Introduction

Functional dyspepsia (FD) is a highly prevalent disorder of gut brain interaction that substantially impairs quality of life. FD is associated with unexplained low grade duodenal inflammation and increased duodenal permeability. Emerging evidence implicates Streptococcus species in the disorder, and we have previously identified a unique strain of Streptococcus salivarius, AGIRA0003, to which specific IgG antibodies were detected in the plasma of FD patients. We hypothesised AGIRA0003 is a pathobiont with the capacity to breach duodenal epithelial barrier defences and translocate into the mucosa, inducing the subtle immune response and symptoms observed in a subset of FD patients.

Aims & Methods

We aimed to investigate the capacity of AGIRA0003 to breach duodenal epithelial defences. Differentiated Caco-2 cell transwell monolayers (n=3), as models of the epithelial barrier, were exposed to 4 strains of cultured S. salivarius, AGIRA0003, ATCC 7073, K12 and M18, with the last 2 being commercially available probiotics, at a multiplicity of infection of 10 for 6 hours. Epithelial permeability was measured by transepithelial electrical resistance (TEER), and tight junction (TJ) protein expression was measured using immunoblot, cytokine secretion by Legendplex bead-based immunoassay, along with bacterial translocation. As a proof of concept, 2-dimensional epithelial transwell monolayer cultures, derived from duodenal spheroids, from an FD patient and outpatient control (OC) were exposed to AGIRA0003.

Results

AGIRA0003 translocated through the Caco-2 monolayer (3hr: 901.5±317.4 CFU/mL) and significantly decreased ΔTEER (-83.85 ± 27.52 Ωxcm², p=0.047) compared to the control (-23.99±7.91 Ωxcm²). In addition, AGIRA0003 disrupted expression of TJ proteins claudin 1 (CLDN1) (fold change (FC) 0.643±0.058, p=0.037), cleaved desmocollin 2 (DSC2 CL) (FC 0.57±0.071, p=0.037) and zonula occludens 1 (ZO-1) (FC 0.55±0.026, p=0.058). AGIRA0003 elicited subtle inflammatory cytokine response from Caco-2 cells including secretion of IP-10 (41.15±8.33 pg/mL, p=0.046) and IL-8 (61.76±36.90 pg/mL, p=0.046). An FD duodenal 2D spheroid epithelial monolayer had decreased levels of CLDN1 (FC to respective media control: 10.13 OC, 0.60 FD) and ZO-1 (FC to respective media control: 4.82 OC, 0.29 FD) compared to an OC following exposure to AGIRA0003.

Conclusion

These investigations implicate AGIRA0003 as a possible pathobiont contributing to FD pathophysiology and may be a suitable target for therapeutic intervention. AGIRA0003 has the capacity to breach Caco-2 epithelial barrier defences, translocate and elicit an inflammatory immune response contributing to the pathogenesis of FD. The exact mechanisms by which AGIRA0003 interacts with epithelia remains to be elucidated and warrants further investigation.

Introduction

Functional dyspepsia (FD) is a chronic gastrointestinal disorder characterised by meal-related symptoms, which a subset of patients attribute to the consumption of gluten-containing foods [1]. Recent evidence suggests that subtle duodenal immune activation is a feature of FD, as evidenced by elevated eosinophils [2] and impaired epithelial barrier function [3]. FD patients also exhibit an altered duodenal mucosa-associated microbiota (d-MAM) when compared to controls [4]. Given the microbiome’s involvement in the digestion of gluten, we hypothesised that the d-MAM determines the capacity for gluten to disrupt the epithelium and drive immune responses in FD patients.

