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Background: Shiga toxin–producing Escherichia coli (STEC) is influenced by seasonality, but there is limited understanding of how specific climatic variables contribute to disease spread. This information aids in understanding disease transmission dynamics and could potentially inform public health modeling. Methods: This retrospective cohort study analyzed public health data from Ontario, Canada, between 2012 and 2021, along with historical climate data from Environment Canada. We employed Seasonal Autoregressive Integrated Moving Average (S-ARIMA) models to assess how temperature and precipitation impact the incidence of STEC infections, measured per 10,000,000 population. Results: The study included 1658 confirmed STEC cases. A significant correlation was found between STEC incidence and climatic variables. Each degree Celsius increase in maximum temperature was associated with a rise of 3 STEC cases per 10,000,000 population (Centers for Disease Control and Prevention (2024)). Additionally, each millimeter of increased precipitation correlated with an increase of 1.1 cases per 10,000,000 population. Conclusions: The findings demonstrate a significant impact of temperature and precipitation on STEC transmission, highlighting the importance of integrating meteorological data into public health surveillance. This integration may help inform public health responses and support healthcare systems in planning for future outbreaks. Further studies are needed to refine predictive models and develop effective early warning systems for clinical settings. [ABSTRACT FROM AUTHOR]

Previous investigations have explored the involvement of wolves in parasitic and viral diseases, but data on the zoonotic bacteria are limited. The aim of this study was to assess the occurrence of bacterial zoonotic agents in 16 wolf (Canis lupus italicus) fecal samples collected in a protected area in Central Italy. Campylobacter spp., Salmonella spp., Yersinia spp., Listeria monocytogenes, and Shiga Toxin-Producing Escherichia coli (STEC) were investigated by culture, while polymerase chain reaction (PCR) was employed to detect Coxiella burnetii, Mycobacterium spp., Brucella spp., and Francisella tularensis. The presence of Extended Spectrum β-Lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae was also evaluated, using selective isolation media and detection of antimicrobial resistance genes. All samples were negative for Campylobacter spp., Salmonella spp., C. burnetii, Mycobacterium spp., Brucella spp., F. tularensis, and carbapenemase-producing Enterobacteriaceae. One sample tested positive for Yersinia aldovae and three for Yersinia enterocolitica BT1A. One L. monocytogenes (serogroup IIa) and one STEC, carrying the stx1 gene, were isolated. Two ESBL isolates were detected: one Serratia fonticola, carrying blaFONA-3/6 gene, and one Escherichia coli, carrying blaCTX-M-1 gene. Both ESBL isolates were resistant to different antimicrobials and therefore classified as multi-drug-resistant. Our data suggest that wolves are potential carriers of zoonotic bacteria and may contribute to the environmental contamination through their feces. [ABSTRACT FROM AUTHOR]

Two distinct stx2f-carrying Escherichia coli (E. coli) strains, isolated from a child with uncomplicated diarrhea fifteen weeks apart, were characterized by combining short- and long-read sequencing to compare their genetic relatedness. One strain was characterized as Shiga toxin-producing E. coli (STEC)/typical enteropathogenic E. coli (tEPEC) O63:H6 with a repertoire of virulence genes including stx2f, eae (α2-subtype), cdt, and bfpA. The other STEC with serotype O157:H16, reported for the first time as stx2f-carrying Escherichia coli in this study, possessed, in addition, eae (ε-subtype) and cdt, amongst other virulence-related genes. BLAST comparison showed that the stx2f-harboring prophage sequences of both strains were highly homologous (99.6% identity and 96.1% coverage). These results were corroborated by core Stx2f phage Multilocus Sequence Typing (cpMLST) as the stx2f-harboring prophages of both isolates clustered together when compared to those of 167 other human stx2f-carrying Escherichia coli. Overall, the stx2f-harboring prophages of the two distinct E. coli strains isolated from the present case were highly similar, suggesting that the stx2f-harboring phage might have been transferred from the STEC/tEPEC O63:H6 strain to the atypical EPEC (aEPEC) O157:H16 strain in the gut of the child. [ABSTRACT FROM AUTHOR]

Abstract Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen with 6,534 annual reported cases in the EU in 2021. This pathotype generally contains strains with smooth LPS with O-antigen serogroup O157 being the predominant serogroup in the US. However, non-O157 STEC serogroups are becoming increasingly prevalent. Here we announce the complete genomes of three newly isolated phages that infect STEC serogroups O103 and O146, namely Escherichia phages vB_EcoP_PAS7, vB_EcoP_PAS59 and vB_EcoP_PAS61. The genome sequences revealed that they belong to three distinct genera, namely the newly proposed genus Cepavirus within the Slopekvirinae subfamily, the genus Suseptimavirus and the genus Uetakevirus, respectively. We identified the tailspikes of phages PAS7 and PAS61 as a primary specificity determinant for the O-antigens O103 and O146, respectively, and predicted their active site in silico.

Previous investigations have explored the involvement of wolves in parasitic and viral diseases, but data on the zoonotic bacteria are limited. The aim of this study was to assess the occurrence of bacterial zoonotic agents in 16 wolf (Canis lupus italicus) fecal samples collected in a protected area in Central Italy. Campylobacter spp., Salmonella spp., Yersinia spp., Listeria monocytogenes, and Shiga Toxin-Producing Escherichia coli (STEC) were investigated by culture, while polymerase chain reaction (PCR) was employed to detect Coxiella burnetii, Mycobacterium spp., Brucella spp., and Francisella tularensis. The presence of Extended Spectrum β-Lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae was also evaluated, using selective isolation media and detection of antimicrobial resistance genes. All samples were negative for Campylobacter spp., Salmonella spp., C. burnetii, Mycobacterium spp., Brucella spp., F. tularensis, and carbapenemase-producing Enterobacteriaceae. One sample tested positive for Yersinia aldovae and three for Yersinia enterocolitica BT1A. One L. monocytogenes (serogroup IIa) and one STEC, carrying the stx1 gene, were isolated. Two ESBL isolates were detected: one Serratia fonticola, carrying blaFONA-3/6 gene, and one Escherichia coli, carrying blaCTX-M-1 gene. Both ESBL isolates were resistant to different antimicrobials and therefore classified as multi-drug-resistant. Our data suggest that wolves are potential carriers of zoonotic bacteria and may contribute to the environmental contamination through their feces.

Two distinct stx2f-carrying Escherichia coli (E. coli) strains, isolated from a child with uncomplicated diarrhea fifteen weeks apart, were characterized by combining short- and long-read sequencing to compare their genetic relatedness. One strain was characterized as Shiga toxin-producing E. coli (STEC)/typical enteropathogenic E. coli (tEPEC) O63:H6 with a repertoire of virulence genes including stx2f, eae (α2-subtype), cdt, and bfpA. The other STEC with serotype O157:H16, reported for the first time as stx2f-carrying Escherichia coli in this study, possessed, in addition, eae (ε-subtype) and cdt, amongst other virulence-related genes. BLAST comparison showed that the stx2f-harboring prophage sequences of both strains were highly homologous (99.6% identity and 96.1% coverage). These results were corroborated by core Stx2f phage Multilocus Sequence Typing (cpMLST) as the stx2f-harboring prophages of both isolates clustered together when compared to those of 167 other human stx2f-carrying Escherichia coli. Overall, the stx2f-harboring prophages of the two distinct E. coli strains isolated from the present case were highly similar, suggesting that the stx2f-harboring phage might have been transferred from the STEC/tEPEC O63:H6 strain to the atypical EPEC (aEPEC) O157:H16 strain in the gut of the child.

