The Protective Effect of Roseburia faecis Against Repeated Water Avoidance Stress-induced Irritable Bowel Syndrome in a Wister Rat Model.

Roseburia faecis, a butyrate-producing, gram-positive anaerobic bacterium, was evaluated for its usefulness against repeated water avoidance stress (WAS)-induced irritable bowel syndrome (IBS) in a rat model, and the underlying mechanism was explored. We divided the subjects into three groups: one without stress exposure, another subjected to daily 1-hour WAS for 10 days, and a third exposed to the same WAS regimen while also receiving two different R. faecis strains (BBH024 or R22-12-24) via oral gavage for the same 10-day duration. Fecal pellet output (FPO), a toluidine blue assay for mast cell infiltration, and fecal microbiota analyses were conducted using 16S rRNA metagenomic sequencing. Predictive functional profiling of microbial communities in metabolism was also conducted. FPO and colonic mucosal mast cell counts were significantly higher in the WAS group than in the control group (male, P = 0.004; female, P = 0.027). The administration of both BBH024 (male, P = 0.015; female, P = 0.022) and R22-12-24 (male, P = 0.003; female, P = 0.040) significantly reduced FPO. Submucosal mast cell infiltration in the colon showed a similar pattern in males. In case of fecal microbiota, the WAS with R. faecis group showed increased abundance of the Roseburia genus compared to WAS alone. Moreover, the expression of a gene encoding a D-methionine transport system substrate-binding protein was significantly elevated in the WAS with R. faecis group compared to that in the WAS (male, P = 0.028; female, P = 0.025) group. These results indicate that R. faecis is a useful probiotic for treating IBS and colonic microinflammation.

Effect of Clostridium butyricum on High-Fat Diet-Induced Intestinal Inflammation and Production of Short-Chain Fatty Acids.

BACKGROUND/AIMS: A high-fat diet (HFD) can cause intestinal inflammation and alter the gut microbiota; probiotics, however, are known to have anti-inflammatory effects. This study aimed to investigate the response of rat colon to HFD and the effect of Clostridium butyricum on HFD-induced intestinal inflammation and production of short-chain fatty acids (SCFAs) according to sex. METHODS: Male and female 6-week-old Fischer-344 rats were fed a chow diet or HFD for 8 weeks, and Biovita or three different concentrations of C. butyricum were orally gavaged. The levels of tight junction proteins (TJPs), inflammatory markers in the ascending colonic mucosa, and bile acids (BAs) and SCFAs in stool were measured. RESULTS: HFD significantly increased the histological inflammation scores and fat proportions. Fecal BA levels were higher in the HFD group than in the control group, with a more prominent increase in deoxycholic acid/cholic acid after probiotics administration in females; however, no statistically significant differences were observed. TJPs showed an opposite response to HFD depending on sex, and tended to increase and decrease after HFD in males and females, respectively. The HFD-reduced TJPs were recovered by probiotics, with some statistical significance in females. HFD-decreased butyric acid in stools appeared to be recovered by probiotics in males, but not in females. The expression of inflammatory markers (TNF-α) was increased by HFD in males and decreased with medium-concentration probiotic supplementation. The opposite was observed in females. MPO was increased by HFD in both sexes and decreased by probiotic supplementation. CONCLUSIONS: The probiotic C. butyricum improved indicators of HFD-induced colonic inflammation such as levels of inflammatory markers and increased the production of SCFAs and the expression of TJPs. These effects tended to be more pronounced in male rats, showing sex difference.

Erratum: The Protective Effect of Roseburia faecis Against Repeated Water Avoidance Stress-induced Irritable Bowel Syndrome in a Wister Rat Model.

[This corrects the article on p. 93 in vol. 28, PMID: 37830115.].

Physiological activity of E. coli engineered to produce butyric acid.

