Emiko Rimbara

Background: Helicobacter pylori was first isolated from humans in 1983. Since then, >50 Helicobacter species have been registered. Helicobacter trogontum was first isolated from rat colonic mucosa in 1996 and has been isolated from pig feces and the livers of aborted sheep. H. trogontum adheres to and invades human cells and secretes factors that may contribute to disease development. Case Summary: A 41-year-old woman, who worked on a pig farm, presented to our hospital with sudden-onset headache, nausea, general fatigue, chills, and fever. Plain computed tomography revealed small lymph nodes in the ileocecal region. Intravenous ceftriaxone, vancomycin, and acyclovir were administered for suspected meningoencephalitis. She was discharged from the hospital on day 9 after her symptoms improved. The cerebrospinal fluid culture was negative. However, an aerobic bottle was positive in one of two blood culture sets. Microscopic examination with Gram staining revealed fusiform gram-negative bacteria. Whole genome sequencing of the NHP16-4001 isolate confirmed H. trogontum infection. Conclusion: Case reports of human infection with H. trogontum are rare. However, H. trogontum has been reported to cause enterocolitis and sepsis in an immunocompetent human, and skin lesions as well as bacteremia in an immunocompromised human. H. trogontum was isolated from pigs, sheep, and mice, suggesting its potential importance as a zoonotic disease. In this case, we hypothesize that bacteremia development was related to contact with pig feces on the patient’s farm. Thus, H. trogontum infection may be considered a zoonosis; however, further reports are warranted for arriving at a definitive conclusion.

Non- Helicobacter pylori Helicobacter species (NHPH) are known to be present in the stomachs of humans and animals and are associated with their health. Here, we report complete genome sequences of type strains of Helicobacter suis , Helicobacter heilmannii , and Helicobacter ailurogastricus , which are major gastric NHPH species associated with human gastric diseases.

To evaluate non-H. pylori Helicobacter (NHPH) infections in Japan, we enrolled 673 consecutive patients who underwent gastric endoscopy during annual medical checkups at 4 hospitals during April 2022-February 2023. We collected intragastric fluid and serum samples to detect NHPH infection by PCR and serologic tests. The prevalence of NHPH was 3% (20/673); 70% (14/20) of patients were infected with H. suis and 30% (6/20) with non-H. suis NHPH species. All 14 H. suis-infected patients were men and had a history of pork offal ingestion. Among non-H. suis NHPH-infected patients, 50% (3/6) owned pet cats, whereas only 22% (145/667) of other patients owned cats. Endoscopic evaluation revealed marbled crack-like gastritis was present in 93% (13/14) of H. suis-infected patients, a significantly higher prevalence than for H. pylori-infected (28.6%) and H. pylori eradication therapy (27.6%) groups. Pork offal ingestion and having pet cats increase risk for Helicobacter spp. infections.

Helicobacter species infecting the stomachs of dogs and cats are potentially pathogenic and have been isolated from patients with gastric diseases. In the present study conducted in Japan, among the nine Helicobacter strains that we isolated from dogs and cats, NHP19-003^T from a dog, and strains NHP19-012^T and NHP21-005^T from cats were identified to be the strains most closely related to Helicobacter heilmannii ASB1^T based on a 16S rRNA comparison (98.7–99.2% similarity with H. heilmannii ASB1^T). However, none of their whole genomes showed more than average nucleotide identity (ANI) threshold value (95–96%) to any Helicobacter species (85.1, 86.7, and 86.6% ANI, respectively, with H. heilmannii ASB1^T), including when compared to each other. Furthermore, NHP19-003^T, NHP19-012^T, and NHP21-005^T exhibited protein profiles different from known gastric Helicobacter species, as revealed by MALDI-TOF MS, indicating that they are novel Helicobacter species. We, thus, propose these novel Helicobacter species as follows: Helicobacter gastrocanis sp. nov. (type strain NHP19-003^T [=JCM 39159^T = DSM 111619^T]), Helicobacter gastrofelis sp. nov. (type strain NHP19-012^T [=JCM 39160^T]) and Helicobacter felistomachi sp. nov. (type strain NHP21-005^T [=JCM 39513^T]). These novel strains have respective GC content values of 48.3, 46.9, and 47.1%. Phylogenetic analysis based on ureAB gene sequences obtained from gastric specimens from 47 dogs and 24 cats in Japan revealed that 29.8% of dogs were infected with H. gastrocanis, while H. gastrofelis infected 44.7% of dogs and 12.5% of cats. Additionally, 10.6% of dogs and 20.8% of cats were infected with H. felistomachi. Animal experiments have confirmed that these three novel species elicit gastric inflammatory responses. This study findings reveal the prevalence of novel gastric Helicobacter species in dogs and cats in Japan and their pathogenicity.