Background: Shiga toxin-producing Escherichia coli (STEC) hemolytic uremic syndrome (HUS) classically presents with diarrhea. Absence of diarrheal prodrome increases suspicion for atypical HUS (aHUS). Inability to obtain a fecal specimen for culture or culture-independent testing limits the ability to differentiate STEC-HUS and aHUS. Case-diagnosis/treatment: Our patient presented with abdominal pain and constipation, and evaluation of pallor led to a diagnosis of HUS. There was a complete absence of diarrhea during the disease course. Lack of fecal specimen for several days delayed testing for STEC. Treatment for atypical HUS was initiated with complement-blockade therapy. PCR-testing for Shiga toxin from fecal specimen later returned positive. Alternative complement-pathway testing did not identify a causative genetic variant or anti-Factor H antibody. A diagnosis of STEC-HUS was assigned, and complement-blockade therapy was stopped. Conclusion: Diagnosis of aHUS remains a diagnosis of exclusion, whereby other causes of HUS are eliminated with reasonable certainty. Exclusion of STEC is necessary and relies on testing availability and recognition of testing limitations. Diarrhea-negative STEC-HUS remains a minority of cases, and future research is needed to explore the clinical characteristics of these patients. [ABSTRACT FROM AUTHOR]

ABSTRACT Two patients with acute gastroenteritis tested positive for Shiga toxin-producing Escherichia coli (STEC) by polymerase chain reaction (PCR), and both strains carried the Shiga toxin 2 encoding gene. Since routine culture using CHROMagar STEC failed to recover these isolates, immunomagnetic separation (IMS) targeting the top six non-O157:H7 serotypes was used for isolate recovery. After two subsequent IMS runs, the STEC strains were isolated from trypticase soy broth with and without overnight enrichment for runs 1 and 2, respectively. Serotyping based on whole-genome sequencing revealed that both patients carried the strain O166:H15 STEC with the stx2 gene. Hence, the magnetic beads used in IMS appeared to have cross-reactivity with other E. coli serotypes. When the STEC isolates from both stools were cultured on CHROMagar STEC and sheep blood agar (BAP), two distinct colony sizes were apparent after overnight incubation. The small and large colonies were picked and separately cultured on both media, and colony growth was observed for 2 weeks at room temperature after an initial overnight incubation at 37°C. After 1 week, the colonies showed concentric ring structures with a darker center and a lighter surrounding on CHROMagar STEC and a “fried egg”-resembling structure with a raised circular center and a flat surrounding on BAP. Both colony types remained morphologically different on CHROMagar STEC throughout the 15 days. However, on BAP, their appearance was comparable by day 7.IMPORTANCEShiga toxin-producing E. coli (STEC) infections can lead to severe complications such as bloody diarrhea and hemolytic uremic syndrome (HUS), especially in young children and the elderly. Strains that carry the shiga toxin 2 gene (stx2), such as O157:H7, have been mostly linked with severe disease outcomes. In recent years, outbreaks caused by non-O157:H7 strains have increased. E. coli O166:H15 has been previously reported causing a gastroenteritis outbreak in 1996 as a non-STEC strain, however the O166:H15 serotype we recovered carried the stx2 gene. It was particularly challenging to isolate this strain from stools by culture. Consequently, we tested immunomagnetic separation for the STEC recovery, which was a novel approach on clinical stools. Virulence genes were included for the characterization of these isolates.

Abstract Background The interaction between bacteriophages and their hosts is intricate and highly specific. Receptor-binding proteins (RBPs) of phages such as tail fibers and tailspikes initiate the infection process. These RBPs bind to diverse outer membrane structures, including the O-antigen, which is a serogroup-specific sugar-based component of the outer lipopolysaccharide layer of Gram-negative bacteria. Among the most virulent Escherichia coli strains is the Shiga toxin-producing E. coli (STEC) pathotype dominated by a subset of O-antigen serogroups. Methods Extensive phylogenetic and structural analyses were used to identify and validate specificity correlations between phage RBP subtypes and STEC O-antigen serogroups, relying on the principle of horizontal gene transfer as main driver for RBP evolution. Results We identified O-antigen specific RBP subtypes for seven out of nine most prevalent STEC serogroups (O26, O45, O103, O104, O111, O145 and O157) and seven additional E. coli serogroups (O2, O8, O16, O18, 4s/O22, O77 and O78). Eight phage genera (Gamaleya-, Justusliebig-, Kaguna-, Kayfuna-, Kutter-, Lederberg-, Nouzilly- and Uetakeviruses) emerged for their high proportion of serogroup-specific RBPs. Additionally, we reveal sequence motifs in the RBP region, potentially serving as recombination hotspots between lytic phages. Conclusion The results contribute to a better understanding of mosaicism of phage RBPs, but also demonstrate a method to identify and validate new RBP subtypes for current and future emerging serogroups.

Simple Summary: Shiga toxin-producing E. coli (STEC) serogroup O157 can cause serious infections in humans, with symptoms ranging from bloody diarrhoea to kidney failure, and, in some instances, can be life-threatening. The natural reservoir for these bacteria is livestock, particularly cattle; however, there is an increasing number of human cases associated with wildlife species such as deer. In Scotland, a human STEC O157 outbreak in 2015 that was associated with the consumption of venison prompted us to investigate the prevalence of STEC O157 in Scottish wild deer species. Although the estimated prevalence was low (0.28%), we found that STEC O157 isolates were shed at high levels from positive deer and that these isolates had the potential to cause severe disease in humans. Furthermore, retrospective analysis identified an isolate from this study as the likely source of another Scottish human outbreak in 2017. Shiga toxin-producing E. coli (STEC) infections associated with wildlife are increasing globally, highlighting many 'spillover' species as important reservoirs for these zoonotic pathogens. A human outbreak of STEC serogroup O157 in 2015 in Scotland, associated with the consumption of venison meat products, highlighted several knowledge gaps, including the prevalence of STEC O157 in Scottish wild deer and the potential risk to humans from wild deer isolates. In this study, we undertook a nationwide survey of wild deer in Scotland and determined that the prevalence of STEC O157 in wild deer is low 0.28% (95% confidence interval = 0.06–0.80). Despite the low prevalence of STEC O157 in Scottish wild deer, identified isolates were present in deer faeces at high levels (>104 colony forming units/g faeces) and had high human pathogenic potential based on whole genome sequencing and virulence gene profiling. A retrospective epidemiological investigation also identified one wild deer isolate from this study as a possible source of a Scottish human outbreak in 2017. These results emphasise the importance of food hygiene practices during the processing of wild deer carcasses for human consumption. [ABSTRACT FROM AUTHOR]

Background: The interaction between bacteriophages and their hosts is intricate and highly specific. Receptor-binding proteins (RBPs) of phages such as tail fibers and tailspikes initiate the infection process. These RBPs bind to diverse outer membrane structures, including the O-antigen, which is a serogroup-specific sugar-based component of the outer lipopolysaccharide layer of Gram-negative bacteria. Among the most virulent Escherichia coli strains is the Shiga toxin-producing E. coli (STEC) pathotype dominated by a subset of O-antigen serogroups. Methods: Extensive phylogenetic and structural analyses were used to identify and validate specificity correlations between phage RBP subtypes and STEC O-antigen serogroups, relying on the principle of horizontal gene transfer as main driver for RBP evolution. Results: We identified O-antigen specific RBP subtypes for seven out of nine most prevalent STEC serogroups (O26, O45, O103, O104, O111, O145 and O157) and seven additional E. coli serogroups (O2, O8, O16, O18, 4s/O22, O77 and O78). Eight phage genera (Gamaleya-, Justusliebig-, Kaguna-, Kayfuna-, Kutter-, Lederberg-, Nouzilly- and Uetakeviruses) emerged for their high proportion of serogroup-specific RBPs. Additionally, we reveal sequence motifs in the RBP region, potentially serving as recombination hotspots between lytic phages. Conclusion: The results contribute to a better understanding of mosaicism of phage RBPs, but also demonstrate a method to identify and validate new RBP subtypes for current and future emerging serogroups. [ABSTRACT FROM AUTHOR]

