Purpose The purpose of this study was to explore if 16S rDNA amplicon sequencing can improve the conventional diagnosis of causative pathogens for bacterial corneal infection. Methods Corneal scraping and conjunctiva and eyelid margin swab samples from infected eyes of patients diagnosed with “bacterial corneal infection” and conjunctiva and eyelid margin swab samples from a random eye of healthy participants were collected. Each swab was used for both aerobic and anaerobic cultures and 16S rDNA amplicon sequencing. The V3 to V4 region of the 16S rDNA was amplified using polymerase chain reaction (PCR) and sequenced on the Illumina HiSeq 2500 Sequencing Platform. Results The overall culture positivity rate for all 72 samples was 69% (72% in the bacterial keratitis group and 67% in the healthy control group), whereas 1719 operational taxonomic units in total were generated using 16S rDNA amplicon sequencing with each sample showing 123 to 337 different genera. Staphylococcus , Corynebacterium , Propionibacterium , and Micrococcus most frequently appeared in culture, whereas Streptococcus, Acinetobacter , and Lactobacillus were the most common genera, with large ratios in 16S rDNA amplicon sequencing. The causative pathogens detected by the two methods were inconsistent for most samples, except for several corneal samples. Conclusions We suggest that a combination of different techniques, such as clinical observation, microscopic analysis, culture, and next-generation sequencing techniques including 16S rDNA amplicon sequencing, should be used to comprehensively analyze pathogens in corneal and external ocular infections. Translational Relevance This paper uses a basic research methodology for studying the microbiome in ocular samples to help improve the diagnostic accuracy of corneal and external ocular infections.

Purpose: To investigate the composition and diversity of the microbiota on the ocular surface of patients with blepharitis in northwestern China via 16S rDNA amplicon sequencing.Methods: Thirty-seven patients with blepharitis divided into groups of anterior, posterior and mixed blepharitis and twenty healthy controls from northwestern China were enrolled in the study. Samples were collected from the eyelid margin and conjunctival sac of each participant. The V3–V4 region of bacterial 16S rDNA in each sample was amplified and sequenced on the Illumina HiSeq 2500 sequencing platform, and the differences in taxonomy and diversity among different groups were compared.Results: The composition of the ocular surface microbiota of patients with blepharitis was similar to that of healthy subjects, but there were differences in the relative abundance of each bacterium. At the phylum level, the abundances of Actinobacteria, Cyanobacteria, Verrucomicrobia, Acidobacteria, Chloroflexi, and Atribacteria were significantly higher in the blepharitis group than in the healthy control group, while the relative abundance of Firmicutes was significantly lower (p < 0.05, Mann-Whitney U). At the genus level, the abundances of Lactobacillus, Ralstonia, Bacteroides, Akkermansia, Bifidobacterium, Escherichia-Shigella, Faecalibacterium, and Brevibacterium were significantly higher in the blepharitis group than in the healthy control group, while the relative abundances of Bacillus, Staphylococcus, Streptococcus, and Acinetobacter were significantly lower in the blepharitis group (p < 0.05, Mann-Whitney U). The microbiota of anterior blepharitis was similar to that of mixed blepharitis but different from that of posterior blepharitis. Lactobacillus and Bifidobacterium are biomarkers of posterior blepharitis, and Ralstonia is a biomarker of mixed blepharitis. There was no significant difference in the ocular surface microbiota between the eyelid margin and conjunctival sac with or without blepharitis.Conclusion: The ocular surface microbiota of patients with blepharitis varied among different study groups, according to 16S rDNA amplicon sequencing analysis. The reason might be due to the participants being from different environments and having different lifestyles. Lactobacillus, Bifidobacterium, Akkermansia, Ralstonia, and Bacteroides may play important roles in the pathogenesis of blepharitis.

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