Helicobacter cinaedi infects the human gut and causes invasive infections such as bacteremia and cellulitis through bacterial translocation. However, the mechanism by which H. cinaedi attaches to host cells and establishes infection remains unclear. This study aimed to investigate the relationship between a novel putative autotransporter protein, H. cinaedi autotransporter protein A (HcaA), and its role in pathogenicity. The cytotoxicity of H. cinaedi infection in colon epithelial cell lines (Caco-2 and HT29) was assessed using a lactate dehydrogenase assay, and it was found that cytotoxicity significantly decreased upon HcaA knockout. Adhesion assays further revealed that the HcaA-knockout strain showed significantly reduced attachment to the human epithelial colorectal adenocarcinoma (Caco-2) cell line compared to that of the wild-type strain. Moreover, the recombinant HcaA protein demonstrated strong adhesion properties to the human monocytic cell line (U937). The adhesive activity was diminished when the Arg-Gly-Asp (RGD) motif in HcaA was replaced with RAD, indicating that the RGD motif in HcaA is crucial for host cell adhesion. To determine the role of HcaA in H. cinaedi infection in vivo , C57BL/6 mice were orally infected with wild-type and HcaA-knockout H. cinaedi strains. Bacterial colonization was assessed 7, 14, and 28 days post-infection. At 7 days post-infection, colonization was significantly lower in mice infected with the HcaA-knockout strain compared to those infected with the wild-type strain. In conclusion, our findings suggest that HcaA, a novel putative autotransporter protein in H. cinaedi , plays a significant role as an adhesin in establishing colonization. IMPORTANCE: Helicobacter species are classified as gastric or enterohepatic according to their habitat. Among enterohepatic Helicobacter species, which inhabit the intestine, colon, and liver, Helicobacter cinaedi has been most frequently isolated from humans. H. cinaedi often causes bacteremia and cellulitis in immunocompromised hosts. Here, we focused on the H. cinaedi autotransporter protein A (HcaA), a novel virulence factor in H. cinaedi . We discovered that HcaA contributes to cell adhesion via its Arg-Gly-Asp motif. Furthermore, in animal experiments, bacterial colonization was reduced in mice infected with HcaA-knockout strains, supporting the hypothesis that HcaA contributes to H. cinaedi adhesion to host cells. Our study provides a novel mechanism for the establishment of H. cinaedi infections and provides new insights into the role of autotransporter proteins in the establishment of Helicobacter infection.

