Hydrophobic adsorptive and hemagglutinating properties ofEscherichia coli possessing colonization factor antigens (CFA/I or CFA/II), type 1 pili,or other pili

Hydrophobic and hemagglutinating activities of piliated enterotoxigenicEscherichia coli possessing colonization factor antigens (CFA)/I and putative CFA/II, strains with type 1 pili, and piliated strains of nonenterotoxigenicE. coli from urinary tract infections were compared. When passed through columns of hydrophobic Phenyl Sepharose in the presence of buffered ammonium sulfate, the strains with CFA adsorbed most strongly. Similarly, the CFA strains showed a tendency to autoagglutinate at a lower (NH₄)₂SO₄ concentration than the other strains studied. The degree of hydrophobicity of the strains tested is in the order CFA/I>CFA/II>type 1 pili>urinary tract strains. Rough variants ofE. coli strains were more hydrophobic than their smooth parents. Electron microscopy showed large numbers of pili on CFA strains, whereas type 1 piliated strains possessed fewer pili. CFA-negative clones possessed few or no, pili and did not adsorb to the gel. A highly piliated mutant strain (PAK/2PfS) ofPseudomonas aeruginosa bound to the Phenyl Sepharose while the poorly piliated wild-type strains did not. Strains, lost their adsorptive capacity after blending, sonication, heating, or trypsin treatment. It is concluded that the hydrophobicity of enteric organisms, as measured by hydrophobic interaction chromatography, is a function of the type and number of pili on the cell surface.     

Effect ofClostridium perfringens phospholipase C (alpha-toxin) on the human diploid fibroblast membrane

Summary Human diploid embryonic lung fibroblasts were cultivated in Eagle's Minimum Essential Medium and labeled with³H-uridine. The release of soluble radioactive substances into the medium was used as an indicator of damage to the cell membrane. The assay method described is simple, sensitive and rapid and allows quantitative estimation of changes in membrane permeability before any morphological damage is observed microscopically. Crude commercial preparations of phospholipase C (E.C. 3.1.4.3.) (40 g/ml) were highly active on the cell membrane but most of the membrane damaging activity was found to be due to contaminating theta-toxin. However, also highly purified phospholipase C caused a membrane damage as measured by release of isotope through the plasma membrane. The release could be increased by including an optimal concentration of calcium ions in the incubation buffer, by treating the cells in a hypotonic medium and by simultaneous treatment with sublytic concentrations of Triton X-100. To our knowledge this is the first report of membrane damage on a live, intact, metabolizing human diploid cell caused by a highly purified phospholipase C. The results are in agreement with a dynamic membrane structure with the polar groups of a part of the phospholipids accessible at the membrane surface.     

Alteration of processive mechanism of native polynucleotide phosphorylase by fixation to solid matrix.

Native $\text{E. coli}$ polynucleotide phosphorylase can be covalently bound to BrCN activated Sepharose. The Sepharose bound enzyme retains 70 % of its initial activity in polymerisation of nucleoside diphosphate. The Km of the enzyme for the polymerisation reaction in comparison to the soluble enzyme is not affected by its linkage to a solid matrix. The phosphorolysis of an hexanucleotide by the Sepharose-bound enzyme is not affected either. However, the rate of phosphorolysis of a long chain polynucleotide is dramatically altered. The Km values for poly(A) or poly(U) are increased by two orders of magnitude. The decrease of affinity for polymeric substrate is accompanied by a significant modification of the known processive mechanism characteristic of the native soluble enzyme.     

