Enhanced infectivity of strains of Helicobacter pylori isolated from children compared with parental strains.

PURPOSE: Intra-familial infection, mother-to-child infection, is considered to be one of the main routes of transmission for Helicobacter pylori, in developed countries such as Japan. A major role for intra-familial spread in the pathogenicity of H. pylori is now beyond controversy, although the major route of transmission remains poorly understood. We performed this study to clarify the factors determining intra-familial transmission. METHODOLOGY: We used several H. pylori strains isolated from family members to compare infectivity. H. pylori K21 and K22 strains were isolated from the father and mother, and the K25 strain was isolated from the third child of the family. Mongolian gerbils were inoculated with H. pylori strains and the infectivity of three strains was compared in each experiment. In addition, the whole genome sequence, adhesion to gastric epithelial cells and the growth of static condition or continuous flow culture among three strains of H. pylori were analysed.Results/Key findings. Most of the colonies were determined as the same molecular type K25 in all of the four grouped animals and H. pylori K25 was observed as the dominant strain. The stronger adhesion capacity of the K25 strain was observed in comparison with the other two strains through in vitro analysis. By assessing the genomic profiles of H. pylori isolates from three strains, identified TnPZ regions were detected only in the K25 strain. CONCLUSION: The infectivity of H. pylori isolates intra-familial infection and animal infection were prescribed by the adhesion capacity and molecular type of each strain.

Influence of Intestinal Indigenous Microbiota on Intrafamilial Infection by Helicobacter pylori in Japan.

Helicobacter pylori is a causative pathogen of chronic gastritis, gastric ulcer disease, and gastric cancer. Humans are known to be a natural host for H. pylori and tend to acquire the pathogen before the age of 5 years. The infection may then persist lifelong if eradication therapy is not applied. One of the modes of transmission of H. pylori is between family members, and therefore, the presence of infected family members is an important risk factor in children. However, other environmental factors have not been fully analyzed. The present study was performed to clarify whether and to what extent intestinal microbiota affect H. pylori intrafamilial infection. The fecal specimens from H. pylori-infected infants and H. pylori-infected and non-infected family members were collected in cohort studies conducted by Sasayama City, Hyogo Prefecture from 2010 to 2013. In total, 18 fecal DNA from 5 families were analyzed. Samples were amplified using 16S rRNA universal primers, and the amplicons were sequenced using the Ion PGM system. Principal-coordinate analysis demonstrated that there was no difference in intestinal microbiota between H. pylori-positive and H. pylori-negative groups. In intrafamilial comparison tests, the Manhattan distance of intestinal microbiota between the H. pylori-infected infant proband and H. pylori-negative mother was nearest in the family with low intestinal microbial diversity. However, in the family with the highest intestinal microbial diversity, the nearest Manhattan distance was shown between the H. pylori-infected infant proband and H. pylori-infected mother. The results in this study showed that the composition of the intestinal microbiota was very similar between members of the same family, and as such, colonization with organisms highly similar to the infected parent(s) may be a risk factor for H. pylori infection in children.

Usefulness of detection of clarithromycin-resistant Helicobacter pylori from fecal specimens for young adults treated with eradication therapy.

BACKGROUND: To prevent Helicobacter pylori infection in the younger generation, it is necessary to investigate the prevalence of antibiotic-resistant H. pylori. OBJECTIVE: The aim of this study was to evaluate the method of PCR-based sequencing to detect clarithromycin (CAM) resistance-associated mutations using fecal samples as a noninvasive method. METHODS: DNA extracted from fecal specimens and isolates from gastric biopsy specimens were collected from patients with H. pylori infection. Antibiotic resistance to CAM was analyzed by molecular and culture methods. The detection rates of CAM resistance-associated mutations (A2142C or A2143G) were compared before and after eradication therapy. RESULTS: With CAM resistance of H. pylori evaluated by antibiotic susceptibility test as a gold standard, the sensitivity and the specificity of gene mutation detection from fecal DNA were 80% and 84.8%, respectively. In contrast, using DNA of isolated strains, the sensitivity and the specificity were 80% and 100%. Of the seven cases in which eradication was unsuccessful by triple therapy including CAM, CAM-resistant H. pylori, and resistance-associated mutations were detected in three cases, CAM-resistant H. pylori without the mutation was detected in two patients, and resistance-associated mutation was only detected in one patient. CONCLUSION: PCR-based sequencing to detect CAM resistance-associated mutations using isolates or fecal samples was useful for finding antibiotic-resistant H. pylori infection. Although the specificity of the detection from fecal samples compared with antibiotic susceptibility testing was lower than that from isolates, this fecal detection method is suitable especially for asymptomatic subjects including children. Further improvement is needed before clinical application.

