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V. I. Shestopalov, D. A. Antonopoulos, D. Miller, A. Iovieno, J. M. Brulc, B. Bates, A. Garoutte, M. Domanus, D. V. Ivanov, V. Slepak; Metagenomic Analysis of Bacterial Community at the Human Conjunctiva. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2409.
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The ocular surface (OS) microbiota has been shown to contribute to the pathogenesis of infectious and immune-mediated diseases of the eye. However, the microbial diversity at the OS remains poorly investigated due to the limits of traditional cultivation techniques. This study aimed to fill this knowledge gap using molecular-based microbial ecology techniques
Total DNA was extracted from conjunctival swabs of the inferior conjunctival fornix of four healthy non contact lens wearing volunteers. Bacterial abundance in these low-biomass samples was assessed using quantitative PCR specific for the 16S rRNA encoding gene. Following multiple displacement amplification of the extracted metagenomic DNA the V4 hypervariable region of the 16S rRNA encoding gene was targeted to produce amplicon libraries for sequencing using the GS-FLX instrument (454 Roche Applied Science). The sequence data were classified according to the bacterial taxonomy and analyzed using a variety of ecological measurements on-line via the Ribosomal Database Project.
We detected a bacterial load averaging 79.8 and 729 bacterial genomes per 1 ng of total DNA, at the conjunctiva and cornea respectively. Microbial community analysis of the conjunctiva revealed an average of 224 distinct bacterial phylotypes per individual (n=4). This amount exceeds what has been cumulatively identified by traditional culture-based techniques by approximately three fold. Among 55 different bacterial genera identified in this study, 22 have never been detected in the eye based on previous cultivation-directed efforts. Remarkably, ten "core" genera were present in volunteers, with six of them accounting to 68% of the bacterial population within the analyzed group. Individual variation (Shannon diversity index) averaged 2.70+0.69 across all samples.
We obtained the first global survey of bacterial population at the conjunctiva. This revealed an unexpectedly diverse population with significant variability between individual. We identified a "core" set of bacterial genera present across all samples. We have applied multiple displacement amplification (MDA) and tag sequencing of 16S rRNA-encoding genes to address the challenges associated with low microbial biomass relative to host material in ocular samples.
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