Aims & Methods

Duodenal biopsies were collected from 10 FD patients and 7 asymptomatic controls. The d-MAM was cultured from biopsies in a habitat simulating media [5] containing 1% wheat-gluten, for up to 48hrs. Microbial cell pellets were frozen for future microbiome sequencing and the cell-free supernatants, containing d-MAM-digested gluten, were collected. Caco2 cells were challenged with the d-MAM-digested gluten, in a Transwell model of the intestinal epithelium (n=3, 2 technical replicates). Transepithelial electrical resistance (TEER) and protein expression of markers of epithelial integrity were measured in Caco2 cells and compared between treatments. Peripheral blood mononuclear cells (PBMCs) from FD patients (n=20) and controls (n=20) were stimulated with pooled samples of FD or control d-MAM-digested gluten, or left untreated, for 44 hours. For comparison to host enzymatic breakdown of gluten, matched PBMCs were also treated with α-chymotrypsin digested, deaminated gluten (CT gluten). The T cell phenotype and presence of activation-induced markers was assessed in PBMCs by flow cytometry.

Results

Challenge of Caco2 cells with gluten digested by the FD d-MAM resulted in a significant increase in Zonula occludens-1 (p=0.007), and reduction in Myd88 (p=0.03), compared to Caco2 cells challenged with control d-MAM-digested gluten. However, TEER readings were not altered between treatment groups in Caco2 experiments. In PBMC experiments, in response to stimulation with FD or control d-MAM-digested gluten, both FD and control PBMCs exhibited heightened expression of activation markers (CD4⁺OX40⁺CD25⁺), when compared to their respective unstimulated cells (p<0.05). When assessing the T-cell phenotype we observed that FD d-MAM-digested gluten promoted an increase in T-helper (Th) 2 cells in both FD (p=0.007) and control (p=0.0001) PBMCs. Meanwhile, control d-MAM-digested gluten stimulation resulted in elevated Th17.1 cells in FD (p<0.0001) and control (p=0.02) PBMCs, when compared to unstimulated cells. Alternatively, CT gluten stimulation did not alter any of the assessed lymphocyte populations in FD or control PBMCs when compared to unstimulated cells, suggesting host digestion of gluten isn’t sufficient to induce immune responses in FD.

Conclusion

Our findings suggest that the d-MAM of FD patients digest gluten in a way that promotes epithelial barrier remodelling and altered immune responses compared to the d-MAM from asymptomatic individuals. The promotion of a Th2 T-cell phenotype by the FD d-MAM is consistent with reports of increased Th2-associated cells, such as eosinophils, in FD patients [6]. These results highlight the likely role of microbiome-gluten interactions in FD, indicating the potential for novel dietary therapies and microbial approaches to management.

References

1. Potter, M., et al., Incidence and prevalence of self-reported non-coeliac wheat sensitivity and gluten avoidance in Australia. Med J Aust, 2020. 212(3): p. 126-131.
2. Shah, A., et al., Duodenal Eosinophils and Mast Cells in Functional Dyspepsia: A Systematic Review and Meta-Analysis of Case-Control Studies. Clinical Gastroenterology and Hepatology, 2022. 20(10): p. 2229-2242.e29.
3. Puthanmadhom Narayanan, S., et al., Duodenal Mucosal Barrier in Functional Dyspepsia. Clin Gastroenterol Hepatol, 2022. 20(5): p. 1019-1028.e3.
4. Shanahan, E.R., et al., Alterations to the duodenal microbiota are linked to gastric emptying and symptoms in functional dyspepsia. Gut, 2023. 72(5): p. 929-938.
5. Schooth, L., Defining the Role of the Mucosa-Associated Microbiota in Digestive Health and Disease Using Novel ex-vivo Combinations of Microbe Culture with Metagenomics. 2024 [Doctoral thesis, The University of Queensland], UQ eSpace.
6. Burns, G.L., et al., Type 2 and type 17 effector cells are increased in the duodenal mucosa but not peripheral blood of patients with functional dyspepsia. Front Immunol, 2022. 13: p. 1051632.

