Bram Flahou

Gastric Helicobacters in Domestic Animals and Nonhuman Primates and Their Significance for Human Health

SUMMARY Helicobacters other than Helicobacter pylori have been associated with gastritis, gastric ulcers, and gastric mucosa-associated lymphoid tissue lymphoma in humans. These very fastidious microorganisms with a typical large spiral-shaped morphology were provisionally designated “H. heilmannii,” but in fact they comprise at least five different Helicobacter species, all of which are known to colonize the gastric mucosa of animals. H. suis, which has been isolated from the stomachs of pigs, is the most prevalent gastric non-H. pylori Helicobacter species in humans. Other gastric non-H. pylori helicobacters colonizing the human stomach are H. felis, H. salomonis, H. bizzozeronii, and the still-uncultivable “Candidatus Helicobacter heilmannii.” These microorganisms are often detected in the stomachs of dogs and cats. “Candidatus Helicobacter bovis” is highly prevalent in the abomasums of cattle but has only occasionally been detected in the stomachs of humans. There are clear indications that gastric non-H. pylori Helicobacter infections in humans originate from animals, and it is likely that transmission to humans occurs through direct contact. Little is known about the virulence factors of these microorganisms. The recent successes with in vitro isolation of non-H. pylori helicobacters from domestic animals open new perspectives for studying these microorganisms and their interactions with the host.

Colonization strategy of Campylobacter jejuni results in persistent infection of the chicken gut.

Although poultry meat is now recognized as the main source of Campylobacter jejuni gastroenteritis, little is known about the strategy used by the bacterium to colonize the chicken intestinal tract. In this study, the mechanism of C. jejuni colonization in chickens was studied using four human and four poultry isolates of C. jejuni. The C. jejuni strains were able to invade chicken primary cecal epithelial crypt cells in a predominantly microtubule-dependent way (five out of eight strains). Invasion of cecal epithelial cells was not accompanied by necrosis or apoptosis in the cell cultures, nor by intestinal inflammation in a cecal loop model. C. jejuni from human origin displayed a similar invasive profile compared to the poultry isolates. Invasiveness of the strains in vitro correlated with the magnitude of spleen colonization in C. jejuni inoculated chicks. The C. jejuni bacteria that invaded the epithelial cells were not able to proliferate intracellularly, but quickly evaded from the cells. In contrast, the C. jejuni strains were capable of replication in chicken intestinal mucus. These findings suggest a novel colonization mechanism by escaping rapid mucosal clearance through short-term epithelial invasion and evasion, combined with fast replication in the mucus.

Butyricicoccus pullicaecorum in inflammatory bowel disease

Objective Many species within the phylum Firmicutes are thought to exert anti-inflammatory effects. We quantified bacteria belonging to the genus Butyricicoccus in stools of patients with ulcerative colitis (UC) and Crohns disease (CD). We evaluated the effect of Butyricicoccus pullicaecorum in a rat colitis model and analysed the ability to prevent cytokine-induced increases in epithelial permeability. Design A genus-specific quantitative PCR was used for quantification of Butyricicoccus in stools from patients with UC or CD and healthy subjects. The effect of B pullicaecorum on trinitrobenzenesulfonic (TNBS)-induced colitis was assessed and the effect of B pullicaecorum culture supernatant on epithelial barrier function was investigated in vitro. Results The average number of Butyricicoccus in stools from patients with UC and CD in active (UC: 8.61 log10/g stool; CD: 6.58 log10/g stool) and remission phase (UC: 8.69 log10/g stool; CD: 8.38 log10/g stool) was significantly lower compared with healthy subjects (9.32 log10/g stool) and correlated with disease activity in CD. Oral administration of B pullicaecorum resulted in a significant protective effect based on macroscopic and histological criteria and decreased intestinal myeloperoxidase (MPO), tumour necrosis factor α (TNFα) and interleukin (IL)-12 levels. Supernatant of B pullicaecorum prevented the loss of transepithelial resistance (TER) and the increase in IL-8 secretion induced by TNFα and interferon γ (IFN gamma) in a Caco-2 cell model. Conclusions Patients with inflammatory bowel disease have lower numbers of Butyricicoccus bacteria in their stools. Administration of B pullicaecorum attenuates TNBS-induced colitis in rats and supernatant of B pullicaecorum cultures strengthens the epithelial barrier function by increasing the TER.

