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Stephanie Wan, Aaron Sullivan, Peyton Shieh, David Evans, Carolyn Bertozzi, Suzanne M J Fleiszig; Ocular Microbiome: Fact or Fiction?. Invest. Ophthalmol. Vis. Sci. 2016;57(12):No Pagination Specified.
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© ARVO (1962-2015); The Authors (2016-present)
Whether the ocular surface hosts a live microbiome remains unresolved. High-throughput sequencing of the microbial 16s ribosomal gene predicts a rich abundance of microbes at the ocular surface. These methods, however, detect bacterial nucleic acid and not live microbes. Here, we tested the hypothesis that the ocular surface does not host a stable, metabolically active microbiome.
Methods involved addition of alkyne functionalized D-alanine (10mM), which incorporates into the cell wall of only metabolically active bacteria and is detected by addition of an azide fluorophore (copper catalyzed click chemistry), imaged using a confocal microscope. To confirm the method works on the eye, 3 mice were sacrificed, eyes enucleated, then deliberately inoculated with viable P. aeruginosa. In other experiments we explored if the eye can be stably colonized when deliberately inoculated. Mice were anesthetized before inoculating one eye with P. aeruginosa, S. aureus or a gram-positive commensal bacterium isolated from the mouse dermis. Mice were sacrificed after 24 h or 3 days. To collect tear fluid, 5μL of sterile PBS was added, then collected with a capillary tube. The conjunctiva and cornea were removed, homogenized, and bacteria enumerated by viable counts (all 3 are culturable).
After inoculation with ~108 CFU of P. aeruginosa, S. aureus, or commensal skin bacteria, >99.99% of the original inoculum was cleared after 24 hours in the cornea, conjunctiva, and tear fluid (P<0.01). Similar results were obtained for P. aeruginosa and S. aureus after 3 days (>99.99% clearance, P < 0.0001) and when using a small inoculum (~104 CFU, >99.75% clearance, P<0.05). The D-alanine label detected metabolically active bacteria if they were deliberately added (11.00 ± 3.30 bacteria/image). Otherwise, metabolically active bacteria were rarely detected on the cornea (0.50 ± 0.27 bacteria/image, p=0.003).
The ocular surface of the healthy mouse quickly eradicated two bacterial pathogens at both high and low inocula, and also commensal skin bacteria, suggesting it is an inhospitable environment for microbes to colonize. Alkyne functionalized D-alanine is a useful novel method to label metabolically active bacteria on the eye. The sparsity of metabolically active bacteria on the mouse cornea suggests the absence of a stable, live microbiome. The presence of non-metabolically active bacteria remains a possibility.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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