greatest commonality with O26:H11 strains from the rest of the UK, followed by France, Italy, and Belgium. Virulence profiles of stx-positive bovine and human strains were similar but more conserved for the stx2a subtype. O26:H11 stx-negative ST29 (n = 17) and ST396 strains (n = 5) were isolated from 19 cattle herds; all were eae-positive, and 10 of these herds yielded strains positive for ehxA, espK, and Z2098, gene markers suggestive of enterohaemorrhagic potential. There was a significant association (p < 0.001) between nucleotide sequence percent identity and stx status for the bacteriophage insertion site genes yecE for stx2 and yehV for stx1. Acquired antimicrobial resistance genes were identified in silico in 12.1% of bovine and 17.7% of human O26:H11 strains, with sul2, tet, aph(3″), and aph(6″) being most common. This study describes the diversity among Scottish bovine O26:H11 strains and investigates their relationship to human STEC infections.

State of the Art Progress in Copper Vanadate Materials for Solar Water Splitting.

The development of a single junction photoelectrode material having specific properties is essential and challenging for the efficient application in solar water splitting for oxygen production and a high value-added product, hydrogen. Moreover, the present material solutions based on binary metal oxides offer limited catalytic activity and hydrogen production efficiency. Therefore, it is paramount to develop and exploit a unique range of materials derived from ternary metal oxides with specifically engineered properties to advance in photoelectrochemical (PEC) water splitting. Among the ternary oxides, copper vanadates offer promising characteristics, such as a narrow bandgap and catalytic surface properties along with favorable band edges for facile oxygen evolution reaction (OER), which is considered the bottleneck step in performing overall water dissociation. Furthermore, the copper vanadates allow the tuning of the stoichiometry through which a wide range of polymorphs and materials could be obtained. This review provides a complete outlook on the range of copper vanadates and the established synthesis approach, morphology, crystal structure, band edge properties, and PEC characterizations. Mainly, the underlying charge dynamic properties, carrier path length, effect of doping, and influence of surface catalysts are discussed. The review concludes that the advancement toward obtaining low-bandgap materials is a main challenge to overcome the limitations for efficient water dissociation to OER and copper vanadates, which offer a promising solution with their unique properties and advantages. Importantly, intense and strategically focused research is vital to overcome the scientific challenges involved in copper vanadates and to explore and exploit new polymorphs to set new efficiency benchmarks and PEC water splitting solutions.

Tuning Binding Strength of Multiple Intermediates towards Efficient pH-universal Electrocatalytic Hydrogen Evolution by Mo8 O26 -NbNx Oy Heterocatalysts.

Developing efficient and robust hydrogen evolution reaction (HER) catalysts for scalable and sustainable hydrogen production through electrochemical water splitting is strategic and challenging. Herein, heterogeneous Mo8 O26 -NbNx Oy supported on N-doped graphene (defined as Mo8 O26 -NbNx Oy /NG) is synthesized by controllable hydrothermal reaction and nitridation process. The O-exposed Mo8 O26 clusters covalently confined on NbNx Oy nanodomains provide a distinctive interface configuration and appropriate electronic structure, where fully exposed multiple active sites give excellent HER performance beyond commercial Pt/C catalyst in pH-universal electrolytes. Theoretical studies reveal that the Mo8 O26 -NbNx Oy interface with electronic reconstruction affords near-optimal hydrogen adsorption energy and enhanced initial H2 O adsorption. Furthermore, the terminal O atoms in Mo8 O26 clusters cooperate with Nb atoms to promote the initial H2 O adsorption, and subsequently reduce the H2 O dissociation energy, accelerating the entire HER kinetics.

Synthesis of Eu3+-Doped NaGd9Si6O26 Sub-Microcrystals from a NaGdF4@SiO2 Structure.

