Prebiotic preferences of human lactobacilli strains in co-culture with bifidobacteria and antimicrobial activity against Clostridium difficile.

AIM: To evaluate robustness, prebiotic utilization of Lactobacillus paracasei F8 and Lactobacillus plantarum F44 in mono- and co-cultures with Bifidobacterium breve 46 and Bifidobacterium animalis sub sp. lactis 8 : 8 and antimicrobial activity of co-culture against Clostridium difficile. METHODS AND RESULTS: The two Lactobacillus strains showed a high acid and bile tolerance. Lactobacillus plantarum F44 showed maximum growth in de Man Rogosa Sharpe basal broth with glucose and lactulose compared to growth in galacto-oligosaccharides (GOS) and isomalto-oligosaccharides (IMOS). In co-culture system, the amylolytic Bif. breve 46 stimulated the growth of a nonamylolytic Lact. paracasei F8, probably by producing intermediate metabolites of starch metabolism. A higher growth of four strains Lact. paracasei F8, Lact. plantarum F44, Bif. breve 46 and Bif. animalis ssp lactis 8 : 8 with different prebiotic combinations was found in a MRSC basal broth with SS (soluble starch) + IMOS + GOS and IMOS + GOS respectively. The two Lactobacillus strains exhibited a high antimicrobial activity against four clinical Cl. difficile strains and a hypervirulent NAP1/027strain and suppressed the toxin titres possibly through the production of organic acids and heat stable antimicrobial proteins when grown on glucose and through the production of acids when grown on prebiotics. Culture supernatants from synbiotic combinations inhibited the growth of the Cl. difficile NAP1/027 strain and its toxin titres. CONCLUSION: Lactobacillus paracasei F8, Lact. plantarum F44 exhibited potential probiotic properties. Further, the two Lactobacillus and two bifidobacteria strains were compatible with each other and exhibited high growth in co-cultures in presence of prebiotics and SS and antimicrobial activity against clinical Cl. difficile strains and a hypervirulent NAPI/027 strain. SIGNIFICANCE AND IMPACT OF THE STUDY: Results are promising for the development of a multi-strain synergistic synbiotic supplement for protection against Cl. difficile infection.

Participation of cell-surface hydrophobins for hemin binding in Helicobacter pylori.

Treatment of Helicobacter pylori cells with several chaotropic agents resulted in different degrees of inhibition in the binding of the bacteria to hemin and Congo-red dye. Polyanions also yielded a >50% inhibitory effect. Furthermore, hydrophobic interaction chromatography was used to determine the relative surface hydrophobicity of cell-associated proteins extracted with 3 mol/L urea, revealing proteins with a significant hydrophobic profile.

DNA of Helicobacter spp. and common gut bacteria in primary liver carcinoma.

BACKGROUND AND AIM: Gastric and enteric Helicobacter species have been associated with the pathogenesis of some extragastric diseases. METHODS: We retrospectively investigated the presence of DNA of Helicobacter species in samples of the cancer and the surrounding tumour-free liver tissues of patients with hepatocellular carcinoma (HCC, n=12) and cholangiocarcinoma (CC, n=13). The patients were from an area with low liver cancer incidence and with low hepatitis B and C prevalence. Patients with a benign liver disease (n=24) were included as controls. Paraffin-embedded liver samples were examined by a Helicobacter genus-specific PCR assay as well as group-specific PCR assays for Enterobacteriaceae, Bacteroides, Lactobacillus and Enterococcus. PCR products of positive samples were characterised by denaturing gradient gel electrophoresis (DGGE) and DNA sequencing. RESULTS: PCR assay detected Helicobacter DNA in seven of 12 (58%) and eight of 13 (62%) normal liver tissue specimens from HCC and CC patients, respectively. Two cancer samples from HCC patients were Helicobacter-positive but none of the CC cancers. In the control group, three of 24 (12.5%) patients with a benign liver condition were positive for Helicobacter species (p<0.01 compared to results of tumour-free liver tissue from the cancer patients). DGGE and DNA sequence analysis showed that 90% of the detected PCR products were "H. pylori-like". DNA of some other enteric bacteria was detected in the liver of one cancer patient and one control (4% of all patients). CONCLUSION: The presence of DNA of Helicobacter species in liver specimens, but not of other common gut bacteria, was associated with human hepatic carcinogenesis.

