Validation of 16S rRNA Gene Sequencing of the Periocular Microbiome and Lack of Alteration by Topical Eyedrops

PURPOSE: Genomic techniques for characterizing the ocular microbiome require further validation. We compared the microbiome of patients’ eyelids through both conventional culture and 16S rRNA analysis and analyzed the impact of eyedrop use on microbiome diversity. METHODS: Ninety-eight patients followed for management of glaucoma or suspicion of glaucoma had eyelid swabs performed with Isohelix MS Mini DNA Swabs (98 participants) and ESwabs (49 participants) for 16S rRNA analysis and conventional culture, respectively. The effect of preservative-containing eyedrops on the microbiomes detected using these two techniques were analyzed and compared across techniques. RESULTS: Forty-five of the 50 (non-unique) genera (90%) identified by conventional culture were also identified by each individual's 16S rRNA analysis within the top 14 most abundant organisms present based on operational taxonomic unit. All conventional cultures performed had at least one or more genera also identified by each participant's 16S rRNA analysis. There was no difference in the conventional culture positivity rate or proportion of participants with a particular genus present on conventional culture based on whether preservative-containing eyedrops were regularly used. Similarly, in eyes using versus not using eyedrops, no differences were observed in the proportions of participants with a particular genus present or the Shannon index as determined by 16S rRNA analysis. CONCLUSIONS: 16S rRNA analysis correlates well with conventional culture results for the eyelid microbiome, with results from neither technique demonstrating an association of microbiome composition and eyedrop use. The clinical relevance of the large numbers of microbes detected via 16S rRNA analysis requires further study. TRANSLATIONAL RELEVANCE: 16S rRNA analysis of the periocular microbiome is consistent with conventional culture and enables further study of physiologic and pathologic ocular processes possibly related to microbiome diversity.