Extracellular Vesicles and Renal Endothelial Cells: A Fatal Attraction in Hemolytic Uremic Syndrome.

This review focuses on typical hemolytic uremic syndrome (HUS), a life-threatening sequela of human infections caused, particularly in children, by Shiga toxin-producing Escherichia coli strains. Thrombotic microangiopathy of the brain and the kidney is the end point of toxin action, resulting in the hallmarks of HUS (ie, thrombocytopenia, anemia, and acute renal failure). A growing body of evidence points to the role of extracellular vesicles released in the blood of patients by toxin-challenged circulating cells (monocytes, neutrophils, and erythrocytes) and platelets, as a key factor in the pathogenesis of HUS. This review provides i) an updated description of the pathogenesis of Shiga toxin-producing E. coli infections; ii) an analysis of blood cell-derived extracellular vesicles, and of their parent cells, as triggering factors in HUS; and iii) a model explaining why Shiga toxin-containing vesicles dock preferentially to the endothelia of target organs.

Transmission Dynamics of Shiga Toxin-Producing Escherichia coli in New Zealand Cattle from Farm to Slaughter.

Cattle are asymptomatic carriers of Shiga toxin-producing Escherichiacoli (STEC) strains that can cause serious illness or death in humans. In New Zealand, contact with cattle feces and living near cattle populations are known risk factors for human STEC infection. Contamination of fresh meat with STEC strains also leads to the potential for rejection of consignments by importing countries. We used a combination of PCR/matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) and whole-genome sequencing (WGS) to evaluate the presence and transmission of STEC on farms and in processing plants to better understand the potential pathways for human exposure and thus mitigate risk. Animal and environmental samples (n = 2,580) were collected from six farms and three meat processing plants in New Zealand during multiple sampling sessions in spring of 2015 and 2016. PCR/MALDI-TOF analysis revealed that 6.2% were positive for "Top 7" STEC. Top 7 STEC strains were identified in all sample sources (n = 17) tested. A marked increase in Top 7 STEC prevalence was observed between calf hides on farm (6.3% prevalence) and calf hides at processing plants (25.1% prevalence). Whole-genome sequencing was performed on Top 7 STEC bacterial isolates (n = 40). Analysis of STEC O26 (n = 25 isolates) revealed relatively low genetic diversity on individual farms, consistent with the presence of a resident strain disseminated within the farm environment. Public health efforts should focus on minimizing human contact with fecal material on farms and during handling, transport, and slaughter of calves. Meat processing plants should focus on minimizing cross-contamination between the hides of calves in a cohort during transport, lairage, and slaughter.IMPORTANCE Cattle are asymptomatic carriers of Shiga toxin-producing E. coli (STEC) strains, which can cause serious illness or death in humans. Contact with cattle feces and living near cattle are known risk factors for human STEC infection. This study evaluated STEC carriage in young calves and the farm environment with an in-depth evaluation of six farms and three meat processing plants over 2 years. An advanced molecular detection method and whole-genome sequencing were used to provide a detailed evaluation of the transmission of STEC both within and between farms. The study revealed widespread STEC contamination within the farm environment, but no evidence of recent spread between farms. Contamination of young dairy calf hides increased following transport and holding at meat processing plants. The elimination of STEC in farm environments may be very difficult given the multiple transmission routes; interventions should be targeted at decreasing fecal contamination of calf hides during transport, lairage, and processing.

Seasonal Prevalence of Shiga Toxin-Producing Escherichia coli on Pork Carcasses for Three Steps of the Harvest Process at Two Commercial Processing Plants in the United States.

