SciCombinator

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Concept: Shiga toxin

154

The ability of Escherichia coli O157:H7 to induce cellular damage leading to disease in humans is related to numerous virulence factors, most notably stx gene encoding Shiga toxin (Stx), carried by a bacteriophage. Loss of the Stx encoding bacteriophage may occur during infection or culturing of the strain. Here, we collected stx-positive and stx-negative variants of E. coli O157:H7/NM (non-motile) isolates from patients with gastrointestinal complaints. Isolates were characterized by whole genome sequencing (WGS) and their virulence properties and phylogenetic relationship were determined. Because of the presence of the eae gene but lack of the bfpA gene, the stx-negative isolates were considered as atypical enteropathogenic E. coli (aEPEC). However, they had similar phenotypic characteristics as the Shiga toxin producing E. coli (STEC) isolates and belonged to the same sequence type ST11. Furthermore, EPEC and STEC isolates shared similar virulence genes, the locus of enterocyte effacement region and plasmids. Core-genome phylogenetic analysis using a gene-by-gene typing approach showed that the sorbitol fermenting (SF) stx-negative isolates clustered together with an SF STEC isolate and one non-sorbitol fermenting (NSF) stx-negative isolate clustered together with NSF STEC isolates. Therefore, these stx-negative isolates were thought either to have lost the Stx phage or to be a progenitor of STEC O157:H7/NM. As detection of STEC infections is often based solely on the identification of the presence of stx genes, these may be misdiagnosed in routine laboratories. Therefore, an improved diagnostic approach is required to manage identification, treatment strategy and prevention of transmission of these potentially pathogenic strains.

Concepts: DNA, Bacteria, Molecular biology, Genome, Escherichia coli, Plasmid, Escherichia coli O157:H7, Shiga toxin

67

Identifying the major sources of risk in disease transmission is key to designing effective controls. However, understanding of transmission dynamics across species boundaries is typically poor, making the design and evaluation of controls particularly challenging for zoonotic pathogens. One such global pathogen is Escherichia coli O157, which causes a serious and sometimes fatal gastrointestinal illness. Cattle are the main reservoir for E. coli O157, and vaccines for cattle now exist. However, adoption of vaccines is being delayed by conflicting responsibilities of veterinary and public health agencies, economic drivers, and because clinical trials cannot easily test interventions across species boundaries, lack of information on the public health benefits. Here, we examine transmission risk across the cattle-human species boundary and show three key results. First, supershedding of the pathogen by cattle is associated with the genetic marker stx2. Second, by quantifying the link between shedding density in cattle and human risk, we show that only the relatively rare supershedding events contribute significantly to human risk. Third, we show that this finding has profound consequences for the public health benefits of the cattle vaccine. A naïve evaluation based on efficacy in cattle would suggest a 50% reduction in risk; however, because the vaccine targets the major source of human risk, we predict a reduction in human cases of nearly 85%. By accounting for nonlinearities in transmission across the human-animal interface, we show that adoption of these vaccines by the livestock industry could prevent substantial numbers of human E. coli O157 cases.

Concepts: Epidemiology, Bacteria, Vaccine, Vaccination, Escherichia coli, Escherichia coli O157:H7, Shiga toxin, Foodborne illness

32

Rapid detection of the foodborne pathogen Escherichia coli O157:H7 is of vital importance for public health worldwide. Among detection methods, reporter phages represent unique and sensitive tools for the detection of E. coli O157:H7 from food as they are host-specific and able to differentiate live cells from dead ones. Upon infection, target bacteria become identifiable since reporter genes are expressed from the engineered phage genome. The E. coli O157:H7 bacteriophage ΦV10 was modified to express NanoLuc luciferase (Nluc) derived from the deep-sea shrimp Oplophorus gracilirostris. Once infected by the ΦV10 reporter phage, E. coli O157:H7 produces a strong bioluminescent signal upon addition of commercial luciferin (Nano-Glo(®)). Enrichment assays using E. coli O157:H7 grown in LB broth with a reporter phage concentration of 1.76 × 10(2) pfu ml(-1) are capable of detecting approximately 5 CFU in 7 hours. Comparable detection was achieved within 9 hours using 9.23 × 10(3) pfu ml(-1) of phage in selective culture enrichments of ground beef as a representative food matrix. Therefore we conclude that this NanoLuc reporter phage assay shows promise for detection of E. coli O157:H7 from food in a simple, fast and sensitive manner.