Presencia de un posible nuevo patógeno gástrico, Candidatus Wohne Africanus, en un paciente venezolano

Reportamos el caso de un paciente que sufre de síntomas recurrentes de dispepsia desde el año 1997, pero no esta infectado por Helicobacter pylori, el agente etiológico de la gastritis en humanos. El diagnostico de H. Pylori fue realizado por tres métodos: histología, test de ureasa rápido en biopsia gástrica y test de HpSA (Meridian Diagnostics, Italy) en heces. La detección de Candidatus W. africanus fue realizada a partir de ADN de jugo gastroesofagal colectado con un Enterotest (HDC, USA), utilizado un ensayo de PCR con cebadores específicos para el genero Helicobacter. El producto amplificado fue analizado mediante un gel de electroforesis en gradiente desnaturalizante (DGGE) y posteriormente fue secuenciado. Encontramos que este paciente está infectado con Candidatus Wolinella africanus, un posible nuevo patógeno del tracto digestivo humano, el cual ha sido reportado por primera y única vez en Diciembre 2003 en pacientes Surafricanos con cáncer de esófago.

We reported the case of a patient who suffers of recurrent symptoms of dyspepsia since 1997, but is not infected by Helicobacter pylori, the etiological agent of gastritis in humans. Diagnose of H. Pylori was made by three methods: histology, fast ureasa test in gastric biopsy and HpSA test (Meridian Diagnostics, Italy) in feces. The detection of Candidatus W. africanus was made from DNA of gastroesophageal juice collected with a Enterotest (HDC, USA), using a PCR test with specific boots for Helicobacter gender. The amplified product was analyzed by means of an electrophoresis gel in denatured gradient (DGGE) and later sequenced. We found that this patient is infected with Candidatus Wolinella africanus, a possible new pathogen of the human digestive tract, which has been reported for the first and only time in December 2003 in south African patients with esophageal cancer.

Serum antibodies to enterohepatic Helicobacter spp. in patients with chronic liver diseases and in a population with high prevalence of H. pylori infection.

BACKGROUND: Enteric Helicobacter species might be a risk factor for chronic liver and biliary tract diseases. AIMS: To analyse serum antibody levels to three enteric Helicobacter species in patients with various biliary tract and chronic liver diseases and compare results with corresponding parameters for an adult population group, known to have a high prevalence of Helicobacter pylori infection, and with healthy blood donors, to explore a possible association of enteric Helicobacter with chronic liver diseases. SUBJECTS: Sera of 90 patients with various chronic liver diseases, 121 Estonian adult persons and 68 blood donors were analysed. METHODS: Sera, previously tested for H. pylori were analysed for IgG to Helicobacter hepaticus, Helicobacter bilis and Helicobacter pullorum. ELISA was initially used for screening and exclusion of negative cases. Sera with positive ELISA results were further analysed by immunoblot. To remove cross-reactive antibodies between H. pylori and the enteric species, sera were pre-absorbed with lysed H. pylori cells. RESULTS: Liver patients showed a significantly higher seroprevalence to H. hepaticus and H. bilis, compared with the adult population group (p=0.0001 and 0.04, respectively), and to H. hepaticus, compared with blood donors (p=0.01). Patients with autoimmune hepatitis showed no significant antibody reactivity to the enteric Helicobacter spp. in contrast to patients with other chronic liver diseases. CONCLUSION: Patients with chronic liver diseases, except autoimmune hepatitis patients, showed increased antibody levels to H. bilis/H. hepaticus compared with the population and blood donors indicating a possible role of enteric Helicobacter in the natural course of chronic liver diseases. Immunoblot seems to be a promising method for serodiagnosis of infections with these fastidious pathogens.

Lectin histochemistry of the gastric mucosa in normal and Helicobacter pylori infected guinea-pigs.

Helicobacter pylori attaches via lectins, carbohydrate binding proteins, to the carbohydrate residues of gastric mucins. Guinea-pigs are a suitable model for a H. pylori infection and thus the carbohydrate composition of normal and H. pylori infected gastric mucosa was investigated by lectin histochemistry. The stomach of all infected animals showed signs of an active chronic gastritis in their mucosa, whereas no inflammation was present in the control animals. The corpus-fundus regions of the controls showed heterogeneous WGA, SNA-I, UEA-I and HPA binding in almost all parts of the gastric glands. While these lectins labelled the superficial mucous cells and chief cells heterogeneously, the staining of the parietal cells was limited to WGA and PHA-L. Mucous neck cells reacted heterogeneously with UEA-I, HPA, WGA and PHA-L. In the antrum, the superficial mucous cells and glands were stained by WGA, UEA-I, HPA, SNA-I or PHA-L. WGA, UEA-I, SNA-I and HPA labelled the surface lining cells strongly. The mucoid glands reacted heterogeneously with WGA, UEA-I, HPA, SNA-I and PHA-L. In both regions, the H. pylori infected animals showed similar lectin binding pattern as the controls. No significant differences in the lectin binding pattern and thus in the carbohydrate composition between normal and H. pylori infected mucosa could be detected, hence H. pylori does not induce any changes in the glycosylation of the mucosa of the guinea-pig. This unaltered glycosylation is of particular relevance for the sialic acid binding lectin SNA-I as H. pylori uses sialic acid binding adhesin for its attachment to the mucosa. As sialic acid binding sites are already expressed in the normal mucosa H. pylori can immediately attach via its sialic acid binding adhesin to the mucosa making the guinea-pig particularly useful as a model organism.