Faecalibacterium prausnitzii (F. prausnitzii) is one of the most abundant bacteria in the human intestine, with its anti-inflammatory effects establishing it as a major effector in human intestinal health. However, its extreme sensitivity to oxygen makes its cultivation and physiological study difficult. F. prausnitzii produces butyric acid, which is beneficial to human gut health. Butyric acid is a short-chain fatty acid (SCFA) produced by the fermentation of carbohydrates, such as dietary fibre in the large bowel. The genes encoding butyryl-CoA dehydrogenase (BCD) and butyryl-CoA:acetate CoA transferase (BUT) in F. prausnitzii were cloned and expressed in E. coli to determine the effect of butyric acid production on intestinal health using DSS-induced colitis model mice. The results from the E. coli Nissle 1917 strain, expressing BCD, BUT, or both, showed that BCD was essential, while BUT was dispensable for producing butyric acid. The effects of different carbon sources, such as glucose, N-acetylglucosamine (NAG), N-acetylgalactosamine (NAGA), and inulin, were compared with results showing that the optimal carbon sources for butyric acid production were NAG, a major component of mucin in the human intestine, and glucose. Furthermore, the anti-inflammatory effects of butyric acid production were tested by administering these strains to DSS-induced colitis model mice. The oral administration of the E. coli Nissle 1917 strain, carrying the expression vector for BCD and BUT (EcN-BCD-BUT), was found to prevent DSS-induced damage. Introduction of the BCD expression vector into E. coli Nissle 1917 led to increased butyric acid production, which improved the strain's health-beneficial effects.

Functional dissection of the phosphotransferase system provides insight into the prevalence of Faecalibacterium prausnitzii in the host intestinal environment.

Faecalibacterium prausnitzii is a dominant member of healthy human colon microbiota, regarded as a beneficial gut bacterium due to its ability to produce anti-inflammatory substances. However, little is known about how F. prausnitzii utilizes the nutrients present in the human gut, influencing its prevalence in the host intestinal environment. The phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) is a widely distributed and highly efficient carbohydrate transport system found in most bacterial species that catalyses the simultaneous phosphorylation and import of cognate carbohydrates; its components play physiological roles through interaction with other regulatory proteins. Here, we performed a systematic analysis of the 16 genes encoding putative PTS components (2 enzyme I, 2 HPr, and 12 enzyme II components) in F. prausnitzii A2-165. We identified the general PTS components responsible for the PEP-dependent phosphotransfer reaction and the sugar-specific PTS components involved in the transport of two carbohydrates, N-acetylglucosamine and fructose, among five enzyme II complexes. We suggest that the dissection of the functional PTS in F. prausnitzii may help to understand how this species outcompetes other bacterial species in the human intestine.

Changes in Cecal Microbiota and Short-chain Fatty Acid During Lifespan of the Rat.

BACKGROUND/AIMS: The gut microbiota regulates intestinal immune homeostasis through host-microbiota interactions. Multiple factors affect the gut microbiota, including age, sex, diet, and use of drugs. In addition, information on gut microbiota differs depending on the samples. The aim of this study is to investigate whether changes in cecal microbiota depend on aging. METHODS: Gut microbiota in cecal contents of 6-, 31-, and 74-week-old and 2-year-old male Fischer-344 rats (corresponding to 5-, 30-, 60-, and 80-year-old humans in terms of age) were analyzed using 16S ribosomal RNA metagenome sequencing and phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) based on the Kyoto Encyclopedia of Genes and Genomes orthology. Moreover, short-chain fatty acid (SCFA) level in cecum and inflammation related factors were measured using real-time quantitative polymerase chain reaction and enzyme linked immunosorbent assay. RESULTS: Alpha and beta diversity did not change significantly with age. At the family level, Lachnospiraceae and Ruminococcaceae, which produce SCFAs, showed significant change in 31-week-old rats: Lachnospiraceae significantly increased at 31 weeks of age, compared to other age groups, while Ruminococcaceae decreased. Butyrate levels in cecum were significantly increased in 31-week-old rats, and the expression of inflammation related genes was increased followed aging. Especially, EU622775_s and EU622773_s, which were highly abundance species in 31-week-old rats, showed significant relationship with butyrate concentration. Enzymes required for producing butyrate-acetyl-CoA transferase, butyryl-CoA dehydrogenase, and butyrate kinase-were not predicted by PICRUSt. CONCLUSIONS: Major bacterial taxa in the cecal lumen, such as Lachnospiraceae, well-known SCFAs-producing family, changed in 31-week-old rats. Moreover, unknown species EU622775_s and EU622773_s showed strong association with cecal butyrate level at 31 weeks of age.

Structural insight into glucose repression of the mannitol operon.

