The Polymorphic Aggregative Phenotype of Shiga Toxin-Producing Escherichia coli O111 Depends on RpoS and Curli.

Escherichia coli O111 is an emerging non-O157:H7 serotype of Shiga toxin-producing E. coli (STEC). We previously reported that outbreak and environmental, but not sporadic-case, strains of STEC O111 share a distinct aggregation phenotype (M. E. Diodati, A. H. Bates, M. B. Cooley, S. Walker, R. E. Mandrell, and M. T. Brandl, Foodborne Pathog Dis 12:235-243, 2015, http://dx.doi.org/10.1089/fpd.2014.1887). We show here the natural occurrence of nonaggregative variants in single STEC O111 strains. These variants do not produce curli fimbriae and lack RpoS function but synthesize cellulose. The deletion of csgBAC or rpoS in an aggregative outbreak strain abolished aggregate formation, which was rescued when curli biogenesis or RpoS function, respectively, was restored. Complementation of a nonaggregative variant with RpoS also conferred curli production and aggregation. These observations were supported by Western blotting with an anti-CsgA antibody. Immunomicroscopy revealed that curli were undetectable on the cells of the nonaggregative variant and the RpoS mutant but were present in large quantities in the intercellular matrix of the assemblages formed by aggregative strains. Sequence analysis of rpoS in the aggregative strain and its variant showed a single substitution of threonine for asparagine at amino acid 124. Our results indicate that the multicellular behavior of STEC O111 is RpoS dependent via positive regulation of curli production. Aggregation may confer a fitness advantage in O111 outbreak strains under stressful conditions in hydrodynamic environments along the food production chain and in the host, while the occurrence of nonaggregative variants may allow the cell population to adapt to conditions benefiting a planktonic lifestyle.

Norovirus binds to blood group A-like antigens in oyster gastrointestinal cells.

AIMS: To determine if histo-blood group antigens (HBGA) present in oyster gastrointestinal (GI) cells mediate accumulation of human noroviruses (NoV) in oyster GI cells. METHODS AND RESULTS: HBGA-specific monoclonal antibodies (MAbs) were used to determine the presence of the corresponding HBGA in oyster GI cells. All oyster samples tested contained type A-like HBGA in GI tissue as measured by ELISA. Recombinant Norwalk virus viral like particles (rNVLP) were bound to plates coated with oyster GI homogenate. The binding was inhibited when rNVLPs were pre-incubated with MAbs specific for type A HBGA, or samples of human saliva from type A individuals. Co-localization of rNVLP and type A-like HBGA, but not type B-like or type H-like HBGA, on GI epithelial cells was observed by immunofluorescent histochemical staining and three-channel confocal scanning laser microscopy. CONCLUSION: Type A-like HBGA is present in oyster GI cells and responsible for binding of rNVLP. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the presence of type A-like HBGA in oyster GI cells and the specific binding of rNVLP to type A-like HBGA on oyster GI cells. The results of this study suggest that human NoV concentrate in oyster GI cells by specific binding to concentrated type A-like HBGA rather than by a nonmolecular entrapment within the tissues.

Production of autoinducer 2 in Salmonella enterica serovar Thompson contributes to its fitness in chickens but not on cilantro leaf surfaces.

Food-borne illness caused by Salmonella enterica has been linked traditionally to poultry products but is associated increasingly with fresh fruits and vegetables. We have investigated the role of the production of autoinducer 2 (AI-2) in the ability of S. enterica serovar Thompson to colonize the chicken intestine and the cilantro phyllosphere. A mutant of S. enterica serovar Thompson that is defective in AI-2 production was constructed by insertional mutagenesis of luxS. The population size of the S. enterica serovar Thompson parental strain was significantly higher than that of its LuxS(-) mutant in the intestine, spleen, and droppings of chicks 12 days after their oral inoculation with the strains in a ratio of 1:1. In contrast, no significant difference in the population dynamics of the parental and LuxS(-) strain was observed after their inoculation singly or in mixtures onto cilantro plants. Digital image analysis revealed that 54% of S. enterica serovar Thompson cells were present in large aggregates on cilantro leaves but that the frequency distributions of the size of aggregates formed by the parental strain and the LuxS(-) mutant were not significantly different. Carbon utilization profiles indicated that the AI-2-producing strain utilized a variety of amino and organic acids more efficiently than its LuxS(-) mutant but that most sugars were utilized similarly in both strains. Thus, inherent differences in the nutrients available to S. enterica in the phyllosphere and in the chicken intestine may underlie the differential contribution of AI-2 synthesis to the fitness of S. enterica in these environments.

Detection on surfaces and in Caco-2 cells of Campylobacter jejuni cells transformed with new gfp, yfp, and cfp marker plasmids.

