Comparison of the ocular surface microbiota between thyroid-associated ophthalmopathy patients and healthy subjects

Abstract

Purpose: Thyroid-associated ophthalmopathy (TAO) is a chronic autoimmune disease. In this study, high-throughput sequencing was used to investigate the diversity and composition of the ocular microbiota in patients with TAO.

Methods: Patients with TAO did not receive treatment for the disease and did not have exposed keratitis. Patients with TAO (TAO group) and healthy individuals (control group) were compared. All samples were swabbed at the conjunctival vault of the lower eyelid. The V3 to V4 region of the 16S rDNA was amplified using polymerase chain reaction and sequenced on the Illumina HiSeq 2500 Sequencing Platform. Statistical analysis was performed to analyze the differences between the groups and the correlation between ocular surface microbiota and the disease. The ocular surface microbiota of patients and healthy individuals were cultured.

Results: The ocular surface microbiota structure of TAO patients changed significantly. The average relative abundance of Bacillus and Brevundimonas increased significantly in the TAO group. Corynebacterium had a significantly decreased relative abundance (P<0.05). Paracoccus, Haemophilus, Lactobacillus, and Bifidobacterium were positively correlated with the severity of clinical manifestations or disease activity (P<0.05). Bacillus cereus and other opportunistic pathogens were obtained by culture from TAO patients.

Conclusions: This study found that the composition of ocular microbiota in patients with TAO was significantly different from that in healthy individuals. The ocular surface opportunistic pathogens, such as Bacillus, Brevundimonas, Paracoccus, and Haemophilus in TAO patients, increase the potential risk of ocular surface infection. The findings of this study provide a new avenue of research into the mechanism of ocular surface in TAO patients.

Keywords: bacteria; high-throughput sequencing; ocular surface; ocular surface microbiota; thyroid-associated ophthalmopathy.

Figure 1

(A) Rarefaction curves of TAO and control samples. The median, and quartile values of the two groups of samples are shown. (B) Alpha diversity (Shannon index) and (C) beta diversity (principal coordinate analysis) are used to measure the diversity and differences between the two groups. (D) Dominant ocular surface microbiota at the phylum level and (E) evolutionary tree are indicated by different colors.

Figure 2

The differences in average relative abundance of dominant bacteria at (A) the phylum level and (B) the genus level (mean relative abundance > 0.50%) were assessed by the Wilcoxon rank-sum test. (C) Linear discriminant analysis was used to verify with significant differences between groups (cutoff = 4.0).

Figure 3

(A) Ocular surface symptoms of different disease activity in the thyroid-associated ophthalmopathy (TAO) patients. (B) A healthy people (left) and a TAO patient with exophthalmos (right). (C) Corneal fluorescein staining results of a healthy person (left) and a TAO patient with moderate corneal epithelial injury (right). (D) Bacteria with higher Spearman’s correlation coefficients with CAS, eyelid retraction, exophthalmos, disease severity, and corneal injury in TAO patients are shown at the genus level. (E) There were significant differences in the average relative abundance of some bacteria within TAO group and between groups according to dry eyes (t-test dichotomous traits and t-test regression coefficients, P<0.05).