The role of microorganisms in biliary tract disease.

The biliary tract is normally sterile, but bile-tolerant bacteria are frequently isolated from patients with cholecystitis. Since the identification of about 25 Helicobacter species, some of which may grow in bile, studies have addressed the role of these organisms in primary biliary cirrhosis, primary sclerosing cholangitis, and cholelithiasis. Most of these bacteria show the presence of Helicobacter DNA or antigens in the bile tract and in liver samples. Altogether, data from studies on biliary and hepatic diseases, as well as pancreatic disorders, suggest that bile-tolerant Helicobacter species may induce a chronic infection with possible malignant transformation

Isolation and characterisation of a 17-kDa staphylococcal heparin-binding protein with broad specificity

A previous study reported the ability of staphylococci to bind heparin and heparin-dependent host growth factors. The present study isolated and identified heparin- and basic fibroblast growth factor (bFGF)-binding surface components of S. epidermidis strain RP12 and S. haemolyticus strain SM 131. The staphylococcal heparin-binding component(s) were purified by affinity chromatography on heparin-Sepharose and a major heparin-binding protein, here designated HBP, was identified by immunoblot in these two coagulase-negative staphylococcal (CNS) species. The HBP was shown to be acidic with an approximate pI of 4.6 and a molecular mass around 17 kDa. The binding of heparin to HBP was inhibited by heparin, fucoidan, pentosan polysulphate and various other sulphated polysaccharides, but not by non-sulphated compounds. However, the purified HBP from both S. epidermidis and S. haemolyticus revealed broad specificity, and also bound bFGF, thrombospondin, von Willebrand factor and, weakly, fibrinogen. The N-terminal sequences of the 17-kDa HBP from S. epidermidis and S. haemolyticus showed only limited identity. Comparison of the first 15 amino acid residues derived from either strain with known sequences in the protein databases revealed no close similarities. Taken together, these results suggest that the adhesion of at least some CNS to host sulphated glycosaminoglycans may be mediated by a previously uncharacterised group of surface proteins

The N-terminal of thrombospondin-1 is essential for coagulase-negative staphylococcal binding

Bacterial binding was studied to determine whether thrombospondin-1 (TSP) acts as a ligand in attachment of coagulase-negative staphylococci (CNS). Twenty-five of 27 CNS strains bound soluble TSP. Staphylococcus epidermidis J9P bound 125I-labelled TSP in a dose-dependent manner. Scatchard plot analysis of the binding of TSP by strain J9P revealed two Kd values of 6.4x10-95muM and 2.9x10-85muM. The binding structures of strain J9P were sensitive to protease and were resistant to heat treatment. Unlabelled TSP and recombinant von Willebrand factor inhibited binding of TSP by strain J9P, but other proteins or monosaccharides did not. Heparin inhibited binding of TSP to strain J9P and two other S. epidermidis strains, BD5703 and BD969. Fusion proteins of the type 1 repeats, type 2 repeats, type 3 repeats and C-terminal domain of TSP or the synthetic Arg-Gly-Asp peptide did not inhibit binding of TSP to bacteria. TSP promoted adhesion of S. epidermidis strains when it was immobilised on polymer surfaces. These results indicate that the specific interaction between CNS and TSP may contribute to bacterial adhesion on biomaterial surfaces. The N-terminal heparin-binding domain of TSP appears to be the major region for recognition by CNS