We have developed two sets of Campylobacter shuttle vectors containing either the gfp (green fluorescent protein), yfp (yellow fluorescent protein), or cfp (cyan fluorescent protein) reporter gene. In one set, the reporter gene is fused to a consensus Campylobacter promoter sequence (P(c)). The other set contains a pUC18 multicloning site upstream of the reporter gene, allowing the construction of transcriptional fusions using known promoters or random genomic fragments. C. jejuni cells transformed with the P(c) fusion plasmids are strongly fluorescent and easily visualized on chicken skin, on plant tissue, and within infected Caco-2 cells. In each C. jejuni strain tested, these plasmids were maintained over several passages in the absence of antibiotic selection. Also, in many C. jejuni strains, >91% of the cells transformed with the P(c) fusion plasmids remained fluorescent after several days. Experiments with yellow fluorescent and cyan fluorescent C. jejuni transformants suggest that aggregates containing two or more strains of C. jejuni may be present in an enrichment broth culture. Colonies arising from these aggregates would be heterologous in nature; therefore, isolation of a pure culture of C. jejuni, by selecting single colonies, from an environmental sample may not always yield a single strain.

Patterning of immobilized antibody layers via photolithography and oxygen plasma exposure.

A novel technique for patterning immobilized antibody layers based upon photolithography and oxygen plasma exposure has been developed. Mouse monoclonal antibodies specific for thiabendazole (a post-harvest fungicide and veterinary anthelmintic) were covalently linked through free amine groups to aminosilanized silicon dioxide films using glutaraldehyde. Immobilized antibody layers were stabilized with sucrose, dehydrated, and stored refrigerated with desiccant. Photolithographic patterning was performed with a positive photoresist with modified bake temperatures and times, selective UV exposure with a contact mask, and aqueous alkaline solubilization of exposed resist. Exposed regions of immobilized antibody were then removed by exposure to a low power, radio frequency oxygen discharge. Residual resist was stripped with acetone. Successful patterning was demonstrated by challenging surfaces with goat anti-mouse antibody conjugated to tetramethylrhodamine isothiocyanate. Sucrose stabilization was necessary for antibody to undergo photoresist processing without loss of binding activity. Challenge with enzyme linked antigen of oxygen plasma exposed antibody layers demonstrated that plasma treatment completely neutralized antibody capture ability. Ellipsometry measurements of oxygen plasma exposed antibody layers indicated complete removal of immobilized antibodies. Fluorescent imaging demonstrated smallest line widths of 2-3 microns.

Effect of heat on the nutritional quality and safety of soybean cultivars.

To evaluate whether soybean strains with reduced levels of trypsin inhibitors have enhanced nutritional and safety characteristics, we measured protease inhibitor content of a standard cultivar (Williams 82) and an isoline (L81-4590) lacking the Kunitz trypsin inhibitor, using enzyme inhibition assays and enzyme-linked immunosorbent assays (ELISA). Less heat was needed to inactivate the remaining trypsin inhibitory activity of the isoline than that of the standard soybean cultivar. In fact, autoclaving (steam heating at 121 degrees C) of the isoline for 20 min resulted in a near zero level of trypsin inhibitor activity, while 20% remained in the Williams 82 sample. Feeding studies with rats showed that the raw soy flour prepared from the isoline was nutritionally superior to the raw flour prepared from the standard variety, as measured by PER and pancreatic weights. Since the content of amino and fatty acids of the flours from both strains was identical and the hemagglutinating activities were within a factor of 2, the increased PER was likely due to the lower level of trypsin inhibitory activity in the isoline. Steam heating the flours for up to 30 min at 121 degrees C progressively increased the PER for both strains. Preliminary screening of several accessions from the USDA Soybean Germplasm Collection showed considerable variation in the content of trypsin inhibitors, sulfur amino acids, and lectins. The BBI content of these cultivars, determined by chymotrypsin inhibition assays, was identical to that found by ELISA. The results indicate that further screening studies could lead to the discovery of soybeans which yield flour that is safe and nutritious, with minimal need for heating.

ELISA analysis of soybean trypsin inhibitors in processed foods.

Soybean proteins are widely used in human foods in a variety of forms, including infant formulas, flour, protein concentrates, protein isolates, soy sauces, textured soy fibers, and tofu. The presence of inhibitors of digestive enzymes in soy proteins impairs the nutritional quality and possibly the safety of soybeans and other legumes. Processing, based on the use of heat or fractionation of protein isolates, does not completely inactivate or remove these inhibitors, so that residual amounts of inhibitors are consumed by animals and humans. New monoclonal antibody-based immunoassays can measure low levels of the soybean Kunitz trypsin inhibitor (KTI) and the Bowman-Birk trypsin and chymotrypsin inhibitor (BBI) and the Bowman-Birk foods. The enzyme-linked immunosorbent assay (ELISA) was used to measure the inhibitor content of soy concentrates, isolates, and flours, both heated and unheated; a commercial soy infant formula; KTI and BBI with rearranged disulfide bonds; browning products derived from heat-treatment of KTI with glucose and starch; and KTI exposed to high pH. The results indicate that even low inhibitor isolates contain significant amounts of specific inhibitors. Thus, infants on soy formula consume about 10 mg of KTI plus BBI per day. The immunoassays complement the established enzymatic assays of trypsin and chymotrypsin inhibitors, and have advantages in (a) measuring low levels of inhibitors in processed foods; and (b) differentiating between the Kunitz and Bowman-Birk inhibitors. The significance of our findings for food safety are discussed.