Rare earth silicate phosphors of high quantum efficiency with a stable performance are promising materials in the fields of display and illumination. The grain sizes of products synthesized via the conventional solid-state reaction method are usually too large to satisfy the requirements of color cast and extraction efficiency in high-resolution light-emitting devices (LEDs). We designed a synthetic route and successfully fabricated rare earth silicate NaGd9Si6O26 (NGSO) sub-microcrystals with a size ranging from 550 to 1200 nm. The reaction mechanism and optical properties were systematically investigated. The quantum efficiency of Eu3+-activated NGSO sub-microcrystals was about 36.6%. The LED encapsulated with these sub-microcrystals showed lower color deviation and higher lumen efficiency and lumen flux compared to that with NGSO fabricated using the conventional solid state reaction method.

The epidemiology of Shiga toxin-producing Escherichia coli O26:H11 (clonal complex 29) in England, 2014-2021.

OBJECTIVES: We aimed to describe the genomic epidemiology of the foodborne gastrointestinal pathogen, Shiga toxin-producing Escherichia coli (STEC) serotype O26:H11 belonging to clonal complex 29 (CC29) in England. METHODS: Between 01 January 2014 and 31 December 2021, 834 human isolates belonging to CC29 were sequenced at the UK Health Security Agency, and the genomic data was integrated with epidemiological data. RESULTS: Diagnoses of STEC O26:H11 in England have increased each year from 19 in 2014 to 144 in 2021. Most isolates had the Shiga toxin subtype profiles stx1a (47%), stx1a,stx2a (n = 24%) or stx2a (n = 28%). Most cases were female (57%), and the highest proportion of cases belonged to the 0-5 age group (38%). Clinical symptoms included diarrhoea (93%), blood-stained stool (48%), and abdominal pain (74%). Haemolytic Uraemic Syndrome (HUS) was diagnosed in 40/459 (9%) cases and three children died. All isolates causing STEC-HUS had stx2a either alone (n = 33) or in combination with stx1a (n = 7). CONCLUSIONS: STEC O26:H11 are a clinically significant, emerging threat to public health in England. Determining the true incidence and prevalence is challenging due to inconsistent national surveillance strategies. Improved diagnostics and surveillance algorithms are required to monitor the true burden, detect outbreaks and to implement effective interventions.

Potencial do polissacarídeo O26 para ser utilizado como antígeno em formulações vacinais contra a-EPEC e STEC

According to the World Health Organization, diarrheal diseases are the second leading cause of mortality in the world in children under 5 years of age, with diarrheagenic Escherichia coli being the main etiological agent of bacterial origin. This group of E. coli is also responsible for outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) in developed countries. Among the main groups of diarrheagenic E. coli are the strains belonging to serogroup O26 that can be categorized as EPEC (enteropathogenic E. coli) and STEC (shiga toxin producing E. coli). Currently, the best way to combat these pathogens is to develop a vaccine that is effective against both categories belonging to serogroup O26. Faced with this problem, this study aimed to determine the potential of the O26 polysaccharide, present in EPEC and STEC strains, to be used as a target antigen in vaccine formulations against these pathogens. For this, we used two different strains of serotype O26:H11, one from the EPEC category and the other from the STEC category. Being EPEC, capsulated bacteria, we determined the ability of anti-O26 antibodies to recognize capsulated and non-encapsulated bacteria. We also verified the ability of these antibodies to recognize bacteria in the presence of biofilm. Finally, we determined the ability of anti-O26 antibodies to increase EPEC phagocytosis by macrophages and inhibit the adhesion of these pathogens to epithelial cells. The results obtained by the ELISA technique showed that the level of recognition of biofilm-forming EPEC by anti-O26 antibodies was equivalent to the recognition of non-biofilm-producing STEC. The results also showed that the antibodies helped macrophages (J774A.1) in the phagocytosis of EPEC and were able to inhibit the adhesion of these bacteria to epithelial cells. In summary, the results indicate that the O26 polysaccharide is a good antigen candidate to be used in vaccine formulations against all E. coli strains belonging to the O26 serogroup, regardless of their virulence mechanism.