The past decade witnessed the emergence in Shiga toxin-producing Escherichia coli (STEC) infections linked to the consumption of unpasteurized milk and raw milk cheese. The virulence of STEC is primarily attributed to the presence of Shiga toxin genes (stxl and stx2) carried by Stx-converting bacteriophages, along with the intimin gene eae. Most of the available information pertains to the "Top 7" serotypes associated with STEC infections. The objectives of this study were to characterize and investigate the pathogenicity potential of E. coli UC4224, a STEC O174:H2 strain isolated from semi-hard raw milk cheese and to develop surrogate strains with reduced virulence for use in food-related studies. Complete genome sequence analysis of E. coli UC4224 unveiled the presence of a Stx1a bacteriophage, a Stx2a bacteriophage, the Locus of Adhesion and Autoaggregation (LAA) pathogenicity island, plasmid-encoded virulence genes, and other colonization facilitators. In the Galleria mellonella animal model, E. coli UC4224 demonstrated high pathogenicity potential with an LD50 of 6CFU/10µL. Upon engineering E. coli UC4224 to generate single and double mutant derivatives by inactivating stxla and/or stx2a genes, the LD50 increased by approximately 1 Log-dose in the single mutants and 2 Log-doses in the double mutants. However, infectivity was not completely abolished, suggesting the involvement of other virulence factors contributing to the pathogenicity of STEC O174:H2. Considering the possibility of raw milk cheese serving as a reservoir for STEC, cheesemaking model was developed to evaluate the survival of UC4224 and the adequacy of the respective mutants as reduced-virulence surrogates. All tested strains exhibited the ability to survive the curd cooking step at 48°C and multiplied (3.4 Log CFU) in cheese within the subsequent 24h. These findings indicate that genomic engineering did not exert any unintended effect on the double stx1-stx2 mutant behaviour, making it as a suitable less-virulent surrogate for conducting studies during food processing. [ABSTRACT FROM AUTHOR]

Shiga toxin (stx)‐producing Escherichia coli (STEC) causes potentially severe gastrointestinal infections. Due to its public health importance, control measures are required, and carriers may need to refrain from work or daycare when the risk of spread to vulnerable people is high. We evaluated the use of direct stool multiplex PCR compared to culture for primary STEC diagnostics and for follow‐up in order to update the national guidelines for STEC monitoring. We analyzed primary and follow‐up samples of 236 STEC PCR‐positive cases at HUSLAB, Helsinki, Finland in 2016–2017, altogether 858 samples. All STEC PCR‐positive samples were inoculated on non‐selective chromogenic agar plates. Culture positivity was confirmed from culture sweeps by PCR. 211 (89%) of the cases were culture positive in their primary sample. Of all primary and follow‐up samples, 499 were PCR positive and of these 450 (90%) were culture positive. PCR‐negative follow‐up samples were available from 125 cases. Of these, 88 cases were followed for at least three consecutive PCR‐negative samples. Two cases (2%) had culture‐positive sample(s) after two consecutive PCR‐negative samples. The median time for STEC clearance was 22–23 days. The laboratory‐developed multiplex PCR test used in this study is a reliable method for STEC diagnostics and follow‐up in a clinical laboratory. When non‐selective methodology is used, the majority of PCR‐positive samples (90%) are also culture positive. Furthermore, only two cases (2%) in our material had two consecutive PCR‐negative samples followed by positive samples. Consequently, to demonstrate the clearance from STEC infection, we consider two PCR‐negative follow‐up samples sufficient. The Finnish national guidelines for STEC monitoring have been updated accordingly. [ABSTRACT FROM AUTHOR]

The past decade witnessed the emergence in Shiga toxin-producing Escherichia coli (STEC) infections linked to the consumption of unpasteurized milk and raw milk cheese. The virulence of STEC is primarily attributed to the presence of Shiga toxin genes (stx1 and stx2) carried by Stx-converting bacteriophages, along with the intimin gene eae. Most of the available information pertains to the “Top 7” serotypes associated with STEC infections. The objectives of this study were to characterize and investigate the pathogenicity potential of E. coli UC4224, a STEC O174:H2 strain isolated from semi-hard raw milk cheese and to develop surrogate strains with reduced virulence for use in food-related studies. Complete genome sequence analysis of E. coli UC4224 unveiled the presence of a Stx1a bacteriophage, a Stx2a bacteriophage, the Locus of Adhesion and Autoaggregation (LAA) pathogenicity island, plasmid-encoded virulence genes, and other colonization facilitators. In the Galleria mellonella animal model, E. coli UC4224 demonstrated high pathogenicity potential with an LD50 of 6 CFU/10 μL. Upon engineering E. coli UC4224 to generate single and double mutant derivatives by inactivating stx1a and/or stx2a genes, the LD50 increased by approximately 1 Log-dose in the single mutants and 2 Log-doses in the double mutants. However, infectivity was not completely abolished, suggesting the involvement of other virulence factors contributing to the pathogenicity of STEC O174:H2. Considering the possibility of raw milk cheese serving as a reservoir for STEC, cheesemaking model was developed to evaluate the survival of UC4224 and the adequacy of the respective mutants as reduced-virulence surrogates. All tested strains exhibited the ability to survive the curd cooking step at 48°C and multiplied (3.4 Log CFU) in cheese within the subsequent 24 h. These findings indicate that genomic engineering did not exert any unintended effect on the double stx1-stx2 mutant behaviour, making it as a suitable less-virulent surrogate for conducting studies during food processing.

Shiga toxin-producing Escherichia coli (STEC) is a zoonosis and food-borne pathogenic bacterium. It is necessary to understand its characteristics and potential pathogenicity and analyze the production ability of Shiga toxin (Stx) in the biofilm state. The traditional PCR was performed to amplify the stx variant gene subtype. The results showed that strain 1 carried stx1c+stx2d subtype, strain 2 carried stx1+stx2f subtype, and strain 3 carried stx2e subtype. The results of multilocus sequence typing (MLST) showed that the three strains were relatively distantly related and possessed genetic diversity. The crystal violet staining test and the cytotoxicity test of Vero cell were used to explore the biofilm-forming ability and Stx-producing ability at 4 ℃, 10 ℃, 25 ℃, and 37 ℃. It showed that temperature significantly affected the biofilm-forming ability and Stx-producing ability. All STEC strains had the strong ability of biofilm formation and Stx production at 37 ℃. The results of the cytotoxicity test of Vero cell showed that strain 2 had the weakest Stx-producing ability under the four different temperature conditions and strain 3 showed strong biofilm-forming ability and Stx-producing ability. Especially at 4 ℃and 10 ℃, strain 3 had the strongest biofilm-forming ability compared to strain 1 and stain 2. Retail meat in China is contaminated with STEC which could be potentially pathogenic. Especially with the discovery of strains with strong biofilm-forming ability and Stx-producing ability, we should strengthen the monitoring of STEC during food processing. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen that causes gastroenteritis and Hemolytic Uremic Syndrome. Cattle are the main animal reservoir, excreting the bacteria in their feces and contaminating the environment. In addition, meat can be contaminated by releasing the intestinal content during slaughtering. Here, we evaluated the safety and immunogenicity of a vaccine candidate against STEC that was formulated with two chimeric proteins (Chi1 and Chi2), which contain epitopes of the OmpT, Cah and Hes proteins. Thirty pregnant cows in their third trimester of gestation were included and distributed into six groups (n = 5 per group): four groups were administered intramuscularly with three doses of the formulation containing 40 µg or 100 µg of each protein plus the Quil-A or Montanide™ Gel adjuvants, while two control groups were administered with placebos. No local or systemic adverse effects were observed during the study, and hematological parameters and values of blood biochemical indicators were similar among all groups. Furthermore, all vaccine formulations triggered systemic anti-Chi1/Chi2 IgG antibody levels that were significantly higher than the control groups. However, specific IgA levels were generally low and without significant differences among groups. Notably, anti-Chi1/Chi2 IgG antibody levels in the serum of newborn calves fed with colostrum from their immunized dams were significantly higher compared to newborn calves fed with colostrum from control cows, suggesting a passive immunization through colostrum. These results demonstrate that this vaccine is safe and immunogenic when applied to pregnant cows during the third trimester of gestation. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen associated with illness ranging from mild diarrhoea to haemolytic uremic syndrome (HUS) or even death. Cross-sectoral data sharing provides an opportunity to gain insight in reservoirs and sources of human infections and starting points for pro-active measures. Nevertheless, phylogenetic clustering of STEC strains from animals, food and human cases is low in the Dutch surveillance system. This is partly due to the substantial contribution of international travel and person-to-person spread in the STEC epidemiology. Furthermore, some STEC strains causing disease in humans may have a human reservoir. Although the main reservoirs and sources are included in the Dutch monitoring programmes, some animals and food products may be under-recognised as potential sources of human infections. More effort in investigating the role of other reservoirs beyond the well-known can provide a better understanding on STEC ecology in general, improving surveillance and source attribution, and ultimately provide better guidance for monitoring and source finding. This also implies having good diagnostics in place and isolates available for typing. Therefore, on the human side of the surveillance, the decision has been made to start isolating STEC at national level.