Binding of von Willebrand factor by coagulase-negative staphylococci

Coagulase-negative staphylococci (CNS) are the most common infectious micro-organisms isolated from prosthetic devices. To determine whether von Willebrand factor (vWF) acts as an adhesin in bacterial recognition, bacterial binding of recombinant vWF (rvWF) was studied. Eleven CNS strains, belonging to S. epidermidis, S. haemolyticus and S. hominis species, bound soluble rvWF, but to a lesser extent than S. aureus. S. epidermidis strain H2-W bound 125I-labelled rvWF in a dose-dependent manner. The binding could be inhibited by unlabelled rvWF and thrombospondin, but not by fibrinogen, vitronectin or the carbohydrates N-acetylgalactoseamine, d-galactose, d-glucose, and d-fucose. Pre-incubation of rvWF with type I collagen and Arg-Gly-Asp-Ser (RGDS) peptides did not inhibit binding, whereas pre-incubation of rvWF with heparin decreased binding significantly. The interaction between CNS and rvWF was sensitive to proteinase treatment of bacterial cells. CNS strains bound to immobilised rvWF an extent greater or equal to the positive control strain S. aureus Cowan I. rvWF binding structures from bacterial cell wall were detected by immunoblot. Cowan I strain had 140-, 90- and 38-kDa binding molecules. S. haemolyticus strain SM131 and S. epidermidis strain H2-W had two (120 and 60 kDa) and five (120, 90, 60, 52 and 38 kDa) binding molecules, respectively. Similar binding structures were formed when cell wall extracts from these strains were incubated with thrombospondin. These results indicate that specific ligand–receptor interaction between CNS and rvWF may contribute to bacterial adhesion and colonisation on biomaterial surfaces. Heparin-binding domains of rvWF might be the crucial regions for bacterial attachment. rvWF and thrombospondin may recognise similar molecules in staphylococcal cell wall extracts

Increased prevalence of seropositivity for non-gastric Helicobacter species in patients with autoimmune liver disease

Various Helicobacter species have been isolated from the stomach, intestinal tract and liver of a variety of mammalian and some avian species, and Helicobacter DNA has been detected in human bile and liver samples. An immunoblot assay was established to analyse serum antibody responses to non-gastric Helicobacter species in patients with autoimmune liver diseases, in comparison with healthy individuals. Sera from 36 patients with primary sclerosing cholangitis (PSC), 21 with primary biliary cirrhosis, 19 with autoimmune chronic hepatitis and 80 blood donors were analysed by immunoblot, using cell-surface proteins from Helicobacter pullorum, Helicobacter bilis and Helicobacter hepaticus as antigens. Prior to testing, sera were cross-absorbed with a whole-cell lysate of Helicobacter pylori. Antibody reactivity to various proteins of these three Helicobacter species was measured by densitometric scanning and results were processed by computer software to estimate antigenic specificity. Results were also compared with antibody response to H. pylori. For H. pullorum, reactivity to at least two of the proteins with molecular masses of 48, 45, 37, 20 and 16 kDa, for H. hepaticus, reactivity to the 76, 30 and 21 kDa proteins and for H. bilis, reactivity to the 22 and 20 kDa proteins, seemed to have high specificity. Positive immunoblot results with sera from patients with PSC to antigens of H. pullorum, H. bilis and H. hepaticus were found in 38, 22 and 25 of cases, respectively, and from patients with other autoimmune liver diseases, in 30, 22 and 22 of cases, respectively. Prevalence of serum antibodies to non-gastric Helicobacter species was significantly higher in patients with autoimmune chronic liver diseases than in healthy blood donors (P < 0.001). Increased antibody levels to enterohepatic Helicobacter species raise questions concerning an infectious role of these emerging bacterial pathogens in human autoimmune liver diseases

Two-year follow-up of Helicobacter pylori infection in C57BL/6 and Balb/cA mice.

Helicobacter pylori infection is associated with chronic gastritis, peptic ulcer disease, gastric adenocarcinoma and MALT lymphoma. We previously found high-grade lymphoma after 13 months' H. pylori infection in C57BL/6 mice. In this study we followed H. pylori infection by three different isolates in C57BL/6 and Balb/cA mice for 23 months. Six-week-old C57BL/6 and Balb/cA mice were infected with H. pylori strains 119p (CagA+, VacA+), SS1 (CagA+, VacA+) and G50 (CagA-, VacA-). Mice were followed at 2 weeks, 10 weeks and 23 months post-inoculation (p.i.) by culture, histopathology and serology. Strain G50 was only reisolated from mice 2 weeks p.i. There was no difference in colonization between strain 119p and SS1 at 10 weeks p.i., whereas SS1 gave 100% colonization versus 119p gave 50% 23 months p.i.. Interestingly, the inflammation score was higher in mice infected with strain 119p than with SS1 10-week p.i., and there were lymphoepithelial lesions in mice infected with strain 119p and G50 but not with SS1 at 23 months post-infection. Eight mice infected with strains 119p and G50 developed gastric lymphoma (grade 5 and 4). One C57BL/6 mouse infected with strain 119p developed hepatocellular carcinoma after 23 months. Immunoblot showed specific bands of 2633 kDa against H. pylori in infected mice, and two mice infected with strain SSI reacted with antibodies to the 120 kDa CagA toxin. Conclusion: A reproducible animal model for H. pylori-induced lymphoma and possibly hepatocellular carcinoma is described. Strain diversity may lead to different outcomes of H. pylori infection

