Evidence for a primate origin of zoonotic Helicobacter suis colonizing domesticated pigs.

Helicobacter suis is the second most prevalent Helicobacter species in the stomach of humans suffering from gastric disease. This bacterium mainly inhabits the stomach of domesticated pigs, in which it causes gastric disease, but it appears to be absent in wild boars. Interestingly, it also colonizes the stomach of asymptomatic rhesus and cynomolgus monkeys. The origin of modern human-, pig- or non-human primate-associated H. suis strains in these respective host populations was hitherto unknown. Here we show that H. suis in pigs possibly originates from non-human primates. Our data suggest that a host jump from macaques to pigs happened between 100 000 and 15 000 years ago and that pig domestication has had a significant impact on the spread of H. suis in the pig population, from where this pathogen occasionally infects humans. Thus, in contrast to our expectations, H. suis appears to have evolved in its main host in a completely different way than its close relative Helicobacter pylori in humans.

Detection of Helicobacter species in the gastrointestinal tract of ringtail possum and koala: possible influence of diet, on the gut microbiota.

The presence of Helicobacter spp. was examined in the liver and in different regions of the gastrointestinal tract (GIT) including the stomach, 3 cm above ileum, ileum, caecum, colon and rectum of 10 ringtail possums (RTPs) and 3 koalas using a combination of microscopy, culture and PCR. Helicobacter was detected in the distal end of the GIT of 7 of 10 RTPs by direct PCR and in all (10/10) RTPs by nested PCR. Five 'S' shaped isolates with bipolar sheathed flagella were isolated from the lower bowel of 3 of the 10 RTPs. 16S rRNA sequence analysis of these 5 isolates confirmed them as potentially novel Helicobacter species. No Helicobacter species were cultured from the koalas, however Helicobacter DNA was detected, in the majority of liver and/or stomach samples of the three koalas and in the colonic region of one koala, using nested PCR. The 16S rRNA gene was sequenced directly from DNA extracted from the homogenised livers and mucus scrapings of the stomach from koala 1 and were confirmed to be Helicobacter species. Based on histopathological examination of sections from the liver and intestine no evidence of infection could be related to the presence of helicobacters in either the RTP or koala. Based on our results, it is possible that diet may influence the detection of Helicobacter species; however this required further investigation.

Detection, isolation, and characterization of helicobacter species from the gastrointestinal tract of the brushtail possum.

The presence of Helicobacter species in Australian marsupials was examined systematically using microscopy, culture, and PCR in different regions of the gastrointestinal tract (GIT) and in the liver of brushtail possums (BTPs) (Trichosurus vulpecula), a common Australian marsupial that feeds on eucalyptus leaves. The spatial distribution of Helicobacter species in the GIT sections also was examined microscopically in silver-stained sections and by fluorescent in situ hybridization (FISH) using a Helicobacter genus-specific probe. Helicobacter species were found colonizing the lower bowel of all BTPs studied. Good agreement was observed between the detection of Helicobacter species using culture and PCR, which was supported by the microscopic examination of silver-stained sections and FISH. The lower bowel of BTPs were colonized by one to three morphologically different (a comma-shaped species with no apparent flagella, a fusiform-shaped species entwined with periplasmic fibers and a bipolar sheathed flagella, and an S-shaped species with bipolar sheathed flagella) and potentially novel Helicobacter species, as well as in one case with a potentially novel Campylobacter species, which was a tightly coiled rod with bipolar unsheathed flagella. The isolation and characterization of these Helicobacter species in BTPs provides important information regarding the specific natural niche of these bacteria and their corelationship within their host, and it increases our understanding of the ecology of Helicobacter species.

Investigation of the immunomodulatory effects of Lactobacillus casei and Bifidobacterium lactis on Helicobacter pylori infection.

