Introduction

The intestinal microbiota is defined as the community of microorganisms inhabiting the gastrointestinal tract, which confer benefits to the organism and play a role in immune system regulation. It has been suggested that the microbiota may play a role in the pathogenesis of gut diseases, including Celiac Disease (CeD), and the response to a gluten free diet (GFD). (1) Intestinal dysbiosis may be a contributing factor to the inflammatory state, thereby hindering resolution of symptoms and complete mucosal healing, particularly in patients who show a lack of clinical and histological improvement despite following a GFD. (2,3).

Aims & Methods

We aimed to provide a preliminary evaluation of the gut microbiota profile from diagnosis to follow-up, classifying patients who respond and do not respond to diet in order to establish a first baseline of the microbial composition and its relationship to the dietary response.
We collected fecal samples from patients enrolled at the Gastroenterology Unit of Azienda Ospedale- Università of Padova, from March to November 2024. All patients needed to be off antibiotics, probiotics and proton-pump inhibitors for at least one month before enrolment. Stool samples were collected and transferred in a buffer solution, and stored at -20°C.
Fecal samples were analyzed by BMR Genomic s.r.l. (Padua, Italy) that sequenced the V3-V4 hypervariable 16S ribosomal RNA region using the Illumina MiSeq. The results were compared to an internal population of healthy subjects (provided by BMR genomics) using Qiime2 tools. Statistical analysis was conducted using STATA 18 software. Categorical variables were compared using the Chi-square test, while non-parametric continuous variables were analysed using the Mann-Whitney test.

Results

We enrolled 71 CeD patients with mean age at diagnosis of 35.76 ± 16.29 years old (50 out of 71 were female). Of the 71 patients, 9 were newly diagnosed with CeD, while 62 were in follow-up. Among follow-up groups, 44 of 62 patients (71%) were classified as responders to diet and 18 (29%) were classified as non- responders, exhibiting classical symptoms, positive serology and histology. We compared the relative abundance of the main bacterial phyla in each sample: a significant decrease in beneficial phyla such as Actinobacteria (p = 0.04) and a decrease close to the significant threshold in Firmicutes (p = 0.06) was observed after the follow-up. This factor could be ascribed to a long-term dietary regime rather than to the disease itself. (4) Proteobacteria were more abundant in follow-up, especially in non-responders. This phylum includes many potentially pathogenic microorganisms belonging to Escherichia, Klebsiella and Shigella genera. Comparing diagnosis and follow-up patients, Clostridium perfringens was significantly more present in patients during follow-up (p=0.02), while Escherichia coli showed greater presence in patients at diagnosis (p=0.035) and in non-responders, consistently with his role in the inflammatory phase of the disease. (5)

Conclusion

Our preliminary data suggest that a long-term GFD does not always restore completely the intestinal dysbiosis present at the diagnosis and could be associated with specific alterations in beneficial bacteria. The presence of abundant Proteobacteria at diagnosis and in non-responder patients may be considered as biomarkers of active state of CeD. Future studies on various biological samples, as well as a more extensive investigation into the impact of long-term GFD, are required to highlight microbiota-immunity relationships involved in development of CeD.

References

1. Zafeiropoulou K, Nichols B, Mackinder M, et al. Alterations in intestinal microbiota of children with celiac disease at the time of diagnosis and on a gluten-free diet. Gastroenterology. 2020;159(6):2039–2051.e20.

2. Freire R, Ferreira R, Almeida LM, et al. Human gut-derived organoids provide model to study gluten response and effects of microbiota-derived molecules in celiac disease. Sci Rep. 2019;9(1):1–12

3. Schiepatti A, Montagnani M, D'Argenio V, et al. Relationship between duodenal microbiota composition, clinical features at diagnosis, and persistent symptoms in adult celiac disease. Front Microbiol. 2021;12:1–12

4. Wacklin P, Nikkilä J, Tuovinen E, et al. Altered duodenal microbiota composition in celiac disease patients suffering from persistent symptoms on a long-term gluten-free diet. Am J Gastroenterol. 2014;109(10):1587–1594

5. Olshan KL, Leonard MM, Serena G, Zomorrodi AR, Fasano A. Gut microbiota in celiac disease: microbes, metabolites, pathways and therapeutics. Expert Rev Clin Immunol. 2020 Nov;16(11):1075-1092.