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen that has a significant impact on public health, with strains possessing the attachment factor intimin referred to as enterohemorrhagic E. coli (EHEC) and associated with life-threatening illnesses. Cattle and beef are considered typical sources of STEC, but their presence in pork products is a growing concern. Therefore, carcasses (n = 1,536) at two U.S. pork processors were sampled once per season at three stages of harvest (poststunning skins, postscald carcasses, and chilled carcasses) and then examined using PCR for Shiga toxin genes (stx), intimin genes (eae), aerobic plate count (APC), and Enterobacteriaceae counts (EBC). The prevalence of stx on skins, postscald, and chilled carcasses was 85.3, 17.5, and 5.4%, respectively, with 82.3, 7.8, and 1.7% of swabs, respectively, having stx and eae present. All stx-positive samples were subjected to culture isolation that resulted in 368 STEC and 46 EHEC isolates. The most frequently identified STEC were serogroups O121, O8, and O91 (63, 6.7, and 6.0% of total STEC, respectively). The most frequently isolated EHEC was serotype O157:H7 (63% of total EHEC). Results showed that scalding significantly reduced (P < 0.05) carcass APC and EBC by 3.00- and 2.50-log10 CFU/100 cm2, respectively. A seasonal effect was observed, with STEC prevalence lower (P < 0.05) in winter. The data from this study show significant (P < 0.05) reduction in the incidence of STEC (stx) from 85.3% to 5.4% and of EHEC (stx plus eae) from 82.3% to 1.7% within the slaughter-to-chilling continuum, respectively, and that potential EHEC can be confirmed present throughout using culture isolation.IMPORTANCE Seven serogroups of STEC are responsible for most (>75%) cases of severe illnesses caused by STEC and are considered adulterants of beef. However, some STEC outbreaks have been attributed to pork products, although the same E. coli are not considered adulterants in pork because little is known of their prevalence along the pork chain. The significance of the work presented here is that it identifies disease-causing STEC, EHEC, demonstrating that these same organisms are a food safety hazard in pork as well as beef. The results show that most STEC isolated from pork are not likely to cause severe disease in humans and that processes used in pork harvest, such as scalding, offer a significant control point to reduce contamination. The results will assist the pork processing industry and regulatory agencies to optimize interventions to improve the safety of pork products.

Genetic and Phenotypic Factors Associated with Persistent Shedding of Shiga Toxin-Producing Escherichia coli by Beef Cattle.

Shiga toxin-producing Escherichia coli (STEC) is a leading cause of foodborne infections. Cattle are an important STEC reservoir, although little is known about specific pathogen traits that impact persistence in the farm environment. Hence, we sought to evaluate STEC isolates recovered from beef cattle in a single herd in Michigan. To do this, we collected fecal grabs from 26 cattle and resampled 13 of these animals at 3 additional visits over a 3-month period. In all, 66 STEC isolates were recovered for genomics and biofilm quantification using crystal violet assays. The STEC population was diverse, representing seven serotypes, including O157:H7, O26:H11, and O103:H2, which are commonly associated with human infections. Although a core genome analysis of 2,933 genes grouped isolates into clusters based on serogroups, some isolates within each cluster had variable biofilm levels and virulence gene profiles. Most (77.8%; n = 49) isolates harbored stx2a, while 38 (57.5%) isolates formed strong biofilms. Isolates belonging to the predominant serogroup O6 (n = 36; 54.5%) were more likely to form strong biofilms, persistently colonize multiple cattle, and be acquired over time. A high-quality single nucleotide polymorphism (SNP) analysis of 33 O6 isolates detected between 0 and 13 single nucleotide polymorphism (SNP) differences between strains, indicating that highly similar strain types were persisting in this herd. Similar findings were observed for other persistent serogroups, although key genes were found to differ among strong and weak biofilm producers. Together, these data highlight the diversity and persistent nature of some STEC types in this important food animal reservoir.IMPORTANCE Food animal reservoirs contribute to Shiga toxin-producing Escherichia coli (STEC) evolution via the acquisition of horizontally acquired elements like Shiga toxin bacteriophages that enhance pathogenicity. In cattle, persistent fecal shedding of STEC contributes to contamination of beef and dairy products and to crops being exposed to contaminated water systems. Hence, identifying factors important for STEC persistence is critical. This longitudinal study enhances our understanding of the genetic diversity of STEC types circulating in a cattle herd and identifies genotypic and phenotypic traits associated with persistence. Key findings demonstrate that multiple STEC types readily persist in and are transmitted across cattle in a shared environment. These dynamics also enhance the persistence of virulence genes that can be transferred between bacterial hosts, resulting in the emergence of novel STEC strain types. Understanding how pathogens persist and diversify in reservoirs is important for guiding new preharvest prevention strategies aimed at reducing foodborne transmission to humans.

