3.1. Dysbiosis of conjunctival microbiome in allergic conjunctivitis
The study cohort was composed of 39 individuals with AC and 48 healthy subjects (Table S1). The group of AC comprised 14 patients with PAC, 7 with SAC, and 18 with VKC (Table S2). Table S1 and Table S2 show the age at sample collection. All the patients were in active stage of AC with inflamed ocular surface. We performed shotgun metagenomic sequencing and obtained on average 0.32 million high quality non-human reads for each individual. A total of 278 species passed our decontamination pipeline (Methods).
Overall, bacteria accounted for the majority of the conjunctival microbiota of both healthy and AC individuals (Figure 1A). We observed the enrichment of eukaryotic virus in a subset of healthy individuals (Kruskal-Wallis rank-sum test P = 0.0014). The prominent viral species in these samples was human beta herpesvirus 7, which is frequently detected and rarely pathogenic in immunocompetent individuals.22 Malassezia Fungi (M.furfur in particular) were abundant in a fraction of patients with SAC/PAC (but not VKC) compared with healthy individuals (Figure 1B;P = 0.0095). IgE mediated hypersensitivity to Malasseziaspecies correlates with the clinical severity of atopic dermatitis (AD).23 Malassezia produces immunogenic proteins that elicit IgE and thus induce pro-inflammatory cytokines and auto-reactive T cells, which contributes to AD pathogenesis.24 Notably, SAC/PAC is dominated by IgE-mediated reactions in contrast to VKC.
The alpha diversity showed no significant difference between healthy and AC individuals (Figure 2A), whereas the Bray-Curtis dissimilarities within AC groups were slightly lower than healthy groups (Figure 2B). The principal coordinates analysis (PCoA) of the species composition showed a clear delineation between healthy and AC participants (Figure 2C), suggesting that dysbiosis of the conjunctival microbiome is associated with AC.
To identify the species that accounts for the dysbiosis, we performed LEfSe analysis on the species profiles and the species with LDA effect size > 4 were displayed in Figure 2D. Numerous species were overabundant on the ocular surface of AC patients compared to healthy subjects (Figure 2E), such as members of Oxalobacteraceae includingJanthinobacterium sp . Marseille (P = 1.6×10-17), Herminiimonas arsenitoxidans(P = 2.3×10-14), and Herminiimonasarsenicoxydans (P = 5.2×10-15). Interestingly, family Oxalobacteraceae was prevalently detected in the conjunctival microbiota of lens wearers25 and contact lens wearing is in turn associated with ocular allergy.26 Rothia was also enriched in samples from participants with AC, including Rothia aeria (P = 1.8×10-4) and Rothia dentocariosa (P = 0.0010). In addition, we detected the enrichment of Moraxella catarrhalis in AC (P = 0.0033). Moraxella catarrhalis in the upper airways is linked with the development or exacerbation of allergic airway inflammation and IL-17 and TNF-α are involved in this process.27 Colonization of M. catarrhalis in the airways leads to a low-grade systemic inflammation that is associated with established asthma.28 This implies that common microbial mechanisms may underlie both ocular allergy and allergic diseases prevalent at other sites.
We next searched for functional differences in the conjunctival microbiome between healthy and AC individuals. Most of the gene families identified using LEfSe were enriched in the metagenome from the AC patients (Figure 3A). For instance, we detected the enrichment of the gene families related to amino acid metabolism such as aspartate kinase (Kruskal-Wallis rank-sum test P = 0.0058), tryptophan synthase (P = 0.012), and glutamine synthetase (P = 0.026). In addition, the gene families encoding thioredoxin (P = 0.012) and thioredoxin reductase (P = 0.026) were more abundant in AC patients compared to healthy individuals.
Exposure to antibiotics has been associated with many allergic diseases.29 Therefore, we further examined the prevalence of antibiotic resistance genes in the conjunctival metagenome of AC patients (Figure 3B). In particular, we observed that the genes resistant to tetracycline is more prevalent in AC patients than healthy individuals, including tetA (Fisher’s exact test P = 0.045), tetB (P = 0.0043), optrA (P = 0.038), vmlR (P = 0.0026), and evgS (P = 0.042). Glycopeptide antibiotic resistance genes such as vanTG(P = 0.016) and vanG (P = 0.037) were detected exclusively in AC samples. Fluoroquinolone antibiotic resistance genes were also more prevalent in the samples from AC patients compared to healthy subjects, including acrF (P = 0.037), efrB(P = 0.031), evgS (P = 0.042), lfrA(P = 0.037), and patA (P = 0.038). This warrants further studies into the relationships between antibiotic use and the risk of ocular allergy.