Prevalence of Helicobacter pylori infection among adult dyspeptic patients in Ethiopia.

In developing countries such as Ethiopia, where chronic gastritis and peptic-ulcer disease are the most common endoscopic findings, it is important to study the association between Helicobacter pylori infection and gastroduodenal diseases. Both invasive and non-invasive diagnostic methods were therefore used to investigate 300, consecutive, adult patients with dyspepsia, from the gastrointestinal clinic of Tikur Anbassa University Hospital, Addis Ababa. The apparent overall prevalence of H. pylori infection varied according to the detection method employed. Culture revealed H. pylori in only 69% of the patients but this pathogen appeared more common when rapid urease tests (71%), PCR-denaturating gradient gel electrophoresis (91%), histopathology (81%), silver staining (75%) or stool-antigen tests (81%) were employed. Antibodies to H. pylori were detected, both by enzyme immuno-assay (EIA) and immunoblotting, in approximately 80% of the patients, whether the antigens used were of a reference strain or from a local isolate of H. pylori. When some of the EIA-positive and EIA-negative sera were cross-absorbed with antigens of Campylobacter jejuni and re-tested by EIA, the H. pylori-positive sera remained positive and the negative sera remained negative. Dyspeptic patients in Ethiopia, like most of those previously observed elsewhere in Africa, are often infected with H. pylori. It is important that the management of these patients should not be hampered by the misinterpretation of the African epidemiology of this pathogen.

Identification of novel immunogenic proteins of Helicobacter pylori by proteome technology.

Cell surface proteins of the human gastric pathogen Helicobacter pylori, reference strain CCUG 17874, were extracted with acid glycine and fractionated by heparin affinity chromatography. The extracts were subsequently analysed using high-resolution two-dimensional gel electrophoresis (2-DE) and immunoblotting. Four proteins of low molecular masses (25-30 kDa) stained by Coomassie R-350, were identified by peptide ESI-MS/MS sequencing after in-gel tryptic digestion. The identified proteins were recognised by sera from H. pylori-infected patients. Two of them are now described for the first time as immunogenic proteins of which one protein was determined to be distinct from all H. pylori proteins previously described. In addition, the specificity of the identified peptides was evaluated using both 1-D and 2-D immunoblotting against a panel of sera from patients with various bacterial infections. The present identification of highly specific antigens of H. pylori will encourage the improvement of serological diagnostic tests to diagnose and monitor H. pylori infection.

Bovine anti-Helicobacter pylori antibodies for oral immunotherapy.

BACKGROUND: Passive immunization with orally administered antibodies against specific pathogens has previously been successfully used therapeutically in both animal and human studies. We employed a similar strategy for experimental treatment of mice infected with the gastric pathogen Helicobacter pylori. METHODS: An anti-H. pylori bovine colostral hyperimmune immunoglobulin preparation (BIC) was generated and its efficacy was tested in different in vitro experiments, such as binding to the Lewis(b) blood group antigen, inhibition of adherence of H. pylori to human gastric mucosa tissue sections in situ and in a haemagglutination assay. The BIC preparation was also given in the drinking water to H. pylori-infected mice. RESULTS: An inhibition of 95% of the binding of H. pylori to Lewis(b) glycoconjugate was observed in vitro. Furthermore, a blocking activity of almost 90% was observed when the BIC was preincubated with H. pylori bacteria. Finally, the BIC preparation inhibited the haemagglutination of H. pylori and human red blood cells. Seven of 40 (17.5%) mice remained infected in the treatment group as compared with 25 of 45 (55.5%) in the control group. Hence, the cure rate was 66%, P = < 0.001. The mean number of colonies in the antibody-treated mice where eradication was not successful was also reduced (P < 0.05). In trials using FVB/N transgenic Lewis(b) expressing mice, a cure rate of 50%-66% was observed. CONCLUSION: Bovine colostral antibodies against H. pylori can be generated in high titres, inhibit binding in vitro and can eradicate or reduce the number of bacteria in infected mice.