A novel von Willebrand factor binding protein expressed by Staphylococcus aureus

When a shotgun phage-display library of Staphylococcus aureus Newman was affinity selected (panned) against recombinant von Willebrand factor (vWf), a novel von Willebrand factor binding protein (vWbp) was found. Experimental data indicate that the interaction between vWbp and vWf is very specific and mediated by a region of 26 aa residues in the C-terminal part of vWbp. vWbp has an N-terminal secretory signal sequence but no cell wall anchoring motif, suggesting a soluble extracellular location. Mature vWbp could be purified from the culture supernatant and the identity of the protein was confirmed by N-terminal sequencing. vWbp migrates with an apparent molecular mass of 66 kDa and the deduced protein consists of 482 aa. The gene encoding vWbp, named vwb, was present in all S. aureus strains investigated

Survival and synergistic growth of mixed cultures of bifidobacteria and lactobacilli combined with prebiotic oligosaccharides in a gastrointestinal tract simulator

Background: Probiotics, especially in combination with non-digestible oligosaccharides, may balance the gut microflora while multistrain preparations may express an improved functionality over single strain cultures. In vitro gastrointestinal models enable to test survival and growth dynamics of mixed strain probiotics in a controlled, replicable manner. Methods: The robustness and compatibility of multistrain probiotics composed of bifidobacteria and lactobacilli combined with mixed prebiotics (galacto-, fructo- and xylo-oligosaccharides or galactooligosaccharides and soluble starch) were studied using a dynamic gastrointestinal tract simulator (GITS). The exposure to acid and bile of the upper gastrointestinal tract was followed by dilution with a continuous decrease of the dilution rate (de-celerostat) to simulate the descending nutrient availability of the large intestine. The bacterial numbers and metabolic products were analyzed and the growth parameters determined. Results: The most acid- and bile-resistant strains were Lactobacillus plantarum F44 and L. paracasei F8. Bifidobacterium breve 46 had the highest specific growth rate and, although sensitive to bile exposure, recovered during the dilution phase in most experiments. B. breve 46, L. plantarum F44, and L. paracasei F8 were selected as the most promising strains for further studies. Conclusions: De-celerostat cultivation can be applied to study the mixed bacterial cultures under defined conditions of decreasing nutrient availability to select a compatible set of strains

High prevalence of Helicobacter Species detected in laboratory mouse strains by multiplex PCR-denaturing gradient gel electrophoresis and pyrosequencing.

Rodent models have been developed to study the pathogenesis of diseases caused by Helicobacter pylori, as well as by other gastric and intestinal Helicobacter spp., but some murine enteric Helicobacter spp. cause hepatobiliary and intestinal tract diseases in specific inbred strains of laboratory mice. To identify these murine Helicobacter spp., we developed an assay based on PCR-denaturing gradient gel electrophoresis and pyrosequencing. Nine strains of mice, maintained in four conventional laboratory animal houses, were assessed for Helicobacter sp. carriage. Tissue samples from the liver, stomach, and small intestine, as well as feces and blood, were collected; and all specimens (n = 210) were screened by a Helicobacter genus-specific PCR. Positive samples were identified to the species level by multiplex denaturing gradient gel electrophoresis, pyrosequencing, and a H. ganmani-specific PCR assay. Histologic examination of 30 tissue samples from 18 animals was performed. All mice of eight of the nine strains tested were Helicobacter genus positive; H. bilis, H. hepaticus, H. typhlonius, H. ganmani, H. rodentium, and a Helicobacter sp. flexispira-like organism were identified. Helicobacter DNA was common in fecal (86%) and gastric tissue (55%) specimens, whereas samples of liver tissue (21%), small intestine tissue (17%), and blood (14%) were less commonly positive. Several mouse strains were colonized with more than one Helicobacter spp. Most tissue specimens analyzed showed no signs of inflammation; however, in one strain of mice, hepatitis was diagnosed in livers positive for H. hepaticus, and in another strain, gastric colonization by H. typhlonius was associated with gastritis. The diagnostic setup developed was efficient at identifying most murine Helicobacter spp