Diversification of the AlpB Outer Membrane Protein of Helicobacter pylori Affects Biofilm Formation and Cellular Adhesion.

Helicobacter pylori is one of the most common causes of bacterial infection in humans, and it forms biofilms on human gastric mucosal epithelium as well as on in vitro abiotic surfaces. Bacterial biofilm is critical not only for environmental survival but also for successful infection. We previously demonstrated that strain TK1402, which was isolated from a Japanese patient with duodenal and gastric ulcers, has high biofilm-forming ability in vitro relative to other strains. In addition, we showed that outer membrane vesicles (OMV) play an important role in biofilm formation. The aim of this study was to analyze which protein(s) in the OMV contributes to biofilm formation in TK1402. We obtained a spontaneous mutant strain derived from TK1402 lacking biofilm-forming ability. The protein profiles of the OMV were compared between this mutant strain and the wild type, and it was found that AlpB, an outer membrane protein in the OMV of the mutant strain, was markedly decreased compared to that of the wild type. Restoration of TK1402 alpB to the mutant strain fully recovered the ability to form biofilm. However, restoration with alpB from other strains demonstrated incomplete recovery of biofilm-forming ability. We therefore inferred that the variable region of AlpB (amino acid positions 121 to 146) was involved in TK1402 biofilm formation. In addition, diversification of the AlpB sequence was shown to affect the ability to adhere to AGS cells. These results demonstrate a new insight into the molecular mechanisms of host colonization by H. pyloriIMPORTANCE Bacterial biofilm is critical not only for environmental survival but also for successful infection. The mechanism of Helicobacter pylori adherence to host cells mediated by cell surface adhesins has been the focus of many studies, but little is known regarding factors involved in H. pylori biofilm formation. Our study demonstrated that AlpB plays an important role in biofilm formation and that this property depends upon the specific sequence of alpB This in turn was shown to be important in the ability to adhere to gastric cells. We anticipate that these results will provide new insight into the molecular mechanisms of H. pylori colonization.

Analysis of intra-familial transmission of Helicobacter pylori in Japanese families.

Intra-familial infection is considered to be one of the main routes of transmission for Helicobacter pylori in Japan. We assessed the genomic profiles of H. pylori isolates from family members by multi-locus sequence typing (MLST) and identified the original strain infecting the index child. A total of 19 isolates from five families were analysed by MLST using seven housekeeping genes and by random amplification of polymorphic DNA (RAPD)-PCR. Phylogenetic analysis was performed using nucleotide sequences of the seven loci. Two or more different types of H. pylori strains were indicated in three (K-1, K-2 and K-5) out of five families. Independent genotypes of H. pylori strains were detected from all members of the other two families suggesting that these strains (K26-28 and K29-33) may be dominant. Mother-to-child transmission of H. pylori was demonstrated in four out of five families, whilst transmission from father-to-child and sibling-to-sibling were demonstrated in two families and one family, respectively.

Analysis of the microbial ecology between Helicobacter pylori and the gastric microbiota of Mongolian gerbils.

Animal models are essential for in vivo analysis of Helicobacter-related diseases. Mongolian gerbils are used frequently to study Helicobacter pylori-induced gastritis and its consequences. The presence of some gastric microbiota with a suppressive effect on H. pylori suggests inhibitory gastric bacteria against H. pylori infection. The aim of the present study was to analyse the microbial ecology between H. pylori and the gastric microbiota of Mongolian gerbils. Gastric mucosa samples of H. pylori-negative and -positive gerbils were orally inoculated to five (Group 1) and six (Group 2) gerbils, respectively, and the gerbils were challenged with H. pylori infection. The colonization rate (40 %) of H. pylori in Group 1 gerbils was lower than the rate (67 %) in Group 2 gerbils. Culture filtrate of the gastric mucosa samples of Group 1 gerbils inhibited the in vitro growth of H. pylori. Three lactobacilli species, Lactobacillus reuteri, Lactobacillus johnsonii and Lactobacillus murinus, were isolated by anaerobic culture from the gerbils in Groups 1 and 2, and identified by genomic sequencing. It was demonstrated that the three different strains of lactobacilli exhibited an inhibitory effect on the in vitro growth of H. pylori. The results suggested that lactobacilli are the dominant gastric microbiota of Mongolian gerbils and the three lactobacilli isolated from the gastric mucosa samples with an inhibitory effect on H. pylori might have an anti-infective effect against H. pylori.