Increased risk of developing atrophic gastritis in patients infected with CagA+ Helicobacter pylori.

BACKGROUND: To clarify the possible role of CagA positive (CagA+) Helicobacter pylori strains in the development of atrophic gastritis, the prevalence of antibodies to H. pylori and CagA (120 kD protein) was studied among subjects with atrophic and non-atrophic gastritis. METHODS: The study population was randomly selected among 12,252 Finnish men who were screened for atrophic corpus gastritis with serum pepsinogen I-assay (S-PGI). S-PGI level was used as a selection criterion. Group A consisted of 295 subjects with S-PGI <25 microg/l (low), group B of 320 subjects with S-PGI 25-100 microg/l (normal) and group C of 338 subjects with S-PGI >100 microg/l (high). Antibodies to H. pylori were measured with EIA and immunoblot analysis and antibodies to CagA with immunoblot analysis. Endoscopical and histological examinations were performed for 203 patients from group A. RESULTS: The prevalence of antibodies to H. pylori was significantly lower in group B than in groups A or C (P < 0.0001, chi-squared test). There was a significant association between the prevalence of antibodies to CagA and the lowered level of S-PGI (P < 0.0001, Jonckheere-Terpstra trend test). There was also a linear decrease in the prevalence of antibodies to CagA as the atrophic corpus gastritis became more severe (P < 0.0001, linear-by-linear trend test). CONCLUSION: The presence of antibodies to CagA seems to be associated with development of atrophic corpus gastritis.

Inhibition of Helicobacter pylori infection by bovine milk glycoconjugates in a BAlb/cA mouse model.

The attachment of Helicobacter pylori to the human gastric mucosa is a complex process involving several specific structures recognised by the cell surface receptors. Sialylated multivalent high mol. wt glycoproteins have been shown to inhibit H. pylori sialic acid-specific haemagglutination. This study explored whether sialylated glycoconjugates from bovine milk could inhibit an experimental H. pylori infection in a mouse model. BALB/cA mice (6-8 weeks old) were inoculated with a mouse-passaged H. pylori strain 317p. Four weeks after infection the mice were given lactoferrin (iron-free LF or 20% iron-saturated LF) or bovine milk fat globule membrane fractions (MFGM or defatted MFGM) orally (400 mg/kg body weight) once daily for 10 days and then killed to examine for bacterial colonisation and gastritis. Mice treated with iron-free LF, 20% iron-saturated LF, MFGM or defatted MFGM showed 30%, 10%, 20% or 20% healing rates, respectively, when compared with the H. pylori-infected control. Gastric colonisation by H. pylori was remarkably decreased in all mice treated with bovine milk glycoconjugates and the inflammation score was also significantly lower in treated mice than in infected control animals. The fact that there was no significant difference between iron-free LF and iron-saturated LF or MFGM and defatted MFGM suggested that iron is not crucial for inhibition of H. pylori by lactoferrin and that the lipid part of MFGM is not important for anti-H. pylori activity. In conclusion, bovine milk glycoconjugates showed potencies to inhibit H. pylori infection in this mouse model and, therefore, could be considered as candidates for non-antibiotic strategies against H. pylori infection in man.

Helicobacter pylori vacuolating cytotoxin binding to a putative cell surface receptor, heparan sulfate, studied by surface plasmon resonance.

The Helicobacter pylori vacuolating cytotoxin or VacA toxin is a major virulence factor in H. pylori infection and type B gastritis. We predicted heparin/heparan sulfate (H/HS) binding properties of the 58-kDa subunit of VacA cytotoxin using bioinformatics tools and showed this by surface plasmon resonance (SPR)-based biosensor studies. Putative H/HS binding peptides were synthesized and binding to HS was shown by SPR in the absence or presence of trifluoroethanol. We found that a recombinant cytotoxin VacA polypeptide binds to surface-immobilized HS and propose that HS might be a receptor/co-receptor for H. pylori VacA cytotoxin.

Infection with cagA- and vacA-positive and -negative strains of Helicobacter pylori in a mouse model.

