Diversity And Prevalence Of Microorganisms At Healthy Ocular Surface

Abstract

Purpose: : Ocular surface (OS) microbiota contribute to infectious diseases of the eye. Comprehensive analysis of microbial diversity at the OS has been impossible because of the limitations of conventional microbial culture techniques. This study aimed to explore true diversity of human OS microbiota using DNA sequencing-based detection and identification of microbes at healthy OS and on contact lenses.

Methods: : We utilized 454 Roche deep sequencing of 16S rRNA gene amplicon libraries and Biome Representational in Silico Karyotyping (BRiSK) to obtain microbial DNA reads. These were classified using ESPRIT, MOTHUR Ribosomal Database Project-II software. Endonuclease-generated fragments were matched to known microbial genomes.

Results: : Deep sequencing of conjunctival rDNA from four subjects yielded 115,003 quality DNA reads corresponding to an average of 221 bacterial species per subject. BRiSK was applied to three conjunctival samples and yielded an average of 434 microbial DNA reads, corresponding to 17 known microbial (bacterial, fungal and viral) species. Significantly, BRiSK analysis of microbiota on used contact lenses detected 28 known microbial species. The combined bacterial community was classified into 5 phyla and 64 distinct genera. In addition, >40% of 16S rRNA-detected bacteria and >90% of BRiSK-detected microbes belonged to unclassified or novel species. Despite high inter-subject variability, 12 known genera were ubiquitous among the analyzed cohort and represented putative "core" of the OS microbiome. The other 52 genera accounted for less than 4% and likely belonged to transient species. Unexpectedly, healthy conjunctiva contained many genera that are commonly identified as ocular surface pathogens.

Conclusions: : Our first DNA sequencing-based survey of healthy OS have revealed unexpectedly diverse microbial community. All analyzed samples contained ubiquitous "core" genera that included commensal, environmental and opportunistic pathogenic bacteria, fungi and viruses.

Keywords: conjunctiva • cornea: epithelium • microbial pathogenesis: experimental studies