A novel isolation protocol and probe-based RT-PCR for diagnosis of gastric infections with the zoonotic pathogen Helicobacter suis.

BACKGROUND: Helicobacter suis is a very fastidious microorganism associated with gastritis, gastric ulcers, and mucosa-associated lymphoid tissue lymphoma in humans. In vitro isolation of this agent from human patients has so far been unsuccessful. MATERIALS AND METHODS: A probe-based real-time PCR (RT-PCR) for the rapid detection of H. suis in gastric biopsies was developed. Secondly, a mouse-passage-based protocol was optimized for isolation of low numbers of viable H. suis bacteria. Mice were inoculated with different numbers of viable H. suis (102 -108 ) and kept for 4 weeks to allow multiplication of this pathogen. RESULTS: The probe-based real-time PCR (RT-PCR) exhibited a high degree of diagnostic specificity and analytical sensitivity, high linear correlations (r2 between 0.995 and 0.999), and high amplification efficiencies (>90%) for H. suis. No cross-reactivity was detected with human, porcine, non-human primate, and murine DNA nor with DNA from other bacteria including Helicobacter spp. and Campylobacter spp. H. suis was successfully re-isolated from the stomach of mice inoculated with at least 104 viable H. suis, using a biphasic medium (pH 5), consisting of Brucella agar with Brucella broth on top, both supplemented with vitox supplement, Campylobacter-selective supplement, amphotericin (5 µg/mL), HCl (0.05%), fetal bovine serum (20%), and linezolid (5 µg/mL). Linezolid was necessary to inhibit proliferation of contaminants, including lactobacilli. CONCLUSION: The methods described above can be implemented for detection or isolation of H. suis from human gastric biopsies.

Gastric de novo Muc13 expression and spasmolytic polypeptide-expressing metaplasia during Helicobacter heilmannii infection.

Helicobacter heilmannii is a zoonotic bacterium that has been associated with gastric disease in humans. In this study, the mRNA expression of mucins in the stomach of BALB/c mice was analyzed at several time points during a 1-year infection with this bacterium, during which gastric disease progressed in severity. Markers for acid production by parietal cells and mucous metaplasia were also examined. In the first 9 weeks postinfection, the mRNA expression of Muc6 was clearly upregulated in both the antrum and fundus of the stomach of H. heilmannii-infected mice. Interestingly, Muc13 was upregulated already at 1 day postinfection in the fundus of the stomach. Its expression level remained high in the stomach over the course of the infection. This mucin is, however, not expressed in a healthy stomach, and high expression of this mucin has so far only been described in gastric cancer. In the later stages of infection, mRNA expression of H(+)/K(+)-ATPase α/ß and KCNQ1 decreased, whereas the expression of Muc4, Tff2, Dmbt1, and polymeric immunoglobulin receptor (pIgR) increased starting at 16 weeks postinfection onwards, suggesting the existence of spasmolytic polypeptide-expressing metaplasia in the fundus of the stomach. Mucous metaplasia present in the mucosa surrounding low-grade mucosa-associated lymphoid tissue (MALT) lymphoma-like lesions was also histologically confirmed. Our findings indicate that H. heilmannii infection causes severe gastric pathologies and alterations in the expression pattern of gastric mucins, such as Muc6 and Muc13, as well as disrupting gastric homeostasis by inducing the loss of parietal cells, resulting in the development of mucous metaplasia.

Diversity in bacterium-host interactions within the species Helicobacter heilmannii sensu stricto.

Helicobacter (H.) heilmannii sensu stricto (s.s.) is a zoonotic bacterium that naturally colonizes the stomach of dogs and cats. In humans, this microorganism has been associated with gastritis, peptic ulcer disease and mucosa associated lymphoid tissue (MALT) lymphoma. Little information is available about the pathogenesis of H. heilmannii s.s. infections in humans and it is unknown whether differences in virulence exist within this species. Therefore, a Mongolian gerbil model was used to study bacterium-host interactions of 9 H. heilmannii s.s. strains. The colonization ability of the strains, the intensity of gastritis and gene expression of various inflammatory cytokines in the stomach were determined at 9 weeks after experimental infection. The induction of an antrum-dominant chronic active gastritis with formation of lymphocytic aggregates was shown for 7 strains. High-level antral colonization was seen for 4 strains, while colonization of 4 other strains was more restricted and one strain was not detected in the stomach at 9 weeks post infection. All strains inducing a chronic active gastritis caused an up-regulation of the pro-inflammatory cytokine IL-1ß in the antrum. A reduced antral expression of H+/K+ ATPase was seen in the stomach after infection with 3 highly colonizing strains and 2 highly colonizing strains caused an increased gastrin expression in the fundus. In none of the H. heilmannii s.s.-infected groups, IFN-γ expression was up-regulated. This study demonstrates diversity in bacterium-host interactions within the species H. heilmannii s.s. and that the pathogenesis of gastric infections with this microorganism is not identical to that of an H. pylori infection.