Genome sequence of Helicobacter suis supports its role in gastric pathology

Helicobacter (H.) suis has been associated with chronic gastritis and ulcers of the pars oesophagea in pigs, and with gastritis, peptic ulcer disease and gastric mucosa-associated lymphoid tissue lymphoma in humans. In order to obtain better insight into the genes involved in pathogenicity and in the specific adaptation to the gastric environment of H. suis, a genome analysis was performed of two H. suis strains isolated from the gastric mucosa of swine. Homologs of the vast majority of genes shown to be important for gastric colonization of the human pathogen H. pylori were detected in the H. suis genome. H. suis encodes several putative outer membrane proteins, of which two similar to the H. pylori adhesins HpaA and HorB. H. suis harbours an almost complete comB type IV secretion system and members of the type IV secretion system 3, but lacks most of the genes present in the cag pathogenicity island of H. pylori. Homologs of genes encoding the H. pylori neutrophil-activating protein and γ-glutamyl transpeptidase were identified in H. suis. H. suis also possesses several other presumptive virulence-associated genes, including homologs for mviN, the H. pylori flavodoxin gene, and a homolog of the H. pylori vacuolating cytotoxin A gene. It was concluded that although genes coding for some important virulence factors in H. pylori, such as the cytotoxin-associated protein (CagA), are not detected in the H. suis genome, homologs of other genes associated with colonization and virulence of H. pylori and other bacteria are present.

Helicobacter suis causes severe gastric pathology in mouse and mongolian gerbil models of human gastric disease.

Background “Helicobacter (H.) heilmannii” type 1 is the most prevalent gastric non-H. pylori Helicobacter species in humans suffering from gastric disease. It has been shown to be identical to H. suis, a bacterium which is mainly associated with pigs. To obtain better insights into the long-term pathogenesis of infections with this micro-organism, experimental infections were carried out in different rodent models. Methodology/Principal Findings Mongolian gerbils and mice of two strains (BALB/c and C57BL/6) were infected with H. suis and sacrificed at 3 weeks, 9 weeks and 8 months after infection. Gastric tissue samples were collected for PCR analysis, histological and ultrastructural examination. In gerbils, bacteria mainly colonized the antrum and a narrow zone in the fundus near the forestomach/stomach transition zone. In both mice strains, bacteria colonized the entire glandular stomach. Colonization with H. suis was associated with necrosis of parietal cells in all three animal strains. From 9 weeks after infection onwards, an increased proliferation rate of mucosal epithelial cells was detected in the stomach regions colonized with H. suis. Most gerbils showed a marked lymphocytic infiltration in the antrum and in the forestomach/stomach transition zone, becoming more pronounced in the course of time. At 8 months post infection, severe destruction of the normal antral architecture at the inflamed sites and development of mucosa-associated lymphoid tissue (MALT) lymphoma-like lesions were observed in some gerbils. In mice, the inflammatory response was less pronounced than in gerbils, consisting mainly of mononuclear cell infiltration and being most severe in the fundus. Conclusions/Significance H. suis causes death of parietal cells, epithelial cell hyperproliferation and severe inflammation in mice and Mongolian gerbil models of human gastric disease. Moreover, MALT lymphoma-like lesions were induced in H. suis-infected Mongolian gerbils. Therefore, the possible involvement of this micro-organism in human gastric disease should not be neglected.