Gastric inflammatory markers and interleukins in patients with functional dyspepsia, with and without Helicobacter pylori infection

Helicobacter pylori is the most important cause of gastritis, peptic ulcers and the development of gastric cancer. The chronic active inflammation is dominated by neutrophils, macrophages, lymphocytes and plasma cells. Several interleukins (IL-8, IL-10 and IFN-gamma) are involved in the inflammatory process in the gastric mucosa. The aim of this study was to investigate the gastric inflammation in patients with functional dyspepsia. Fifty-three consecutive patients were included and antral biopsies were obtained for histology, culture and immunohistochemistry. The sections were examined for the interleukins IL-4, IL-6, IL-8, IL-10 and IFN-gamma as well as for the cell markers CD4, CD8, CD14, Cd19, CD25 and CD30. Only CD4 and CD19 were significantly increased in patients with increased gastric inflammation and increased density of H. pylori. However, several of the examined markers (IFN-gamma, IL-8, IL-10 and CD14) showed a non-significant trend to be increased in patients with extensive gastric inflammation and high density of H. pylori. Therefore, an arbitrary index (IM11) for all the 11 immunological markers was made as an average value for each of the four morphological groups. For the four morphologically different groups of patients the values were 0.49, 0.77, 0.86 and 1.25, respectively. Significant increases in the index from none to moderate antral inflammation as well as the density of H. pylori were found (p<0.001). By using an index of inflammatory markers trends can be summarized and thereby significant which may be of importance when gastric inflammation is investigated in children and patients with functional dyspepsia.     

Characterization of the PCR inhibitory effect of bile to optimize real-time PCR detection of Helicobacter species

The inhibitory effect of human and porcine bile samples to detect Helicobacter DNA was studied by adding different concentrations of bile samples to PCR mixtures of six thermostable DNA polymerases containing cagA specific primers and Helicobacter pylori DNA. PCR products were amplified by using the Rotorgene system and SYBR Green I. Among the six DNA polymerases tested, rTth had the lowest sensitivity to bile inhibitors, whereas Taq and Tfl had the highest sensitivity. Bile proteins did not inhibit AmpliTaq DNA polymerase, whereas the fraction containing mainly bile acids and their salts inhibited the amplification capacity of AmpliTaq. Heating human bile at 98 degrees C and adding casein and formamide to the reaction mixture reduced the PCR inhibitory effect of bile. Therefore, a pre-PCR treatment based on dilution and heating of bile, adding casein and formamide to the reaction mixture of rTth DNA polymerase was found efficient to amplify DNA directly in bile.     

Formation of Bacteriolytic Enzymes in Batch and Continuous Culture of Staphylococcus aureus

The formation of bacteriolytic enzymes of Staphylococcus aureus, with special reference to strain M18, was investigated under a variety of conditions. The bacteriolytic activity was tested by using whole cells of Micrococcus lysodeikticus as a substrate. Complex media were required for production, and a Casein Hydrolysate-Yeast Extract medium (CCY(I)) was superior to Brain Heart Infusion and Trypticase Soy Broth. The optimal pH level for production was 7.0. Effective oxygenation and exchange of the beta-glycerophosphate of the CCY(I) medium for glucose increased the rates of growth and autolysis and the rate of appearance of extracellular bacteriolytic enzymes. However, the extracellular lytic activity decreased more rapidly at the end of the growth period than under the standard culture conditions. The appearance of inhibitor(s), probably derived from autolysis, might be responsible for this rapid decrease. The highest yields were obtained in a continuous process in which the activity was almost twice that of batch cultures grown under the same conditions. The bacteriolytic activity produced in continuous culture had a considerably increased stability in the purification process. The advantage of producing unstable bacterial proteins in continuous culture under controlled growth conditions is discussed.     

High-affinity binding of laminin by Helicobacter pylori: Evidence for a lectin-like interaction

Abstract Laminin, the major glycoprotein of basement membranes, was shown to be bound by the human gastric pathogen Helicobacter pylori . Binding of ¹²⁵I-laminin by strain 17874 was time-dependent, specific and saturable. Scatchard analysis of specific binding indicated about 2000 binding sites per cell with a dissociation constant of 8.5 pM. Treatment of the cells by heat (80°) and with proteolytic enzymes drastically reduced laminin binding, suggesting that the laminin receptors are surface proteins. Some highly glycosylated glycoproteins inhibited laminin binding by 50%. Furthermore, N -acetylneuraminyllactose decreased laminin binding by 70% and neuraminidase treatment of laminin by 50%, while a recombinant B1 chain of laminin, containing high-mannose type oligosaccharides, inhibited binding by only 25%. This suggests that terminal sialic acids on laminin compete for a specific sugar binding protein(s) on H. pylori cells.     