Disclosure

JP, EH, CN, SS, SR, MS, KH, GB, and KD have no disclosures to report.
NT: Financial support from: BluMaiden (microbiome advisory board) (2021), Comvita Mānuka Honey (2021) (digestive health), Biocodex (functional dyspepsia tool), Microba (microbiome advisory board) outside the submitted work. In addition, Dr. Talley has a patent Nepean Dyspepsia Index (NDI) 1998, Biomarkers of IBS licensed, a patent Licensing Questionnaires Talley Bowel Disease Questionnaire licensed to Mayo/Talley, a patent issued, “Diagnostic marker for functional gastrointestinal disorders” Australian Patent Application WO2022256861A1via the University of Newcastle and UniQuest (University of Queensland) and ”Methods and compositions for treating age-related neurodegenerative disease associated with dysbiosis” US Application No. 63/537,725. Dr. Talley is supported by funding from the National Health and Medical Research Council (NHMRC) to the Centre for Research Excellence in Digestive Health and he holds an NHMRC Investigator grant.
SK: Grants from National Health and Medical Research Council (Ideas Grant and Centre for Research Excellence), grants from Viscera Labs (Research contract), grants from Microba Life Science (Research contract), personal fees from Gossamer Bio, personal fees from Anatara Lifescience, personal fees from Immuron, personal fees from Microba Life Science.

Introduction

Microplastics are unavoidable in our current environment and can enter the human body through ingestion, inhalation, and skin contact. Gut microbes play a role in plastic degradation, breaking down polymers into smaller molecules through enzymatic processes like depolymerization and oxidation, which can then be used as a carbon source (1). However, there is mounting alarming evidence that implicates microplastics in causing oxidative stress, DNA damage, and chronic inflammation, potentially leading to various health issues, including gastrointestinal disorders (2-4). In individuals with digestive disorders such as DGBI (disorders of gut-brain interaction), microplastics may exacerbate symptoms by disrupting gut microbiome and immune responses. Previously, within a functional dyspepsia (FD) cohort we identified a strong positive correlation between plastic degrading Exiguobacterium genus (5) within the duodenal mucosa and mucosa Th2 immune cells (6).

Aims & Methods

We aimed to investigate the differential abundance of microbes and metabolic pathways known to degrade plastics in patients with FD. Previously, we have published the 16S duodenal mucosa associated microbiome sequence data for FD (n=17) and asymptomatic control (n=11) groups (6), in addition we conducted a systematic review and retrieved 1 raw duodenal mucosa-associated microbiome 16S sequence dataset (FD n=61, controls n=38) for re-analysis with a consistent bioinformatic pipeline. This included quality assessment, demultiplexing and denoising with QIIME2 and taxonomic assignment with SILVA database. Functional differences in the microbiome were predicted with PICRUSt2. Differentially abundant plastic utilising genes were assessed in R, after CLR-transformation.

Results

As the two 16S upper gastrointestinal mucosa microbiome FD studies were sequenced separately, and the batch effect are pronounced we could not combine them for pooled analysis. When we examined the first study for predicted metabolic pathways for differential polyethylene terephthalate (PET) utilization when comparing FD patients to controls in the duodenum mucosa-associated microbiome, we did not identify any significant differences. An increase in the "partial TCA cycle (obligate autotrophs)" was observed in the gastric mucosa-associated microbiome of FD patients vs controls; PET by-products have been linked to the TCA cycle (7).
The study retrieved through our systematic review identified several significantly increased predicted functional polyethylene-associated genes, primarily from later stages of degradation. The findings revealed that the gene 3-oxoadipate enol-lactonase [EC:3.1.1.24] was significantly increased in FD patients. Furthermore, the genes 5-carboxymethyl-2-hydroxymuconic-semialdehyde dehydrogenase [EC:1.2.1.60] and 5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylase/2-hydroxyhepta-2,4-diene-1,7-dioate isome were significantly increased in controls. Finally, 5-carboxymethyl-2-hydroxymuconate isomerase [EC:5.3.3.10] was also increased in controls though not significantly.