Non-Helicobacter pylori Helicobacter Species in the Human Gastric Mucosa: A Proposal to Introduce the Terms H. heilmannii Sensu Lato and Sensu Stricto

Dear Editor, Helicobacter (H.) pylori is by far the most prevalent Helicobacter species in the human stomach. However, already in the early days of H. pylori research, pathologists examining stomach biopsies have reported the presence of morphologically distinct, typically long spiral shaped bacteria. These gastric non-H. pylori helicobacters were originally referred to as Gastrospirillum hominis and later as H. heilmannii which was further subdivided in two taxa, types 1 and 2 [1]. A general characteristic of these bacteria is that these are very fastidious microorganisms which, as far as we know, have been cultured from the gastric mucosa of only two human patients [2,3]. Although the name H. heilmannii has for many years been used to refer to the long spiral shaped bacteria in the human stomach, it was not formally recognized as a valid species name until recently. Gastric non-H. pylori helicobacters actually comprise several Helicobacter species, all of them known to colonize the gastric mucosa of animals. Microorganisms previously referred to as H. heilmannii type 1 are identical to H. suis, a species colonizing the stomachs of pigs. The former H. heilmannii type 2 does not represent a single species, but rather a group of species all of them known to colonize the gastric mucosa of dogs and cats: H. felis, H. bizzozeronii, H. salomonis, H. cynogastricus, H. baculiformis and a bacterium which was in 2004 given the provisional name ‘‘Candidatus H. heilmannii’’ because, at that time, it could not be cultured in vitro [1,4]. Only recently have in vitro cultures been obtained from the latter microorganism, resulting in the formal and valid description of H. heilmannii as a novel Helicobacter species [5]. Unfortunately, this will further add to the already existing confusion on the nomenclature of this complex and expanding group of gastric microorganisms. Even for specialists in the field, it is not always easy or even impossible to figure out which bacterial species some of the papers on gastric non-H. pylori helicobacters exactly deal with. We expect that clinicians and clinical bacteriologists will continue to use the name H. heilmannii to refer to the group of long spiral shaped microorganisms although according to taxonomic rules it represents only one member of this group. To avoid further confusion, we propose to introduce the terms H. heilmannii sensu lato (H. heilmannii s.l.) and H. heilmannii sensu stricto (H. heilmannii s.s.). H. heilmannii s.l. may then be used to refer to the whole group of non-H. pylori helicobacters detected in the human or animal stomach, for instance when only results of histopathology, electron microscopy or crude taxonomic data are available. The name H. heilmannii s.s. or the other species names should be used whenever bacteria are really identified to the species level. Although differences in morphology between different non-H. pylori helicobacters have been described, it is not an accurate method for species identification since variation in morphology within a species occurs and different species may be morphologically very similar. Sequencing of the 16S or 23S rRNA-encoding genes allows differentiation of H. suis from the other gastric non-H. pylori helicobacters mentioned above, but it cannot distinguish between H. felis, H. bizzozeronii, H. salomonis, H. cynogastricus, H. baculiformis and H. heilmannii s.s. [1]. Although for differentiation between these species, sequencing of the hsp60 or gyrB gene is useful, sequencing of the urease A and B genes seems currently to be the most suitable method since sequences of these genes are available for all H. heilmannii s.l. species [1,3,4]. Whole cell protein profiling may also be useful but it is only applicable if pure in vitro cultures are available, which is a serious drawback for these fastidious microorganisms [1]. H. bizzozeronii is the only H. heilmannii s.l. species isolated from the human gastric mucosa thus far [2,3], although PCR-based screening studies revealed that human patients are more often colonized with other species, including H. suis [1]. For describing new species of the genus Helicobacter, a polyphasic approach is necessary and the recommendations by Dewhirst et al. [6] for required phenotypic and molecular data should be followed.

Gastric epithelial cell death caused by Helicobacter suis and Helicobacter pylori γ‐glutamyl transpeptidase is mainly glutathione degradation‐dependent

