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Shiga toxin-producing Escherichia coli (STEC) are diverse bacteria, with seven serogroups (O26, O45, O103, O111, O121, O145, O157; "Top 7") of interest due to their predominance in human disease. Confirmation of STEC relies on a combination of culturing, immunological and molecular assays, but no single gold standard for identification exists. In this study, we compared analysis of STEC between three independent laboratories (LAB) using different methodologies. In LAB A, colonies of Top 7 were picked after serogroup-specific immunomagnetic separation of feces from western-Canadian slaughter cattle. A fraction of each colony was tested by PCR (stx1, stx2, eae, O group), and Top 7 isolates were saved as glycerol stocks (n = 689). In LAB B, a subsample of isolates (n = 171) were evaluated for stx1 and stx2 using different primer sets. For this, approximately half of the PCR were performed using original DNA template provided by LAB A and half using DNA extracted from sub-cultured isolates. All Top 7 isolates were sub-cultured by LAB A and shipped to LAB C for traditional serotyping (TS) to determine O and H groups, with PCR-confirmation of virulence genes using a third set of primers. By TS, 76% of O groups (525/689) matched PCR-determined O groups. Lowest proportions (p < 0.05) of O group matches between PCR and TS (62.6% and 69.8%) occurred for O26 and O45 serogroups, respectively. PCR-detection of stx differed most between LAB A and LAB C. Excluding isolates where O groups by PCR and TS did not match, detection of stx1 was most consistent (p < 0.01) for O111 and O157:H7/NM. In contrast, for O45 and O103, stx1 was detected in >65% of isolates by LAB A and <5% by LAB C. Stx2 was only detected by LAB C in isolates of serogroups O121, O145, and O157:H7/NM. LAB B also detected stx2 in O26 and O157:H12/H29, while LAB A detected stx2 in all serogroups. Excluding O111 and O157:H7/NM, marked changes in stx detection were observed between initial isolation and sub-cultures of the same isolate. While multiple explanations exist for discordant O-typing between PCR and TS and for differences in stx detection across labs, these data suggest that assays for STEC classification may require re-evaluation and/or standardization. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate the impact of soluble (10% sugar beet pulp) versus insoluble dietary fiber (15% low fat corn DDGS) with or without the addition of exogenous carbohydrases on serum acute phase proteins (APP) and digesta VFA in weaned piglets challenged with enterotoxigenic Escherichia coli (ETEC). Sixty newly weaned piglets (6.9 ± 0.07 kg) were preselected for the F18 ETEC sensitivity genotype. Pigs were randomly assigned to 1 of 6 treatments (n=10/trt) including: 1) PC: non-challenged, 2) NC: F18 ETEC challenged, 3) SF: NC + soluble fiber, 4) IF: NC + insoluble fiber, and 5, 6) SFE and IFE: SF or IF with exogenous enzymes (xylanase, ß-glucanase, and pectinase). Pigs were housed individually and then orally challenged with ETEC on d 7 (0 day post inoculation, dpi) post weaning. Blood samples were collected on dpi -1, 3, and at necropsy (dpi 7 or 8) to determine circulating concentrations of APP. Colon contents were collected at necropsy for VFA analysis. Data were analyzed using PROC GLIMMIX (SAS 9.4) with pen as the experimental unit. There was no difference in serum haptoglobin or C-reactive protein (CRP) between PC and NC on any collection day (P>0.10). On dpi 3, enzyme supplementation (SFE+IFE) decreased haptoglobin concentration regardless of fiber source (P<0.05). Serum haptoglobin on dpi 3 was lower in SFE, but not PC, SF, IF, or IFE, compared to NC (0.11 vs. 0.40 mg/mL; P<0.05). Fiber and carbohydrases did not affect CRP compared to NC on any collection day (P>0.10). Regardless of dietary treatment, pigs had higher serum haptoglobin and CRP levels on dpi -1 than dpi 3 and at necropsy (P<0.05). Pigs in the PC group had higher total VFA (107.3 vs. 87.1 uM/g; P<0.05) and tended (P<0.10) to have a higher concentration of acetic and propionic acid in the colon than those in NC. Soluble fiber increased colonic acetic acid, butyric acid, and total VFA concentration compared to NC (P<0.05). Insoluble fiber, regardless of enzyme addition, had no impact on any colonic VFA (P>0.10). In conclusion, SFE may reduce serum APP compared to other diets. Soluble fiber, but not IF, regardless of enzyme addition, may have improved colonic VFA production during an ETEC challenge. This may stimulate gut health and help the pig respond to ETEC, which may partly explain our previous findings that soluble fiber improved growth rate and reduced ileal epithelial E. coli attachment. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate the effects of 2 direct-fed microbial blends (DFM1 and DFM2) on fecal score, shedding, and growth performance of piglets challenged with F18 enterotoxigenic Escherichia coli (ETEC). Seventy-two piglets weaned at approximately 21-d of age (Ave BW = 6.47 ± 0.21 kg) were blocked by initial body weight in a randomized complete block design using 4 treatments: 1) PC: Non-challenged positive control, 2) NC: F18 ETEC challenged negative control, 3) NC + DFM1 or 4) NC+DFM2. Pigs were housed two pigs per pen to record BW and feed intake on d 0, 7, and 17. Pigs were either sham-infected with saline or orally challenged with ETEC on d 7 (0 d post-inoculation, dpi). Fecal swabs were collected pre- and post-challenge to evaluate ETEC shedding score (SS) using a categorical scale ranging from 0 – 4, with a higher score representing increased shedding. Feces were visually scored (FS) pre-challenge and daily post-challenge using a categorical scale as follows: 0 = solid; 3 = liquid. Growth performance data were analyzed using PROC MIXED (SAS 9.4) with initial body weight as a covariate. Time course data were analyzed as repeated measures in PROC GLIMMIX. Considering the 10 d period post-challenge, pigs on the NC, DFM1 and DFM2 had lower final BW (8.99, 8.72, 9.56 vs 10.58 kg for NC, DFM1, DFM2 and PC, respectively; P < 0.05) and lower ADG (181, 152, 229 vs 386 g/d for NC, DFM1, DFM2 and PC, respectively; P < 0.05). The PC pigs also ate more feed than pigs on the other treatments (P < 0.05). The G:F of NC pigs (0.542) and pigs fed DFM1 (0.506) was poorer than that of the PC pigs (0.817; P < 0.05); pigs receiving DFM2 (0.647) were intermediate in G:F ratio. The average PC SS was lower than NC (0 vs. 2.14; P < 0.01). The DFM2 treatment increased SS on 2 dpi (3.08 vs. 2.46; P < 0.05) and decreased SS on 7 dpi (2.25 vs. 3.24; P < 0.01) compared to NC. Overall FS for PC were lower compared to the challenged treatments (P < 0.01). In conclusion, the challenge model was successful as evidenced by the difference between PC and NC; the results also provide some evidence that while DFM1 appeared to provide no benefit, dietary supplementation with DFM2 tended to show promise in terms of growth performance. [ABSTRACT FROM AUTHOR]