Conclusion

Our recent microbiome analysis reveals a significant enrichment of PET-degrading microbes colonising the upper gastrointestinal tract of patients with FD compared to controls. These findings provide new insights into the microbial and immune landscape of FD patients, highlighting potential microbial biomarkers and immune associations that warrant further investigation.

References

1. Yang X-G, Wen P-P, Yang Y-F, Jia P-P, Li W-G, Pei D-S. 2023. Plastic biodegradation by in vitro environmental microorganisms and in vivo gut microorganisms of insects. Frontiers in Microbiology
2. Jayavel S, Govindaraju B, Michael JR, Viswanathan B. 2024. Impacts of micro and nanoplastics on human health. Bulletin of the National Research Centre
3. Cheng Y, Yang Y, Bai L, Cui J. 2024. Microplastics: an often-overlooked issue in the transition from chronic inflammation to cancer. J Transl Med
4. Han Q, Gao X, Wang S, Wei Z, Wang Y, Xu K, Chen M. 2023. Co-exposure to polystyrene microplastics and di-(2-ethylhexyl) phthalate aggravates allergic asthma through the TRPA1-p38 MAPK pathway. Toxicology Letters
5. Parthasarathy A, Miranda RR, Eddingsaas NC, Chu J, Freezman IM, Tyler AC, Hudson AO. 2022. Polystyrene Degradation by Exiguobacterium sp. RIT 594: Preliminary Evidence for a Pathway Containing an Atypical Oxygenase. Microorganisms
6. Hoedt EC, Burns GL, Kang S, Bruce J, Morrison M, Keely S, Talley NJ. 2025. Altered Duodenal Mucosa-Associated Microbiota and Immune Profiles in Functional Dyspepsia: A Study of Host-Microbiome Homeostasis. bioRxiv doi:10.1101/2025.04.10.648308:2025.04.10.648308.
7. Carr CM, Clarke DJ, Dobson ADW. 2020. Microbial Polyethylene Terephthalate Hydrolases: Current and Future Perspectives. Front Microbiology

Disclosure

Keely reports consultancy and positions held on advisory boards for: Gossamer Bio (Scientific Advisory Board), Anatara Lifescience (Scientific Advisory Board), Microba Life Science (Consultancy) and Immuron Ltd. (Consultancy).
Talley reports personal fees from Biocodex (FD) (2024), Brown University (fiber and laxation), Microba (microbiome), outside the submitted work. NJT has a patent Licensing Questionnaires Talley Bowel Disease Questionnaire licensed to Mayo/Talley and Nepean Dyspepsia Index (NDI) 1998, patent “Diagnostic marker for functional gastrointestinal disorders” Australian Provisional Patent Application 2021901692, “Methods and compositions for treating age-related neurodegenerative disease associated with dysbiosis” US Application No. 63/537,725.

Introduction

Functional dyspepsia (FD) is a disorder of gut-brain interaction (DGBI) characterised by relapsing and remitting gastro-duodenal symptoms, in the absence of identifiable pathology via routine clinical screening. An association between FD and wheat-containing foods has been reported in observational studies, and recent work demonstrates increased gut-homing T cells in peripheral blood of FD patients reporting sensitivity to wheat, compared to those without wheat sensitivity and controls. We have also recently shown increased populations of mucosal lymphocytes with a T helper (Th)2- and Th17-like phenotype in FD patients compared to controls, however, a mucosal adaptive lymphocyte response specific to wheat has not been demonstrated.

Aims & Methods

We aimed to examine whether proteins present in wheat could provoke a response from FD duodenal lymphocytes isolated from mucosal biopsies. Lamina propria mononuclear cells (LPMCs) were isolated from duodenal biopsies from 50 FD patients (38 female, 46.8±16.8 years) and 23 controls (11 female, 52.1±13.1 years). LPMCs were exposed to gluten (0.2mg/mL) or gliadin (0.2mg/mL) for 24 hours. Flow cytometry was performed to phenotype lymphocytes using a surface marker panel. Quantitative polymerase chain reaction (qPCR) was used to measure expression of the gliadin-associated T-cell receptor alpha variant (TRAV)26-2.