The gut microbes, Enterococcus and Escherichia-Shigella, affect the responses of heart valve replacement patients to the anticoagulant warfarin.

Numerous algorithms based on patient genetic variants have been established with the aim of reducing the risk of GI bleeding and thromboembolism during warfarin administration. However, approximately 35 % of individual warfarin sensitivity still remains unexplained. Few of warfarin algorithms take into account gut microbiota profiles. The identification of certain microbiome will provide new targets and new strategies for reducing the risk of bleeding and thromboembolism during warfarin administration. In this study, we collected plasma and stool samples from 200 inpatients undergoing heart valve replacement (HVR), which were classified as low responder (LR), high responder (HR) and normal responder (NR). Significant differences were observed in the diversity and relative abundance of the gut microbiota among the three groups. The genus Escherichia-Shigella was enriched significantly in the LRs (P = 3.189e-11), while the genus Enterococcus was enriched significantly in the HRs (P = 1.249e-11). The amount of VK2 synthesized by gut microbiota in LR group was much higher than that in HR group (P = 0.005). Whole genome shotgun sequencing indicated that the relative abundance of enzymes and modules associated with VK biosynthesis was significantly higher in LRs than in HRs or NRs. The 12 microbial markers were identified through tenfold cross-validation with a random forest model. The results provided a new microbial diagnostic model that can be used to inform modulation of warfarin dosage on the basis of patient intestinal flora composition.

Biofilm formation by South African non-O157 Shiga toxigenic Escherichia coli on stainless steel coupons.

This study examined the biofilm-forming ability of six non-O157 Shiga-toxin-producing Escherichia coli (STEC) strains: O116:H21, wzx-Onovel5:H19, O129:H21, O129:H23, O26:H11, and O154:H10 on stainless steel coupons after 24, 48, and 72 h of incubation at 22 °C and after 168 h at 10 °C. The results of crystal violet staining revealed that strains O129:H23 and O154:H10 were able to form biofilms on both the submerged surface and the air-liquid interface of coupons, whereas strains O116:H21, wzx-Onovel5:H19, O129:H21, and O26:H11 formed biofilm only at the air-liquid interface. Viable cell counts and scanning electron microscopy showed that biofilm formation increased (p < 0.05) over time. The biofilm-forming ability of non-O157 STEC was strongest (p < 0.05) at 22 °C after 48 h of incubation. The strongest biofilm former regardless of temperature was O129:H23. Generally, at 10 °C, weak to no biofilm was observed for isolates O154:H10, O116:H21, wzx-Onovel5:H19, O26:H11, and O129:H21 after 168 h. This study found that temperature affected the biofilm-forming ability of non-O157 STEC strains. Overall, our data indicate a high potential for biofilm formation by the isolates at 22 °C, suggesting that non-O157 STEC strains could colonize stainless steel within food-processing facilities. This could serve as a potential source of adulteration and promote the dissemination of these potential pathogens in food.

Bacteriophage biocontrol of Shiga toxigenic Escherichia coli (STEC) O145 biofilms on stainless steel reduces the contamination of beef.