Carbon catabolite repression is a regulatory mechanism to ensure sequential utilization of carbohydrates and is usually accomplished by repression of genes for the transport and metabolism of less preferred carbon compounds by a more preferred one. Although glucose and mannitol share the general components, enzyme I and HPr, of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) for their transport, glucose represses the transport and metabolism of mannitol in a manner dependent on the mannitol operon repressor MtlR in Escherichia coli. In a recent study, we identified the dephosphorylated form of HPr as a regulator determining the glucose preference over mannitol by interacting with and augmenting the repressor activity of MtlR in E. coli. Here, we determined the X-ray structure of the MtlR-HPr complex at 3.5 Å resolution to understand how phosphorylation of HPr impedes its interaction with MtlR. The phosphorylation site (His15) of HPr is located close to Glu108 and Glu140 of MtlR and phosphorylation at His15 causes electrostatic repulsion between the two proteins. Based on this structural insight and comparative sequence analyses, we suggest that the determination of the glucose preference over mannitol solely by the MtlR-HPr interaction is conserved within  the Enterobacteriaceae family.

Gut microbiota and butyrate level changes associated with the long-term administration of proton pump inhibitors to old rats.

The association between adverse effects of PPI and gut microbiota in old age has yet to be elucidated. We assessed changes in the gut microbiota and butyrate levels following the long-term administration of PPIs to old rats and investigated their associations. F344 aged male rats were fed a PPI-supplemented diet for 50 weeks. The ileal microbiota was analysed by metagenomic sequencing of the 16S rRNA, while the butyrate concentration was measured by high-performance liquid chromatography. We observed a significant decrease in microbial diversity following PPI administration in the 2-year-old rats but not in the 74-week-old rats. PPI treatment reduced both commensal bacteria and opportunistic pathogens, particularly in the 2-year-old rats. Enterotypes comprising the majority of the control samples were enriched in Lactobacillus, while other enterotypes in the PPI group were dominated by Turicibacter or Romboutsia. The PPI treatment reduced the butyrate concentrations in the intestines and colons of 74-week-old rats compared to the control group. The abundance of Lactobacillus significantly correlated with butyrate concentrations in 74-week-old rats. In conclusion, long-term administration of PPIs alters the gut microbiota and butyrate concentrations in rats, particularly in old age, which may be an underlying mechanism of PPI-induced adverse effects such as pseudomembranous colitis.

Polar landmark protein HubP recruits flagella assembly protein FapA under glucose limitation in Vibrio vulnificus.

How motile bacteria recognize their environment and decide whether to stay or navigate toward more favorable location is a fundamental issue in survival. The flagellum is an elaborate molecular device responsible for bacterial locomotion, and the flagellum-driven motility allows bacteria to move themselves to the appropriate location at the right time. Here, we identify the polar landmark protein HubP as a modulator of polar flagellation that recruits the flagellar assembly protein FapA to the old cell pole, thereby controlling its activity for the early events of flagellar assembly in Vibrio vulnificus. We show that dephosphorylated EIIAGlc of the PEP-dependent sugar transporting phosphotransferase system sequesters FapA from HubP in response to glucose and hence inhibits FapA-mediated flagellation. Thus, flagellar assembly and motility is governed by spatiotemporal control of FapA, which is orchestrated by the competition between dephosphorylated EIIAGlc and HubP, in the human pathogen V. vulnificus.

Rat Intestinal Acetic Acid and Butyric acid and Effects of Age, Sex, and High-fat Diet on the Intestinal Levels in Rats.

BACKGROUND: High-fat diet is known to be implicated in the pathogenesis of various metabolic disorders related to an inflammatory response. The aim of this study was to investigate the influence of high-fat diet for intestinal acetic acid and butyric acid concentrations which are related to inflammation-associated colon cancer risk. METHODS: Both male and female rats of 6, 31, 74 and 104-week of age were fed chow diet or high-fat diet for 8 weeks. Body weight and food intake were measured weekly during the feeding period. Intestinal acetic acid and butyric acid levels were measured by high-performance liquid chromatography from luminal contents of ileum and cecum. RESULTS: Male rats showed greater weight change than female rats in every age. Calorie-adjusted food intake was also higher in male rats compared to female rats. Male rats showed similar intake of food in every age while 31-week old female rats showed increased intake, which was decreased at 74-week and 104-week of age. The ileal acetic acid concentration was increased in male rats fed high-fat diet, while female rats fed high-fat diet showed no significant change in the ileal acetic acid level. On the other hand, butyric acid almost disappeared in high-fat diet fed rats regardless of sex. CONCLUSIONS: High-fat diet increases the intestinal acetic acid concentration while reducing the butyric acid concentration which may account for increased risk of inflammation-associated colon cancer.