Shiga toxin-producing E. coli (STEC) infections associated with wildlife are increasing globally, highlighting many ‘spillover’ species as important reservoirs for these zoonotic pathogens. A human outbreak of STEC serogroup O157 in 2015 in Scotland, associated with the consumption of venison meat products, highlighted several knowledge gaps, including the prevalence of STEC O157 in Scottish wild deer and the potential risk to humans from wild deer isolates. In this study, we undertook a nationwide survey of wild deer in Scotland and determined that the prevalence of STEC O157 in wild deer is low 0.28% (95% confidence interval = 0.06–0.80). Despite the low prevalence of STEC O157 in Scottish wild deer, identified isolates were present in deer faeces at high levels (>104 colony forming units/g faeces) and had high human pathogenic potential based on whole genome sequencing and virulence gene profiling. A retrospective epidemiological investigation also identified one wild deer isolate from this study as a possible source of a Scottish human outbreak in 2017. These results emphasise the importance of food hygiene practices during the processing of wild deer carcasses for human consumption.

Lactose utilization is one of the general biochemical characteristics of Escherichia coli, and the lac operon is responsible for this phenotype, which can be detected on lactose-containing media, such as MacConkey agar, after 24 h of incubation. However, some Shiga toxin-producing E. coli (STEC) O121:H19 strains exhibit an unusual phenotype called delayed lactose utilization (DLU), in which lactose utilization can be detected after 48 h of cultivation but not after only 24 h of cultivation. Insertion of an insertion sequence (IS), IS600, into the lacZ gene appears to be responsible for the DLU phenotype, and exposure to lactose has been reported to be necessary to observe this phenotype, but the mechanism underlying these phenomena remains to be elucidated. Here, we performed detailed analyses of the lactose utilization abilities of a set of O121:H19 strains and their mutants and found that ISexcision enhancer (IEE)-mediated excision of IS600 reactivates the lacZ gene and that the selective proliferation of IS-cured subclones in lactose-supplemented culture medium is responsible for the expression of the DLU phenotype. In addition, we analyzed the patterns of IS insertion into the lacZ and iee genes in the global O121:H19 population and revealed that while there are O121:H19 strains or lineage/sublineages that contain the IS insertion into iee or intact lacZ and thus do not show the DLU phenotype, most currently circulating O121:H19 strains contain IS600-inserted lacZ and intact iee and thus exhibit this phenotype. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing Escherichia coli (STEC) O145:H28 can cause severe disease in humans and is a predominant serotype in STEC O145 environmental isolates. Here, comparative genomics was applied to a set of clinical and environmental strains to systematically evaluate the pathogenicity potential in environmental strains. While the core genes-based tree separated all O145:H28 strains from the non O145:H28 reference strains, it failed to segregate environmental strains from the clinical. In contrast, the accessory genes-based tree placed all clinical strains in the same clade regardless of their genotypes or serotypes, apart from the environmental strains. Loss-of-function mutations were common in the virulence genes examined, with a high frequency in genes related to adherence, autotransporters, and the type three secretion system. Distinct differences in pathogenicity islands LEE, OI-122, and OI-57, the acid fitness island, and the tellurite resistance island were detected between the O145:H28 and reference strains. A great amount of genetic variation was detected in O145:H28, which was mainly attributed to deletions, insertions, and gene acquisition at several chromosomal "hot spots". Our study demonstrated a distinct virulence gene repertoire among the STEC O145:H28 strains originating from the same geographical region and revealed unforeseen contributions of loss-of-function mutations to virulence evolution and genetic diversification in STEC. [ABSTRACT FROM AUTHOR]

The prevalence of Shiga toxin-producing Escherichia coli O157 (STEC O157) strains in wild deer and boar in Japan was investigated. STEC O157 strains were isolated from 1.9% (9/474) of the wild deer and 0.7% (3/426) of the wild boar examined. Pulsed-field gel electrophoresis (PFGE) analysis classified the wild deer and boar strains into five and three PFGE patterns, respectively. The PFGE pattern of one wild boar strain was similar to that of a cattle strain that had been isolated from a farm in the same area the wild boar was caught, suggesting that a STEC O157 strain may have been transmitted between wild boar and cattle. Clade analysis indicated that, although most of the strains were classified in clade 12, two strains were classified in clade 7. Whole-genome sequence (WGS) analysis indicated that all the strains carried mdfA, a drug resistance gene for macrolide antibiotics, and also pathogenicity-related genes similar to those in the Sakai strain. In conclusion, our study emphasized the importance of food hygiene in processing meat from Japanese wild animals for human consumption. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing Escherichia coli (STEC) and Salmonella enterica are important foodborne pathogens capable of forming both single- and multi-species biofilms. In this study, the mono- and dual-species biofilms were formed by STEC O113:H21 and Salmonella enterica serovar Choleraesuis 10708 on stainless steel in the presence of beef juice over 5 d at 22 °C. The dual-species biofilm mass was substantially (p < 0.05) greater than that produced by STEC O113:H21 or S. Choleraesuis 10708 alone. However, numbers (CFU/mL) of S. Choleraesuis 10708 or STEC O113:H21 cells in the dual-species biofilm were (p < 0.05) lower than their respective counts in single-species biofilms. In multi-species biofilms, the sensitivity of S. Choleraesuis 10708 to the antimicrobial peptide WK2 was reduced, but it was increased for STEC O113:H21. Visualization of the temporal and spatial development of dual-species biofilms using florescent protein labeling confirmed that WK2 reduced cell numbers within biofilms. Collectively, our results highlight the potential risk of cross-contamination by multi-species biofilms to food safety and suggest that WK2 may be developed as a novel antimicrobial or sanitizer for the control of biofilms on stainless steel. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing Escherichia coli (STEC) is a global foodborne bacterial pathogen that is often accountable for colon disorder or distress. STEC commonly induces severe diarrhea in hosts but can cause critical illnesses due to the Shiga toxin virulence factors. To date, there have been a significant number of STEC serotypes have been evolved. STECs vary from nausea and hemorrhoid (HC) to possible lethal hemolytic-based uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP). Inflammation-based STEC is usually a foodborne illness with Shiga toxins (Stx 1 and 2) thought to be pathogenesis. The STEC’s pathogenicity depends significantly on developing one or more Shiga toxins, which can constrain host cell protein synthesis leading to cytotoxicity. In managing STEC infections, antimicrobial agents are generally avoided, as bacterial damage and discharge of accumulated toxins are thought the body. It has also been documented that certain antibiotics improve toxin production and the development of these species. Many different groups have attempted various therapies, including toxin-focused antibodies, toxin-based polymers, synbiotic agents, and secondary metabolites remedies. Besides, in recent years, antibiotics’ efficacy in treating STEC infections has been reassessed with some encouraging methods. Nevertheless, the primary role of synbiotic effectiveness (probiotic and prebiotic) against pathogenic STEC and other enteropathogens is less recognized. Additional studies are required to understand the mechanisms of action of probiotic bacteria and yeast against STEC infection. Because of the consensus contraindication of antimicrobials for these bacterial pathogens, the examination was focused on alternative remedy strategies for STEC infections. The rise of novel STEC serotypes and approaches employed in its treatment are highlighted.