Shiga toxigenic Escherichia coli (STEC) can form biofilms and frequently cause serious foodborne illnesses. A strain of STEC O145:H25 (EC19990166) known to be a strong biofilm former was used to evaluate the efficacy of bacteriophage AZO145A against biofilms formed on stainless steel (SS) coupons. Exposure of STEC O145:H25 to phage AZO145A (1010 PFU/mL) for 2 h resulted in a 4.0 log10 reduction (P < 0.01) of planktonic cells grown in M9 broth at 24 °C for 24 h, while reductions were 2.0 log10 CFU/mL if these cells were grown for 48 h or 72 h prior to phage treatment. STEC O145 biofilms formed on SS coupons for 24, 48 and 72 h were reduced (P < 0.01) 2.9, 1.9 and 1.9 log10 CFU/coupon by phages. STEC O145 cells in biofilms were readily transferred from the surface of the SS coupon to beef (3.6 log10 CFU/coupon) even with as little as 10 s of contact with the meat surface. However, transfer of STEC O145 cells from biofilms that formed on SS coupons for 48 h to beef was reduced (P < 0.01) by 3.1 log10 CFU by phage (2 × 1010 PFU/mL) at 24 °C. Scanning electron microscopy revealed that bacterial cells within indentations on the surface of SS coupons were reduced by phage. These results suggest that bacteriophage AZO145A could be effective in reducing the viability of biofilm-adherent STEC O145 on stainless steel in food industry environments.

Effects of Beef Juice on Biofilm Formation by Shiga Toxin-Producing Escherichia coli on Stainless Steel.

Shiga toxin-producing Escherichia coli (STEC) are a leading cause of foodborne illnesses worldwide, with beef and beef products as a common food reservoir. STEC strains may be present in beef-processing environments in the form of biofilms. The exudate of raw beef, also referred to as beef juice, has been identified as an important source of bacterial contamination on food-processing surfaces. This study applied beef juice as a food-based model to study its effects on biofilm formation of six STEC isolates on stainless steel. Crystal violet staining and cell enumeration demonstrated that beef juice inhibited the biofilm formation of strains O113, O145, and O91 up to 24 h at 22°C, but that biofilm increased (p < 0.05) thereafter over 72 h. Biofilms formed by O157, O111, and O45 were not affected by the addition of beef juice over the whole incubation period. Electron microscopy showed that the morphology of biofilm cells was altered and more extracellular matrix was produced with beef juice than with M9 medium. The present study demonstrated that beef juice residues on stainless steel can enhance biofilm formation of some STEC strains. Thorough and frequent cleaning of meat residues and exudate during meat production and handling is critical to reduce STEC biofilm formation even at 13°C.

Prevalence of Shiga-toxigenic and atypical enteropathogenic Escherichia coli in untreated surface water and reclaimed water in the Mid-Atlantic U.S.

The microbial quality of irrigation water has increasingly become a concern as a source of contamination for fruits and vegetables. Non-traditional sources of water are being used by more and more growers in smaller, highly diversified farms in the Mid-Atlantic region of the U.S. Shiga-toxigenic E. coli (STEC) have been responsible for several outbreaks of infections associated with the consumption of leafy greens. Our study evaluated the prevalence of the "big seven" STEC serogroups and the associated enterohemorrhagic E. coli (EHEC) virulence factors (VF) genes in conventional and nontraditional irrigation waters in the Mid-Atlantic region of the U.S. Water samples (n = 510) from 170 sampling events were collected from eight untreated surface water sites, two wastewater reclamation facilities, and one vegetable processing plant, over a 12-month period. Ten liters of water were filtered through Modified Moore swabs (MMS); swabs were then enriched into Universal Pre-enrichment Broth (UPB), followed by enrichment into non-O157 STEC R&F broth and isolation on R & F non-O157 STEC chromogenic plating medium. Isolates (n = 2489) from enriched MMS from water samples were screened for frequently reported STEC serogroups that cause foodborne illness: O26, O45, O103, O111, O121, O145, and O157, along with VF genes stx1, stx2, eae, and ehxA. Through this screening process, STEC isolates were found in 2.35% (12/510) of water samples, while 9.0% (46/510) contained an atypical enteropathogenic E. coli (aEPEC) isolate. The eae gene (n = 88 isolates) was the most frequently detected EHEC VF of the isolates screened. The majority of STEC isolates (stx1 or stx2) genes mainly came from either a pond or reclamation pond water site on two specific dates, potentially indicating that these isolates were not spatially or temporally distributed among the sampling sites. STEC isolates at reclaimed water sites may have been introduced after wastewater treatment. None of the isolates containing eae were determined to be Escherichia albertii. Our work showed that STEC prevalence in Mid-Atlantic untreated surface waters over a 12-month period was lower than the prevalence of atypical EPEC.