BACKGROUND: Lactobacillus and Bifidobacterium species have shown beneficial effects in the treatment of Helicobacter pylori infection; however, the mechanisms behind such effects are not fully understood. In this study, we have investigated the immunomodulatory effects of probiotics in a mouse model of H. pylori infection. MATERIALS AND METHODS: H. pylori-infected C57BL/6 mice were treated with L. casei L26, B. lactis B94, or no probiotics for 5 weeks, respectively. Mice not infected with H. pylori were included as normal controls. Gastric histology, protein levels of interleukin (IL)-1beta, IL-10, IL-12/23p40, and H. pylori colonization density in the gastric tissues, as well as H. pylori-specific antibodies were examined. RESULTS: In mice receiving L. casei L26 and B. lactis B94, gastric neutrophil infiltration and IL-1beta were significantly decreased and IL-10 was significantly increased as compared with mice receiving no probiotics. In mice receiving B. lactis B94, IL-12/23p40 was significantly increased and H. pylori IgG was significantly reduced as compared with mice receiving no probiotics. No significant difference of H. pylori colonization was observed among the three groups of mice. CONCLUSION: The reduced level of IL-1beta and neutrophil infiltration observed in mice infected with H. pylori following treatment with L. casei L26 and B. lactis B94 resulted from a modulation of immune response rather than a decrease of H. pylori colonization. Furthermore, B. lactis B94 has the intrinsic ability to promote a Th1 immune response through an increase in IL-12/IL-23.

Gene expression profiling in Helicobacter-induced MALT lymphoma with reference to antigen drive and protective immunization.

We have previously shown that long-term infection of BALB/c mice with gastric Helicobacter species results in the development of histopathological lesions that resemble those seen in patients diagnosed with gastric mucosa associated lymphoid tissue (MALT) lymphoma. This paper describes analysis of this disease at the molecular level through the use of microarray technology and immunohistochemical staining. We were able to monitor the genetic changes in the gastric mucosa characterized by distinct transcriptional signatures and correlate these with histological changes as the infection progressed from a chronic inflammatory infiltrate through to MALT lymphoma. This model system also enabled us to further dissect the role of antigen presentation and prophylactic immunization in the disease process. Antimicrobial therapy to eradicate the antigen correlated with significant reduction in pathology and major changes in the gene expression profile. Subsequent reintroduction of the antigen resulted in rapid tumor development which correlated with an increase in aggressively proliferating cells and changes in the cellular composition of the tumor. The response in vaccinated animals showed that the protected animals exhibited a strikingly different transcriptional profile compared to those of non-protected or control mice, indicating that the vaccination targeted the appropriate site leaving a long-lasting signature. The genes which were most significantly up-regulated included a number of adipocyte-specific factors, such as fat-cell specific cytokines and adipocyte surface markers. This study allowed for us to highlight the significance of antigen presentation in this disease and to hypothesis mechanisms associated with protective immunity.

Identification and characterisation of ssrA in members of the Helicobacter genus.

Bacterial ssrA encodes tmRNA that functions both as a tRNA and an mRNA to rescue the stalled ribosome on defective mRNAs. In this study, ssrA was identified in four gastric species of Helicobacters and four enterohepatic species of Helicobacters. The tag peptide of 14 amino acids encoded by ssrA showed a pattern of Val(1)Ala(13) in gastric species, a pattern of Ala(1)Val(13 )in enterohepatic species, in contrast to the pattern of Ala(1)Ala(13) in W. succinogenes and C. jejuni, which are closely related to helicobacters. Phylogenetic analysis and the patterns of the tag peptide suggest that the Helicobacter genus could be separated into two genera. High conservation of ssrA in H. pylori was observed. The annotated ORF HP0784 in H. pylori, which largely overlaps ssrA, is unlikely to be functional. H. pylori ssrA interestingly expressed a large and a small tmRNA molecule.

Urea, fluorofamide, and omeprazole treatments alter helicobacter colonization in the mouse gastric mucosa.

