The microbiome is the collective term for all microorganisms and their genetic material present in a specific environment 4). The ocular surface microbiome (OSM) refers to the community of bacteria, fungi, and viruses that normally reside on the conjunctiva and cornea 2). The flora of the eyelids and eyelashes is considered part of the skin microbiome.
Compared to other body surfaces and mucous membranes, the healthy ocular surface has an extremely low microbial load and is termed “paucimicrobial” 4). Approximately 0.05 bacteria per conjunctival cell are detected, which is about 1/150th of that on facial skin or oral mucosa 4). This low microbial load is attributed to the selective pressure of antimicrobial enzymes (lysozyme, lactoferrin, defensins) in tears and the physical removal mechanisms of blinking and tear reflex 4).
Commensal microorganisms on the ocular surface competitively inhibit the colonization of pathogens and contribute to the maturation and regulation of local immunity 1)2). When this balance is disrupted, it is called dysbiosis, and associations with various ocular diseases have been reported.
Note that a precise definition of the “core microbiome” (the set of microorganisms commonly present in a specific environment) has not yet been established 4). Technical challenges in low-biomass environments and data variability make standardization difficult.
The ocular surface microbiome is the totality of bacteria, fungi, and viruses that normally reside on the conjunctiva and cornea. It is involved in maintaining homeostasis and defending against pathogens, and its imbalance (dysbiosis) is associated with many ocular diseases. For details, see the “Pathophysiology” section.
Integrated data from five studies using metagenomic shotgun sequencing show that bacteria account for an average of 91% of ocular surface microorganisms, viruses for an average of 5%, and fungi and other eukaryotes for an average of 4% 4).
Bacteria
Three major phyla: Proteobacteria (average 45%), Actinobacteria (average 23%), and Firmicutes (average 19%). Consistently detected regardless of method 4).
Most abundant genus: Corynebacterium (detected in 17/18 studies, weighted average 11%). Followed by Pseudomonas, Staphylococcus, Streptococcus, and Acinetobacter 4).
Culture method: Coagulase-negative staphylococci (CNS) are most frequently isolated, followed by Corynebacterium and Propionibacterium 3).
Viruses
TTV (Torque teno virus): The most predominant virus on the ocular surface. Detected in 86.3% of healthy conjunctiva 4).
Bacteriophages: Viruses that infect bacteria and regulate the density and distribution of bacterial populations 4).
Others: MSRV, HERV-K (human endogenous retrovirus K), MCV, and HPV are detected at low frequencies 4).
Fungi
Two major phyla: Basidiomycota (average 78.67%) and Ascomycota (average 19.54%) 4).
Core fungi: Malassezia (74.65%) present in over 80% of all subjects. Followed by Rhodotorula, Davidiella, Aspergillus, and Alternaria 4).
Opportunistic pathogens: Fusarium, Aspergillus, and Malassezia are also present in healthy eyes.
Disruption of the ocular surface microbiome is associated with many eye diseases. The following table shows major diseases and changes in bacterial flora.
| Disease | Increased genera | Decreased genera |
|---|---|---|
| Bacterial keratitis | Streptococcus, Pseudomonas | — |
| Meibomian gland dysfunction | Staphylococcus, Sphingomonas | Corynebacterium |
| Stevens-Johnson syndrome | Pseudomonas, Acinetobacter | — |
In patients with Stevens-Johnson syndrome (SJS), the positive culture rate in the conjunctiva is 60%, significantly higher than the 10% in healthy individuals 3). 16S rRNA sequencing has reported increased alpha diversity and increases in Lactobacillus, Bacteroides, Pseudomonas, Staphylococcus, and Acinetobacter 3).
The conjunctival microbiome of diabetic patients has higher diversity than that of healthy individuals, with an increase in Acinetobacter and predominance of Proteobacteria 5).
Factors that alter the composition of the ocular surface microbiome are diverse.
Contact lens wear increases skin-related bacteria such as Pseudomonas and Acinetobacter on the ocular surface microbiome, while decreasing normal commensal bacteria. Lenses act as a vehicle for transferring skin microbes to the eye, potentially increasing the risk of microbial keratitis.
The traditional culture method has a long history but low sensitivity. The culture positivity rate for healthy ocular surfaces is only 10–13% 3). The advantage of the culture method is that it can detect only viable bacteria, but it cannot detect most difficult-to-culture bacteria, viruses, or fungi 4).
This method involves PCR amplification of the 16S rRNA gene followed by sequencing 4). It can detect more than three times the diversity of the culture method 3). However, it targets only bacteria and cannot detect viruses or fungi. PCR amplification bias may affect the results 4).