Segundo a Organização Mundial da Saúde, as doenças diarreicas são a segunda maior causa de mortalidade no mundo em crianças menores de 5 anos, sendo Escherichia coli diarreiogênicas, o principal agente etiológico de origem bacteriana. Esse grupo de E. coli também é responsável por surtos de diarreia com sangue e síndrome hemolítica urêmica (SHU) em países desenvolvidos. Dentre os principais grupos de E. coli diarreiogênicas encontram-se as linhagens pertencentes ao sorogrupo O26 que podem ser categorizadas como EPEC (E. coli enteropatogênica) e STEC (E. coli produtoras de toxina shiga). Atualmente, a melhor maneira de se combater esses patógenos é o desenvolvimento de uma vacina que seja eficaz contra as duas categorias pertencentes ao sorogrupo O26. Diante dessa problemática, esse estudo visou determinar o potencial do polissacarídeo O26, presente nas cepas de EPEC e STEC de ser utilizado como antígeno alvo em formulações vacinais contra esses patógenos. Para isso utilizamos duas linhagens diferentes do sorotipo O26:H11, sendo uma da categoria EPEC e outra da categoria STEC. Sendo as EPEC bactérias capsuladas, determinamos a capacidade de anticorpos anti-O26 de reconhecer bactérias capsuladas e não capsuladas. Verificamos também, a capacidade desses anticorpos de reconhecer bactérias na presença de biofilme. Finalmente, determinamos a capacidade dos anticorpos anti-O26 de aumentar a fagocitose de EPEC por macrófagos e inibir a adesão desses patógenos a células epiteliais. Os resultados obtidos pela técnica de ELISA mostraram que o nível de reconhecimento de EPEC formadora de biofilme pelos anticorpos anti-O26 foi equivalente ao reconhecimento de STEC não produtoras de biofilme. Os resultados também mostraram que os anticorpos auxiliaram macrófagos (J774A.1) na fagocitose de EPEC e também foram capazes de inibir a adesão dessas bactérias a células epiteliais. Em resumo, os resultados indicam que o polissacarídeo O26 é um bom candidato a antígeno para ser utilizado em formulações vacinais contra todas as linhagens de E. coli pertencentes ao sorogrupo O26 independentemente do mecanismo de virulência das mesmas.

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

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

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

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

Low-Temperature Virus vB_EcoM_VR26 Shows Potential in Biocontrol of STEC O26:H11.

Shiga toxin-producing Escherichia coli (STEC) O26:H11 is an emerging foodborne pathogen of growing concern. Since current strategies to control microbial contamination in foodstuffs do not guarantee the elimination of O26:H11, novel approaches are needed. Bacteriophages present an alternative to traditional biocontrol methods used in the food industry. Here, a previously isolated bacteriophage vB_EcoM_VR26 (VR26), adapted to grow at common refrigeration temperatures (4 and 8 °C), has been evaluated for its potential as a biocontrol agent against O26:H11. After 2 h of treatment in broth, VR26 reduced O26:H11 numbers (p < 0.01) by > 2 log10 at 22 °C, and ~3 log10 at 4 °C. No bacterial regrowth was observed after 24 h of treatment at both temperatures. When VR26 was introduced to O26:H11-inoculated lettuce, ~2.0 log10 CFU/piece reduction was observed at 4, 8, and 22 °C. No survivors were detected after 4 and 6 h at 8 and 4 °C, respectively. Although at 22 °C, bacterial regrowth was observed after 6 h of treatment, O26:H11 counts on non-treated samples were >2 log10 CFU/piece higher than on phage-treated ones (p < 0.02). This, and the ability of VR26 to survive over a pH range of 3-11, indicates that VR26 could be used to control STEC O26:H11 in the food industry.

Pathogenic potential of Escherichia coli O157 and O26 isolated from young Belgian dairy calves by recto-anal mucosal swab culturing.