Curli fimbriae confer shiga toxin-producing Escherichia coli a competitive trait in mixed biofilms.

Shiga toxin-producing Escherichia coli (STEC) is one of the most common causal agents of foodborne illness linked to fresh leafy vegetables. Here, we investigated the impact of spinach-associated microorganisms on proliferation and biofilm formation of STEC O157:H7 on stainless steel surfaces at temperatures related to produce production and postharvest processing environments. Although a proliferation of inoculated pathogen cells in spinach leaf wash water was detected at all temperatures examined, the impact of spinach-associated microorganisms on the proliferation of E. coli O157:H7 was observed at 10 °C and 26 °C, but not at 4 °C. The inhibition of E. coli O157:H7 growth by spinach-associated microorganisms indicated a competition between the pathogen and spinach indigenous microflora. A significant decrease of the pathogen population in mixed biofilms was observed only at 26 °C for curli-deficient strain MQC43, but not for curli-expressing strain MQC57. Deletion of curli genes in a curli-expressing strain resulted in a phenotype similar to that of MQC43 in mixed biofilms; however, this deficiency was rescued when curli biogenesis was restored in the curli-deletion mutant strain. Our data support that curli confer E. coli O157:H7 a competitive trait in mixed biofilms, presumably through the interaction between STEC and the biofilm-proficient microorganisms associated with spinach leaves.

[Effects of the culture medium and the methodology applied on the biofilm formation of 2diarrheagenic Escherichia coli strains]. / Efectos del medio de cultivo y de la metodología aplicada sobre la formación de biopelículas de 2cepas de Escherichia coli diarreagénicas.

The ability to form biofilms of pathogenic microorganisms in a wide variety of environments, surfaces and conditions constitute an important risk, both for the food industry and for public health. The aim of this work was to evaluate and to compare the effects of the methodology applied and the culture medium used on the ability of a non-O157 verotoxigenic Escherichia coli strain and an enteropathogenic strain to form biofilm on polystyrene surface. Two methodological variants were tested in static culture and culture mediums with different composition were used. The results showed that both strains were able to form a greater biofilm under culture in LB supplemented with glucose, with medium replacement at 24h and the quantification of the biofilm carried out at 48h of incubation. These conditions could be used in future studies on biofilm formation.

Conditional Function of Autoaggregative Protein Cah and Common cah Mutations in Shiga Toxin-Producing Escherichia coli.

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-of-function 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.

Decreased STEC shedding by cattle following passive and active vaccination based on recombinant Escherichia coli Shiga toxoids.

The principal virulence factor of Shiga toxin (Stx)-producing Escherichia coli (STEC), the eponymous Stx, modulates cellular immune responses in cattle, the primary STEC reservoir. We examined whether immunization with genetically inactivated recombinant Shiga toxoids (rStx1MUT/rStx2MUT) influences STEC shedding in a calf cohort. A group of 24 calves was passively (colostrum from immunized cows) and actively (intra-muscularly at 5th and 8th week) vaccinated. Twenty-four calves served as unvaccinated controls (fed with low anti-Stx colostrum, placebo injected). Each group was divided according to the vitamin E concentration they received by milk replacer (moderate and high supplemented). The effective transfer of Stx-neutralizing antibodies from dams to calves via colostrum was confirmed by Vero cell assay. Serum antibody titers in calves differed significantly between the vaccinated and the control group until the 16th week of life. Using the expression of activation marker CD25 on CD4+CD45RO+ cells and CD8αhiCD45RO+ cells as flow cytometry based read-out, cells from vaccinated animals responded more pronounced than those of control calves to lysates of STEC and E. coli strains isolated from the farm as well as to rStx2MUT in the 16th week. Summarized for the entire observation period, less fecal samples from vaccinated calves were stx1 and/or stx2 positive than samples from control animals when calves were fed a moderate amount of vitamin E. This study provides first evidence, that transfer to and induction in young calves of Stx-neutralizing antibodies by Shiga toxoid vaccination offers the opportunity to reduce the incidence of stx-positive fecal samples in a calf cohort.