Helicobacter (H.) suis is the most prevalent non‐H. pylori Helicobacter species colonizing the stomach of humans suffering from gastric disease. In the present study, we aimed to unravel the mechanism used by H. suis to induce gastric epithelial cell damage. H. suis lysate induced mainly apoptotic death of human gastric epithelial cells. Inhibition of γ‐glutamyl transpeptidase (GGT) activity present in H. suis lysate and incubation of AGS cells with purified native and recombinant H. suis GGT showed that this enzyme was partly responsible for the observed apoptosis. Supplementation of H. suis or H. pylori GGT‐treated cells with glutathione strongly enhanced the harmful effect of both enzymes and resulted in the induction of oncosis/necrosis, demonstrating that H. suis and H. pylori GGT‐mediated degradation of glutathione and the resulting formation of glutathione degradation products play a direct and active role in the induction of gastric epithelial cell death. This was preceded by an increase of extracellular H2O2 concentrations, generated in a cell‐independent manner and causing lipid peroxidation. In conclusion, H. suis and H. pylori GGT‐mediated generation of pro‐oxidant glutathione degradation products brings on cell damage and causes apoptosis or necrosis, dependent on the amount of extracellular glutathione available as a GGT substrate.

An experimental Helicobacter suis infection causes gastritis and reduced daily weight gain in pigs

Helicobacter suis is a zoonotically important bacterium, that has been associated with gastritis and ulcerative lesions of the pars oesophagea of the stomach in pigs. Its exact role in these pathologies, however, still remains controversial. Therefore, a total of 29 medicated early weaned piglets were inoculated intragastrically or orally, with a total of 2 × 10(9) viable H. suis bacteria and the effect on gastric pathology and weight gain was determined. Twenty-three medicated early weaned piglets were inoculated with a sterile culture medium and used as sham-inoculated controls. The animals were euthanized between 28 and 42 days after inoculation. Infected animals showed a more severe gastritis compared to the control group. There was also a significant reduction of approximately 60 g per day (10%) in weight gain in H. suis inoculated animals compared to the sham-inoculated control animals. In conclusion, this study demonstrates for the first time that a pure in vitro culture of H. suis not only causes gastritis but also a marked decrease of the daily weight gain in experimentally infected pigs.

The local immune response of mice after Helicobacter suis infection: strain differences and distinction with Helicobacter pylori

Helicobacter (H.) suis colonizes the stomach of pigs and is the most prevalent gastric non-H. pylori Helicobacter species in humans. Limited information is available on host immune responses after infection with this agent and it is unknown if variation in virulence exists between different H. suis strains. Therefore, BALB/c and C57BL/6 mice were used to compare colonization ability and gene expression of various inflammatory cytokines, as determined by real-time PCR, after experimental infection with 9 different H. suis strains. All strains were able to persist in the stomach of mice, but the number of colonizing bacteria at 59 days post inoculation was higher in stomachs of C57BL/6 mice compared to BALB/c mice. All H. suis strains caused an upregulation of interleukin (IL)-17, which was more pronounced in BALB/c mice. This upregulation was inversely correlated with the number of colonizing bacteria. Most strains also caused an upregulation of regulatory IL-10, positively correlating with colonization in BALB/c mice. Only in C57BL/6 mice, upregulation of IL-1β was observed. Increased levels of IFN-γ mRNA were never detected, whereas most H. suis strains caused an upregulation of the Th2 signature cytokine IL-4, mainly in BALB/c mice. In conclusion, the genetic background of the murine strain has a clear impact on the colonization ability of different H. suis strains and the immune response they evoke. A predominant Th17 response was observed, accompanied by a mild Th2 response, which is different from the Th17/Th1 response evoked by H. pylori infection.

Survival of Helicobacter suis bacteria in retail pig meat

Helicobacter (H.) suis colonizes the gastric mucosa of pigs world-wide and is the most prevalent non-Helicobacter pylori Helicobacter species in humans. This agent might be transmitted to humans by manipulation or consumption of contaminated pork. H. suis is a very fastidious micro-organism and is extremely difficult to isolate. Therefore, we developed a non-culture dependent, quantitative detection method allowing differentiation of viable from dead H. suis bacteria in pork. This was established by a combination of ethidium bromide monoazide (EMA) treatment and real-time (RT)-PCR. This EMA RT-PCR was applied to 50 retail pork samples. In two samples, viable H. suis bacteria were detected. Sequence analysis of the obtained PCR products confirmed the presence of H. suis DNA. Viable H. suis bacteria persisted for at least 48h in experimentally contaminated pork. In conclusion, consumption of contaminated pork may constitute a new route of transmission for H. suis infections in humans.