Helicobacter pylori-associated gastritis in mice is host and strain specific.

The vacA and cagA geno- and phenotypes of two mouse-adapted strains of Helicobacter pylori, SS1 and SPM326, were determined. The SS1 strain, which had the cagA+ and vacA s2-m2 genotype, induced neither vacuole formation in HeLa cells nor interleukin-8 (IL-8) production in KATO III cells. In contrast, H. pylori SPM326, with the cagA+ and vacA s1b-m1 genotype, induced vacuoles as well as IL-8 production in vitro. Furthermore, a spontaneous mutant of SPM326, which produced a vacuolating cytotoxin but was not able to induce IL-8 production (SPM326/IL-8(-)), was detected. C57Bl/6 and BALB/c mice were infected with these three strains to investigate the colonization pattern and the effect on the immune response in vivo. The SS1 strain colonized the stomachs of all mice in large numbers which remained constant over time. Colonization with the SPM326/IL-8(+) and SPM326/IL-8(-) strains was lesser, or even absent, and decreased over time. At 5 weeks postinoculation all three H. pylori strains induced a mild increase of neutrophil count in the gastric corpus of C57Bl/6 mice, which disappeared by 12 weeks. At both 5 and 12 weeks postinoculation C57Bl/6 mice colonized with SPM326/IL-8(+) showed an increased expression of major histocompatibility complex (MHC) class II antigen in the cardia which was accompanied by an increased number of T cells. C57Bl/6 mice that were infected with SS1 and SPM326/IL-8(-) did not show chronic inflammation. BALB/c mice colonized with SS1 and SPM326/IL-8(-) also showed an increase in neutrophil count at 5 weeks, which normalized again by 12 weeks postinoculation. At this time point SS1-infected mice showed inflammation in the corpus and antrum. At these sites an increased expression of MHC class II antigens and an increased number of T cells were observed. Although small lymphoid follicles were already observed 5 weeks after inoculation with SS1, their incidence as well as their number was increased at 12 weeks. These results show that inflammation induced by H. pylori depends both on the bacterial strain and the host.

Neutrophil-activating protein mediates adhesion of Helicobacter pylori to sulfated carbohydrates on high-molecular-weight salivary mucin.

The in vitro binding of surface-exposed material and outer membrane proteins of Helicobacter pylori to high-molecular-weight salivary mucin was studied. We identified a 16-kDa surface protein which adhered to high-molecular-weight salivary mucin. This protein binds specifically to sulfated oligosaccharide structures such as sulfo-Lewis a, sulfogalactose and sulfo-N-acetyl-glucosamine on mucin. Sequence analysis of the protein proved that it was identical to the N-terminal amino acid sequence of neutrophil-activating protein. Moreover, this adhesin was able to bind to Lewis x blood group antigen.

Human serum antibody response against iron-repressible outer membrane proteins of Helicobacter pylori.

In Helicobacter pylori, in vitro iron limitation induces the expression of several iron repressible outer membrane proteins (IROMPs), which are not expressed under normal growth conditions. To substantiate their proposed role in virulence of H. pylori, we determined whether these IROMPs are also expressed in vivo. Therefore, we tested whether sera of patients with H. pylori infection contained antibodies against IROMPs. All sera from 20 H. pylori positive patients showed a clear immune response against a 77 kDa heme-binding IROMP in an immunoblot assay. Antibody responses against the other IROMPs were also found, but with lower frequencies. Serum samples from 18 patients negative for H. pylori infection did not show any immunoreactivity with IROMPs. These results indicate that the IROMPs of H. pylori are immunogenic and are expressed in vivo.

Mechanism of clarithromycin resistance in clinical isolates of Helicobacter pylori.

Seventy-three Helicobacter pylori-positive patients were treated with a combination of clarithromycin and ranitidine in order to eradicate the bacterium. Eradication was successful in 79.5%. In 15 patients eradication failed, and in 11 cases this was due to clarithromycin resistance. In one patient the infecting strain was resistant at the onset of treatment, while in the remaining 10 patients resistance developed during therapy. These isolates had also become resistant to various other antibiotics. Random amplified polymorphic DNA and restriction fragment end-labeling analysis of the isolates showed close genetic relatedness between pre- and post-treatment isolates, indicating that resistance was the result of selection of variants of the infecting strain rather then infection with an exogenous resistant strain. Nucleotide sequence comparisons revealed that all resistant isolates had a single base pair mutation in the 23S rRNA. Since this single point mutation results in co-resistance to various antibiotics at high frequencies, caution should be taken when using clarithromycin as a single antibiotic.

Utilization of haem from the haptoglobin-haemoglobin complex by Bacteroides fragilis.