Evaluation of a novel cocktail of six lytic bacteriophages against Shiga toxin-producing Escherichia coli in broth, milk and meat.

Phages are potentially useful as antimicrobial agents in food, especially cocktails of different phages which may prevent the development of bacterial resistance. Biocontrol assays with a six-phage cocktail, which is lytic against DH5α, an enteropathogenic (EPEC) and two Shiga-toxigenic (STEC) Escherichia coli strains, were performed in Hershey-Mg broth, milk and meat at refrigerated (4 °C), room (24 °C) and abusive (37 °C) temperatures. At 4 °C, cell counts were significantly lower (2.2-2.8 log10 CFU/mL) when E. coli strains (∼109 CFU/mL) were challenged against the phage cocktail (∼109 PFU/mL) in Hershey-Mg broth after 24 h. However, reductions were higher (3.2-3.4 log10 CFU/mL) after a 48 h exposure for all the strains tested. In addition, reduction values reached up to 3.4 log10 CFU/mL (24 °C) and 3.6 log10 CFU/mL (37 °C) in challenge tests after 24 h, though the reductions achieved were slightly lower after 48 h for the four E. coli strains tested. In milk, the cocktail was highly effective since bacterial counts were below the detection limit (<101 CFU/mL) at 4 °C, while the reductions ranged from 2 to 4 log10 CFU/mL at 24 °C after a 24 h exposure. At 37 °C, DH5α was eliminated within 2 h, and an average cell decrease of 4 log10 CFU/mL was observed for the three pathogenic strains tested. When the assays were performed in meat, biocontrol values ranged from 0.5 to 1.0 log10 CFU/mL after 48 h at 4 °C, while a higher cell inactivation was achieved at 24 °C (2.6-4.0 log10 CFU/mL) and 37 °C (3.0-3.8 log10 CFU/mL). Furthermore, higher inactivation values for O157:H7 STEC (1.55 ±â€¯0.35 log10 CFU/mL) at 4 °C were obtained in meat when incubation was extended up to 6 days. As a conclusion, our six-phage cocktail was highly effective at 24 °C and 37 °C, though less effective at 4 °C in both food matrices evaluated. Thus, it might be applied against pathogenic EPEC and STEC strains to prevent foodborne diseases especially when the cold chain is lost.

A 10-year analysis of VTEC microbiological clearance times, in the under-six population of the Midlands, Ireland.

Verotoxin-producing Escherichia coli (VTEC) is a significant problem in the under-six population in the Midlands, Ireland. VTEC spreads by person-to-person transmission and children attending childcare facilities are excluded until they achieve two consecutive negative stool samples. This report analyses 10 years data on the number of days children under the age of six take to microbiologically clear VTEC. We identified from our data that the median clearance time for VTEC was 39 days, interquartile range (IQR) 27-56 days, maximum clearance time 283 days. At 70 days from onset of infection, 90% of children had cleared the infection. These findings were slightly more prolonged but consistent with international literature on VTEC clearance times for children. Asymptomatic children cleared VTEC infection significantly faster (median time 25 days IQR 13-43 days) than symptomatic children (median time 43 days IQR 31-58 days). Symptomatic children older than 1 year of age cleared VTEC infection significantly faster (median time 42 days IQR 31-57) than symptomatic children year under 1 year (median time 56 days IQR 35-74 days). This report identifies clear data which can be used to more accurately advise parents on time periods required to achieve microbiological clearance from VTEC.

Real-time genomic investigation underlying the public health response to a Shiga toxin-producing Escherichia coli O26:H11 outbreak in a nursery.