Determination of protein phosphorylation by polyacrylamide gel electrophoresis.

Phosphorylation is the most important modification for protein regulation; it controls many signal transduction pathways in all organisms. While several tools to detect phosphorylated proteins have been developed to study a variety of basic cellular processes involving protein phosphorylation, these methods have several limitations. Many proteins exhibit a phosphorylation-dependent electrophoretic mobility shift (PDEMS) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the molecular mechanism responsible for this phenomenon has been elucidated recently. The method for detecting phosphorylated proteins can be simplified by the application of the PDEMS. Herein, we present a novel simple method to detect protein phosphorylation, which is based on the construction of a variant protein displaying a PDEMS. The PDEMS of proteins is caused by the distribution of negatively charged amino acids around the phosphorylation site, i.e. an electrophoretic mobility shift (EMS)-related motif (ΘX1-3ΘX1-3Θ, where Θ corresponds to an acidic or phosphorylated amino acid and X represents any amino acid). The EMS-related motif can be constructed by the introduction of a negative charge by phosphorylation; it results in the decreased binding of SDS to the proteins, consequently inducing the retardation of the mobility of the protein during SDS-PAGE. Based on these molecular analyses of the PDEMS, a protein with the EMSrelated motif is designed and used to determine the in vivo phosphorylation state of the protein. This method may be used as a general strategy to easily measure the ratio of protein phosphorylation in cells.

Effect of the RNA pyrophosphohydrolase RppH on envelope integrity in Escherichia coli.

The bacterial enzyme RppH initiates mRNA decay by removing pyrophosphate from 5΄-triphosphorylated mRNA. Escherichia coli RppH has promiscuous substrate specificity, but relatively few transcripts are affected by loss of RppH. The phenotypic analysis of the rppH mutant is required for understanding the physiological role of RppH, but the phenotype of the rppH mutant has not yet been determined. In this study, we provide several phenotypes of the rppH mutant associated with envelope integrity. Through phenotype analysis and drug susceptibility testing, we found that the rppH mutant is sensitive to a variety of chemicals including antibiotics, and is also significantly sensitive to envelope stresses, such as osmotic stress, ethanol and sodium dodecyl sulfate. All phenotypes of the rppH mutant were caused by loss of its enzymatic activity. The rppH mutant exhibited increased envelope permeability, compared to wild-type cells. In contrast, an increase of RppH activity significantly inhibited the growth of wild-type cells under low-temperature conditions. In conclusion, various phenotypes of the rppH mutant propose that RppH is associated with regulation of envelope integrity.

Analysis of Gastric Body Microbiota by Pyrosequencing: Possible Role of Bacteria Other Than Helicobacter pylori in the Gastric Carcinogenesis.

BACKGROUND: Gastric microbiota along with Helicobacter pylori (HP) plays a key role in gastric disease. The aim of our study is to investigate the difference of human gastric microbiota between antrum and body according to disease (control vs. gastric cancer) and HP status. METHODS: Each antrum and body biopsy was collected from 12 subjects at Seoul National University Bundang Hospital. Gastric microbiota was analyzed by bar-coded 454 pyrosequencing of the 16S rRNA gene. Twelve subjects consisted of HP-negative control (n = 2), HP-negative cancer (n = 2), HP-positive control (n = 3), and HP-positive cancer (n = 5). The analysis was focused on non-HP urease-producing bacteria (UB) and non-HP nitrosating or nitroreducing bacteria (NB) between antrum and body. RESULTS: Gastric body samples showed higher diversity compared to gastric antrum mucosa samples but there was no significant difference. The mean of operational taxonomic units was higher in HP(-) cancer than HP(+) cancer (antrum, 273.5 vs. 228.2, P = 0.439; body, 585.5 vs. 183.2, P = 0.053). The number of non-HP UB and non-HP NB was higher in HP(-) cancer groups than the others. These differences were more pronounced in the body (P = 0.051 and P = 0.081, respectively). Analysis of overlap of non-HP UB and non-HP NB revealed the higher composition of Streptococcus pseudopneumoniae, S. parasanguinis, and S. oralis in HP(-) cancer groups than the others, only in the body (P = 0.030) but not in the antrum (P = 0.123). CONCLUSIONS: Higher diversity and higher composition of S. pseudopneumoniae, S. parasanguinis, and S. oralis in HP(-) cancer group than the other groups in the body suggest that analysis of microbiota from body mucosa could be beneficial to identify a role of non-HP bacteria in the gastric carcinogenesis.

