Isolation and characterization of Helicobacter suis from human stomach

Abstract

Helicobacter suis, a bacterial species naturally hosted by pigs, can colonize the human stomach in the context of gastric diseases such as gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Because H. suis has been successfully isolated from pigs, but not from humans, evidence linking human H. suis infection to gastric diseases has remained incomplete. In this study, we successfully in vitro cultured H. suis directly from human stomachs. Unlike Helicobacter pylori, the viability of H. suis decreases significantly on neutral pH; therefore, we achieved this using a low-pH medium for transport of gastric biopsies. Ultimately, we isolated H. suis from three patients with gastric diseases, including gastric MALT lymphoma. Successful eradication of H. suis yielded significant improvements in endoscopic and histopathological findings. Oral infection of mice with H. suis clinical isolates elicited gastric and systemic inflammatory responses; in addition, progression of gastric mucosal metaplasia was observed 4 mo postinfection. Because H. suis could be isolated from the stomachs of infected mice, our findings satisfied Koch's postulates. Although further prospective clinical studies are needed, H. suis, like H. pylori, is likely a gastric pathogen in humans. Furthermore, comparative genomic analysis of H. suis using complete genomes of clinical isolates revealed that the genome of each H. suis isolate contained highly plastic genomic regions encoding putative strain-specific virulence factors, including type IV secretion system-associated genes, and that H. suis isolates from humans and pigs were genetically very similar, suggesting possible pig-to-human transmission.

Keywords: Helicobacter suis; gastric diseases; zoonosis.

Fig. 1.

Scanning electron micrograph of H. suis strain NHP19-4003 from a human patient.

Fig. 2.

Endoscopic and histological images from H. suis–infected patients. (A) Endoscopic images obtained from patient A and patient B pre- and posteradication. Patient A: Nodular gastritis from the antrum to the angle and the mucosal thickening in the angle of the stomach, endoscopically observed preeradication, improved 9 mo after eradication. Patient B: The open and linear multiple gastric ulcers observed before eradication disappeared. Histological examination of gastric biopsies obtained from patient A (B) and patient B (C) pre- and posteradication. Patient A: Preeradication, diffuse infiltration of lymphocytes and lymphoepithelial lesion (arrow) were observed by H&E staining. Immunostaining of infiltrating lymphocytes within the follicle was positive for CD20 and negative for CD3. Improvement of diffuse infiltration of lymphocytes and improvement of lymphoepithelial lesions were confirmed 3 mo after eradication. Patient B: Improvement of an infiltration of neutrophils observed by H&E staining pretreatment was confirmed posteradication. In both patients, bacteria with spiral morphology very different from that of H. pylori were observed by Giemsa staining.

Fig. 3.

Effects of H. suis infection in mice. (A) Relative bacterial number of H. suis in the mouse stomach. Bars indicate means ± SD. (B) Number of lymph follicles observed in stomach sections from H. suis–infected and control mice. (C) H&E staining of a lymph follicle (arrows) observed in mice infected with H. suis SNTW101c. (D) H&E, Alcian blue–PAS, TFF2, and TFF3 staining of stomach sections from H. suis–infected and control mice. Lymphocyte infiltration in mucosa and muscularis mucosae (arrows) was observed in H. suis–infected mice. Acidic mucus, stained blue with Alcian blue–PAS (arrows), was observed in H. suis–infected mice. Expression of TFF2 and TFF3 was observed at the bases of glands (arrows) in H. suis–infected mice. (E) Scoring of neutrophil and lymphocyte infiltration in muscularis mucosae and mucosa and mucosal metaplasia of stomach sections from H. suis–infected and control mice. Bars indicate means ± SD. (F) Percentage of glands expressing TFF2 or TFF3 in H. suis–infected and control mice. (G) TFF2- or TFF3-expressing glands of stomach from H. suis NHP19-4004–infected mice. Most glands expressed both TFF2 and TFF3 (black arrows), and rare glands (blue arrows) expressed only TFF2. In each of the H. suis–infected samples, the relative bacterial number of H. suis in the stomach of NHP19-4003– or NHP19-4004–infected mice was compared with that of SNTW101c–infected mice by one-way ANOVA with Dunnett’s test. Scores for cell infiltration observed in stomach sections in H. suis–infected mice were compared with those in control mice by two-way ANOVA with Sidak’s multiple comparisons test. Numbers of lymph follicles and scores for mucosal metaplasia in H. suis–infected mice were compared with those in controls by one-way ANOVA with Holm–Sidak’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4.

Comparative analysis based on whole-genome data of H. suis isolates. (A) Pangenome analysis using genomes from the indicated 16 isolates. Phylogenetic trees generated using core genome alignments. Sequence type (ST), host, year of isolation, number of coding sequences, and gene presence/absence matrix are shown. Bar lengths represent the number of substitutions per site in the core genome. The pangenome of H. suis consisted of 2,392 genes, including 1,306 core genes (≥99.0% conserved in the isolates) and 1,086 accessory genes (<99.0% conserved in the isolates). (B) Linear sequence comparison of chromosomes of five isolates. Four genomic regions corresponding to PZs in NHP19-4003 are shown.