Microbiome Characterization in Retina and Choroid of Patients with Age-related Macular Degeneration

Abstract

Purpose : To determine whether DNA or RNA from microbial organisms are present in the retina, choroid, or other ocular tissues of healthy human eyes and eyes with age-related macular degeneration (AMD).

Methods : Eyes were retrieved, tissues were dissected, and DNA and RNA were purified from individuals with or without AMD. DNA from 20 different bacteria served as a positive control, and purified water and DNA extraction elution buffer were negative controls. DNA from retina and choroid were PCR amplified for sequences encoding bacterial 16S ribosomal RNA (rRNA) genes. The PCR amplicons were sequenced from both ends and the resulting sequences were classified according to a bacterial taxonomy tree. Separately, RNA from each specimen was sequenced using the standard Ribo-Depletion RNA Sequencing protocol (60 million reads per sample) and searched for viral RNA sequences.

Results : Our PCR-based assay was capable of amplifying 16S rRNA genes in retina or choroidal tissues spiked with as little as 1 picogram of DNA from an individual bacterial species. The 16S sequences from 78 to 104 genera of bacteria were found in water controls added at different experimental steps. In pristine, non-spiked tissues, 162 different genera of bacteria were identified in retina and choroid from eyes of unaffected individuals, and 236 genera in retina and choroid from eyes with AMD. RNA sequences from adenoviruses, bromoviruses, and virgaviridae were detected in both controls and ocular samples.

Conclusions : Bacterial 16S rRNA genes can be detected in the retina and choroid from eyes with AMD. There was no clear association of a particular bacterial species in the retina and choroid from eyes with AMD versus retinas and choroid from unaffected eyes. The data from this study indicated that to potentially see whether specific microbes are significantly associated with AMD, hundreds of samples per disease state would be required. Furthermore, contamination at different stages of procurement and sample preparation must be reduced to prevent misleading conclusions.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.