Shiga toxin-producing Escherichia coli (STEC) strain RM13514 is a clinical isolate linked to the 2010 romaine lettuce-associated outbreak in the U.S. The genes encoding a type II restriction and modification system, PstI R-M, are located in a prophage genome that is also encoding Shiga toxin. In-frame deletion of the PstI R-M genes or dam, encoding a DNA adenine methylase, in strain RM13514 were generated, resulting in two mutant strains MQC599 and MQC602, respectively. The mutant strain MQC599 exhibited a similar growth rate as the wild-type (WT) strain RM13514 when grown in Luria-Bertani (LB) broth at 37 °C. In contrast, the growth of mutant strain MQC602 was significantly slower than either RM13514 or MQC599. Genes transcriptionally regulated by the PstI R-M system or by Dam were examined by the RNA-Seq based comparative transcriptomics. The total RNA was extracted from cells of each strain grown in LB broth at exponential and stationary phases. Three biological replicates were collected for each strain. After removal of ribosomal RNA, the mRNAs were converted to cDNAs followed by Illumina sequence library construction. For strains RM13514 and MQC599, six libraires were generated for each, three from the cells in the exponential growth phase and three from the cells in the stationary phase. For strain MQC602, three additional libraries were constructed from the cells in the early exponential growth phase. The resulting 21 libraries were combined in equal amounts and sequenced on an Illumina HighSeq 2000 instrument with the Paired End 100 bp (PE100) read format, generating a total of 45.83 Gbp sequence reads. This set of sequence data is available in the NCBI SRA database under the BioProject accession number PRJNA684587. This set of transcriptomic data provides information on methylation-mediated epigenetic regulation in STEC, an important foodborne pathogen that is frequently associated with large epidemic outbreaks and can cause life-threatening disease in humans [1]. This set of data will be useful for researchers who are interested in physiology and pathogenicity of foodborne pathogens or in the fundamental mechanisms of epigenetic regulation in bacteria.

Shiga toxin-producing Escherichia coli (STEC) is a global foodborne bacterial pathogen that is often accountable for colon disorder or distress. STEC commonly induces severe diarrhea in hosts but can cause critical illnesses due to the Shiga toxin virulence factors. To date, there have been a significant number of STEC serotypes have been evolved. STECs vary from nausea and hemorrhoid (HC) to possible lethal hemolytic-based uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP). Inflammation-based STEC is usually a foodborne illness with Shiga toxins (Stx 1 and 2) thought to be pathogenesis. The STEC's pathogenicity depends significantly on developing one or more Shiga toxins, which can constrain host cell protein synthesis leading to cytotoxicity. In managing STEC infections, antimicrobial agents are generally avoided, as bacterial damage and discharge of accumulated toxins are thought the body. It has also been documented that certain antibiotics improve toxin production and the development of these species. Many different groups have attempted various therapies, including toxin-focused antibodies, toxin-based polymers, synbiotic agents, and secondary metabolites remedies. Besides, in recent years, antibiotics' efficacy in treating STEC infections has been reassessed with some encouraging methods. Nevertheless, the primary role of synbiotic effectiveness (probiotic and prebiotic) against pathogenic STEC and other enteropathogens is less recognized. Additional studies are required to understand the mechanisms of action of probiotic bacteria and yeast against STEC infection. Because of the consensus contraindication of antimicrobials for these bacterial pathogens, the examination was focused on alternative remedy strategies for STEC infections. The rise of novel STEC serotypes and approaches employed in its treatment are highlighted. [ABSTRACT FROM AUTHOR]

The subtilase cytotoxin (SubAB) is secreted by certain Shiga toxin-producing Escherichia coli (STEC) strains and is composed of the enzymatically active subunit SubA and the pentameric binding/transport subunit SubB. We previously demonstrated that SubA (10 µg/ml), in the absence of SubB, binds and intoxicates the human cervix cancer-derived epithelial cell line HeLa. However, the cellular and molecular mechanisms underlying the cytotoxic activity of SubA in the absence of SubB remained unclear. In the present study, the cytotoxic effects mediated by SubA alone were investigated in more detail in HeLa cells and the human colon cancer cell line HCT116. We found that in the absence of SubB, SubA (10 µg/ml) is internalized into the endoplasmic reticulum (ER), where it cleaves the chaperone GRP78, an already known substrate for SubA after its canonical uptake into cells via SubB. The autonomous cellular uptake of SubA and subsequent cleavage of GRP78 in cells is prevented by treatment of cells with 10 µM brefeldin A, which inhibits the transport of protein toxins into the ER. In addition, by analyzing the SubA mutant SubAΔC344, we identified the C-terminal SEEL motif as an ER-targeting signal. Conclusively, our results strongly suggest that SubA alone shares the same intracellular transport route and cytotoxic activity as the SubAB holotoxin. [ABSTRACT FROM AUTHOR]

Two patients with acute gastroenteritis tested positive for Shiga toxin-producing Escherichia coli (STEC) by polymerase chain reaction (PCR), and both strains carried the Shiga toxin 2 encoding gene. Since routine culture using CHROMagar STEC failed to recover these isolates, immunomagnetic separation (IMS) targeting the top six non-O157:H7 serotypes was used for isolate recovery. After two subsequent IMS runs, the STEC strains were isolated from trypticase soy broth with and without overnight enrichment for runs 1 and 2, respectively. Serotyping based on whole-genome sequencing revealed that both patients carried the strain O166:H15 STEC with the stx2 gene. Hence, the magnetic beads used in IMS appeared to have cross-reactivity with other E. coli serotypes. When the STEC isolates from both stools were cultured on CHROMagar STEC and sheep blood agar (BAP), two distinct colony sizes were apparent after overnight incubation. The small and large colonies were picked and separately cultured on both media, and colony growth was observed for 2 weeks at room temperature after an initial overnight incubation at 37°C. After 1 week, the colonies showed concentric ring structures with a darker center and a lighter surrounding on CHROMagar STEC and a "fried egg"-resembling structure with a raised circular center and a flat surrounding on BAP. Both colony types remained morphologically different on CHROMagar STEC throughout the 15 days. However, on BAP, their appearance was comparable by day 7., Importance: Shiga toxin-producing E. coli (STEC) infections can lead to severe complications such as bloody diarrhea and hemolytic uremic syndrome (HUS), especially in young children and the elderly. Strains that carry the shiga toxin 2 gene ( stx2 ), such as O157:H7, have been mostly linked with severe disease outcomes. In recent years, outbreaks caused by non-O157:H7 strains have increased. E. coli O166:H15 has been previously reported causing a gastroenteritis outbreak in 1996 as a non-STEC strain, however the O166:H15 serotype we recovered carried the stx2 gene. It was particularly challenging to isolate this strain from stools by culture. Consequently, we tested immunomagnetic separation for the STEC recovery, which was a novel approach on clinical stools. Virulence genes were included for the characterization of these isolates., Competing Interests: The authors declare no conflict of interest.

Shiga toxin-producing Escherichia coli (STEC) and Listeria monocytogenes are worldwide recognized zoonotic pathogens. Recent reports have emerged about the circulation of antimicrobial-resistant STEC and L. monocytogenes isolates. To assess the frequency of antimicrobial resistance and related genes in these pathogens, we studied 45 STEC and 50 L. monocytogenes isolates locally recovered from different sources. Antimicrobial susceptibility testing was performed by disk-diffusion method, and the genomic sequences of three selected STEC and from all 50 L. monocytogenes isolates were analyzed for antibiotic resistance genes. Four STEC and three L. monocytogenes isolates were phenotypically resistant to at least one of the antibiotics tested. Resistance genes aph(3″)-Ib, aph(3′)-Ia, aph(6)-Id, blaTEM−1B, sul2, mef (A), and tet(A) were found in a human STEC ampicillin-resistant isolate. All L. monocytogenes isolates harbored fosX, lin, mdrL, lde fepA, and norB. Overall resistance in L. monocytogenes and STEC was low or middle. However, the high load of resistance genes found, even in susceptible isolates, suggests that these pathogens could contribute to the burden of antimicrobial resistance.

