April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Metagenomic Analysis of Bacterial Community at the Human Conjunctiva
Author Affiliations & Notes
  • V. I. Shestopalov
    Bascom Palmer Eye Institute,
    Univ of Miami Miller Sch of Med, Miami, Florida
  • D. A. Antonopoulos
    Institute for Genomics and Systems Biology, Argonne National Laboratory, Argonne, Illinois
  • D. Miller
    Bascom Palmer Eye Institute,
    Univ of Miami Miller Sch of Med, Miami, Florida
  • A. Iovieno
    Bascom Palmer Eye Institute,
    Univ of Miami Miller Sch of Med, Miami, Florida
  • J. M. Brulc
    Institute for Genomics and Systems Biology, Argonne National Laboratory (Argonne, IL), Argonne, Illinois
  • B. Bates
    Institute for Genomics and Systems Biology, Argonne National Laboratory, Argonne, Illinois
  • A. Garoutte
    Institute for Genomics and Systems Biology, Argonne National Laboratory, Argonne, Illinois
  • M. Domanus
    Institute for Genomics and Systems Biology, Argonne National Laboratory, Argonne, Illinois
  • D. V. Ivanov
    Bascom Palmer Eye Institute,
    Univ of Miami Miller Sch of Med, Miami, Florida
  • V. Slepak
    Molecular Pharmacology,
    Univ of Miami Miller Sch of Med, Miami, Florida
  • Footnotes
    Commercial Relationships  V.I. Shestopalov, None; D.A. Antonopoulos, None; D. Miller, None; A. Iovieno, None; J.M. Brulc, None; B. Bates, None; A. Garoutte, None; M. Domanus, None; D.V. Ivanov, None; V. Slepak, None.
  • Footnotes
    Support  1R21EY020613-01 (D.I.), 1RO1EY018666 (V.S.); seed funding from the Dept. of Ophthalmology; NIH center grant P30 EY014801 and an unrestricted RPB grant to Dept. Ophthalmol.
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 2409. doi:
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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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : 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

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: conjunctiva • microbial pathogenesis: experimental studies • detection 
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