Objectives In 2012 and 2014 the Norwegian monitoring programme for antimicrobial resistance in the veterinary and food production sectors (NORM-VET) showed that 124 of a total of 406 samples (31%) of Norwegian retail chicken meat were contaminated with extended-spectrum cephalosporin-resistant Escherichia coli . The aim of this study was to compare selected cephalosporin-resistant E. coli from humans and poultry to determine their genetic relatedness based on whole genome sequencing (WGS). Methods Escherichia coli representing three prevalent cephalosporin-resistant multi-locus sequence types (STs) isolated from poultry ( n = 17) were selected from the NORM-VET strain collections. All strains carried an IncK plasmid with a bla CMY-2 gene. Clinical E. coli isolates ( n = 284) with AmpC-mediated resistance were collected at Norwegian microbiology laboratories from 2010 to 2014. PCR screening showed that 29 of the clinical isolates harboured both IncK and bla CMY-2 . All IncK/ bla CMY-2 -positive isolates were analysed with WGS-based bioinformatics tools. Results Analysis of single nucleotide polymorphisms (SNP) in 2.5 Mbp of shared genome sequences showed close relationship, with fewer than 15 SNP differences between five clinical isolates from urinary tract infections (UTIs) and the ST38 isolates from poultry. Furthermore, all of the 29 clinical isolates harboured IncK/ bla CMY-2 plasmid variants highly similar to the IncK/ bla CMY-2 plasmid present in the poultry isolates. Conclusions Our results provide support for the hypothesis that clonal transfer of cephalosporin-resistant E. coli from chicken meat to humans may occur, and may cause difficult-to-treat infections. Furthermore, these E. coli can be a source of AmpC-resistance plasmids for opportunistic pathogens in the human microbiota. [ABSTRACT FROM AUTHOR]

The article reports on a cluster of 10 Shiga toxin-producing Escherichia coli (STEC) O157:H7 infections identified by the U.S. Centers for Disease Control and Prevention (CDC) PulseNet with pulsed-field gel electrophoresis (PFGE) pattern combinations. It discusses the STEC O17:H7 outbreak linked to dough mix in the U.S. in 2016, which serves a reminder that flour might be contaminated with pathogens and might pose human health risks when consumed raw or undercooked.