The general PTS component HPr determines the preference for glucose over mannitol.

Preferential sugar utilization is a widespread phenomenon in biological systems. Glucose is usually the most preferred carbon source in various organisms, especially in bacteria where it is taken up via the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The currently proposed model for glucose preference over non-PTS sugars in enteric bacteria including E. coli is strictly dependent on the phosphorylation state of the glucose-specific PTS component, enzyme IIAGlc (EIIAGlc). However, the mechanism of the preference among PTS sugars is largely unknown in Gram-negative bacteria. Here, we show that glucose preference over another PTS sugar, mannitol, is absolutely dependent on the general PTS component HPr, but not on EIIAGlc, in E. coli. Dephosphorylated HPr accumulates during the transport of glucose and interacts with the mannitol operon regulator, MtlR, to augment its repressor activity. This interaction blocks the inductive effect of mannitol on the mannitol operon expression and results in the inhibition of mannitol utilization.

Erratum: Analysis of Gastric Body Microbiota by Pyrosequencing: Possible Role of Bacteria Other Than Helicobacter pylori in the Gastric Carcinogenesis.

[This corrects the article on p. 115 in vol. 22, PMID: 28698866.].

Impact of Long-Term Proton Pump Inhibitor Therapy on Gut Microbiota in F344 Rats: Pilot Study.

BACKGROUND/AIMS: To evaluate changes in gut microbiota composition following long-term proton pump inhibitor (PPI) treatment. METHODS: Twenty-four-week-old F344 rats were fed diets with (n=6) or without (n=5) lansoprazole for 50 weeks. Profiles of luminal microbiota in the terminal ileum were then analyzed. Pyrosequencing of the 16S rRNA gene was performed using an FLX genome sequencer (454 Life Sciences/Roche). RESULTS: Rats treated with lansoprazole showed significantly reduced body weights compared to controls (lansoprazole-treated rats and controls, 322.3±15.3 g vs 403.2±5.2 g, respectively, p<0.001). However, stool frequencies and consistencies did not differ between the two groups. The composition of the gut microbiota in lansoprazole-treated rats was quite different from that of the controls. In the controls, the microbiota profiles obtained from the terminal ileum showed a predominance of Proteobacteria (93.9%) due to the abundance of Escherichia and Pasteurella genera. Conversely, lansoprazole-treated rats showed an elevated population of Firmicutes (66.9%), which was attributed to an increased ratio of Clostridium g4 to Lactobacillus genera. CONCLUSIONS: This preliminary study suggests that long-term administration of PPI may cause weight loss and changes to the microbiota in the terminal ileum.

Glucose induces delocalization of a flagellar biosynthesis protein from the flagellated pole.

To survive in a continuously changing environment, bacteria sense concentration gradients of attractants or repellents, and purposefully migrate until a more favourable habitat is encountered. While glucose is known as the most effective attractant, the flagellar biosynthesis and hence chemotactic motility has been known to be repressed by glucose in some bacteria. To date, the only known regulatory mechanism of the repression of flagellar synthesis by glucose is via downregulation of the cAMP level, as shown in a few members of the family Enterobacteriaceae. Here we show that, in Vibrio vulnificus, the glucose-mediated inhibition of flagellar motility operates by a completely different mechanism. In the presence of glucose, EIIA(Glc) is dephosphorylated and inhibits the polar localization of FapA (flagellar assembly protein A) by sequestering it from the flagellated pole. A loss or delocalization of FapA results in a complete failure of the flagellar biosynthesis and motility. However, when glucose is depleted, EIIA(Glc) is phosphorylated and releases FapA such that free FapA can be localized back to the pole and trigger flagellation. Together, these data provide new insight into a bacterial strategy to reach and stay in the glucose-rich area.