Results

In response to gliadin (but not gluten) stimulation, the effector Th2-like population was increased in FD LPMCs compared to controls (16.34 ± 13.17 vs 25.83 ± 14.87, p = 0.01) and unstimulated FD LPMCs (19.40 ± 14.35 vs 25.83 ± 14.87, p = 0.04). Duodenal gene expression of TRAV26-2 was decreased in FD compared to controls (0.013 ± 0.015 vs 0.003 ± 0.005, p = 0.04). We identified a positive association between gene expression of this T cell receptor variant and LPMC effector Th17-like cell populations in FD patients (rs = 0.3733, p = 0.04), but not controls (rs = 0.2238, p = 0.485) following exposure to gluten, but not gliadin.

Conclusion

Our findings in this observational study suggest differential T cell responses to gluten and gliadin in a subset of FD patients, and suggest Th2 responses previously reported in FD may occur in response to food components such gliadin. We also show that FD patients have reduced expression of TRAV26-2, previously associated with T cell responses against gliadin in celiac disease. TRAV26-2 expression correlated with Th17-like populations in FD patients, suggesting that specific TCR variants in may be associated with an immune response to the consumption of wheat-containing foods in FD.

Introduction

Functional dyspepsia (FD) is a common gastrointestinal disorder, affecting 10–20% of the population. Although Helicobacter pylori (Hp) eradication is recommended in FD, its therapeutic benefit is modest, and no clear correlation exists between eradication success and symptom improvement. Emerging evidence suggests that microbiota changes may contribute to symptom relief beyond Hp clearance. Since antibiotics and acid-suppressing agents—both integral to eradication regimens—can significantly affect the gut microbiota, it is uncertain whether the observed clinical improvement stems primarily from the elimination of H. pylori, or from microbiota shifts induced by the treatment itself.

Aims & Methods

We aimed to investigate the effects of antibiotics and a potassium-competitive acid blocker on microbiome changes in FD patients with or without Hp infection, and to examine their association with symptom improvement. Four Hp(+) and eight Hp(-) patients with FD, diagnosed based on the Rome IV criteria, were prospectively enrolled. All patients were treated with tegoprazan 50 mg, amoxicillin 1000 mg, and clarithromycin 500 mg, twice daily for 10 days, regardless of Hp status. Hp infection was determined using a rapid urease test and a serum Hp IgG test. Symptom changes were assessed using the Korean version of the Nepean Dyspepsia Index (NDI-K) at baseline, 3 months, and 6 months after eradication. Biopsy samples were taken from the antrum, duodenal bulb, and second portion of the duodenum at baseline and 3 months after eradication. Microbiome profiling via 16S rRNA sequencing was performed to assess alpha and beta diversity, and LEfSe was used to identify discriminative bacterial taxa. Eosinophil counts were also measured at baseline and 3 months after eradication.

Results

At baseline, the median NDI-K total score was 71.5 in the Hp(+) group and 33.5 in the Hp(-) group. Three months after eradication therapy, the median scores decreased to 26.5 and 12.0, respectively. This reduction in symptom scores persisted through six months, with median NDI-K total scores of 21.0 and 11.0, respectively. In the Hp(+) group, the ACE and Chao1 alpha indices in the duodenal bulb were significantly decreased at 3 months after eradication compared with baseline. All indices of alpha diversity in the antrum and duodenal second portion were decreased at 3 months after eradication compared with baseline, although the changes were not statistically significant. In the Hp(-) group, alpha diversity in the antrum and duodenum tended to decrease at 3 months after eradication compared with baseline. At the phylum level, Proteobacteria were predominant in both groups, followed by Firmicutes, Bacteroidetes, and Actinobacteria. In the antrum of the Hp(-) group, Proteobacteria decreased in relative abundance from 59.9% at V2 to 48.7% at V4. In contrast, Firmicutes showed a tendency to increase from 17.7% at V2 to 26% at V4. LEfSe analysis identified multiple species-level differences in microbial composition between the two groups. Median eosinophil counts decreased in the duodenal bulb and the second portion of the duodenum at 3 months after eradication, regardless of Hp status.

