Paucibacterial Microbiome and Resident DNA Virome of the Healthy Conjunctiva

Abstract

Purpose: To characterize the ocular surface microbiome of healthy volunteers using a combination of microbial culture and high-throughput DNA sequencing techniques.

Methods: Conjunctival swab samples from 107 healthy volunteers were analyzed by bacterial culture, 16S rDNA gene deep sequencing (n = 89), and biome representational in silico karyotyping (BRiSK; n = 80). Swab samples of the facial skin (n = 42), buccal mucosa (n = 50), and environmental controls (n = 27) were processed in parallel. 16S rDNA gene quantitative PCR was used to calculate the bacterial load in each site. Bacteria were characterized by site using principal coordinate analysis of metagenomics data. BRiSK data were analyzed for presence of fungi and viruses.

Results: Corynebacteria, Propionibacteria, and coagulase-negative Staphylococci were the predominant organisms identified by all three techniques. Quantitative 16S PCR demonstrated approximately 0.1 bacterial 16S rDNA/human actin copy on the ocular surface compared with greater than 10 16S rDNA/human actin copy for facial skin or the buccal mucosa. The conjunctival bacterial community structure is distinct compared with the facial skin (R = 0.474, analysis of similarities P = 0.0001), the buccal mucosa (R = 0.893, P = 0.0001), and environmental control samples (R = 0.536, P = 0.0001). 16S metagenomics revealed substantially more bacterial diversity on the ocular surface than other techniques, which appears to be artifactual. BRiSK revealed presence of torque teno virus (TTV) on the healthy ocular surface, which was confirmed by direct PCR to be present in 65% of all conjunctiva samples tested.

Conclusions: Relative to adjacent skin or other mucosa, healthy ocular surface microbiome is paucibacterial. Its flora are distinct from adjacent skin. Torque teno virus is a frequent constituent of the ocular surface microbiome. (ClinicalTrials.gov number, NCT02298881.).

Figure 1

Quantification of 16S rRNA gene in healthy subjects. Detection of 16S rRNA gene normalized to human β-actin in (A) the conjunctiva comparing with the buccal mucosa and the facial skin, (B) the fornix of the upper eyelid compared with the lower eyelid, (C) the conjunctiva between men and women, and (D) the conjunctiva in subjects younger 30 years and older than 60 years. P values were calculated using Wilcoxon rank test and Kruskal-Wallis with Dunn's test for multiple comparisons.

Figure 2

Quantification of 16S rRNA gene between the left and right eyes in healthy subjects. No statistical significant difference existed in the normalized 16S rRNA gene to human β-actin in (A) the upper eyelids (N = 77), and (B) the lower eyelids (N = 73) of the right and left eyes. P values were calculated using Wilcoxon rank test and Kruskal-Wallis with Dunn's test for multiple comparisons.

Figure 3

Distribution of confidence heuristic values between conjunctiva and environmental control samples. See text for description of confidence heuristic.

Figure 4

Differences in bacterial community structure, number of genera, and diversity in the various body sites in healthy subjects. Bacterial community structures of the conjunctiva, skin, buccal mucosa, and control samples are represented by NMDS. There were significant differences in the bacterial communities of the healthy conjunctiva and the skin (R = 0.474, ANOSIM P = 0.0001), healthy conjunctiva and the buccal mucosa (R = 0.893, P = 0.0001), and healthy conjunctiva and the control samples (R = 0.536, P = 0.0001).

Figure 5

Detection of bacteria and viruses by BRiSK and quantification of TTV in healthy conjunctiva samples. (A) Microbes detected by BRiSK in 80 conjunctiva samples from 80 healthy subjects. (B) Distribution histogram of torque teno virus (TTV) copies in 80 healthy conjunctiva samples. (C) Torque teno virus–specific PCR products in healthy conjunctiva samples. Five samples on left of plot were TTV-positive and five on right were TTV-negative.