Abstract
Purpose :
Vernal keratoconjunctivitis (VKC) is a severe form of ocular disease possibly leading to visual impairments. Microbial exposure has been postulated to have a crucial role in the development of hypersensitivity disorders. The purpose of the study is to apply 16S metagenomics based on next-generation sequencing (NGS) to provide the first VKC-associated microbioma data.
Methods :
In spring-summer 2019 a total of 42 conjunctival swabs were collected from 15 active VKC patients (10 males, 5 females) and from 16 healthy, age-matched volunteers (CT) (10 males, 6 females). Total DNA was extracted from the swabs and the V3-V4 16S rRNA region was amplified using degenerate primers designed to cover the diversity of bacterial and archaea species. The amplified DNA amplicons were multiplexed and sequenced on an Illumina MiSeq platform with 2x300 bp paired-end mode. Quality-trimmed reads were further filtered to remove chimeric reads and clustered into operational taxonomic units (OTUs). OTUs were taxonomically classified using the Greengenes 16S rRNA database.
Results :
DNA extraction and PCR amplification provided positive results for 17 out of 42 tested samples. High throughput Illumina sequencing resulted in 1.37 million clean reads with a range of 58-106k reads per sample. Read denoising with DADA2 tool resulted in a mean of 45.9k non-chimeric reads per sample (25-61k in range), which were exploited to identify 1,363 OTUs. At the phylum level, the relative abundances of Firmicutes (37.1% vs. 19.67%) Actinobacteria (7.3% vs. 3.7%), Bacteroidetes (7.2% vs 2.7%) and Fusobacteria (2.4% vs 0.9%) were significantly higher in VKC vs CT samples (p< 0.0001), while the abundance of Proteobacteria (41.5% vs. 67.6%) was lower in VKC than CT (p< 0.0001). At the genus level, the abundances of Staphylococcus (11.4% vs 9.4%), Streptococcus (14.4% vs. 4.8%) and Acinetobacter (5.0% vs 0.004%) were significantly higher in VKC patients vs CT (p< 0.0001), while the abundance of Neissariae (1.5% vs. 8.6%) and Pseudomonas (23.6% vs 54.7%) were significantly lower in VKC (p< 0.0001).
Conclusions :
This study explored the ocular surface bacterial microbiome of active VKC patients using NGS. Various degrees of conjunctival bacteria microbiota imbalance were found in VKC conjunctiva suggesting that conjunctival dysbiosis may play a role in VKC.
This is a 2020 ARVO Annual Meeting abstract.