The article discusses a study which investigated the Stx-2 subunit A (Stx-2A) bacteriophages in unrelated enteroaggregative Escherichia coli (EAEC) O104:H4 strains that produce Shiga toxins (EAEC-STEC). Topics discussed the morphologic features of Stx-2A bacteriophages, major genetic differences between the bacteriophages and evolution of EAEC-STEC O104:H4 by uptake of a distinct type of Stx-2A bacteriophage.

A letter to the editor is presented in response to the article "Ten-yeartrends and risk factors for non- O157 Shiga toxin-producing Escherichiacoli found through Shiga toxin testing," by J.L. Hadler, P. Clogher, and S. Hurd in the 2011 issue.

Engineering of efflux pumps is a promising way to improve host's tolerance to biofuels such as medium-chain alcohols (CmOHs); however, this strategy is restricted by poor understanding of the efflux pumps engaged in extrusion of solvents. In this study, several Escherichia coli mutants of multidrug transporters were evaluated for isoprenol tolerance. Susceptible phenotypes were observed in the mutants with individual deletion of six transporters, AcrD, EmrAB, MacAB, MdtBC, MdtJI and YdiM, whereas inactivation of AcrAB transporter resulted in an improved tolerance to isoprenol and other CmOHs. AcrAB is the major transporter forming tripartite transperiplasmic complex with outer membrane channel TolC for direct extrusion of toxic molecules in E. coli. The AcrAB inactivation enables to enhance TolC availability for the multidrug transporters associated with extrusion of CmOHs and increase the tolerance to CmOHs including isoprenol. It is assumed that outer membrane channel TolC plays an important role in extrusion of isoprenol and other CmOHs. [ABSTRACT FROM AUTHOR]

WcaJ is an Escherichia coli membrane enzyme catalysing the biosynthesis of undecaprenyl-diphosphate-glucose, the first step in the assembly of colanic acid exopolysaccharide. WcaJ belongs to a large family of polyisoprenyl-phosphate hexose-1-phosphate transferases (PHPTs) sharing a similar predicted topology consisting of an N-terminal domain containing four transmembrane helices (TMHs), a large central periplasmic loop, and a C-terminal domain containing the fifth TMH (TMH-V) and a cytosolic tail. However, the topology of PHPTs has not been experimentally validated. Here, we investigated the topology of WcaJ using a combination of LacZ/PhoA reporter fusions and sulfhydryl labelling by PEGylation of novel cysteine residues introduced into a cysteine-less WcaJ. The results showed that the large central loop and the C-terminal tail both reside in the cytoplasm and are separated by TMH-V, which does not fully span the membrane, likely forming a "hairpin" structure. Modelling of TMH-V revealed that a highly conserved proline might contribute to a helix-break-helix structure in all PHPT members. Bioinformatic analyses show that all of these features are conserved in PHPT homologues from Gram-negative and Gram-positive bacteria. Our data demonstrate a novel topological configuration for PHPTs, which is proposed as a signature for all members of this enzyme family. [ABSTRACT FROM AUTHOR]

Isotopic composition of reactants affects the rates of chemical and biochemical reactions. As a rule, enrichment of heavy stable isotopes leads to progressively slower reactions. But the recent isotopic resonance hypothesis suggests that the dependence of the reaction rate upon the enrichment degree is not monotonous. Instead, at some 'resonance' isotopic compositions, the kinetics increases, while at 'off-resonance' compositions the same reactions progress slower. To test the predictions of this hypothesis for the elements C, H, N and O, we designed a precise (standard error ±0.05%) experiment that measures the parameters of bacterial growth in minimal media with varying isotopic composition. A number of predicted resonance conditions were tested, with significant enhancements in kinetics discovered at these conditions. The combined statistics extremely strongly supports the validity of the isotopic resonance phenomenon (p ≪ 10−15). This phenomenon has numerous implications for the origin of life studies and astrobiology, and possible applications in agriculture, biotechnology, medicine, chemistry and other areas. [ABSTRACT FROM AUTHOR]