Helicobacter cinaedi infects the human gut and causes invasive infections such as bacteremia and cellulitis through bacterial translocation. However, the mechanism by which H. cinaedi attaches to host cells and establishes infection remains unclear. This study aimed to investigate the relationship between a novel putative autotransporter protein, H. cinaedi autotransporter protein A (HcaA), and its role in pathogenicity. The cytotoxicity of H. cinaedi infection in colon epithelial cell lines (Caco-2 and HT-29) was assessed using a lactate dehydrogenase assay, and it was found that cytotoxicity significantly decreased upon HcaA knockout. Adhesion assays further revealed that the HcaA-knockout strain showed significantly reduced attachment to the human epithelial colorectal adenocarcinoma cell line (Caco-2) compared to that of the wild-type strain. Moreover, the recombinant HcaA protein demonstrated strong adhesion properties to the human monocytic cell line (U937). The adhesive activity was diminished when the RGD motif in HcaA was replaced with RAD, indicating that the RGD motif in HcaA is crucial for host cell adhesion. To determine the role of HcaA in H. cinaedi infection in vivo , C57BL/6 mice were orally infected with wild-type and HcaA-knockout H. cinaedi strains. Bacterial colonization was assessed 7, 14, and 28 days post-infection. At 7 days post-infection, colonization was significantly lower in mice infected with the HcaA-knockout strain compared to those infected with the wild-type strain. In conclusion, our findings suggest that HcaA, a novel putative autotransporter protein in H. cinaedi , plays a significant role as an adhesin in establishing colonization. IMPORTANCE: Helicobacter species are classified as gastric or enterohepatic according to their habitat. Among enterohepatic Helicobacter species, which inhabit the intestine, colon and liver, H. cinaedi has been most frequently isolated from humans. H. cinaedi often causes bacteremia and cellulitis in immunocompromised hosts. Here, we focused on the H. cinaedi autotransporter protein A (HcaA), a novel virulence factor in H. cinaedi . We discovered that HcaA contributes to cell adhesion via its RGD motif. Furthermore, in animal experiments, bacterial colonization was reduced in mice infected with HcaA-knockout strains, supporting the hypothesis that HcaA contributes to H. cinaedi adhesion to host cells. Our study provides a novel mechanism for the establishment of H. cinaedi infections and provides new insights into the role of autotransporter proteins in the establishment of Helicobacter infection.

Background: Eradication treatment for Helicobacter pylori gastritis is covered by national health insurance since 2013 in Japan. However, eradication failure due to the increase of antimicrobial resistance has become a serious problem. The present study aims to establish a reference panel of Japanese H. pylori strains for antimicrobial susceptibility testing.Method: A total of 28 strains were collected from 4 medical facilities in Japan. Antimicrobial susceptibility tests (ASTs) to clarithromycin (CLR), amoxicillin (AMX), and metronidazole (MNZ), were used to select standard reference strains. Complete genome sequences were also determined.Results: Three H. pylori strains (JSHR3, JSHR6 and JSHR31) were selected as standard reference strains by the Japanese Society for Helicobacter Research (JSHR). The minimum inhibitory concentrations (MICs) of the antibiotics against these 3 strains by agar dilution method with Brucella‐based horse‐serum‐containing agar medium were as follows: JSHR3 (CLR 16 μg/ml, AMX 0.032 μg/ml and MNZ 4 μg/ml), JSHR6 (CLR 0.016 μg/ml, AMX 0.032 μg/ml and MNZ 4 μg/ml), and JSHR31 (CLR 16 μg/ml, AMX 1 μg/ml and MNZ 64 μg/ml).Conclusions: A reference panel of H. pylori JSHR strains was established. The panel consisted of JSHR6, which was antibiotic‐susceptible, JSHR3, which was CLR‐resistant, and JSHR31, which was multi‐resistant. This reference panel will be essential for standardized ASTs before the optimal drugs are selected for eradication treatment.

Significance: This study directly demonstrates virulence-associated features ofHelicobacter suisinfection using isolates obtained from human stomachs.H. suisis the second-most-prevalentHelicobacterspecies in the human stomach, but diagnostic tests forHelicobacter pylorioften fail to diagnoseH. suis. H. suisdoes not have orthologs of either cytotoxin-associated gene A (CagA) or vacuolating cytotoxin A (VacA), two major virulence factors inH. pylori, suggesting that the molecular pathogenicity ofH. suisand clinical manifestations caused byH. suisinfection are different from those caused byH. pylori. This study paves the way for epidemiological research aimed at identifying the causal relationships betweenH. suisand gastric diseases and developing diagnostic methods.