Escherichia coli carrying prophage with genes that encode for Shiga toxins are categorized as Shiga toxin-producing E. coli (STEC) pathotype. Illnesses caused by STEC in humans, which are often foodborne, range from mild to bloody diarrhea with life-threatening complications of renal failure and hemolytic uremic syndrome and even death, particularly in children. As many as 158 of the total 187 serogroups of E. coli are known to carry Shiga toxin genes, which makes STEC a major pathotype of E. coli. Seven STEC serogroups, called top-7, which include O26, O45, O103, O111, O121, O145, and O157, are responsible for the majority of the STEC-associated human illnesses. The STEC serogroups, other than the top-7, called “non-top-7” have also been associated with human illnesses, more often as sporadic infections. Ruminants, particularly cattle, are principal reservoirs of STEC and harbor the organisms in the hindgut and shed in the feces, which serves as a major source of food and water contaminations. A number of studies have reported on the fecal prevalence of top-7 STEC in cattle feces. However, there is paucity of data on the prevalence of non-top-7 STEC serogroups in cattle feces, generally because of lack of validated detection methods. The objective of our study was to develop and validate 14 sets of multiplex PCR (mPCR) assays targeting serogroup-specific genes to detect 137 non-top-7 STEC serogroups previously reported to be present in cattle feces. Each assay included 7–12 serogroups and primers were designed to amplify the target genes with distinct amplicon sizes for each serogroup that can be readily identified within each assay. The assays were validated with 460 strains of known serogroups. The multiplex PCR assays designed in our study can be readily adapted by most laboratories for rapid identification of strains belonging to the non-top-7 STEC serogroups associated with cattle.

Shiga toxin-producing Escherichia coli (STEC) is a zoonotic foodborne pathogen associated with hemolytic uremic syndrome (HUS) that vary in their ability to cause disease in humans. STEC represents a serious problem for public health and Argentina is the country with the highest HUS incidence worldwide. Non-LEE effector (nle) genes, present on pathogenicity islands (PAIs), encode translocated substrates of the type III secretion system (T3SS), which could have an important role in STEC virulence. Particularly, nleB is one of the main effector genes proposed as a virulence marker that is involved in the action of T3SS during the STEC infection. NleB inhibits the inflammatory response of the host cell allowing the bacteria to persist in the first stage of the infection. In order to identify the potential risk of STEC strains for public health, the aim of this study was to evaluate and compare basal nleB transcription of 24 STEC strains belonging to 10 serotypes isolated from cattle, food and patients. The results showed differences in nleB transcription among strains. Some non-O157:H7 strains presented transcription levels above the control, an O157:H7 HUS-producing strain. On the other hand, no significant differences were found in basal transcription levels associated with origin or serotype but differences were found between HUS and non-HUS strains. These differences in nleB transcription may be of importance in STEC pathogenesis and could help to differentiate high and low virulence STEC strains.

Shiga toxin-producing Escherichia coli (STEC) O145:H28 can cause severe disease in humans and is a predominant serotype in STEC O145 environmental isolates. Here, comparative genomics was applied to a set of clinical and environmental strains to systematically evaluate the pathogenicity potential in environmental strains. While the core genes-based tree separated all O145:H28 strains from the non O145:H28 reference strains, it failed to segregate environmental strains from the clinical. In contrast, the accessory genes-based tree placed all clinical strains in the same clade regardless of their genotypes or serotypes, apart from the environmental strains. Loss-of-function mutations were common in the virulence genes examined, with a high frequency in genes related to adherence, autotransporters, and the type three secretion system. Distinct differences in pathogenicity islands LEE, OI-122, and OI-57, the acid fitness island, and the tellurite resistance island were detected between the O145:H28 and reference strains. A great amount of genetic variation was detected in O145:H28, which was mainly attributed to deletions, insertions, and gene acquisition at several chromosomal “hot spots”. Our study demonstrated a distinct virulence gene repertoire among the STEC O145:H28 strains originating from the same geographical region and revealed unforeseen contributions of loss-of-function mutations to virulence evolution and genetic diversification in STEC.

Escherichia coli carrying prophage with genes that encode for Shiga toxins are categorized as Shiga toxin-producing E. coli (STEC) pathotype. Illnesses caused by STEC in humans, which are often foodborne, range from mild to bloody diarrhea with life-threatening complications of renal failure and hemolytic uremic syndrome and even death, particularly in children. As many as 158 of the total 187 serogroups of E. coli are known to carry Shiga toxin genes, which makes STEC a major pathotype of E. coli. Seven STEC serogroups, called top-7, which include O26, O45, O103, O111, O121, O145, and O157, are responsible for the majority of the STEC-associated human illnesses. The STEC serogroups, other than the top-7, called "non-top-7" have also been associated with human illnesses, more often as sporadic infections. Ruminants, particularly cattle, are principal reservoirs of STEC and harbor the organisms in the hindgut and shed in the feces, which serves as a major source of food and water contaminations. A number of studies have reported on the fecal prevalence of top-7 STEC in cattle feces. However, there is paucity of data on the prevalence of non-top-7 STEC serogroups in cattle feces, generally because of lack of validated detection methods. The objective of our study was to develop and validate 14 sets of multiplex PCR (mPCR) assays targeting serogroup-specific genes to detect 137 non-top-7 STEC serogroups previously reported to be present in cattle feces. Each assay included 7–12 serogroups and primers were designed to amplify the target genes with distinct amplicon sizes for each serogroup that can be readily identified within each assay. The assays were validated with 460 strains of known serogroups. The multiplex PCR assays designed in our study can be readily adapted by most laboratories for rapid identification of strains belonging to the non-top-7 STEC serogroups associated with cattle. [ABSTRACT FROM AUTHOR]

Escherichia coli strains carrying Shiga toxins 1 and 2 (stx 1 and stx 2), intimin (eae), and hemolysin (ehx A) production genes were found in grass shoot, rhizosphere soil, and stable manure samples from a small-scale cattle farm located at the center of Netherlands, using cultivation-dependent and -independent microbiological detection techniques. Pasture land with grazing heifers in the first year of sampling in 2014 and without grazing cattle in 2015 was physically separated from the stable that housed rose calves during both years. Manure from the stable was applied to pasture via injection into soil once per year in early spring. Among a variety of 35 phylogenetic distinctly related E. coli strains, one large group consisting of 21 closely resembling E. coli O150:H2 (18), O98:H21 (2), and O84:H2 (1) strains, all belonging to phylogenetic group B1 and carrying all screened virulence traits, was found present on grass shoots (10), rhizosphere soil (3), and stable manure (8) in 2014, but not anymore in 2015 when grazing heifers were absent. Presence and absence of these strains, obtained via enrichments, were confirmed via molecular detection using PCR-NALFIA in all ecosystems in both years. We propose that this group of Shiga toxin-producing E. coli phylogenetic group B1 strains was originally introduced via stable manure injection into the pasture. Upon grazing, these potential pathogens proliferated in the intestinal track systems of the heifers resulting in defecation with higher loads of the STEC strain onto the grass cover. The STEC strain was further smeared over the field via the hooves of the heifers resulting in augmentation of the potential pathogen in the pasture in 2014, whereas in 2015, in the absence of heifers, no augmentation occurred and only a more diverse group of potentially mild virulent E. coli phylogenetic group A and B1 strains, indigenous to pasture plants, remained present. Via this model, it was postulated that human pathogens can circulate between plants and farm animals, using the plant as an alternative ecosystem. These data indicate that grazed pasture must be considered as a potential carrier of human pathogenic E. coli strains and possibly also of other pathogens. [ABSTRACT FROM AUTHOR]