Shiga toxin-producing Escherichia coli (STEC) is a significant cause of gastrointestinal infection and the haemolytic-uremic syndrome (HUS). STEC outbreaks are commonly associated with food but animal contact is increasingly being implicated in its transmission. We report an outbreak of STEC affecting young infants at a nursery in a rural community (three HUS cases, one definite case, one probable case, three possible cases and five carriers, based on the combination of clinical, epidemiological and laboratory data) identified using culture-based and molecular techniques. The investigation identified repeated animal contact (animal farming and petting) as a likely source of STEC introduction followed by horizontal transmission. Whole genome sequencing (WGS) was used for real-time investigation of the incident and revealed a unique strain of STEC O26:H11 carrying stx2a and intimin. Following a public health intervention, no additional cases have occurred. This is the first STEC outbreak reported from Israel. WGS proved as a useful tool for rapid laboratory characterization and typing of the outbreak strain and informed the public health response at an early stage of this unusual outbreak.

Nationwide investigation of Shiga toxin-producing Escherichia coli among cattle in Japan revealed the risk factors and potentially virulent subgroups.

A nationwide study of Shiga toxin-producing Escherichia coli (STEC) was performed to determine the prevalence, characteristics and risk factors for fecal shedding of STEC among cattle in Japan. Information on rearing practices was also collected to identify risk factors for fecal shedding of STEC. STEC was isolated from 24·1% of samples (133/551) collected from 59·1% of farms (65/110). Bayesian clustering using the virulence marker profiles of the isolates subdivided the isolates into four genetically distinct groups, two of which corresponded to eae- or saa-positive STEC, which can cause severe disease in human. Both STEC groups exhibited characteristic phylogeny and virulence marker profiles. It is noteworthy that the tellurite resistance gene was not detected in all saa-positive STEC isolates, suggesting that the standard isolation method using tellurite might lead to an underestimation of the prevalence of saa-positive STEC. A multivariate logistic regression model using epidemiological information revealed a significantly (P < 0·01) high odds ratio on STEC fecal shedding in tie-stall housing and a low odds ratio in flat feed box and mechanical ventilation. Information on isolate characteristics of the two major pathotypes and risk factors in rearing practices will facilitate the development of preventative measures for STEC fecal shedding from cattle.

Biofilm formation, invasiveness and colicinogeny in locus of enterocyte and effacement negative O113:H21 Shigatoxigenic Escherichia coli.

AIMS: Although Shiga toxins (Stx) are well-established virulence traits of O113:H21 Shigatoxigenic Escherichia coli (STEC) strains, a shortage in the knowledge of other virulence properties that may contribute to pathogenesis may exist in this serotype. This study investigated biofilm, invasiveness and colicinogeny capabilities in O113:H21 STEC isolated in Brazil, mostly from animal reservoirs. A search for genes that were reported to participate in the process of biofilm formation was also performed. METHODS AND RESULTS: The 34 O113:H21 STEC isolates analysed were assayed for biofilm production in polystyrene microplates. Genes for biofilm were investigated by PCR. Invasion of cell lineages was assessed in gentamicin protection assays and colicinogeny was investigated by phenotypic tests. Fifty per cent of the strains were biofilm formers, and 35% exhibited an invasive behaviour. The pattern of distribution of biofilm-related genes did not correlate with biofilm phenotypes observed, and a high percentage of the investigated strains were able to secrete colicins. CONCLUSION: Ability to form biofilm, invasiveness and colicinogeny is demonstrated for the first time in a collection of O113:H21 STEC. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to express three additional phenotypes besides Stx production may be a factor influencing the pathogenicity and persistence potential of O113:H21 STEC.

Studies on occurrence, characterisation and decontamination of emerging pathogenic Escherichia coli (STEC, ETEC and EIEC) in table eggs.

