Conjunctival microbiome changes associated with fungal keratitis: metagenomic analysis

Abstract

Aim: To investigate the ocular surface microbiome profile of patients with fungal keratitis (FK) through bacterial 16S rDNA sequencing.

Methods: The swab samples were collected from 8 patients with FK (Group 1 from the corneal ulcer, Group 2 from the conjunctival sac of the infected eyes, and Group 3 from the conjunctival sac of the fellow eyes) and 10 healthy eyes (Group 4 from the conjunctival sac). Bacterial 16S rDNA V4-V5 region sequencing was performed to characterize the bacterial communities on the ocular surfaces of the patients with FK.

Results: Our metagenomic data showed that 97% of the sequence reads were categorized into 245 distinct bacterial genera, with 67.75±7.79 genera detected in Group 1, 73.80±13.44 in Group 2, 74.57±14.14 in Group 3, and 89.60±27.49 in Group 4. Compared with the healthy eyes (Group 4), both infected (Groups 1 and 2) and fellow eyes (Group 3) of the patients with FK showed reduced bacterial diversity and altered ocular surface microbiota compositions, with lower abundance of Corynebacterium and Staphylococcus and higher abundances of Pseudomonas, Achromobacter, Caulobacter and Psychrobacter.

Conclusion: Our report depicts the altered ocular surface bacterial community structures both in the affected and fellow eyes of patients with FK. These changes may contribute to the pathogenesis of FK or the increased risk for FK.

Keywords: 16S rDNA; bacteria; conjunctiva microbiota; fungal keratitis; metagenomics.

Figure 1. The common or unique OTUs between different samples

The common or unique OTUs in different groups were presented by Venn diagram. Group 1: Corneal scraping samples from infected eyes of patients with FK; Group 2: Conjunctival specimens from infected eyes of patients with FK; Group 3: Conjunctival specimens from fellow eyes of patients with FK; Group 4: Conjunctival specimens from healthy eyes as control.

Figure 2. The alpha diversity in the ocular surface of different groups

The alpha diversity was measured by observed species (A) and Shannon diversity index (B) using rarefaction curves. Group 1: Corneal scraping samples from infected eyes of patients with FK; Group 2: Conjunctival specimens from infected eyes of patients with FK; Group 3: Conjunctival specimens from fellow eyes of patients with FK; Group 4: Conjunctival specimens from healthy eyes as control.

Figure 3. The principal coordinate analysis plot of conjunctival microbial communities from different groups

The analysis was derived from weighted Unifrac distances of 16S rDNA gene between microbial communities of the 4 groups. Values in parentheses on the axis labels indicated the percentage variation for each axis. The samples were clustered based on patients with or without FK. Group 1: corneal scraping samples from infected eyes of patients with FK; Group 2: Conjunctival specimens from infected eyes of patients with FK; Group 3: Conjunctival specimens from fellow eyes of patients with FK; Group 4: Conjunctival specimens from healthy eyes as control.

Figure 4. The composition and relative abundances of the top 20 genera in the four groups

Relative abundances plots of the top 20 genera were analyzed and presented using stack picture. Group 1: Corneal scraping samples from infected eyes of patients with FK; Group 2: Conjunctival specimens from infected eyes of patients with FK; Group 3: Conjunctival specimens from fellow eyes of patients with FK; Group 4: Conjunctival specimens from healthy eyes as control.

Figure 5. The relative abundances of top 20 genera in different groups

The relative abundances of top 20 genera in different groups were analyzed and present by heat map, and values in color key indicated the relative abundance of each genus. Group 1: Corneal scraping samples from infected eyes of patients with FK; Group 2: Conjunctival specimens from infected eyes of patients with FK; Group 3: Conjunctival specimens from fellow eyes of patients with FK; Group 4: Conjunctival specimens from healthy eyes as control.