Although a causal relationship has long been suspected between H. pylori infection and cardiovascular disease, haematologic disease, and metabolic syndrome, the roles of commensal microbiota in these diseases have remained obscure . This study found that H. pylori infection significantly influenced the relative abundance of three phyla and 10 genera in the duodenal microbiota and that the altered duodenal microbiota was characterised by increased Neisseria abundance and an enhanced impact of Gammaproteobacteria. The abundance of multiple commensal microbial metabolic pathways was also significantly altered, suggesting that H. pylori altered aspects of microbial metabolites that may affect host biofunctions. Many studies have investigated the gut microbiota before and after therapy for various diseases [6, 19]. Although many comparative studies on the gut microbiota have associated differences in the gut microbiota with certain diseases, the results remain inadequate, especially for factors originating in different microbiota that are substantial etiological effectors .
Although a study comparing biopsied gastric tissue with and without H. pylori reported differences in diversity , the present α- and β-diversity analyses in this limited sample size revealed no significant differences between H. pylori-positive and -negative groups (Supplementary Fig. 2). Although the results of the diversity analyses indicate no differences, this does not necessarily indicate that an identical abundance or representation of bacterial species exists in each group. In fact, the duodenal bacterial community structures differed at the phylum level between the two groups, with a greater abundance of Proteobacteria and a lower abundance of Actinobacteria and TM7 (Saccharibacteria) in the H. pylori-positive group. The greater abundance of Proteobacteria is a predictable natural consequence because H. pylori belongs to Epsilonproteobacteria, a subclass of Proteobacteria. However, the bacterial community structure at the genus level and LEfSe results suggested that H. pylori infection altered the microbial features by increasing the abundance of Neisseria and enhancing the impact of Gammaproteobacteria in the duodenum (Table 1 and Fig. 2). The increase in Neisseria in the duodenum is probably related to the gastric acid output owing to atrophic gastritis induced by H. pylori. Intraoral indigenous bacteria in the genus Neisseria are not generally highly pathogenic, except for Neisseria gonorrhoeae and Neisseria meningitidis, which respectively cause gonorrhoea and meningitis . However, excessive Neisseria proliferation in the duodenum may be pathogenic through changes in microbial community structure [21, 22]. The subclass Gammaproteobacteria comprises several medically important bacteria, such as Enterobacteriaceae, Vibrionaceae, and Pseudomonadaceae.
Many studies on the relationships between H. pylori infections and extra-gastric diseases have identified increased short-chain fatty acid (SCFA) production induced by the proliferation of Bacteroidetes . These SCFAs induce the release of gut hormones such as peptide YY and glucose like peptide-1, activation of host metabolic pathways, mucosal immune response, and inflammation [19, 23, 24]. This study revealed that H. pylori did not significantly change the abundance of Bacteroidetes in the duodenum (Fig. 1). The LDA scores also indicated that taxa belonging to Bacteroidetes did not significantly affect duodenal microbial features (Fig. 2). These findings were consistent with previous analyses of duodenal aspirates . Although the hypothesis that increased SCFA production causes various diseases is attractive, SCFAs are generated primarily through the fermentation of nonhost-digestible dietary fibres by the colonic microbiota. Therefore, other factors associated with upper gastrointestinal microbial functions should be considered.
The KEGG pathway analysis showed that 12 bacterial metabolic pathways were affected by the presence or absence of H. pylori infection. Two pathways that were upregulated in the H. pylori-positive group, synthesis and degradation of ketone bodies (ko00072) and ether lipid metabolism (ko00565), are important for degradation of fatty acids, butyrate and acetic acid synthesis, and the production of phosphocholine or seminolipid, which functions in the maintenance of mucosal integrity and immune homeostasis [25, 26].
Notably, the ko-abundance of the tryptophan metabolic pathway (ko00380) was significantly greater in the H. pylori-positive group, suggesting that an abnormal tryptophan supply from the intestine impaired serotonin production. Serotonin is a paracrine messenger expressed primarily in enterochromaffin cells and enteric neurons. This information would help to clarify the causal relationship between H. pylori infection, the duodenal microbiota, and the pathophysiology of functional dyspepsia [27–29]. In addition, serotonin production issues may alter local serotonin concentrations in portal blood, which can also affect the gut-liver axis .