The exo-xis region of lambdoid phages contains open reading frames and genes that appear to be evolutionarily important. However, this region has received little attention up to now. In this study, we provided evidence that ea22, the largest gene of this region, favors the lysogenic pathway over the lytic pathway in contrast to other characterized exo-xis region genes including ea8.5, orf61, orf60a, and orf63. Our assays also suggest some functional analogies between Ea22 and the phage integrase protein (Int). While it is unsurprising that Ea22 operates similarly in both λ and Stx phages, we have observed some distinctions that may arise from considerable sequence dissimilarity at the carboxy termini of each protein. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing Escherichia coli are pathogenic bacteria able to form biofilms both on abiotic surfaces and on food, thus increasing risks for food consumers. Moreover, biofilms are difficult to remove and more resistant to antimicrobial agents compared to planktonic cells. Bacteriophages, natural predators of bacteria, can be used as an alternative to prevent biofilm formation or to remove pre-formed biofilm. In this work, four STEC able to produce biofilm were selected among 31 different strains and tested against single bacteriophages and two-phage cocktails. Results showed that our phages were able to reduce biofilm formation by 43.46% both when used as single phage preparation and as a cocktail formulation. Since one of the two cocktails had a slightly better performance, it was used to remove pre-existing biofilms. In this case, the phages were unable to destroy the biofilms and reduce the number of bacterial cells. Our data confirm that preventing biofilm formation in a food plant is better than trying to remove a preformed biofilm and the continuous presence of bacteriophages in the process environment could reduce the number of bacteria able to form biofilms and therefore improve the food safety.

Shiga toxin-producing Escherichia coli (STEC) cause illnesses ranging from mild diarrhea to ischemic colitis and hemolytic uremic syndrome (HUS); serogroup O157 is the most common cause. We describe the epidemiology and transmission routes for U.S. STEC outbreaks during 2010–2017. Health departments reported 466 STEC outbreaks affecting 4769 persons; 459 outbreaks had a serogroup identified (330 O157, 124 non-O157, 5 both). Among these, 361 (77%) had a known transmission route: 200 foodborne (44% of O157 outbreaks, 41% of non-O157 outbreaks), 87 person-to-person (16%, 24%), 49 animal contact (11%, 9%), 20 water (4%, 5%), and 5 environmental contamination (2%, 0%). The most common food category implicated was vegetable row crops. The distribution of O157 and non-O157 outbreaks varied by age, sex, and severity. A significantly higher percentage of STEC O157 than non-O157 outbreaks were transmitted by beef (p = 0.02). STEC O157 outbreaks also had significantly higher rates of hospitalization and HUS (p < 0.001).

Abstract In the United States, Shiga toxin-producing Escherichia coli (STEC) O157 and six non-O157 serogroups O26, O45, O103, O111, O121 and O145 are considered adulterants in non-intact beef. Further, Salmonella is responsible for one of the highest numbers of foodborne infections worldwide. Multiple foods, especially meats, are routinely tested for these pathogens using methods like PCR. However, with such a large group of organisms, multiplexing using probe-based PCR assays is expensive due to the need for differently labeled oligonucleotide probes and sophisticated instrumentation. The aim of this study was to design low-cost multiplex real-time PCR assays for the detection of seven STEC serogroups, stx 1 , stx 2 genes and virulent Salmonella. Two multiplex real-time PCR melt curve assays with internal amplification controls (IAC) were standardized. The first assay detected E. coli O121, E. coli O145, E. coli O157, stx 1 , and stx 2. The second assay targeted E. coli O26, E. coli O111, E. coli O103, E. coli O45, and Salmonella. Ground beef and beef trim inoculated with 5–27 CFU/325 g of STEC and 9–36 CFU/325 g of Salmonella could be detected following an 8–10 h enrichment at 40 °C ± 2 °C in buffered peptone water containing 8 mg/L vancomycin. The assays showed reproducible results for beef products with different fat contents. These assays do not rely on fluorescent-labeled probes or immunomagnetic beads, yet accurately detect seven STEC serogroups, seven stx gene subtypes and Salmonella , making them suitable for routine testing of STEC and Salmonella in beef. Highlights • Multiplex melting curve PCR to detect STEC, stx subtypes and Salmonella were developed. • The assays targeted E. coli O121, O145, O157, O26, O111, O103, O45, Salmonella , stx 1 , stx 2. • Beef samples inoculated with 5–27 CFU/325 g of beef could be detected after 8–12 h enrichment. • The assays complied with USDA, FSIS recommendations for these pathogens. [ABSTRACT FROM AUTHOR]

High pressure processing (HPP) was evaluated to inactivate Shiga toxin-producing Escherichia coli (STEC) in raw meatballs. Ground meat (>90% lean) was inoculated (ca. 7.0 log CFU/g) with a rifampicin-resistant cocktail of eight STEC strains (O26:H11, O45:H2, O103:H2, O104:H4, O111:H-, O121:H19, O145:NM, and O157:H7). Inoculated ground beef, ground veal, or a mixture of ground beef, pork, and veal were separately mixed with liquid whole eggs and seasonings, shaped by hand into meatballs (40 g each), and stored at −20 or at 4 °C for at least 18 h. Samples were then exposed to 400 or 600 MPa for 0 to 18 min. There were no differences (p > 0.05) in pathogen reduction related to the species of meat used or for meatballs that were refrigerated (0.9 to 2.9 log CFU/g) compared to otherwise similar meatballs that were stored frozen (1.0 to 3.0 log CFU/g) prior to HPP treatment. However, less time was needed to achieve a ≥ 2.0 log CFU/g reduction at 600 MPa (1 to 3 min) compared to 400 MPa (at least 9 min). This work provides new and practically useful information on the use of HPP to inactivate STEC in raw meatballs.

Lambdoid bacteriophages form a group of viruses that shares a common schema of genome organization and lifecycle. Some of them can play crucial roles in creating the pathogenic profiles of Escherichia coli strains. For example, Shiga toxin-producing E. coli (STEC) acquired stx genes, encoding Shiga toxins, via lambdoid prophages (Stx phages). The results obtained so far present the evidence for the relation between the exo-xis region of the phage genome and lambdoid phage development, however molecular mechanisms of activities of the exo-xis genes' products are still unknown. In view of this, we decided to determine the influence of the uncharacterized open reading frame orf63 of the exo-xis region on lambdoid phages development using recombinant prophages, λ and Stx phage Φ24B. We have demonstrated that orf63 codes for a folded protein, thus, it is a functional gene. NMR spectroscopy and analytical gel filtration were used to extend this observation further. From backbone chemical shifts, Orf63 is oligomeric in solution, likely a trimer and consistent with its small size (63 aa.), is comprised of two helices, likely intertwined to form the oligomer. We observed that the deletion of phage orf63 does not impair the intracellular lambdoid phage lytic development, however delays the time and decreases the efficiency of prophage induction and in consequence results in increased survival of E. coli during phage lytic development. Additionally, the deletion of phage orf63 negatively influences expression of the major phage genes and open reading frames from the exo-xis region during prophage induction with hydrogen peroxide. We conclude, that lambdoid phage orf63 may have specific functions in the regulation of lambdoid phages development, especially at the stage of the lysis vs. lysogenization decision. Besides, orf63 probably participates in the regulation of the level of expression of essential phage genes and open reading frames from the exo-xis region during prophage induction.

