The ocular surface (OS), like other mucosal sites, hosts a diverse microbiome. However, the impact of hyperglycemia associated with diabetes on OS microbial composition remains poorly understood. In this study, we established type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) models in C57BL/6J mice by administering high-dose streptozotocin (STZ) for T1DM and low-dose STZ combined with a high-fat diet for T2DM. The OS microbiome was characterized and analyzed using 16S rRNA sequencing. The results showed that neither T1DM nor T2DM significantly affected microbial richness compared to normal mice
however, T2DM led to a significant reduction in microbial diversity. This reduction in microbial diversity in T2DM is consistent with known microbial dysbiosis in diabetes, which may contribute to the pathogenesis of ocular complications such as dry eye disease and diabetic retinopathy. Community composition analysis identified Proteobacteria, Pelagibacterium, and Aliihoeflea as the core OS bacteria in normal mice. Diabetes significantly altered the OS microbial composition at various taxonomic levels. Specifically, T1DM was associated with 9 signature bacterial species, including Oceanospirillales, Bacillales, Halomonas, unclassified_f_Lachnospiraceae, and Anoxybacillus. T2DM exhibited 17 bacterial markers, including Firmicutes, Staphylococcus, Corynebacterium, and Parasutterella. Functional prediction of the microbiota using PICRUSt2 indicated potential impairments in carbohydrate metabolism due to diabetes. In conclusion, diabetic mice exhibit severe dysregulation of their OS microbiota, and restoring microbial balance in diabetic patients may represent a promising strategy for preventing and treating diabetic OS pathologies.