1. Escherichia coli is one of the most common facultative anaerobic species present in the gastrointestinal tract of animals and human beings. Usually they occur as commensals, but some serotypes can cause significant illnesses in humans as well as mammals and birds. 2. The occurrence of E. coli in different categories of table eggs collected from markets was evaluated. Isolates were analysed for the presence of virulence genes, antibiotic susceptibility pattern and efficacy of peracetic acid and chlorine for the purpose of decontaminating table eggs. 3. Significant differences were observed in the occurrence of E. coli between different groups viz. processed (cleaned, washed, sanitised and packed eggs), unprocessed (un-cleaned, un-sanitised and loose eggs) and free range (eggs obtained from backyard poultry) table eggs. Overall, E. coli occurred in table eggs at 28.6% with 22.9, 29.2 and 50.0% occurrence in processed, unprocessed and free-range table eggs, respectively. 4. A total of 24 isolates of E. coli were obtained and screened for virulence genes viz. STH, SLT1/2 and INVE genes. Of the 24 isolates recovered, 10 typeable isolates belonged to O141, O119, O9, O120 and O101 serotypes, while the remaining 14 were untypeable. Antibiograms of the isolates showed multiple antimicrobial resistance (MAR) index in the range of 0.13-0.40. 5. Peracetic acid (PAA) and chlorine (CL) were studied for their sanitisation efficacy; concentrations of 100 mg/kg of PAA and 200 mg/kg of CL completely inactivated E. coli over the egg surface and also resulted in 2.58 and 2.38 log reduction in total viable counts (TVC), respectively. 6. The presence of virulence-associated shiga-like toxin (SLT1/2) and invasion E (INVE) genes and antimicrobial resistance among the emerging serotypes of pathogenic E. coli isolated from table eggs has public health implications. It underscores the need to implement better management practices across the production systems and marketing channels to produce E. coli-free wholesome eggs for consumers.

AB5 Preassembly Is Not Required for Shiga Toxin Activity.

UNLABELLED: Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of foodborne illness, including the life-threatening complication hemolytic-uremic syndrome. The German outbreak in 2011 resulted in nearly 4,000 cases of infection, with 54 deaths. Two forms of Stx, Stx1 and Stx2, differ in potency, and subtype Stx2a is most commonly associated with fatal human disease. Stx is considered to be an AB5 toxin. The single A (enzymatically active) subunit inhibits protein synthesis by cleaving a catalytic adenine from the eukaryotic rRNA. The B (binding) subunit forms a homopentamer and mediates cellular association and toxin internalization by binding to the glycolipid globotriaosylceramide (Gb3). Both subunits are essential for toxicity. Here we report that unlike other AB5 toxin family members, Stx is produced by STEC as unassembled A and B subunits. A preformed AB5 complex is not required for cellular toxicity or in vivo toxicity to mice, and toxin assembly likely occurs at the cell membrane. We demonstrate that disruption of A- and B-subunit association by use of A-subunit peptides that lack enzymatic activity can protect mice from lethal doses of toxin. Currently, no treatments have been proven to be effective for hemolytic-uremic syndrome. Our studies demonstrate that agents that interfere with A- and B-subunit assembly may have therapeutic potential. Shiga toxin (Stx) produced by pathogenic Escherichia coli is considered to be an AB5 heterohexamer; however, no known mechanisms ensure AB5 assembly. Stx released by E. coli is not in the AB5 conformation and assembles at the receptor interface. Thus, unassembled Stx can impart toxicity. This finding shows that preventing AB5 assembly is a potential treatment for Stx-associated illnesses. IMPORTANCE: Complications due to Shiga toxin are frequently fatal, and at present, supportive care is the only treatment option. Furthermore, antibiotic treatment is contraindicated due to the ability of antibiotics to amplify bacterial expression of Shiga toxin. We report, contrary to prevailing assumptions, that Shiga toxin produced by STEC circulates as unassembled A and B subunits at concentrations that are lethal to mice. Similar to the case for anthrax toxin, assembly occurs on receptors expressed on the surfaces of mammalian target cells. Disruption of Shiga toxin assembly by use of A-subunit peptides that lack enzymatic activity protects mice from lethal challenge with Shiga toxin, suggesting a new approach for development of therapeutics.