Increased expression of genes involved in uptake and degradation of murein tripeptide under nitrogen starvation in Escherichia coli.

Peptidoglycan (also known as murein) is an important envelope component of bacteria, and its turnover usually takes place at considerable levels during normal growth. Amino sugars and murein tripeptide resulting from murein degradation are used for resynthesis of peptidoglycan or as self-generated nutrients or energy sources for cell growth. PgrR (regulator of peptide glycan recycling; formerly YcjZ) was recently identified as a repressor of several genes participating in uptake and degradation of murein tripeptide. In this study, we identified the ycjG gene involved in murein tripeptide degradation as a new direct target of PgrR. The expression of PgrR-regulated genes including ycjY, mppA, mpaA and ycjG was repressed in the presence of a good nitrogen source, but their expression increased under poor nitrogen conditions. Under nitrogen starvation, the pgrR mutant cells exhibited faster growth than wild-type cells, implying that derepression of genes under the control of PgrR may help cells overcome nitrogen limitation. Therefore, these results suggest that nitrogen starvation induces derepression of PgrR-controlled genes involved in uptake and degradation of murein tripeptide, and this may stimulate the utilization of murein tripeptide as a nitrogen source.

Comparison of Gastric Microbiota Between Gastric Juice and Mucosa by Next Generation Sequencing Method.

BACKGROUND: Not much is known about the role of gastric microbiota except for Helicobacter pylori in human health and disease. In this study, we aimed to detect human gastric microbiota in both gastric mucosa and gastric juice by barcoded 454-pyrosequencing of the 16S rRNA gene and to compare the results from mucosa and juice. METHODS: Gastric biopsies and stomach juices were collected from 4 subjects who underwent standard endoscopy at Seoul National University Bundang Hospital. Gastric microbiota of antral mucosa, corpus mucosa samples, and gastric fluids were analyzed by barcoded 454-pyrosequencing of the 16S rRNA gene. The analysis focused on bacteria, such as H. pylori and nitrosating or nitrate-reducing bacteria. RESULTS: Gastric fluid samples showed higher diversity compared to that of gastric mucosa samples. The mean of operational taxonomic units was higher in gastric fluid than in gastric mucosa. The samples of gastric fluid and gastric mucosa showed different composition of phyla. The composition of H. pylori and Proteobacteria was higher in mucosa samples compared to gastric fluid samples (H. pylori, 66.5% vs. 3.3%, P = 0.033; Proteobacteria, 75.4% vs. 26.3%, P = 0.041), while Actinobacteria, Bacteroidetes, and Firmicutes were proportioned relatively less in mucosa samples than gastric fluid. However there was no significant difference. (Actinobacteria, 3.5% vs. 20.2%, P = 0.312; Bacteroidetes, 6.0% vs. 14.8%, P = 0.329; Firmicutes, 12.8% vs. 33.4%, P = 0.246). CONCLUSIONS: Even though these samples were small, gastric mucosa could be more effective than gastric fluid in the detection of meaningful gastric microbiota by pyrosequencing.

Analysis of Gastric Microbiota by Pyrosequencing: Minor Role of Bacteria Other Than Helicobacter pylori in the Gastric Carcinogenesis.

BACKGROUND: Little is known about the role of gastric microbiota except for Helicobacter pylori (HP) in human health and disease. We compared the differences of human gastric microbiota according to gastric cancer or control and HP infection status and assessed the role of bacteria other than HP. METHODS: Gastric microbiota of 63 antral mucosal and 18 corpus mucosal samples were analyzed by bar-coded 454 pyrosequencing of the 16S rRNA gene. Antral samples were divided into four subgroups based on HP positivity in pyrosequencing and the presence of cancer. The analysis was focused on bacteria other than HP, especially nitrosating or nitrate-reducing bacteria (NB). The changes of NB in antral mucosa of 16 subjects were followed up. RESULTS: The number of NB other than HP (non-HP-NB) was two times higher in the cancer groups than in the control groups, but it did not reach statistical significance. The number of non-HP-NB tends to increase over time, but this phenomenon was prevented by HP eradication in the HP-positive control group, but not in the HP-positive cancer group. CONCLUSION: We could not find the significant role of bacteria other than HP in the gastric carcinogenesis.