AIMS: The purpose of this study was to investigate the occurrence of Escherichia coli O157 and O26 on Belgian dairy cattle farms, the presence of virulence genes in the confirmed isolates and the association of E. coli O26 presence with calf diarrhoea. METHODS AND RESULTS: In total, 233 recto-anal mucosal swabs (RAMS) were obtained from healthy and diarrheic dairy calves on three farms, each alternately visited three consecutive times. RAMS were analysed for presence of E. coli O157 and O26, and stx1, stx2 and eae virulence genes. Overall, 19% of RAMS tested positive for E. coli O157, while 31% tested positive for E. coli O26. The majority of isolates possessed both stx and eae, denoting a high pathogenic potential to humans. While both serogroups persisted at farm level, persistence within the same animal over time appeared to be relatively rare. Interestingly, E. coli O26 was already abundantly present at a younger age compared to E. coli O157. Calf diarrhoea could not be associated with presence of E. coli O26. CONCLUSIONS: Young dairy calves are important on-farm reservoirs of potentially pathogenic E. coli O157 and O26. A role of E. coli O26 in calf diarrhoea could not be confirmed. SIGNIFICANCE AND IMPACT OF THE STUDY: O157 and O26 are responsible for the majority of human STEC infections. Gaining more epidemiological information regarding their occurrence and persistence on cattle farms will contribute to a better understanding of STEC ecology and risk of human transmission.

Application of QMRA to MAR operations for safe agricultural water reuses in coastal areas.

A pathogenic Escherichia coli (E.coli) O157:H7 and O26:H11 dose-response model was set up for a quantitative microbial risk assessment (QMRA) of the waterborne diseases associated with managed aquifer recharge (MAR) practices in semiarid regions. The MAR facility at Forcatella (Southern Italy) was selected for the QMRA application. The target counts of pathogens incidentally exposed to hosts by eating contaminated raw crops or while bathing at beaches of the coastal area were determined by applying the Monte Carlo Markov Chain (MCMC) Bayesian method to the water sampling results. The MCMC provided the most probable pathogen count reaching the target and allowed for the minimization of the number of water samplings, and hence, reducing the associated costs. The sampling stations along the coast were positioned based on the results of a groundwater flow and pathogen transport model, which highlighted the preferential flow pathways of the transported E. coli in the fractured coastal aquifer. QMRA indicated tolerable (<10-6 DALY) health risks for bathing at beaches and irrigation with wastewater, with 0.4 infectious diseases per year (11.4% probability of occurrence) associated with the reuse of reclaimed water via soil irrigation even though exceeding the E. coli regulation limit of 10 CFU/100 mL by five times. The results show negligible health risk and insignificant impacts on the coastal water quality due to pathogenic E. coli in the wastewater used for MAR. However, droughts and reclaimed water quality can be considered the main issues of MAR practices in semiarid regions suggesting additional reclaimed water treatments and further stress-tests via QMRAs by considering more persistent pathogens than E. coli.

Overall changes in the transcriptome of Escherichia coli O26:H11 induced by a subinhibitory concentration of ciprofloxacin.

AIMS: The goal was to explore the effects of subinhibitory concentration (SIC) (0·5 MIC = 20 µg l-1 ) of ciprofloxacin on the transcriptome of enterohaemorrhagic Escherichia coli O26:H11 isolate by 60 minutes of exposure. MATERIALS AND RESULTS: We used a combination of comparative genomic and transcriptomic (RNAseq) analyses. The whole genome of the E. coli O26:H11 #30934 strain of bovine origin was sequenced and assembled. This genome was next used as reference for the differential gene expression analysis. A whole-genome-based analysis of 36 publicly available E. coli O26:H11 genomes was performed to define the core and the accessory transcriptome of E. coli O26:H11. Using RNAseq and RT-qPCR analysis we observed overexpression of the SOS response and of T3SS effectors, together with the inhibition of specific motility-associated genes. Among the large set of transposases present, only three were activated, suggesting moderate transposition of genes with low doses of ciprofloxacin. Our results illustrated that transcriptional repressors, such as the CopG family protein, belonging to the core genome of E. coli O26:H11, are altered in response to fluoroquinolone exposure. The gene ontology enrichment analysis showed SIC of ciprofloxacin induced binding functions and catalytic activities, including mostly transferase and hydrolase proteins. The amino acid pathways involved in metabolic processes were significantly enhanced after the treatment. CONCLUSIONS: Although the core genome of E. coli O26:H11 constituted only 54·5% of the whole genome, we demonstrated that most differentially expressed genes were associated with the core genome of E. coli O26:H11, and that effects on the mobile genetic element, phage, and plasmid-related genes were rare. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time the effect of low dose of ciprofloxacin on the core transcriptome of E. coli O26:H11 was described. The effects on the main biological functions and protein classes including transcriptional regulators were illustrated.