Comparative analysis of gastric bacterial microbiota in Mongolian gerbils after long-term infection with Helicobacter pylori.

Quantitative (qt) real time PCR using 16SrDNA primers is useful for determination of the bacterial composition of the gastric microbiota in Mongolian gerbils. The aim of this study was to determine the change in the gastric microbiota after long-term infection with Helicobacter pylori. One year after inoculation with H. pylori, five gerbils were determined as H. pylori-positive and 6 gerbils H. pylori-negative by culture and real time qt PCR methods. The gastric microbiota of each group of gerbils was also compared with that of 6 gerbils uninfected with H. pylori. DNA from the Atopobium cluster, Bifidobacterium spp., Clostridium coccoides group, Clostridium leptum subgroup, Enterococcus spp. and Lactobacillus spp. were detected in the gastric mucus of both infected and uninfected gerbils. In contrast, Eubacterium cylindroides group and Prevotella spp. were detected only in H. pylori-negative gerbils. The numbers of C. leptum subgroup, C. coccoides group and Bifidobacterium spp. in gastric mucus of H. pylori-negative Mongolian gerbils were significantly lower than those in non-infected gerbils. The results obtained suggest that the composition of gastric indigenous microbiota in Mongolian gerbils may be disturbed by long-term infection with H. pylori, and that these changes may in fact inhibit H. pylori infection.

Destructive effects of butyrate on the cell envelope of Helicobacter pylori.

Helicobacter pylori can be found in the oral cavity and is mostly detected by the use of PCR techniques. Growth of H. pylori is influenced by various factors in the mouth, such as the oral microflora, saliva and other antimicrobial substances, all of which make colonization of the oral cavity by H. pylori difficult. In the present study, we analysed the effect of the cell supernatant of a representative periodontal bacterium Porphyromonas gingivalis on H. pylori and found that the cell supernatant destroyed the H. pylori cell envelope. As P. gingivalis produces butyric acid, we focused our research on the effects of butyrate and found that it significantly inhibited the growth of H. pylori. H. pylori cytoplasmic proteins and DNA were detected in the extracellular environment after treatment with butyrate, suggesting that the integrity of the cell envelope was compromised and indicating that butyrate has a bactericidal effect on H. pylori. In addition, levels of extracellular H. pylori DNA increased following treatment with the cell supernatant of butyric acid-producing bacteria, indicating that the cell supernatant also has a bactericidal effect and that this may be due to its butyric acid content. In conclusion, butyric acid-producing bacteria may play a role in affecting H. pylori colonization of the oral cavity.

Analysis of outer membrane vesicle protein involved in biofilm formation of Helicobacter pylori.

Helicobacter pylori is one of the most common causes of bacterial infection in humans. Infection with H. pylori is closely associated with gastritis and peptic ulcers and is a risk factor for gastric cancer and mucosa-associated lymphoid tissue lymphoma. H. pylori forms biofilms on glass surfaces at the air-liquid interface in in-vitro batch cultures. We previously reported that strain TK1402 showed a strong biofilm-forming ability in vitro. We also suggested the outer membrane vesicles (OMV) produced by strain TK1402 might be related to its biofilm forming ability. In the present study, we analyzed the protein profile of the OMV produced by strain TK1402 and found a unique 22-kDa protein in TK1402 OMV cultured for 2-3 days. In addition, this protein could not be detected in the OMVs produced by other H. pylori strains. These results suggest that the 22-kDa protein is involved in effective biofilm formation by strain TK1402.

In vitro and in vivo effects of the Mongolian drug Amu-ru 7 on Helicobacter pylori growth and viability.

Amu-ru 7, a Mongolian folk medicine, is used to treat digestive diseases such as gastritis and gastric and duodenal ulcers. We examined the effect of Amu-ru 7 on the growth and viability of Helicobacter pylori in vivo and in vitro. By the agar dilution method, the MIC of Amu-ru 7 for H. pylori strains was shown to be 100-200 µg/mL with a MIC(90) of 200 µg/mL. Two hundred micrograms per milliliter of Amu-ru 7 exhibited potent bactericidal activity against H. pylori in the stationary phase of growth 6 hr after treatment. Amu-ru 7 inhibited the growth of both AMPC-resistant and CAM-resistant strains, and also had a combined effect with AMPC on AMPC-resistant strain 403. The Amu-ru 7 inhibited biofilm formation by H. pylori and induced morphological changes, such as bleb-like formation and shortening of the cell. Although colonization of the stomach of the Mongolian gerbil by H. pylori was not cured by treatment with Amu-ru 7, both the mean number of H. pylori colonized and the colonization rate were decreased in Amu-ru 7 treated gerbils. These results suggest the effectiveness Amu-ru 7 as an adjunct therapy for eradication therapies consisting of a PPI combined with antibiotics.

