Miyuki Matsuda

Discovery of novel mutations for clarithromycin resistance in Helicobacter pylori by using next-generation sequencing

OBJECTIVES Resistance to clarithromycin is the most important factor causing failure of Helicobacter pylori eradication. Although clarithromycin resistance is mainly associated with three point mutations in the 23S rRNA genes, it is unclear whether other mutations are associated with this resistance. METHODS Two types of clarithromycin-resistant strains (low- and high-resistance strains) were obtained from clarithromycin-susceptible H. pylori following exposure to low clarithromycin concentrations. The genome sequences were determined with a next-generation sequencer. Natural transformation was used to introduce the candidate mutations into strain 26695. Etest and an agar dilution method were used to determine the MICs. RESULTS High-resistance strains contained the mutation A2143G in the 23S rRNA genes, whereas low-resistance strains did not. There were seven candidate mutations in six genes outside of the 23S rRNA genes. The mutated sequences in hp1048 (infB), hp1314 (rpl22) and the 23S rRNA gene were successfully transformed into strain 26695 and the transformants showed an increased MIC of and low resistance to clarithromycin. The transformants containing a single mutation in infB or rpl22 (either a 9 bp insertion or a 3 bp deletion) or the 23S rRNA gene showed low MICs (0.5, 2.0, 4.0 and 32 mg/L, respectively) while the transformants containing double mutations (mutation in the 23S rRNA genes and mutation in infB or rpl22) showed higher MICs (>256 mg/L). CONCLUSIONS Next-generation sequencing can be a useful tool for screening mutations related to drug resistance. We discovered novel mutations related to clarithromycin resistance in H. pylori (infB and rpl22), which have synergic effects with 23S rRNA resulting in higher MICs.

Complete Genome Sequences of Eight Helicobacter pylori Strains with Different Virulence Factor Genotypes and Methylation Profiles, Isolated from Patients with Diverse Gastrointestinal Diseases on Okinawa Island, Japan, Determined Using PacBio Single-Molecule Real-Time Technology.

ABSTRACT We report the complete genome sequences of eight Helicobacter pylori strains isolated from patients with gastrointestinal diseases in Okinawa, Japan. Whole-genome sequencing and DNA methylation detection were performed using the PacBio platform. De novo assembly determined a single, complete contig for each strain. Furthermore, methylation analysis identified virulence factor genotype-dependent motifs.

Virulence genes of Helicobacter pylori in the Dominican Republic

Although the incidence of gastric cancer in the Dominican Republic is not high, the disease remains a significant health problem. We first conducted a detailed analysis of Helicobacter pylori status in the Dominican Republic. In total, 158 patients (103 females and 55 males; mean age 47.1±16.2 years) were recruited. The status of H. pylori infection was determined based on four tests: rapid urease test, culture test, histological test and immunohistochemistry. The status of cagA and vacA genotypes in H. pylori was examined using PCR and gene sequencing. The overall prevalence of H. pylori infection was 58.9 %. No relationship was found between the H. pylori infection rate and the age range of 17-91 years. Even in the youngest group (patients aged <29 years), the H. pylori infection rate was 62.5 %. Peptic ulcer was found in 23 patients and gastric cancer was found in one patient. The H. pylori infection rate in patients with peptic ulcer was significantly higher than that in patients with gastritis (82.6 versus 54.5 %, P<0.01). The cagA-positive/vacA s1m1 genotype was the most prevalent (43/64, 67.2 %). Compared with H. pylori-negative patients, H. pylori-positive patients showed more severe gastritis. Furthermore, the presence of cagA was related to the presence of more severe gastritis. All CagA-positive strains had Western-type CagA. In conclusion, we found that H. pylori infection is a risk factor for peptic ulcer in the Dominican Republic. Patients with cagA-positive H. pylori could be at higher risk for severe inflammation and atrophy.