Conclusion

Hp eradication therapy resulted in gut microbiota alterations and symptom improvement, regardless of H. pylori status. Duodenal microbial diversity tended to decrease after treatment, and this pattern was observed in both Hp(+) and Hp(–) groups, although the magnitude of change differed. These microbiota shifts may have contributed to the observed improvement in symptoms.

References

1. Ford, Alexander C., et al. "Functional dyspepsia." The Lancet 396.10263 (2020): 1689-1702.
2. Kim, Sung Eun, et al. "Prevalence and risk factors of functional dyspepsia in health check-up population: a nationwide multicenter prospective study." Journal of neurogastroenterology and motility 24.4 (2018): 603.
3. Tan, Victoria PY, et al. "Randomised clinical trial: rifaximin versus placebo for the treatment of functional dyspepsia." Alimentary pharmacology & therapeutics 45.6 (2017): 767-776.
4. Imhann, Floris, et al. "Proton pump inhibitors affect the gut microbiome." Gut 65.5 (2016): 740-748.
5. Oh, Jung Hwan, et al. "Clinical practice guidelines for functional dyspepsia in Korea." Journal of neurogastroenterology and motility 26.1 (2020): 29.

Disclosure

This study was supported by research funding from HK inno.N.

Introduction

STW 5, a herbal medicinal product with well characterized pharmacological action, has been shown in several RTCs to be an effective treatment in disorders of the gut-brain interaction (DGBI) as functional dyspepsia (FD) [1, 2]. The aim of the present study was to examine whether changes in gut microbiota induced in an experimental model of FD could be prevented by STW 5.

Aims & Methods

FD was induced in rats by subjecting them to neonatal maternal separation followed by subjecting them to restraint stress for 90 minutes/day for one week. During that week, one group (8 animals) was treated with the liquid form of STW 5 (5 ml/Kg) and one with its vehicle at the same dose level. Animals were sacrificed 24 h after the last drug administration. Fecal samples were taken from the cecum. Genomic DNA was isolated and changes in selected bacterial phyla and genera were assessed using quantitative Real Time-PCR (qPCR). The main phyla studied under the influence of stress in the model were the Bacteroidetes, Firmicutes, Fusibacterium and Actinobacteria.

Results

FD led to a 50% drop in the Lactobacilli population but to a two-fold rise in Proteobacteria abundance but did not affect significantly the changes in Bacteroidetes. Furthermore, the Methanobrevibacter population was raised more than two-fold while the genomic DNA concentration in Bifidobacterium dropped from ca. 3 ng/g feces to nearly nil.

Conclusion

In general, STW 5 tended to ameliorate and protect against the changes induced by the stress model. The results lend further support to the use of STW 5 in FD and IBS by providing evidence that it acts at least in part through influencing beneficially the changes in intestinal microbiota induced by stress. Taken together, the findings shed more light on mechanisms involved in the therapeutic action of STW 5 in gastro-intestinal disorders.

References

1. Malfertheiner P. STW 5 (Iberogast) Therapy in Gastrointestinal Functional Disorders. Dig Dis. 2017, 35 Suppl 1: 25-29
2. Allescher H-D, Burgell R, Malfertheiner P, Mearin F. Multi-target Treatment for Irritable Bowel Syndrome with STW 5: Pharmacological Modes of Action. Gastrointest Liver Dis. 2020;29: 227-233

Disclosure

OK and HA-K are empolyees of Steigerwald Arzneimittelwerk GmbH, Darmstadt, Germany. MTK has been obtaining travel support and speakers fees from Steigerwald Arzneimittelwerk GmbH, Darmstadt, Germany.