Analysis of the microflora in the stomach of Mongolian gerbils infected with Helicobacter pylori.

BACKGROUND AND AIMS: Mongolian gerbils are frequently used to study Helicobacter pylori-induced gastritis and its consequences. The presence of some gastric flora with a suppressive effect on H. pylori suggests inhibitory microflora against H. pylori infection. The aim of the present study was to analyze the microflora in the stomach of Mongolian gerbils with H. pylori infection. METHODS: H. pylori ureA was detected by polymerase chain reaction (PCR) in the fecal samples of infected Mongolian gerbils. H. pylori was isolated from the gastric mucosa of the gerbils by microaerophilic cultivation. Gastric microflora were isolated by aerobic and anaerobic culture, and the identification of gastric bacterial species was performed by API20E and API20A. RESULTS: Oral administration of H. pylori TK1402 induced colonization and gastric inflammation of the stomach of the Mongolian gerbils. According to the frequency of detection of H. pylori ureA in fecal samples, the gerbils were divided into three groups (frequently detected, moderately detected and infrequently detected). According to the analysis of the gastric microflora in the frequently and infrequently detected groups, Lactobacillus spp. and Eubacterium limosum were isolated from the former and latter group, respectively. CONCLUSION: Some gastric flora, such as Lactobacillus spp., may inhibit colonization by H. pylori.

Assessment of in vitro biofilm formation by Helicobacter pylori.

BACKGROUND AND AIMS: Biofilms are surface-bound communities of bacterial cells that are implicated in their survival. As with various bacteria studied to date, Helicobacter pylori can have an alternate lifestyle as a biofilm. We previously reported that strain TK1402 showed a strong biofilm-forming ability in vitro. However, the mechanisms of its biofilm development remain unclear. We analyzed the basic characteristics of the biofilm-forming ability in strain TK1402. METHODS: In order to characterize the biofilm-forming ability of the H. pylori strains, auto-aggregation, motility and hydrophobicity, which are important factors in biofilm formation by other bacteria, were analyzed. Further, we tested whether cell growth participated in biofilm formation in strain TK1402. RESULTS: There were no significant differences in the auto-aggregation, motility and hydrophobicity of strain TK1402 compared with the other strains. On the other hand, pre-culture of this strain for 24-48 h resulted in decreased biofilm formation. CONCLUSION: TK1402 is a strong biofilm-forming strain of H. pylori in Brucella broth supplemented with 7% fetal calf serum. It is possible that biofilm-forming cell growth is a principal factor in biofilm development.

Vertical Helicobacter pylori transmission from Mongolian gerbil mothers to pups.

To identify the time frame and route of mother-to-child Helicobacter pylori infection, a Mongolian gerbil model was used. Four-week-old female Mongolian gerbils were infected with H. pylori, and then mated with uninfected males 2 months after infection. The offspring were sacrificed weekly after birth, and then serum, mother's milk from the stomach and gastric tissues were obtained from pups. Anti-H. pylori antibody titres were measured in sera and maternal milk using an ELISA. The stomach was cut in two in the sagittal plane, and then H. pylori colonization in mucosa was confirmed by culture and real-time RT-PCR in one specimen and by immunochemical staining in the other. Faeces and oral swabs were obtained from infected mothers, and H. pylori 16S rRNA was measured using real-time RT-PCR. H. pylori was not identified in cultures from the gastric mucosa of pups delivered by infected mothers, but H. pylori 16S rRNA was detected from 4 weeks after birth, suggesting that Mongolian gerbil pups become infected via maternal H. pylori transmission from 4 weeks of age. The anti-H. pylori antibody titre in sera of pups from infected mothers was maximum at 3 weeks of age and then rapidly decreased from 4 weeks of age. High antibody titres in mother's milk were detected during the suckling period, and GlcNAcalpha was detectable at 2-4 weeks of age, but disappeared as the offspring aged. Thus H. pylori seems to infect Mongolian gerbil pups from 4 weeks of age, in parallel with decreasing GlcNAcalpha expression in the gastric mucosa. These results suggested that H. pylori infection of Mongolian gerbil pups occurs via faecal-oral transmission from an infected mother.