Establishment of a reference panel of Helicobacter pylori strains for antimicrobial susceptibility testing.

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.

Effects of Helicobacter pylori and Nitrate-Reducing Bacteria Coculture on Cells.

Helicobacter pylori infection is an important risk factor for developing gastric cancer. However, only a few H. pylori-infected people develop gastric cancer. Thus, other risk factors aside from H. pylori infection may be involved in gastric cancer development. This study aimed to investigate whether the nitrate-reducing bacteria isolated from patients with atrophic gastritis caused by H. pylori infection are risk factors for developing atrophic gastritis and gastric neoplasia. Nitrate-reducing bacteria were isolated from patients with atrophic gastritis caused by H. pylori infection. Among the isolated bacteria, Actinomyces oris, Actinomyces odontolyticus, Rothia dentocariosa, and Rothia mucilaginosa were used in the subsequent experiments. Cytokine inducibility was evaluated in monocytic cells, and mitogen-activated protein kinase (MAPK) activity and cell cycle were assessed in the gastric epithelial cells. The cytotoxicities and neutrophil-inducing abilities of the Actinomyces and Rothia species were enhanced when cocultured with H. pylori. Th1/Th2-related cytokines were also expressed, but their expression levels differed depending on the bacterial species. Moreover, H. pylori and Actinomyces activated MAPK (ERK and p38) and affected cell cycle progression. Some nitrate-reducing bacteria cocultured with H. pylori may promote inflammation and atrophy by inducing cytokine production. In addition, the MAPK activation and cell cycle progression caused by these bacteria can contribute to gastric cancer development.

Serodiagnosis and Bacterial Genome of Helicobacter pylori Infection.

The infection caused by Helicobacter pylori is associated with several diseases, including gastric cancer. Several methods for the diagnosis of H. pylori infection exist, including endoscopy, the urea breath test, and the fecal antigen test, which is the serum antibody titer test that is often used since it is a simple and highly sensitive test. In this context, this study aims to find the association between different antibody reactivities and the organization of bacterial genomes. Next-generation sequences were performed to determine the genome sequences of four strains of antigens with different reactivity. The search was performed on the common genes, with the homology analysis conducted using a genome ring and dot plot analysis. The two antigens of the highly reactive strains showed a high gene homology, and Western blots for CagA and VacA also showed high expression levels of proteins. In the poorly responsive antigen strains, it was found that the inversion occurred around the vacA gene in the genome. The structure of bacterial genomes might contribute to the poor reactivity exhibited by the antibodies of patients. In the future, an accurate serodiagnosis could be performed by using a strain with few gene mutations of the antigen used for the antibody titer test of H. pylori.