This method fragments and sequences all DNA in a sample. It can simultaneously detect bacteria, viruses, fungi, and archaea, and also allows functional profiling 4). However, in low-biomass environments, contamination from short fragments (including contamination from DNA extraction kits, known as “kitome”) is a problem 4).
The characteristics of each method are summarized below.
| Method | Target | Advantages/Limitations |
|---|---|---|
| Culture method | Viable bacteria only | Low sensitivity (positivity rate 10–13%) |
| 16S rRNA | Bacteria only | High sensitivity but PCR bias exists |
| Shotgun | All microorganisms | Comprehensive but susceptible to contamination |
In low-biomass samples, host DNA depletion or microbiome enrichment is necessary. Selective host cell lysis can increase relative bacterial DNA content up to 10-fold 4). In the future, establishment of standardized protocols including positive and negative controls is required 4).
There is currently no established treatment specifically targeting dysbiosis of the ocular surface microbiome. Current management is divided into lifestyle modifications and investigational interventions.
There are reports that oral probiotics improve tear secretion and TBUT in dry eye, but these are research-stage findings and not standard treatment. Immune modulation via the gut-eye axis has been proposed as a mechanism of action. For details, see the “Pathophysiology” section.
Homeostasis of the ocular surface is maintained by multilayered defense mechanisms2)4).
Physical and chemical defenses
Blinking and tear reflex: Mechanically removes microorganisms4).
Antimicrobial tear proteins: Lysozyme, lactoferrin, mucin, and defensins inhibit bacterial growth4). 9% of the tear proteome is involved in antimicrobial function4).
Epithelial tight junctions: Tight junctions between corneal and conjunctival epithelial cells form a physical barrier2).
Immunological defenses
CALT: Conjunctiva-associated lymphoid tissue. Follicular structures containing dendritic cells, B cells, and T cells, responsible for both immune tolerance and immune surveillance2).
Secretory IgA: Produced by IgA-producing plasma cells in the lacrimal gland and conjunctiva. Inhibits pathogen adhesion and coats commensal bacteria in a non-inflammatory manner2).
γδT cells, MAIT cells, NKT cells: Non-conventional T cells present in the epithelium, bridging innate and adaptive immunity2).
Corynebacterium mastitidis is a non-pathogenic commensal bacterium frequently found on the ocular surface 2). It stimulates conjunctival γδT cells to induce secretion of IL-17 and IL-22 2).
TLR2, TLR4, and TLR5 in the corneal epithelium are localized intracellularly at the wing cell and basal cell levels, not on the surface 2). This strategic positioning maintains an “immunosilence” state, preventing unnecessary inflammatory responses upon contact with commensal bacteria 2). When the epithelium is damaged, these TLRs become activated and trigger downstream inflammatory cascades.
The following pathways have been proposed through which gut microbiota dysbiosis induces ocular surface diseases 2):
In animal models, the ocular surface of germ-free mice shows decreased goblet cell density and epithelial damage, which is improved by fecal transplantation 5).
C. mastitidis is a non-pathogenic commensal bacterium that stimulates conjunctival γδT cells to induce secretion of IL-17 and IL-22. This leads to production of antimicrobial peptides, recruitment of neutrophils, and strengthening of the epithelial barrier, preventing pathogen colonization.
Kittipibul et al. (2021) compared the conjunctival microbiome of 20 eyes from Stevens-Johnson syndrome patients and 20 eyes from healthy controls 3). By culture method, 60% of the Stevens-Johnson syndrome group were positive, compared to 10% of the healthy group. 16S rRNA sequencing identified Pseudoalteromonadaceae, Vibrionaceae, Burkholderiaceae, and Enterobacteriaceae as the core microbiome in the Stevens-Johnson syndrome group. The culture-positive group had significantly higher disease severity scores (p=0.016).
Peter et al. (2023) reported the association between the healthy ocular surface microbiome and the tear proteome 4). Among 2172 tear proteins, 9% were involved in antimicrobial function. Amino acid metabolic pathways are noted as a point of contact between ocular surface bacteria and tear composition, suggesting that Corynebacterium may contribute to amino acid metabolism.
Bacteriophages are major regulators of bacterial populations and are being investigated as therapeutic alternatives to antibiotics 4). Phages of the family Siphoviridae have also been detected on the ocular surface, and may become future therapeutic targets 4).
Treatment of ocular surface diseases through manipulation of the gut microbiota is being studied 2). Clinical trials of FMT in patients with Sjögren’s syndrome are underway, but further research is needed to elucidate the pathophysiological link between the gut microbiota and the ocular surface 5).
Establishing standardized analysis protocols in low-biomass environments is an urgent priority 4). Multi-layered analyses integrating metagenomics, proteomics, and metabolomics are expected to deepen the functional understanding of the ocular surface microbiome 3).
