The aim of this study is to investigate the structural characteristics of gut microbiota in patients with primary moderate-to-severe dry eye disease (DED) and analyze their correlation with ocular surface functional parameters.

A total of 30 patients (30 eyes) with moderate-to-severe DED who attended the Department of Ophthalmology of the Second Affiliated Hospital of Baotou Medical College from May 2024 to September 2025, and 30 age-and gender-matched healthy controls (30 eyes) recruited during the same period were enrolled. Among the DED patients, there were 15 males (15 eyes) and 15 females (15 eyes), aged 22 to 67 years, with a mean age of (41.5±14.3) years. Among the controls, there were 15 males (15 eyes) and 15 females (15 eyes) with a mean age of (42.5±16.5) years (ranging from 20 to 69 years). The ocular surface disease index (OSDI) questionnaires were collected from all subjects. Non-invasive tear break-up time (NIBUT-f), non-invasive average break-up time (NIBUT-av), tear meniscus height (TMH), and conjunctival redness were measured using an ocular surface comprehensive analyzer, and the Schirmer Ⅰ test was performed. Fecal samples were collected, and 16S ribosomal RNA gene high-throughput sequencing was used to analyze the Alpha diversity, Beta diversity, and species composition of gut microbiota. Measurement data conforming to normal distribution, including OSDI score, NIBUT-av, TMH, and Schirmer Ⅰ test values, were expressed as ±s, with comparisons between groups performed using the independent samples t-test. Measurement data not conforming to normal distribution were expressed as [M(P25, P75)], with comparisons between groups performed using the Mann-Whitney U test. Count data were expressed as rates (%), with comparisons between groups performed using the χ² test. The overall structure of gut microbiota between the two groups was analyzed using permutational multivariate analysis of variance. Spearman′s rank correlation analysis and linear regression models were applied to explore the associations between the relative abundance of differential bacterial genera and ocular surface clinical parameters.

No statistically significant differences were observed between the two groups in age or gender distribution (t=-0.25, χ²=0.00, P>0.05). The OSDI score, conjunctival redness score, NIBUT-f, NIBUT-av, TMH, and Schirmer I test values in the DED group were (44.93±6.11) points, (2.75±0.53) points, (3.70±0.64) s, (6.34±0.54) s, (0.10±0.03) mm, and (3.61±1.93) mm/5 min, respectively, while those in the control group were (10.76±5.50) points, (1.60±0.51) points, (13.74±1.55) s, (16.73±0.69) s, (0.26±0.04) mm, and (13.45±1.32) mm/5 min, respectively, with statistically significant differences between the two groups (t=22.76, 8.52, 32.76, 64.81, 17.29, 23.02; P<0.05). Alpha diversity analysis showed that the Chao1 index reflecting species richness and the Shannon index reflecting species diversity in the DED group were 91 (71 to 107) and 2.61 (2.37 to 3.06), respectively, while those in the control group were 160 (149.5 to 170) and 3.41 (3.25 to 3.73), respectively, with statistically significant differences between the two groups (Z=-6.34, -5.87; P<0.05). Principal coordinate analysis based on the Bray-Curtis distance matrix revealed that the DED group and control group samples formed distinct independent clusters in spatial distribution, with a clear boundary in the intergroup microbial community structure. The first principal coordinate explained 30.4% of the total variance, and the second principal coordinate explained 11.8% of the total variance. Permutational multivariate analysis of variance showed a statistically significant difference in the overall gut microbiota structure between the two groups (R²=0.267, P<0.05). At the phylum level, the gut microbiota of both groups was dominated by Firmicutes and Bacteroidetes. The relative abundances of Proteobacteria and Firmicutes in the DED group and control group were 20.2% vs 2.1% and 49.8% vs 68.2%, respectively, with statistically significant differences (t=5.01, 3.48; P<0.05). At the order and family levels, the increase in Proteobacteria abundance was mainly driven by the significant elevation of Enterobacterales and Enterobacteriaceae. The relative abundances of Enterobacteriaceae and Bacteroidaceae in the DED group and control group were 17.7% vs 0.7% and 24.9% vs 12.5%, respectively, with statistically significant differences (t=4.66, 3.48; P<0.05). The abundance of Ruminococcaceae under Firmicutes in the DED group and control group was 10.2% and 27.1%, respectively, with a statistically significant difference (t=6.62, P<0.05). At the genus level, the relative abundances of Bacteroides, Parabacteroides, and Faecalibacterium in the DED group and control group were 26.4% vs 1.5%, 12.9% vs 0.4%, and 7.6% vs 23.8%, respectively, with statistically significant differences (t=3.57, 3.81, 7.47; P<0.05). At the species level, Escherichia coli and Bacteroides vulgatus were identified as the key species driving the structural differences between the two groups, with abundances of 15.8% vs 3.3% and 0.02% vs 0.3%, respectively, showing statistically significant differences (t=4.53, 4.47; P<0.05). The relative abundance of short-chain fatty acid-producing Faecalibacterium prausnitzii in the DED group and control group was 3.9% and 8.8%, respectively, with a statistically significant difference (t=3.45, P<0.05). Spearman rank correlation analysis and linear regression analysis revealed that, in terms of subjective symptoms, the relative abundances of Butyricimonas and Faecalibacterium were significantly negatively correlated with the OSDI score (r=-0.75, -0.60; P<0.05); the relative abundance of Parabacteroides was significantly positively correlated with the OSDI score (r=0.50, P<0.05). Regarding objective ocular surface parameters, the relative abundance of Prevotellaceae was significantly positively correlated with NIBUT-av (r=0.78, P<0.05); the relative abundance of Butyricimonas was significantly positively correlated with TMH (r=0.67, P<0.05); and the relative abundance of Prevotellaceae was significantly positively correlated with Schirmer I test values (r=0.77, P<0.05). In most control subjects, Firmicutes was the dominant phylum, while its relative abundance decreased in the DED group. Meanwhile, the relative abundance of Proteobacteria in the DED group was higher than that in the control group. The gut microbiota structure of DED patients showed a shift, characterized by increased Proteobacteria and decreased Firmicutes. The abundances of potential opportunistic pathogens such as Bacteroides and Parabacteroides were increased in the DED group, while beneficial bacteria such as Faecalibacterium and Ruminococcus, which produce short-chain fatty acids, were markedly reduced.

Patients with primary moderate-to-severe DED exhibit significant gut microbiota dysbiosis, characterized by increased relative abundance of opportunistic pathogens and decreased abundance of beneficial bacteria such as butyrate-producing genera. The alterations in specific gut microbiota are closely associated with the exacerbation of subjective ocular surface symptoms, decreased tear film stability, and reduced tear secretion. Modulating gut microbiota balance may emerge as a potential novel target for DED intervention.