Prevalence of Helicobacter pylori infection and atrophic gastritis in patients with dyspeptic symptoms in Myanmar

AIM To survey the detailed analyses for Helicobacter pylori (H. pylori) infection and gastric mucosal status in Myanmar. METHODS A total of 252 volunteers with dyspeptic symptoms (155 female and 97 male; mean age of 43.6 ± 14.2 years) was participated in Yangon and Mandalay. The status of H. pylori infection was determined based on 5 different tests including rapid urease test, culture, histology, immunohistochemistry and serology. Histological scores were evaluated according to the update Sydney system and the Operative Link for Gastritis Assessment system. Pepsinogen (PG) I and PG II were measured using enzyme-linked immunosorbent assays. RESULTS The overall prevalence of H. pylori infection was 48.0%. There was no relationship between age and infection rate. Even in young group (less than 29 years old), the H. pylori infection rate was relatively high (41.9%). The prevalence of H. pylori infection was significantly higher in Yangon than that of Mandalay. H. pylori infection was significantly associated with the presence of gastric mucosal atrophy. All 7 subjects with peptic ulcer were infected with H. pylori. Although H. pylori-positive subjects showed stronger gastritis than H. pylori-negative subjects, most cases had mild gastritis. CONCLUSION We revealed the prevalence of H. pylori infection in patients with dyspeptic symptoms in Myanmar. The H. pylori infection was a risk factor for peptic ulcer and stronger gastritis.

Extremely low Helicobacter pylori prevalence in North Sulawesi, Indonesia and identification of a Maori-tribe type strain: a cross sectional study

BackgroundSulawesi in Indonesia has a unique geographical profile with assumed separation from Sundaland. Studies of Helicobacter pylori in this region are rare due to the region’s rural location and lack of endoscopy equipment. Indirect methods are, therefore, the most appropriate for measuring H. pylori infection in these areas; with the disposable gastric brush test, we can obtain gastric juice as well as small gastric tissue samples for H. pylori culture. We investigated the prevalence of H. pylori infection and evaluated human migration patterns in the remote areas of North Sulawesi.MethodsWe recruited a total of 251 consecutive adult volunteers and 131 elementary school children. H. pylori infection was determined by urine antibody test. A gastric brush test was used to culture H. pylori. We used next-generation and polymerase chain reaction based sequencing to determine virulence factors and multi-locus sequence typing (MLST).ResultsThe overall H. pylori prevalence was only 14.3% for adults and 3.8% for children, and 13.6% and 16.7% in Minahasanese and Mongondownese participants, respectively. We isolated a single H. pylori strain, termed -Manado-1. Manado-1 was East Asian type cagA (ABD type), vacA s1c-m1b, iceA1 positive/iceA2 negative, jhp0562-positive/-(1,3) galT-negative, oipA ‘on’, and dupA-negative. Phylogenetic analyses showed the strain to be hspMaori type, a major type observed in native Taiwanese and Maori tribes.ConclusionsOur data support that very low H. pylori infection prevalence in Indonesia. Identification of hspMaori type H. pylori in North Sulawesi may support the hypothesis that North Sulawesi people migrated from north.

Prevalence of two homologous genes encoding glycosyltransferases of Helicobacter pylori in the United States and Japan

Background and Aim:  jhp0562 and β‐(1,3)galT (jhp0563) of Helicobacter pylori have been suggested as novel virulent factors; however, the clinical associations and functions of these genes remain unclear. We examined the prevalence of jhp0562, β‐(1,3)galT, and cagA in the United States (US) and Japanese populations.

The Prevalence of Helicobacter pylori Virulence Factors in Bhutan, Vietnam, and Myanmar Is Related to Gastric Cancer Incidence

Gastric cancer is a significant health problem in Asia. Although the prevalence of Helicobacter pylori infection is similar in Bhutan, Vietnam, and Myanmar, the incidence of gastric cancer is highest in Bhutan, followed by Vietnam and Myanmar. We hypothesized that H. pylori virulence factors contribute to the differences. The status of cagA, vacA, jhp0562, and β-(1,3)galT(jhp0563) was examined in 371 H. pylori-infected patients from Bhutan, Vietnam, and Myanmar. Each virulence factor could not explain the difference of the incidence of gastric cancer. However, the prevalence of quadruple-positive for cagA, vacA s1, vacA m1, and jhp0562-positive/β-(1,3)galT-negative was significantly higher in Bhutan than in Vietnam and Myanmar and correlated with gastric cancer incidence. Moreover, gastritis-staging scores measured by histology of gastric mucosa were significantly higher in quadruple-positive strains. We suggest that the cagA, vacA s1, vacA m1, and jhp0562-positive/β-(1,3)galT-negative genotype may play a role in the development of gastric cancer.

