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Olof H. Sundin, Ted W Reid, Kelly Mitchell, Coby Ray, McCartney David, Elisa Morales, B. Matthew Fagan, Sophia N. Hantzopulos, Peter Lin, Michael F. Maldonado; The Conjunctiva has a Sparse Endogenous Flora that is Distinct from that of the Peri-Ocular Skin. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5614. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Knowledge concerning the types and numbers of bacteria on the normal conjunctival surface is a crucial starting point for understanding ocular infections and related pathology. Our hypothesis is that the eye has an endogenous, self-renewing bacterial population. We have found that characterizing this very sparse flora by Next Generation DNA sequence analysis is feasible, but poses special technical challenges.
We recruited 29 volunteers and collected samples from both inferior palpebral and nasal bulbar conjunctiva using sterile nylon swabs. From 9 of these, we also collected swab samples of peri-ocular skin. Centrifugation concentrated intact bacteria, and DNA was extracted by mechanical disruption, then analyzed by pan-bacterial quantitative PCR. The Illumina MiSeq sequencer and its software was used for metagenomic analysis of 16S rRNA genes. Low levels of microbial DNA made it necessary to first PCR amplify a 963 bp segment of 16S rRNA gene that spanned variable regions V1 through V5. This was followed by a standard Illumina 16S rRNA gene metagenomic workflow, which involved bacterial identification using a V3-V4 segment nested inside the first amplimer.
Roughly 3,000 bacterial genomes were collected from each eye. Over 70% of this bacterial DNA pelleted, indicating that it was packaged in cells, which we are examining microscopically by in situ hybridization. Only 3% of these cells produce colonies in aerobic culture. Sequencing of controls revealed 5% contamination with Alpha Proteobacteria from extraction kits and other reagents. When these contaminants were subtracted, the most abundant genera were 50% Staphylococcus, 20% Corynebacterium, 4% Propionobacter, 2% Streptococcus 2% Anaerococcus, and 1% Micrococcus. The most common ocular species was Staphylococcus epidermidis, at 4%. Peri-ocular skin flora of the same individual was populated by similar genera, but had different species, many of which were completely absent from the eye.
Most bacterial DNA recovered from the eye is packaged in cells, and its metagenomic characterization reflects a broader diversity than the species recovered by culture. Its species composition suggests that the ocular surface has a distinctive endogenous flora, one that is not simply the result of bulk transfers from the skin. The origins of this flora and its variation between individuals remain to be determined.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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