BACKGROUND: Helicobacter pylori is a causative agent of gastric and duodenal ulcers and gastric cancer. Its urease enzyme allows survival in acid conditions and drives bacterial intracellular metabolism. We aimed to investigate the role of urease in determining the intragastric distribution of Helicobacter species in vivo. MATERIALS AND METHODS: The C57BL/6 mouse model of gastritis was used for infection with Helicobacter felis (CS1) or H. pylori (SS1). Urease-modulating compounds urea and/or fluorofamide (urease inhibitor) were administered to mice over 7 days. Concurrent gastric acid inhibition by omeprazole was also examined. Bacterial distribution in the antrum, body, antrum/body, and body/cardia transitional zones was graded "blindly" by histologic evaluation. Bacterial colony counts on corresponding tissue were also conducted. RESULTS: Urease inhibition by fluorofamide decreased H. pylori survival in most gastric regions (p < .05); however, there were no marked changes to H. felis colonization after this treatment. There was a consistent trend for decreased antral colonization, and an increase in antrum/body transitional zone and body colonization with excess 5% or 6% (w/v) urea treatment. Significant reductions of both Helicobacter species were observed with the co-treatment of urea and fluorofamide (p < .05). Collateral treatment with omeprazole did not alter H. pylori colonization patterns caused by urea/fluorofamide. CONCLUSIONS: Urease perturbations affect colonization patterns of Helicobacter species. Combined urea and fluorofamide treatment reduced the density of both Helicobacter species in our infection model.

The role of antigenic drive and tumor-infiltrating accessory cells in the pathogenesis of helicobacter-induced mucosa-associated lymphoid tissue lymphoma.

Gastric B-cell lymphoma of mucosa-associated lymphoid tissue type is closely linked to chronic Helicobacter pylori infection. Most clinical and histopathological features of the tumor can be reproduced by prolonged Helicobacter infection of BALB/c mice. In this study, we have addressed the role of antigenic stimulation in the pathogenesis of the lymphoma by experimental infection with Helicobacter felis, followed by antibiotic eradication therapy and subsequent re-infection. Antimicrobial therapy was successful in 75% of mice and led to complete histological but not "molecular" tumor remission. Although lympho-epithelial lesions disappeared and most gastric lymphoid aggregates resolved, transcriptional profiling revealed the long-term mucosal persistence of residual B cells. Experimental re-introduction of Helicobacter led to very rapid recurrence of the lymphomas, which differed from the original lesions by higher proliferative indices and more aggressive behavior. Immunophenotyping of tumor cells revealed massive infiltration of lesions by CD4(+) T cells, which express CD 28, CD 69, and interleukin-4 but not interferon-gamma, suggesting that tumor B-cell proliferation was driven by Th 2-polarized, immunocompetent, and activated T cells. Tumors were also densely colonized by follicular dendritic cells, whose numbers were closely associated with and predictive of treatment outcome.

Mucispirillum schaedleri gen. nov., sp. nov., a spiral-shaped bacterium colonizing the mucus layer of the gastrointestinal tract of laboratory rodents.

The mammalian gastrointestinal tract is covered by a layer of mucus that can harbour a range of bacterial species specifically adapted to colonize this ecological niche. Examination of 110 bacterial isolates cultivated from the gastrointestinal tract of 23 mice revealed the presence of a subgroup of 30 isolates that did not correspond genetically with genera commonly associated with this site, i.e. members of the epsilon-Proteobacteria such as Helicobacter and Campylobacter species. Instead this group of isolates was found to lie within the phylum Deferribacteres, a completely distinct lineage in the domain Bacteria. There was a high level of consensus in results obtained from the phenotypic and genotypic characterization of a number of the isolates, which showed they were distinct from other members of the Deferribacteres. As such, they are proposed to constitute a new genus and species, Mucispirillum schaedleri gen. nov., sp. nov. These organisms are anaerobic, Gram-negative, spiral-shaped rods with bipolar flagella. The type strain is HRI I17(T) (= ATCC BAA-1009(T) = ACM 5223(T)).

Description of 'Candidatus Helicobacter heilmannii' based on DNA sequence analysis of 16S rRNA and urease genes.