Binding to erythrocyte membrane glycoproteins and carbohydrate specificity of F41 fimbriae of enterotoxigenic Escherichia coli

Abstract Haemagglutination of enterotoxigenic Escherichia coli (ETEC) possessing F41 fimbriae was found to be inhibited by N -acetylgalactosamine. Other monosaccharides, such as N -acetylglucosamine, galactose and fucose were also inhibitors, although less effective than N -acetylgalactosamine. Purified F41 fimbriae bound to glycoproteins of human erythrocytes and glycophorin was found to act as an erythrocyte receptor for F41.     

A novel multi‐strain probiotic and synbiotic supplement for prevention of Clostridium difficile infection in a murine model

The protective effect of a multi‐strain probiotic and synbiotic formulation was evaluated in C57BL/6 mice infected with Clostridium difficile (CD) NAP1/027. Antibiotic‐treated mice were divided into the following four groups: Group 1, fed with a synbiotic formulation consisting of Lactobacillus plantarum F44, L. paracasei F8, Bifidobacterium breve 46, B. lactis 8:8, galacto‐oligosaccharides, isomalto‐oligosaccharides, and resistant starch; Group 2, fed with the same four probiotic strains as Group 1; Group 3, fed with the same prebiotic supplements as Group 1 for 7 days before CD infection; and Group 4 (control group) antibiotic treated and infected with NAP1/027 strain. Feces and cecal contents were collected for microbial cell viability, quantitative PCR (qPCR), toxin analyses and histopathology. Synbiotics‐ and probiotics‐fed mice showed a significant increase in total bifidobacteria (P < 0.05). The total lactobacilli count was increased in Group 1. Tests for cecal toxins were negative in Group 2 mice, whereas one sample each from Group 1 and 3 was positive. qPCR of cecal contents showed significant reduction in NAP1/027 DNA copies in Groups 1 and 2 and significantly higher numbers of B. breve 46, L. plantarum F44, and L. paracasei F8 in Groups 1 and 2 (P < 0.05); these changes were much less pronounced in Groups 3 and 4. Our findings indicate that the newly developed synbiotic or multi‐strain probiotic formulation confers protection against NAP1/027 infection in C57BL/6 mice. This holds promise for performing human studies.     

Description of Pyramimonas diskoicola sp. nov. and the importance of the flagellate Pyramimonas (Prasinophyceae) in Greenland sea ice during the winter–spring transition

Pyramimonas Schmarda is a genus of unicellular green flagellates, recorded in marine water and sea ice samples. Pyramimonas is within the prey size range of the most important protozoan grazers in Disko Bay, West Greenland, where this study took place. Despite the potential ecological importance, little is known about the occurrence of the genus. The aim of this study was to explore the biomass of Pyramimonas in developing stages of sea ice and in the water column. Pyramimonas colonized the early stages of sea ice, and the highest percent of Pyramimonas biomass was found in grease ice. The biomass of Pyramimonas was more than a magnitude higher within sea ice compared to the surface water. The results illustrate that Pyramimonas from the ice is an important contributor to the plankton community prior to the spring bloom. An undescribed species, Pyramimonas diskoicola sp. nov., was found. Based on morphology and ultrastructure, combined with molecular phylogeny inferred from the small-subunit SSU rDNA and the large-subunit chloroplast-encoded rbcL, the species was placed in subgenus Vestigifera. The cells possessed four flagella, measured 8.3 ± 2.6 μm in length and 5.1 ± 0.8 μm in width, and were characterized by an uplifted quadrant in the center of the box scales, not seen at any other Pyramimonas species. The phylogenetic analyses indicated P. diskoicola to be closely related to other polar sea ice species of Pyramimonas.