A quantitative real time PCR assay to detect and enumerate Escherichia coli O157 and O26 serogroups in sheep recto-anal swabs.

A quantitative PCR method is described for the detection and quantification of E. coli O157 and O26 in sheep recto-anal junction swabs. The method incorporated a short enrichment step (5 h) and the use of a developed standard calibration curve relating the real time PCR cycle threshold (Ct) values to the initial concentration of pathogen in the sheep sample.

Outbreak of Shiga toxin-producing Escherichia coli (STEC) O26 paediatric haemolytic uraemic syndrome (HUS) cases associated with the consumption of soft raw cow's milk cheeses, France, March to May 2019.

We report an outbreak of Shiga toxin-producing Escherichia coli (STEC) associated paediatric haemolytic uraemic syndrome linked to the consumption of raw cow's milk soft cheeses. From 25 March to 27 May 2019, 16 outbreak cases infected with STEC O26 (median age: 22 months) were identified. Interviews and trace-back investigations using loyalty cards identified the consumption of raw milk cheeses from a single producer. Trace-forward investigations revealed that these cheeses were internationally distributed.

Whole genome sequencing to characterize shiga toxin-producing Escherichia coli O26 in a public health setting.

BACKGROUND: Shiga-toxin producing Escherichia coli (STEC) O26:H11 is the second most common cause of severe diarrhea and hemolytic uremic syndrome worldwide. The implementation of whole genome sequencing (WGS) enhances the detection and in-depth characterization of these non-O157 STEC strains. The aim of this study was to compare WGS to phenotypic serotyping and pulse field gel electrophoresis (PFGE) for characterization of STECO26 strains following a zoonotic outbreak from cattle to humans. METHODS AND RESULTS: This study evaluated seven E. coli strains; two strains isolated from two children with gastrointestinal symptoms and five strains from five calves suspected as the source of infection. Six of these isolates were serotyped phenotypically and by WGS as E. coli O26:H11 while one bovine isolate could be serotyped only by WGS as E. coli O182:H25. Stx1 was detected in two human- and two bovine-isolates using PCR and WGS. Using WGS, all four STECO26 isolates belong to sequence type (ST) 21 while the two stx1 negative E. coli O26 were ST29. All four STECO26 isolates were indistinguishable by PFGE. However, the data generated by WGS linked the two human STECO26 isolates to only one bovine STECO26 strain by having identical high-quality single nucleotide polymorphisms (hqSNPs) and identical virulence factor profiles while the remaining bovine STECO26 isolate differed by 7 hqSNPs and lacked virulence factor toxB. CONCLUSIONS: These data demonstrated that WGS provided significant information beyond traditional epidemiological tools allowing for comprehensive characterization of the STEC. Using this approach, WGS was able to identify the specific source of infection in this study.

A Comparative Analysis of the Metabolomic Response of Electron Beam Inactivated E. coli O26:H11 and Salmonella Typhimurium ATCC 13311.

