Characterization of the normal equine conjunctival bacterial community using culture-independent methods

Background The equine conjunctival microbiota has often been reported to be dominated by Gram-positive species such as Staphylococcus sp., Bacillus sp., and Corynebacterium sp. However, traditional culture-based methods can only recover a fraction of the bacterial species present in the sample. Objectives This pilot study aimed at exploring the diversity of the equine conjunctival microbiota using culture-independent methods. Study design Eight horses were included in this study, and only eyes with normal ophthalmic examination (n = 15 eyes) were sampled. Methods Conjunctival biopsies (culture-independent) were collected, and DNA was extracted from the tissues. Bacterial communities in conjunctival biopsies were characterized by next-generation sequencing of the 16S rRNA genes. Individual reads were ascribed to operational taxonomic units (OTUs) using BLASTn and Greengenes databases. Species richness, evenness, and Good's coverage were determined for each conjunctiva-associated microbial community. Results Culture-independent samples produced a total of 329 bacterial OTUs. The main OTUs identified in the study belonged to the Gram-negative species Ralstonia mannitolilytica (88.0%), Nicoletella semolina (3.3%), and Pseudomonas tolaasii (1.5%). Conclusions Contrary to previously published data based on culture-dependent methods, the horse eye microbial community was dominated by Gram-negative bacteria of the phylum Proteobacteria.