The pathways of linoleic (ko00591) and α-linolenic (ko00592) acid metabolism were also upregulated in the H. pylori-positive group (Table 2). Such upregulation may cause an imbalance between ω-3 and ω-6 fatty acids and affect the arachidonic acid cascade associated with inflammation . The biotin metabolic pathway (ko00780) was also upregulated in the H. pylori-positive group. Bacteria synthesise biotin, which is an indispensable essential cofactor for fatty acid biosynthesis. Vitamin A production may also be affected by H. pylori because the biosynthetic pathway of the vitamin A precursor, carotenoid (ko00906), was upregulated in the H. pylori-positive group [32, 33]. The phenylpropanoid biosynthesis pathway (ko00940) was also upregulated significantly in this group. However, the physiological significance of this upregulation in humans is difficult to determine because the roles of metabolites (such as chavicol, eugenol, lignin) originating in this pathway have not been fully elucidated. The terpenoid and steroid biosynthesis pathway (ko01062) was upregulated in the H. pylori-positive group. This could extensively affect host functions because terpenoids are steroid precursors and closely related to cytochrome P450 that functions as an oxidase in terpenoid biosynthesis [34, 35].
Yap et al.  found 45 upregulated and 551 downregulated serum metabolites 18 months after H. pylori eradication. The affected metabolites were mapped to various biochemical pathways, including tryptophan metabolism, biosynthesis of unsaturated fatty acids, and linoleic acid metabolism. Although whether these alterations affect host biofunctions remains obscure, our findings confirmed that the metabolomic findings reported by Yap et al. originated from microbial metabolic pathways affected by H. pylori infection.
In this study, we have several study limitations and issues. First, we could not exclude the possibility that some subjects in the H. pylori-negative group might have already experienced significant changes in the structure and biofunctions of the commensal duodenal microbiota due to previous H. pylori infections. In fact, nine patients with a history of infection and eradication were included in the H. pylori-negative group (Supplementary Information 1). To eliminate this concern, it is necessary to analyse changes in microbial features before and after H. pylori eradication therapy in the same subject.
In addition, we could not comprehensively evaluate the effect of gastric acid on the duodenal microbiota because we did not quantify gastric acid secretion. The extent of gastric mucosal atrophy caused by H. pylori infection depends on various factors, such as age, duration of infection, differences between individual immune responses, and the number of bacteria. The gastric acid output depends on the extent of gastric mucosal atrophy, and the extent of atrophic gastritis is closely related to a history of H. pylori infection . In fact, the endoscopic findings in this study indicated that an extended atrophic change was likely to be observed in the H. pylori-positive group. Moreover, the duodenal microbiota might be affected by a decrease in gastric acid output.
Another limitation is that contamination with gastric microbiota could not be completely ruled out because of the sampling method. A concern has been raised that aspirate samples include only floating microbiota, which may have little to do with host biofunctions, and that the microbiota originating from biopsy samples (mucosa-associated microbial community structure) inhabit the mucosa [38, 39]. Our findings suggest that microbial metabolite production may fluctuate depending on changes in commensal duodenal microbiota and this phenomenon may affect host biofunctions. These action mechanisms may not depend on areas inhabited by microbes, such as duodenal juice or mucosa, because microbial metabolites act as chemical effectors. This study focused on changes in duodenal bacterial flora caused by the presence of H. pylori rather than direct changes in the duodenal bacterial flora.
Finally, differential abundance (DA) analysis methods for microbiome data are controversial in terms of consistency and reliability. For example, some have pointed out that sometimes the false discovery rate could not be controlled in the LEfSe analysis, which was used in this study . The problem originates in the biases due to differences in sampling fractions among collected samples, and it would be difficult to correct the biases adequately. Recently, the Analysis of Compositions of Microbiomes with Bias Correction (ANCOM-BC) has been proposed as a solution to overcome this shortcoming and is considered having a potential as a more reliable DA analysis method for microbiome data .
In conclusion, H. pylori infection changed the aspects of the microbiota in the descending part of the duodenum. This dysbiosis, characterised primarily by the upregulation of microbial metabolic pathways, altered commensal microbial biofunctions, which may affect host biofunctions. The gut microbiota can be regarded as an independent organ within the gut lumen, and an investigation of biofunctions originating from this “commensal bacterial organ” would help elucidating the aetiology of various diseases.