Concepts: Bacteria, Genome, Escherichia coli, Plasmid, Escherichia coli O157:H7, Shiga toxin, Diarrhea, Foodborne illness

28

The formation of a thin antibody film on a glass surface using pneumatic spray was investigated as a potential immobilization technique for capturing pathogenic targets. Goat-Escherichia coli O157:H7 IgG films were made by pneumatic spray and compared against the avidin-biotin bridge immobilized films by assaying with green fluorescent protein (GFP) transformed E. coli O157:H7 cells and fluorescent reporter antibodies. Functionality, stability, and immobilization of the films were tested. The pneumatic spray films had lower fluorescence intensity values than the avidin-biotin bridge films but resulted in similar detection for E. coli O157:H7 at 10(5)-10(7)cells/ml sample concentrations with no detection of non-E. coli O157:H7 strains. Both methods also resulted in similar percent capture efficiencies. The results demonstrated that immobilization of antibody via pneumatic spray did not render the antibody non-functional and produced stable antibody films. The amount of time necessary for immobilization of the antibody was reduced significantly from 24h for the avidin-biotin bridge to 7 min using the pneumatic spray technique, with additional benefits of greatly reduced use of materials and chemicals. The pneumatic spray technique promises to be an alternative for the immobilization of antibodies on glass slides for capturing pathogenic targets and use in biosensor type devices.

Concepts: Immune system, Protein, Bacteria, Fluorescence spectroscopy, Green fluorescent protein, Escherichia coli, Escherichia coli O157:H7, Shiga toxin

27

Lemon juice, a major source of acidulant citric acid, is frequently used in the preparation of ethnic foods. Raw or partially cooked meats are marinated with lemon juice in the preparation of a popular Chamorro dish called kelaguen, which is, unfortunately, strongly associated with foodborne illness outbreaks in Guam. We investigated the efficacy of lemon juice in reducing numbers of Escherichia coli O157:H7, Salmonella Enteritidis, and Listeria monocytogenes at stationary phase during marination. Beef inoculated with a three-strain mixture of E. coli O157:H7, S. Enteritidis, or L. monocytogenes at 10(6)CFU/mL was marinated with lemon juice from 0.2 to 10mL/g for 48h at 28°C. The decline of the pathogens during marination exhibited various degrees of deviation from first-order kinetics. Based on calculations with both linear regression and Weibull models, the decimal reduction time (4-D values) over the range of lemon concentrations was 366-5.1h for E. coli O157:H7, 282-2.4h for S. Enteritidis, and 104-2.4h for L. monocytogenes, indicating that E. coli O157:H7 was the most lemon-juice-resistant of the three. The pathogen reduction time (log 4-D values) plotted against undissociated titratable citric acid exhibited a biphasic pattern. The pathogen reduction time (log 4-D or δ values) was linearly correlated with the pH of the marinating beef (R(2)=0.92 to 0.98). The Z(pH) values (pH dependence of death rate) with beef marination were 1.03 for E. coli O157:H7, 0.92 for S. Enteritidis, and 1.29 for L. monocytogenes, indicating that L. monocytogenes was the most pH resistant of the three. L. monocytogenes exhibited less resistance to lemon juice than S. Enteritidis at pH of 3.5-4.4 but more resistance at pH of 2.6-2.8. In addition, at 4°C, all three pathogens exhibited 4-D values 1.7-4.1 times greater than those at 24°C at 5mL lemon juice/g beef. In conclusion, the usual beef marinating practice for kelaguen preparation (<0.5mL lemon juice/g beef for 1-12h) did not sufficiently inactivate E. coli O157:H7, S. Enteritidis, and L. monocytogenes to meet minimum food-safety requirements. To reduce the risk of kelaguen-associated foodborne illness, kelaguen preparation must include heat treatment in addition to marination with lemon juice.

Concepts: Bacteria, Escherichia coli, Escherichia coli O157:H7, Shiga toxin, Citric acid, Diarrhea, Foodborne illness, Foodborne illnesses

26

Enterohemorrhagic Escherichia coli (EHEC) are anthropozoonotic agents that range third among food-borne pathogens respective to their incidence and dangerousness in the European Union. EHEC are Shiga-toxin producing E. coli (STEC) responsible for foodborne poisoning mainly incriminated to the consumption of contaminated beef meat. Among the hundreds of STEC serotypes identified, EHEC mainly belong to O157:H7 but non-O157 can represent 20 to 70% of EHEC infections per year. Seven of those serogroups are especially of high-risk for human health, i.e. O26, O45, O103, O111, O121, O145 and O104. While meat can be contaminated all along the food processing chain, EHEC contamination essentially occurs at the dehiding stage of slaughtering. Investigating bacterial colonization to the skeletal-muscle extracellular matrix (ECM) proteins, it appeared that environmental factors influenced specific and non-specific bacterial adhesion of O157 and non-O157 EHEC as well as biofilm formation. Importantly, mechanical treatment (i.e. shaking, centrifugation, pipetting and vortexing) inhibited and biased the results of bacterial adhesion assay. Besides stressing the importance of the protocol to investigate bacterial adhesion to ECM proteins, this study demonstrated that the colonization abilities to ECM proteins vary among EHEC serogroups and should ultimately be taken into consideration to evaluate the risk of contamination for different types of food matrices.

