Metagenomic sequencing of Sjögren syndrome and healthy gut microbiota reveals differential bacterial species that correlate with disease severity

Abstract

Purpose : A growing number of studies show that homeostasis of the ocular surface is impacted by the intestinal microbiome, and several 16S sequencing studies have demonstrated dysbiosis of the intestinal microbiota in Sjögren Syndrome (SS) patients. In this study, we utilized metagenomic sequencing to perform a deeper analysis of the intestinal microbiome to identify differential bacterial species using stools collected from sex- and age-matched subjects.

Methods : Age-matched healthy (n = 20), dry eye (n = 4) and SS (n = 7) female patients were enrolled at the BCM Alkek Eye Center. Ocular disease severity was scored following International Dry Eye Workshop guidelines. Patients were supplied with stool collection kits and submitted samples within 24 hours post-collection. High quality DNA was prepared for metagenomics sequencing from stool aliquots using DNEasy Powersoil Pro kits. Sequencing, annotation, and initial analysis were performed by Diversigen with their BoosterShot Shotgun Sequencing service. We further analyzed the data using the vegan and randomForest packages in R statistical software.

Results : Shannon alpha diversity and observed operational taxonomic units (OTUs) were significantly decreased in SS (P<0.05). Observed OTUs inversely correlated with ocular severity score (Spearman’s r=-0.37, P<0.05). Using Bray-Curtis beta diversity analysis, we found significant separation between healthy and SS groups (PERMANOVA R²=0.08, P<0.05), but not between healthy and dry eye groups. We investigated taxonomic differences using the Random Forest machine learning algorithm. At the phylum level, Bacteroidetes (P<0.05) and Actinobacteria (P<0.01) contributed the most to community differences. At the species level, SS patients were distinguished by significant increases in Bacteroides caecimuris, Mediterranea masilliensis, Bacteroides coprophilus and Clostridium_sp_7_3_54FAA and a significant decrease in Bifidobacterium bifidum compared to healthy controls (all P<0.01), and these species changes correlated with disease severity.

Conclusions : Our metagenomic data confirm that the SS gut microbiome is less diverse, and that reduced diversity correlates with increased ocular disease severity. In addition, we have identified specific bacterial species that are either depleted or enriched in SS patients and correlate with ocular surface disease severity.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.