While Helicobacter pylori is accepted as the major bacterial agent of gastric disease in humans, some patients and many animals are infected with a larger, tightly helical-shaped bacterium previously referred to as 'Helicobacter heilmannii' or 'Gastrospirillum hominis'. Taxonomic classification of these bacteria has been hampered by the inability to cultivate them in vitro and by the inadequate discriminatory power of 16S rRNA gene sequence analysis. This study describes the detection and phylogenetic analysis of 26 different gastrospirillum isolates from humans and animals, which incorporates sequence data based on the 16S rRNA and urease genes. Fifteen gastrospirilla detected in humans, primates and pigs clustered with 'Candidatus Helicobacter suis', thus expanding the host range for this organism. By comparison, based on 16S rRNA data, the remaining 11 gastrospirilla could not be differentiated from Helicobacter felis, Helicobacter bizzozeronii and Helicobacter salomonis. However, urease gene sequence analysis allowed for the discrimination of this latter group into four discrete clusters, three of which contained the above recognized species. The fourth cluster contained isolates from human and feline hosts, and should provisionally be considered a unique bacterial species, for which the name 'Candidatus Helicobacter heilmannii' is proposed.

Chronic Helicobacter pylori infection with Sydney strain 1 and a newly identified mouse-adapted strain (Sydney strain 2000) in C57BL/6 and BALB/c mice.

The mouse model of Helicobacter pylori-induced disease using Sydney strain 1 (SS1) has been used extensively in Helicobacter research. Herein we describe the isolation and characterization of a new mouse-colonizing strain for use in comparative studies. One strain capable of persistent mouse colonization was isolated from a total of 110 clinical isolates and is named here SS2000 (Sydney strain 2000). Genome typing revealed a number of differences between SS1 and SS2000 as well as between them and the respective original clinical isolates. In particular, SS2000 lacked the entire cag pathogenicity island, while SS1 contained all 27 genes of the island. C57BL/6 and BALB/c mice were infected with SS1 or SS2000 or were treated with broth medium (controls). After 6 months host-specific effects were evident, including lower colonization levels in the BALB/c animals. Few pathological differences were observed between SS1- and SS2000-infected animals. However, by 15 months postinfection, SS1-infected C57BL/6 mice had developed more severe gastritis than the SS2000-infected animals. In contrast SS2000-infected BALB/c mice showed increased accumulation of mucosa-associated lymphoid tissue compared to those infected with SS1. This improved comparative model of H. pylori-induced disease allowed dissection of both host and strain effects and thus will prove useful in further studies.

Comparative chemical and biological characterization of the lipopolysaccharides of gastric and enterohepatic helicobacters.

BACKGROUND: The lipopolysaccharide of Helicobacter pylori plays an important role in colonization and pathogenicity. The present study sought to compare structural and biological features of lipopolysaccharides from gastric and enterohepatic Helicobacter spp. not previously characterized. MATERIALS AND METHODS: Purified lipopolysaccharides from four gastric Helicobacter spp. (H. pylori, Helicobacter felis, Helicobacter bizzozeronii and Helicobacter mustelae) and four enterohepatic Helicobacter spp. (Helicobacter hepaticus, Helicobacter bilis, 'Helicobacter sp. flexispira' and Helicobacter pullorum) were structurally characterized using electrophoretic, serological and chemical methods. RESULTS: Structural insights into all three moieties of the lipopolysaccharides, i.e. lipid A, core and O-polysaccharide chains, were gained. All species expressed lipopolysaccharides bearing an O-polysaccharide chain, but H. mustelae and H. hepaticus produced truncated semirough lipopolysaccharides. However, in contrast to lipopolysaccharides of H. pylori and H. mustelae, no blood group mimicry was detected in the other Helicobacter spp. examined. Intra-species, but not interspecies, fatty acid profiles of lipopolysaccharides were identical within the genus. Although shared lipopolysaccharide-core epitopes with H. pylori occurred, differing structural characteristics were noted in this lipopolysaccharide region of some Helicobacter spp. The lipopolysaccharides of the gastric helicobacters, H. bizzozeronii and H. mustelae, had relative Limulus amoebocyte lysate activities which clustered around that of H. pylori lipopolysaccharide, whereas H. bilis, 'Helicobacter sp. flexispira' and H. hepaticus formed a cluster with approximately 1000-10,000-fold lower activities. H. pullorum lipopolysaccharide had the highest relative Limulus amoebocyte lysate activity of all the helicobacter lipopolysaccharides (10-fold higher than that of H. pylori lipopolysaccharide), and all the lipopolysaccharides of enterohepatic Helicobacter spp. were capable of inducing nuclear factor-Kappa B(NF-kappaB) activation. CONCLUSIONS: The collective results demonstrate the structural heterogeneity and pathogenic potential of lipopolysaccharides of the Helicobacter genus as a group and these differences in lipopolysaccharides may be indicative of adaptation of the bacteria to different ecological niches.