Concepts: Bacteria, Extracellular matrix, Escherichia coli, Escherichia coli O157:H7, Shiga toxin, Diarrhea, Foodborne illness, Enterohemorrhagic

26

The genetic differences of enterohemorrhagic Escherichia coli O157 strains isolated from humans in three widely-separated areas in Japan were analyzed to provide information on possible geographic aspects of O157 pathogenicity.

Concepts: Genetics, Bacteria, Biology, Escherichia coli, Escherichia coli O157:H7, Shiga toxin, Foodborne illness, Enterohemorrhagic

26

A simple immunoenzymatic enterohemorrhagic Escherichia coli (EHEC) colony check (ECC) assay was developed for the presumptive identification of priority EHEC colonies isolated on plating media from enrichment broth cultures of foods. With this approach, lipopolysaccharide extracted from a colony is spotted on the grid of a polymyxin-coated polyester cloth strip, and bound E. coli serogroup O26, O45, O103, O111, O121, O145, and O157 antigens are subsequently detected by sequential reactions with a pool of commercially available peroxidase-conjugated goat antibodies and tetramethylbenzidine substrate solution. Each strip can accommodate up to 15 colonies, and test results are available within 30 min. Assay performance was verified using colonies from a total of 73 target EHEC isolates covering the range of designated priority serogroups (all of which were reactive), 41 nontarget E. coli isolates including several nontarget Shiga toxin-producing E. coli serogroups (all unreactive), and 33 non-E. coli strains (all unreactive except two bacterial strains possessing O-antigenic structures in common with those of the priority EHEC). The ECC assay was reactive with target colonies grown on several types of selective and nonselective plating media designed for their cultivation. These results support the use of the ECC assay for high-throughput screening of colonies isolated on plating media for detecting priority EHEC strains in foods.

Concepts: Bacteria, Escherichia coli, Escherichia coli O157:H7, Shiga toxin, Diarrhea, Foodborne illness, Enterohemorrhagic, The Grid

25

We experienced an outbreak of enterohemorrhagic Escherichia coli (EHEC) colitis. The purpose of this study was to reveal the computed tomographic (CT) findings on EHEC colitis.

Concepts: Bacteria, Escherichia coli, Escherichia coli O157:H7, Shiga toxin, Diarrhea, Foodborne illness, Enterohemorrhagic

25

The objectives of this study were to determine the influence of a symbiotic arbuscular mycorrhizal (AM) fungus on persistence of Salmonella and enterohemorrhagic Escherichia coli O157:H7 (EHEC) within soil, and survival within Romaine lettuce. Romaine seedlings were grown with or without AM fungi. Soil surrounding plants was inoculated with ca. 8log CFU/plant of either Salmonella enterica or E. coli EHEC composites. Samples (soil, root, and shoot) were analyzed on days 1, 8, 15 and 22 for Salmonella and EHEC by direct plating and selective enrichment. Twenty-four hours after inoculation, populations of Salmonella and EHEC, respectively, were 4.20 and 3.24log CFU/root, 2.52 and 1.17log CFU/shoot, and 5.46 and 5.17log CFU/g soil. By selective enrichment, samples tested positive for Salmonella or EHEC at day 22 at rates of 94 and 68% (shoot), 97 and 56% (root), and 100 and 75% (soil), respectively, suggesting that Salmonella has a greater propensity for survival than EHEC. Salmonella populations in soil remained as high as 4.35log CFU/g by day 22, while EHEC populations dropped to 1.12log CFU/g in the same amount of time. Ninety-two percent of all Romaine leaves in our study were positive for internalized Salmonella from days 8 to 22 and remained as high as 1.26log CFU/shoot on day 22 in AM fungi+Romaine plants. There were no differences (P>0.05) between the survival of either pathogen based on the presence or absence of mycorrhizal fungi. Results of this study suggest that AM fungi do not affect the internalization and/or survival of either S. enterica or E. coli O157:H7 in Romaine lettuce seedlings. Our results should provide Romaine lettuce farmers confidence that the presence and/or application of AM fungi to crop soil is not a contributing factor to the internalization and survival of Salmonella or E. coli O157:H7 within Romaine lettuce plants.

Concepts: Bacteria, Escherichia coli, Escherichia coli O157:H7, Shiga toxin, Diarrhea, Foodborne illness, Hamburger, Enterohemorrhagic