Carotenoids are widely distributed natural pigments that are excellent antioxidants acting in photoprotection. They are typically solubilized in membranes or attached to proteins. In cyanobacteria, the photoactive soluble Orange Carotenoid Protein (OCP) is involved in photoprotective mechanisms as a highly active singlet oxygen and excitation energy quencher. Here we describe a method for producing large amounts of holo-OCP in E.coli. The six different genes involved in the synthesis of holo-OCP were introduced into E. coli using three different plasmids. The choice of promoters and the order of gene induction were important: the induction of genes involved in carotenoid synthesis must precede the induction of the ocp gene in order to obtain holo-OCPs. Active holo-OCPs with primary structures derived from several cyanobacterial strains and containing different carotenoids were isolated. This approach for rapid heterologous synthesis of large quantities of carotenoproteins is a fundamental advance in the production of antioxidants of great interest to the pharmaceutical and cosmetic industries. [ABSTRACT FROM AUTHOR]

Enterotoxigenic Escherichia coli (ETEC) infection is of global importance because of its high morbidity and mortality rate in humans and animals. ETEC has been the principal agent causing the traveler's diarrhea in humans and post-weaning diarrhea in pigs. Intestinal innate immunity is the first line of defense in enteric bacterial infections. The pathogenicity of ETEC is due to the fimbriae and heat labile (LT) and heat stable (ST) toxins. LT has two subunits A and B. Porcine intestinal epithelial cells (IPEC-J2) are a suitable in vitro model for studying ETEC infections. This study was conducted to analyze the changes in the gene expression of proinflammatory cytokines and chemokines upon stimulation of IPEC-J2 cells with different strains of ETEC and LT toxins for 3 hrs. We used wild type ETEC strains with K88ac fimbriae 3030-2 (O157:K87 LT+, STb+), 2534-86 (O8:K87: NM: LT-I+, STb+), 1836-2 (LT_, ST-, astA+), and G58-1, a non pathogenic, non-fimbriated, wild-type (O101:K28: NM, LT_), at multiplicity of infection 10:1. LT and LTb were used at 100 ng/ml. G58-1 and cells with media alone served as negative controls. The changes in the gene expression of proinflammatory cytokines such as IL-1α, IL-1β, TNF-α, IL-6 and chemokine, IL-8, were quantified relative to porcine specific cyclophilin-A. We found that the 3030-2 strain decreased IL-6 and increased TNF-α expression at 3h. Strain 1836-2 increased IL-8 and LTb decreased IL-8 gene expressions. Further investigations will help in understanding more about enteric mucosal innate immune responses to ETEC, which may potentially contribute to its prevention and treatment. [ABSTRACT FROM AUTHOR]

Shiga toxin (Stx)-producing Escherichia coli (STEC)-induced enteropathic HUS (eHUS) is a major cause of acute kidney injury in children and substantial morbidity and mortality in elderly patients. Systemic intestinal absorption of Stx and rapid uptake, through its glycolipid receptor (Gb3), by small vessel endothelial cells, are essential steps in the pathophysiology of STEC disease. HUS is characterized by intravascular hemolytic anemia, thrombocytopenia and acute kidney injury (AKI) that develop abruptly within a week of onset of STEC diarrhea/colitis. Subtle thrombotic changes, attributed to Stx-mediated endothelial injury, may not be limited to HUS. Current treatment of STEC disease targets gastrointestinal, hematological, vascular and renal complications. It includes isotonic volume replacement/expansion, red blood cell and platelet transfusion and, for severe AKI, hemo- or peritoneal dialysis. Plasma exchange is not indicated for eHUS. Novel strategies are being designed for disease prevention or amelioration, including STEC-component vaccines (Stx, protective antigens), toxin neutralizers (Stx-neutralizing monoclonal antibodies [STmAb], Gb3 mimics), and small molecules that block Stx-induced, pathogenic cellular pathways of cell activation/apoptosis. Receptor mimics and STmAb, given parenterally up to 48–72 h after oro-gastric infection, protect experimental animals from otherwise lethal outcomes. Phase II/III mAb studies are planned; however, the narrow, hypothetical therapeutic window makes treatment trials challenging. [ABSTRACT FROM AUTHOR]

Verocytotoxin (VT)-producing Escherichia coli (VTEC) infection is associated with a spectrum of clinical manifestations that includes diarrhea, hemorrhagic colitis, and the hemolytic uremic syndrome (HUS). The occurrence of HUS in a minority of individuals in outbreaks of VTEC infection is a function of several pathogen and host factors. Pathogen factors include the inoculum size and serotype of the infecting strain, horizontally acquired genetic elements known as pathogenicity islands, and probably the VT type. Host factors that increase the risk of developing HUS include age, pre-existing immunity, gastric acidity, the use of antibiotics and anti-motility agents, and, probably, stress and genetic factors that modulate host response to infection, such as innate immunity and toxin receptor type, expression, and distribution. A better understanding of the pathogen and host determinants of HUS can aid in the development of more effective public health strategies to reduce the risk of developing HUS. [ABSTRACT FROM AUTHOR]