To study the role of cytotoxin-associated protein (cagA) and vacuolating cytotoxin (vacA) in Helicobacter pylori infection in an experimental murine model, mice were infected with seven strains with different cagA and vacA status. Groups of 10 NMRI mice were challenged and were killed 5 weeks later. In a second study, 20 mice were challenged with a mixture of the same seven strains and killed 1, 3, 15 and 17 weeks post-inoculation. All seven strains were found to colonize the mice for the 5-week experimental period. Animals infected with vacA-positive strains, regardless of cagA status, showed an elevation of antibody titers. Two cagA-negative and vacA-positive strains and one cagA- and vacA-positive strain were found to 'take over' in the mixed infection as analyzed by the randomly amplified polymorphic DNA-polymerase chain reaction technique and in one mouse stomach we found coexistence of two of the strains. We found no evidence of the different strains colonizing different parts of the stomach.

A potential double role of anti-Lewis X antibodies in Helicobacter pylori-associated gastroduodenal diseases.

In this study, we found Lewis X (Le(x)) determinants on 68% of Helicobacter pylori isolates from patients with chronic gastroduodenal diseases. Anti-Le(x) IgG were detected more frequently in the sera from dyspeptic children and adults (45 and 46%), with or without proved (culture) H. pylori infection, than in the sera from healthy individuals (14% and 25%). In contrast, the prevalence of anti-Le(x) IgM was higher in the groups of healthy individuals than in the groups of dyspeptic patients. Moreover, anti-Le(x) monoclonal antibody of IgM class enhanced the uptake of Le(x)(+) but not Le(x)(-) H. pylori isolates by phagocytes. In the sera from some dyspeptic patients, we detected Le(x)-anti-Le(x) IgG immune complexes (Le(x) ICs). There was a great difference between children and adults as regards the presence of Le(x) ICs. The immune complexes were found in the sera from nine out of 29 (27%) H. pylori-infected and three out of eight (37%) uninfected adult dyspeptic patients. In comparison, Le(x)-anti-Le(x) IgG ICs were detected only for two out of 18 (11%) H. pylori-infected children. Le(x) ICs were not found in the sera from healthy individuals. Our results suggest that anti-Le(x) IgM may play a protective role in H. pylori infections. In contrast, anti-Le(x) IgG and particularly Le(x)-anti-Le(x) IgG ICs might contribute to the pathogenesis of chronic H. pylori infections.

Seropositivity to Helicobacter pylori heat shock protein 60 is strongly associated with intensity of chronic inflammation, particularly in antrum mucosa: an extension of an 18-year follow-up study of chronic gastritis in Saaremaa, Estonia.

Helicobacter pylori is a cause of chronic gastritis and leads to development of atrophy in some cases. There is evidence that the heat shock protein 60 (HSP60) of H. pylori is involved in induction of chronic inflammation. Seroprevalence of IgG antibodies to H. pylori HSP60 in an adult cohort from Saaremaa, Estonia (68 persons, median age 57 years), with a high prevalence of antibodies to cell surface proteins of H. pylori (92%) and a well characterized dynamics of chronic gastritis in an 18-year follow-up study, was tested using purified H. pylori HSP60 at a concentration of 1 microg ml(-1) with ELISA. The state of the gastric mucosa and the presence of H. pylori in histological sections in the samples of 1979 and 1997 were assessed in accordance with the Sydney system. Seropositivity for H. pylori HSP60 was 65%. Immunological response to H. pylori HSP60 is associated with the morphological presence of H. pylori in the antrum and corpus (P=0.01) and is strongly correlated with the grade of chronic inflammation, particularly in the antrum mucosa (r=0.34; P=0.003; OR=5.97 (95% CI 1.21-29.3)), but is not associated with development of atrophy during 18 years of follow-up, or with the activity of gastritis. This finding supports the evidence that immunological response to H. pylori HSP60 may play a role in triggering of the inflammatory process in the gastric mucosa.

Comparative study of Helicobacter pylori infection in guinea pigs and mice - elevation of acute-phase protein C3 in infected guinea pigs.

Eighteen Dunkin-Hartley guinea pigs and 50 NMRI mice were inoculated with Helicobacter pylori and the infection followed by culture, histopathology, antibody response, and plasma levels of the acute-phase proteins albumin, C3, and transferrin for up to 7 weeks. The immune response to H. pylori surface proteins was studied by an enzyme immunoassay (EIA) and Western immunoblot and the plasma levels of albumin, C3, and transferrin were analyzed by single radial immunodiffusion. Guinea pigs had a more severe gastritis and a higher EIA immune response than NMRI mice. Serum C3 levels were elevated in infected guinea pigs after 3 and 7 weeks indicating a systemic inflammatory response and a possible link between H. pylori infection and extragastric manifestations such as vasculitis associated with atherosclerosis. Serum cholesterol levels were analyzed in guinea pigs at 7 weeks and indicated a higher level in H. pylori-infected than in control animals, but this difference was not statistically significant.