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Alexander Tuzhikov, Qunfeng Dong, Alexander Panchin, Onsiri Thanathanee, Nabeel Shalabi, David Nelson, Lakshmi Akileswaran, Russell Van Gelder, Terrence O'Brien, Valery Shestopalov; Keratitis-induced changes to the homeostatic microbiome at the human cornea. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2891.
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Indigenous microflora can contribute to ocular surface (OS) infections, the major causes of corneal blindness in the US and world-wide. We analyzed microbial composition on the OS of healthy and infected human eyes using DNA sequencing.
We utilized 454 Roche sequencing of 16S rRNA gene libraries and Biome Representational in Silico Karyotyping (BRiSK) to analyze the OS microbiome. Phylogenetic classification was performed using ESPRIT, MOTHUR and classification algorithm of the RDP-II database. 16S reads, rejected by RDP-II tools, were classified by a novel TUIT algorithm utilizing GenBank database. Between-group comparison was used to analyze diversity, relative abundance, gender- and infection-related changes.
The eyes of both genders were examined; 17 healthy and 4 eyes with ulcerative bacterial keratitis eyes were analyzed. The application of TUIT algorithm allowed us to classify 29% of reads rejected by RDP-II to the genus and 1% to the species levels, which substantially increased the percentage of identified reads. At the phylum level, the homeostatic community of the healthy cornea is dominated by Proteobacteria, Actinobacteria, Furmicutes and trace amounts of Cyanobacteria, and Bacteoidetes. At the genus level Propionibacterium, Staphilococcus and Bradyrhizobium showed the highest prevalence in healthy eyes. Between-group comparison revealed that the composition is heavily influenced by gender. More than 90% of all reads belong to five most prevalent genera in women and two genera in men. Bacterial keratitis caused depletion of homeostatic and the onset of stable pathological biome dominated by Pseudomonas aerugenosa and a defined cohort of satellites, indicating multibacterial type of infection. BRiSK corroborated deep 16S sequences and additionally identified a number of phage species present in both normal flora and enhanced in microbial keratitis.
Our results obtained with the two sequencing approaches and novel bioinformatics suggest that homeostatic corneal microbiome correlates with OS health, while the onset of pathology disrupts this community.
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