Immunisation against Helicobacter felis infection protects against the development of gastric MALT Lymphoma.

UNLABELLED: The formation of mucosa-associated lymphoid tissue (MALT) in response to Helicobacter pylori infection is closely associated with the development of primary gastric MALT lymphoma. AIM: To examine whether immunisation against Helicobacter felis can protect against development of MALT lymphoma. RESULTS: The majority of control infected mice demonstrated MALT formation (13/15) and five developed lymphoma. Fifteen immunised mice were protected against bacterial challenge, of which only five had evidence of MALT formation and none developed lymphoma. Interestingly, of the four mice in which immunisation failed, all developed MALT and two of these had lymphoma. CONCLUSION: Effective immunisation against Helicobacter infection can protect against gastric MALT lymphoma. To our knowledge this is the first demonstration of vaccination protecting against a bacteria-induced malignancy.

Protective immunity against Helicobacter is characterized by a unique transcriptional signature.

Immunization with a whole-cell sonicate vaccine of Helicobacter felis in conjunction with cholera toxin as a mucosal adjuvant induces long-term protective immunity in a majority of laboratory mice. We have combined gene expression profiling and immunohistochemical analysis on a set of immunized animals to better understand the mechanism of protection. The stomachs of protected animals exhibited a strikingly different transcriptional profile compared with those of nonprotected or control mice, indicating that vaccination targets the appropriate site and leaves a molecular signature. Among the genes whose up-regulation is significantly correlated with protection are a number of adipocyte-specific factors. These include the fat-cell-specific cytokines adipsin, resistin, and adiponectin and the adipocyte surface marker CD36. Interestingly, potentially protective T and B lymphocytes can be found embedded in the adipose tissue surrounding protected stomachs but never in control or unprotected stomachs. Adipsin-specific immunohistochemical staining of protected stomach sections further revealed molecular cross-talk between adjacent lymphoid and adipose cell populations. We propose a mechanism of protection that involves the effector responses of either or both lymphocyte subclasses as well as the previously unappreciated paracrine functions of adipose tissue surrounding the resident lymphocytes.

Animal models of Helicobacter pylori infection and disease.

The acceptance of Helicobacter pylori as a major human pathogen has necessitated the development of animal models to help elucidate the pathogenic mechanisms of this bacterium and aid in the development of improved strategies for the treatment of gastric disease. Appropriate models, utilising a range of animal species, have been developed to examine factors such as the influence of host responses and bacterial factors in disease development and the success of new therapeutic regimens, including vaccination, to cure infection.

Gastric transitional zones, areas where Helicobacter treatment fails: results of a treatment trial using the Sydney strain mouse model.