Relationship Between the Helicobacter pylori Icea Gene and Clinical Outcomes in Western Countries: Meta-Analysis

Background: Although the iceA (induced by contact with epithelium) allelic types of Helicobacter pylori (H. pylori) have been reported to be associated with peptic ulcer, the importance of the iceA gene on clinical outcomes is conflicting in subsequent studies. The aim of this study was to estimate the magnitude of the risk for clinical outcomes associated with iceA gene in Western countries (Europe, North and South America). Methods: A literature search was performed using the PubMed and EMBASE databases for articles published through November 2010, using the following text words: 1) iceA, iceA1 or iceA2, and 2) pylori or Helicobacter. We did not include abstract alone or unpublished articles. The following criteria were applied to select fully published case-control studies examining the relationship between the iceA gene and clinical outcomes (gastritis, peptic ulcer including gastric ulcer and duodenal ulcer, and gastric cancer) in adult population; the presence of the iceA gene was examined by polymerase chain reaction (PCR); original articles published in English. Studies were excluded if raw data were not presented. Subgroup analysis was performed by geographic area. Results: Nineteen studies with a total of 2,218 patients were identified in the search. Infection with the iceA1-positive H. pylori increased the risk for peptic ulcer by 1.27fold (95% confidence interval [CI], 1.01-1.59) overall. However, the test of heterogeneity was significant among these studies. Sensitivity analysis showed that the presence of the iceA1 gene was significantly associated with peptic ulcer (odds ratio [OR] = 1.28, 95% CI = 1.02-1.61) overall. Those of the iceA2 gene was inversely associated with peptic ulcer especially in Europe (OR = 0.55, 95% CI = 0.40-0.76). This trend was the same as in case of duodenal ulcer. There was no association between the presence of the iceA gene and gastric ulcer and gastric cancer. Seven studied examined the correlation with cagA gene, and only two showed the positive correlation. Publication bias did not exist. Conclusion: Our meta-analysis confirmed the importance of the presence of the iceA gene for duodenal ulcer in Western countries, especially in Europe although the significance was small. It is possible that the iceA gene is discriminating factor for peptic ulcer independent on cagA gene.

Long Type dupA but Not Short Type dupA Associated With Peptic Ulcer Disease in Japan

Background and aims:Helicobacter pylori dupA (duodenal ulcer promoting) gene was reported as a virulence factor that induced duodenal ulcer and had a suppressive action on gastric cancer. However, the association of the dupA gene on clinical outcomes is different in different populations. Full-sequenced data of H. pylori showed that the length of the dupA gene is depends on the strain; Shi470 and G27 have approximately 600bp longer than that of other strains (e.g., strain J99) in the C-terminal region of the dupA gene. This suggests that the dupA gene has two genotypes: long and short type. In addition, previous studies indicated that some strains have a presence of the mutation (insertion or mutation) in the dupA gene, which created a premature stop codon . In this study, we aimed to examine the dupA genotypes (long type or short type), presence of the mutation, and examined the association with clinical outcomes in Japanese population. Methods: A total of 388 patients (97 with gastritis, 137 with duodenal ulcer [DU], 121 with gastric ulcer [GU], and 24 with gastric cancer [GC]) were included in this study. The status of the dupA genes was examined by polymerase chain reaction. The oligonucleotide primers for the dupA typing were designed according to the dupA gene sequence deposited in Genbank. First primers were designed to include the additional region of C-terminal of the dupA gene (long type dupA). Second primers were designed to include the conventional dupA gene (short type dupA). We also sequenced full-length dupA gene and evaluated the presence of mutation. Results: The prevalence of short type dupA gene alone was not significantly different among four groups (gastritis 14.4%, DU 8.0%, GU 6.5%, and GC 4.2%, respectively). Interestingly, the prevalence of the long type dupA gene in strains from DU was significantly higher than that from gastritis (19.7 v.s. 9.3%, p = 0.02). Those of GU, GC were also significantly higher than that of gastritis (25.8, 25.0 and 9.3%, p = 0.0001, p = 0.03, respectively). Full-length sequence of the dupA gene was completed in 33 strains. Among them, point mutation lead to stop codon was found in only one strain. Conclusions: The long type but not the short type dupA gene can be used as discriminating factor of peptic ulcer and gastric cancer from gastritis in Japan. These observations suggest that only strains that are intact dupA-positive might be involved in gastroduodenal diseases.