Ionizing radiation such as Electron beam (EB) and gamma irradiation inactivate microbial cells preventing their multiplication. These cells, however, are structurally intact and appear to have residual metabolic activity. We were interested in understanding the metabolic pathways that were still functional in EB-inactivated cells. Therefore, the primary objective of this study was to compare the metabolites accumulating in EB-inactivated pathogens E. coli 026:H11 and S. Typhimurium immediately after EB inactivation and 24 h post inactivation. Defined aliquots (109 CFU/mL) of E. coli O26-H11 (TW 1597) and S. Typhimurium (ATCC 13311) suspended in phosphate-buffered saline were exposed to lethal EB doses of 3 kGy and 2 kGy, respectively. Complete inactivation (inability of cells to multiply) was confirmed by traditional plating methods. An untargeted analysis of the primary metabolites accumulating in un-irradiated (control) cells, EB-inactivated cells immediately after irradiation, and EB-inactivated cells that were incubated at room temperature for 24 h post EB inactivation was performed using gas chromatography/mass spectrometry. A total of 349 different metabolites were detected in the EB-inactivated S. Typhimurium and E. coli O26:H11 cells, out of which, only 50% were identifiable. In S. Typhimurium, 98 metabolites were expressed at statistically different concentrations (P < 0.05) between the three treatment groups. In E. coli O26:H11, 63 metabolites were expressed at statistically different concentrations (P < 0.05) between the three treatment groups. In both these pathogens, the ß-alanine, alanine, aspartate, and glutamate metabolic pathways were significantly impacted (P < 0.01). Furthermore, the metabolomic changes in EB-inactivated cells were amplified significantly after 24 h storage at room temperature in phosphate-buffered saline. These results suggest that EB-inactivated cells are very metabolically active and, therefore, the term Metabolically Active yet Non-culturable is an apt term describing EB-inactivated bacterial cells.

Use of Genomics to Investigate Historical Importation of Shiga Toxin-Producing Escherichia coli Serogroup O26 and Nontoxigenic Variants into New Zealand.

Shiga toxin-producing Escherichia coli serogroup O26 is an important public health pathogen. Phylogenetic bacterial lineages in a country can be associated with the level and timing of international imports of live cattle, the main reservoir. We sequenced the genomes of 152 E. coli O26 isolates from New Zealand and compared them with 252 E. coli O26 genomes from 14 other countries. Gene variation among isolates from humans, animals, and food was strongly associated with country of origin and stx toxin profile but not isolation source. Time of origin estimates indicate serogroup O26 sequence type 21 was introduced at least 3 times into New Zealand from the 1920s to the 1980s, whereas nonvirulent O26 sequence type 29 strains were introduced during the early 2000s. New Zealand's remarkably fewer introductions of Shiga toxin-producing Escherichia coli O26 compared with other countries (such as Japan) might be related to patterns of trade in live cattle.

Shiga Toxin Type 1a (Stx1a) Reduces the Toxicity of the More Potent Stx2a In Vivo and In Vitro.

Shiga toxin (Stx)-producing Escherichia coli (STEC) causes foodborne outbreaks of bloody diarrhea. There are two major types of immunologically distinct Stxs: Stx1a and Stx2a. Stx1a is more cytotoxic to Vero cells than Stx2a, but Stx2a has a lower 50% lethal dose (LD50) in mice. Epidemiological data suggest that infections by STEC strains that produce only Stx2a progress more often to a life-threatening sequela of infection called hemolytic-uremic syndrome (HUS) than isolates that make Stx1a only or produce both Stx1a and Stx2a. In this study, we found that an E. coli O26:H11 strain that produces both Stx1a and Stx2a was virulent in streptomycin- and ciprofloxacin-treated mice and that mice were protected by administration of an anti-Stx2 antibody. However, we discovered that in the absence of ciprofloxacin, neutralization of Stx1a enhanced the virulence of the strain, a result that corroborated our previous finding that Stx1a reduces the toxicity of Stx2a by the oral route. We further found that intraperitoneal administration of the purified Stx1a B subunit delayed the mean time to death of mice intoxicated with Stx2a and reduced the cytotoxic effect of Stx2a on Vero cells. Taken together, our data suggest that Stx1a reduces both the pathogenicity of Stx2 in vivo and cytotoxicity in vitro.