Possession of specialized iron acquisition systems is a prerequisite for the survival of pathogenic bacteria in their host. The purpose of this study was to determine whether Bacteroides fragilis, a clinically important Gram-negative anaerobic bacterium, possesses a specific haem-uptake system. Growth studies indicated that this microorganism can utilize haem from either haemoglobin or haptoglobin-haemoglobin as its sole source of iron. Iron-repressible haem-binding protein complexes (HBP complexes), involved in the uptake of haem from haptoglobin-haemoglobin were detected by means of lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS-PAGE). Four polypeptides of approximately 60, 58, 49 and 35 kDa, which are part of these HBP complexes, were identified as haem-binding proteins. A 44 kDa iron-repressible outer-membrane protein is needed for a functional HBP complex, but the exact role of this protein in the uptake of haem is still unknown.

Human immune response to an iron-repressible outer membrane protein of Bacteroides fragilis.

Under conditions of iron starvation, Bacteroides fragilis expresses various iron-repressible outer membrane proteins (IROMPs). A 44-kDa protein appears to be one of the major outer membrane proteins (OMPs) in B. fragilis under iron stress and plays a role in heme uptake by this bacterium. To determine whether the 44-kDa IROMP of B. fragilis is expressed in vivo and whether this protein is immunogenic, we used Western immunoblotting to examine serum samples from patients with an infection caused by Bacteroides species. All the serum samples from patients and from normal controls showed reactivity with several proteins of B. fragilis. Only serum samples from patients infected with B. fragilis showed immunoreactivity with the 44-kDa protein. We also used a rat infection model to study the immune response against this protein during the process of an intra-abdominal infection in these animals. During the first 8 days of infection a gradual increase of antibodies to the 44-kDa protein in the rat was detected. These results suggest that the 44-kDa IROMP is expressed in vivo, since it induces an antibody response in patients and animals. We also analyzed 85 strains of the B. fragilis group for the presence of proteins antigenically related to the B. fragilis 44-kDa protein. The data indicate that this protein was conserved in B. fragilis strains and was absent in the other bacterial strains tested.

Iron-regulated outer membrane protein of Bacteroides fragilis involved in heme uptake.

Under iron starvation, Bacteroides fragilis expresses various iron-regulated outer membrane proteins. In this study, a deferrated minimal medium was used in growth experiments, and the role of one of these iron-regulated outer membrane proteins (a 44-kDa protein) in an iron uptake mechanism which acquires iron from heme compounds was elucidated. When a specific 44-kDa protein antiserum was used in a medium with heme as the only iron source, growth inhibition was observed. These results demonstrate that the 44-kDa outer membrane protein plays an important role in the uptake of heme in B. fragilis.

Influence of rosmarinic acid on opsonization and intracellular killing of Escherichia coli and Staphylococcus aureus by porcine and human polymorphonuclear leucocytes.

The influence of rosmarinic acid on the function of porcine and human polymorphonuclear leucocytes was tested. Rosmarinic acid inhibited the chemiluminescence of PMNL, induced by preopsonized Zymosan or phorbol myristate acetate. The killing of Escherichia coli was inhibited by rosmarinic acid at a concentration of 2 mM, but not that of Staphylococcus aureus. The inhibition of the killing was due to an impaired opsonization, caused by an adverse influence of rosmarinic acid on complement activation. Direct effects of rosmarinic acid on the killing mechanisms of PMNL were not observed.

Pathogenic synergy between Escherichia coli and Bacteroides fragilis or B. vulgatus in experimental infections: a non-specific phenomenon.

The virulence of Bacteroides fragilis and B. vulgatus for mice was compared in a skin-infection model. These strains were also tested for pathogenic synergy in mixed infections with Escherichia coli. Strains of B. fragilis were generally more virulent than strains of B. vulgatus and, with one exception, the effect of Bacteroides strains in mixed infections merely reflected their inherent virulence.

Pathogenic synergy between Escherichia coli and Bacteroides fragilis: studies in an experimental mouse model.

An animal model is described for quantitative evaluation of pathogenic synergy between Escherichia coli and Bacteroides fragilis in which adjuvants were not required for abscess formation. Two sets of strains of E. coli and B. fragilis isolated from human wound infections were tested. Pathogenic synergy was observed in only one of the two combinations and was dependent on properties of E. coli.

The role of different K antigens of Escherichia coli in phagocytosis by polymorphonuclear leukocytes.

The importance of K antigens from six strains of Escherichia coli for the interaction with polymorphonuclear leukocytes (PMNL) was studied. The major factor influencing this interaction was the ability of strains to activate complement by the classical route during opsonisation, this process being reduced for most K-positive strains. Interference of K antigens with the functioning of common pili as adhesions of eukaryotic cells was not observed nor a toxic effect of K antigens on PMNL.

K antigens of Escherichia coli and virulence in urinary-tract infection: studies in a mouse model.

The importance of K antigens of Escherichia coli as virulence factors was studied by comparing groups of mice given either strains of E. coli isolated from urinary tract infection in humans or mutant strains differing only in the absence of the K antigen. K antigens proved to be of minor importance for mouse nephropathogenicity; however, with the exception of the K(A) antigen, they contributed substantially to deaths attributed to more general infection. Possible mechanisms for the virulence of strains with K antigens are discussed in terms of the bactericidal effect of serum and phagocytosis.