Current combination therapies cure Helicobacter pylori infection in 75 to 85% of cases. However, many treatment failures are not explained by antibiotic resistance. Our goal was to explore treatment failures under in vivo conditions by using the H. pylori Sydney strain (SS1) mouse model. Mice infected with H. pylori (SS1) were treated with monotherapies or combination therapies used in human trials. Bacterial levels and distribution of organisms within the stomach were assessed 24 h after treatment to determine clearance and location of treatment failures and 29 days after treatment to determine cure rates. Except for treatment with metronidazole, mono- and dual therapies did not cure infection but resulted in decreases in bacterial levels and differences in distribution within the stomach. In cases of treatment failure when clarithromycin was used, omeprazole and dual therapy with omeprazole and amoxicillin resulted in organisms being cleared from the antrum, but organisms remained in the antrum-body transitional zone. The triple therapies of OMC and bismuth subcitrate, metronidazole, and tetracycline were successful in eradicating infection. Except for metronidazole monotherapy and triple therapy with OAC, there was good correlation between the Sydney strain mouse model and humans with respect to the success of antimicrobial therapy. The antrum-body transitional zone was identified as a sanctuary site in treatment failure. This could result from antimicrobial agents not functioning effectively at this site or bacteria in this location expressing products that protect them against antimicrobial agents. This is the first demonstration of a possible sanctuary site as a reason for failure of therapy.

Distinct gene expression profiles characterize the histopathological stages of disease in Helicobacter-induced mucosa-associated lymphoid tissue lymphoma.

Long-term colonization of humans with Helicobacter pylori can cause the development of gastric B cell mucosa-associated lymphoid tissue lymphoma, yet little is known about the sequence of molecular steps that accompany disease progression. We used microarray analysis and laser microdissection to identify gene expression profiles characteristic and predictive of the various histopathological stages in a mouse model of the disease. The initial step in lymphoma development is marked by infiltration of reactive lymphocytes into the stomach and the launching of a mucosal immune response. Our analysis uncovered molecular markers of both of these processes, including genes coding for the immunoglobulins and the small proline-rich protein Sprr 2A. The subsequent step is characterized histologically by the antigen-driven proliferation and aggregation of B cells and the gradual appearance of lymphoepithelial lesions. In tissues of this stage, we observed increased expression of genes previously associated with malignancy, including the laminin receptor-1 and the multidrug-resistance channel MDR-1. Finally, we found that the transition to destructive lymphoepithelial lesions and malignant lymphoma is marked by an increase in transcription of a single gene encoding calgranulin AMrp-8.

In vivo behavior of a Helicobacter pylori SS1 nixA mutant with reduced urease activity.

Helicobacter pylori mutants devoid of urease activity fail to colonize the gastric mucosa of mice; however, the effect of decreased levels of urease on colonization has not been examined. The nixA gene, required for full urease activity, encodes a cytoplasmic membrane nickel transporter that imports nickel ions and leads to incorporation of nickel ions into apourease. A nixA mutant of the Sydney strain of H. pylori (SS1) was constructed by disruption of the nixA gene with a kanamycin resistance cassette. This mutant retained only half the urease activity of the wild-type (wild-type) SS1 strain. C57BL/6j (n = 75) and BALB/c (n = 75) mice were inoculated independently with the wild-type or the nixA strain. The level and distribution of colonization were assessed by bacterial colony counts and histological grading at 4, 12, and 24 weeks postinfection. Colonization levels of the nixA strain in BALB/c mice were significantly lower compared with SS1 (P = 0.005), while colonization in C57BL/6j mice was similar for both the wild-type and mutant strains. Subtle differences in colonization of the different regions of the stomach, determined by microscopic grading, were observed between wild-type SS1 and the nixA strain in BALB/c mice. On the contrary, when C57BL/6j (n = 35) and BALB/c (n = 35) mice were coinfected with the wild-type and nixA strains simultaneously, the nixA mutant failed to colonize and was outcompeted by the wild-type SS1 strain, which established normal levels of colonization. These results demonstrate the importance of the nixA gene for increasing the fitness of H. pylori for gastric colonization. Since nixA is required for full urease activity, the decreased fitness of the nixA mutant is likely due to reduced urease activity; however, pleiotropic effects of the mutation cannot be completely ruled out.