Forming biofilm is a strategy utilized by Shiga toxin-producing Escherichia coli (STEC) to survive and persist in food processing environments. We investigated the biofilm-forming potential of STEC strains from 10 clinically important serogroups on stainless steel at 22 °C or 13 °C after 24, 48, and 72 h of incubation. Results from crystal violet staining, plate counts, and scanning electron microscopy (SEM) identified a single isolate from each of the O113, O145, O91, O157, and O121 serogroups that was capable of forming strong or moderate biofilms on stainless steel at 22 °C. However, the biofilm-forming strength of these five strains was reduced when incubation time progressed. Moreover, we found that these strains formed a dense pellicle at the air-liquid interface on stainless steel, which suggests that oxygen was conducive to biofilm formation. At 13 °C, biofilm formation by these strains decreased (P < 0.05), but gradually increased over time. Overall, STEC biofilm formation was most prominent at 22 °C up to 24 h. The findings in this study identify the environmental conditions that may promote STEC biofilm formation in food processing facilities and suggest that the ability of specific strains to form biofilms contributes to their persistence within these environments.

Cah is a calcium-binding autotransporter protein involved in autoaggregation and biofilm formation. Although cah is widespread in Shiga toxin-producing Escherichia coli (STEC), we detected mutations in cah at a frequency of 31.3% in this pathogen. In STEC O157:H7 supershedder strain SS17, a large deletion results in a smaller coding sequence, encoding a protein lacking the C-terminal 71 amino acids compared with Cah in STEC O157:H7 strain EDL933. We examined the function of Cah in biofilm formation and host colonization to better understand the selective pressures for cah mutations. EDL933-Cah played a conditional role in biofilm formation in vitro: it enhanced E. coli DH5α biofilm formation on glass surfaces under agitated culture conditions that prevented autoaggregation but inhibited biofilm formation under hydrostatic conditions that facilitated autoaggregation. This function appeared to be strain dependent since Cah-mediated biofilm formation was diminished when an EDL933 cah gene was expressed in SS17. Deletion of cah in EDL933 enhanced bacterial attachment to spinach leaves and altered the adherence pattern of EDL933 to bovine recto-anal junction squamous epithelial (RSE) cells. In contrast, in trans expression of EDL933 cah in SS17 increased its attachment to leaf surfaces, and in DH5α, it enhanced its adherence to RSE cells. Hence, the ecological function of Cah appears to be modulated by environmental conditions and other bacterial strain-specific properties. Considering the prevalence of cah in STEC and its role in attachment and biofilm formation, cah mutations might be selected in ecological niches in which inactivation of Cah would result in an increased fitness in STEC during colonization of plants or animal hosts IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) harbors genes encoding diverse adhesins, and many of these are known to play an important role in bacterial attachment and host colonization. We demonstrated here that the autotransporter protein Cah confers on E. coli DH5α cells a strong autoaggregative phenotype that is inversely correlated with its ability to form biofilms and plays a strain-specific role in plant and animal colonization by STEC. Although cah is widespread in the STEC population, we detected a mutation rate of 31.3% in cah, which is similar to that reported for rpoS and fimH. The formation of cell aggregates due to increased bacterium-to-bacterium interactions may be disadvantageous to bacterial populations under conditions that favor a planktonic state in STEC. Therefore, a loss-offunction mutation in cah is likely a selective trait in STEC when autoaggregative properties become detrimental to bacterial cells and may contribute to the adaptability of STEC to fluctuating environments. [ABSTRACT FROM AUTHOR]

Lambdoid bacteriophages form a group of viruses that shares a common schema of genome organization and lifecycle. Some of them can play crucial roles in creating the pathogenic profiles of Escherichia coli strains. For example, Shiga toxin-producing E. coli (STEC) acquired stx genes, encoding Shiga toxins, via lambdoid prophages (Stx phages). The results obtained so far present the evidence for the relation between the exo-xis region of the phage genome and lambdoid phage development, however molecular mechanisms of activities of the exo-xis genes' products are still unknown. In view of this, we decided to determine the influence of the uncharacterized open reading frame orf63 of the exo-xis region on lambdoid phages development using recombinant prophages, λ and Stx phage 824B. We have demonstrated that orf63 codes for a folded protein, thus, it is a functional gene. NMR spectroscopy and analytical gel filtration were used to extend this observation further. From backbone chemical shifts, Orf63 is oligomeric in solution, likely a trimer and consistent with its small size (63 aa.), is comprised of two helices, likely intertwined to form the oligomer. We observed that the deletion of phage orf63 does not impair the intracellular lambdoid phage lytic development, however delays the time and decreases the efficiency of prophage induction and in consequence results in increased survival of E. coli during phage lytic development. Additionally, the deletion of phage orf63 negatively influences expression of themajor phage genes and open reading frames from the exo-xis region during prophage induction with hydrogen peroxide. We conclude, that lambdoid phage orf63 may have specific functions in the regulation of lambdoid phages development, especially at the stage of the lysis vs. lysogenization decision. Besides, orf63 probably participates in the regulation of the level of expression of essential phage genes and open reading frames from the exo-xis region during prophage induction. [ABSTRACT FROM AUTHOR]

This study examined the potential pathogenicity of Shiga toxin-producing Escherichia coli (STEC) in feces of sika deer by PCR binary typing (P-BIT), using 24 selected STEC genes. A total of 31 STEC strains derived from sika deer in 6 prefectures of Japan were O-serotyped and found to be O93 (n=12), O146 (n=5), O176 (n=3), O130 (n=3), O5 (n=2), O7 (n=1), O96 (n=1), O116 (n=1), O141 (n=1), O157 (n=1) and O-untypable (n=1). Of the 31 STEC strains, 13 carried both stx1 and stx2, 5 carried only stx1, and 13 carried one or two variants of stx2. However, no Stx2 production was observed in 3 strains that carried only stx2: the other 28 strains produced the appropriate Stx. P-BIT analysis showed that the 5 O5 strains from two wild deer formed a cluster with human STEC strains, suggesting that the profiles of the presence of the 24 P-BIT genes in the deer strains were significantly similar to those in human strains. All of the other non-O157 STEC strains in this study were classified with strains from food, domestic animals and humans in another cluster. Good sanitary conditions should be used for deer meat processing to avoid STEC contamination, because STEC is prevalent in deer and deer may be a potential source of STEC causing human infections. [ABSTRACT FROM AUTHOR]

The aim of this study was to compare the growth of non-O157 Shiga toxin-producing Escherichia coli (STEC) with E. coli O157:H7 in ground beef and modified Tryptic Soy Broth (TSB). Additionally, the performance of three available models (ComBase, Huang et al. model, and Cepeda et al. model), for predicting STEC growth in ground beef was evaluated. To achieve this, eleven groups of STEC, including E. coli O157:H7, were inoculated into irradiated ground beef (7% or 27% fat) or modified TSB, and then subjected to programmed water baths to follow time-varying sinusoidal temperature profiles for 300 h (low temperature, 5–15 °C) and 25 h (high temperature, 10–40 °C). The growth data of STEC in ground beef were fitted to the three models, and model performance indices such as mean relative percentage error (MRPE), mean absolute relative error (MARE), root mean squared error (RMSE), bias factor (BF) and accuracy factor (AF) were calculated to assess their accuracy in predicting STEC growth. The study found that the growth of non-O157 STEC was similar to that of E. coli O157:H7, indicating that growth models for E. coli O157:H7 can be used to predict the growth of non-O157 STEC. The fat content did not significantly affect STEC growth in ground beef. Among the three models tested, the Huang et al. model underestimated the growth of all STEC in ground beef, with an RMSE of up to 2.14 log CFU/g. The ComBase model also under-predicted STEC growth in ground beef at high temperatures (10–40 °C) and could not predict growth at temperatures below 10 °C, where STEC might grow slowly. In contrast, the Cepeda et al. model accurately predicted STEC growth in ground beef and is considered an conservative growth model for STEC in commercial ground beef products. In summary, this study provides important insights into the growth of non-O157 STEC and E. coli O157:H7 in ground beef and their modeling, which can aid in predicting the safety of commercial ground beef products. • Growth of non-O157 STEC with E. coli O157:H7 was compared in ground beef and modified TSB. • The effect of fat content on STEC growth was investigated. • Three models for predicting STEC growth in ground beef were evaluated. [ABSTRACT FROM AUTHOR]