The “Eyes” Have It, Too! A Closer Look at the Ocular Microbiome

While the ocular surface was late to the microbiome table, a growing body of evidence suggests that it should not be overlooked. Read on to learn how these microbes are measured, their role in ophthalmic disease, and how contact lenses may alter their microenvironment.

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Researchers are constantly finding new and impactful connections between the microbiota and our physiology. In recent years, the field of microbiome research has exploded. I’ve covered a lot of it at ChrisKresser.com, including connections between the microbiota and the skin, allergies, thyroid, autoimmune disease, brain, food cravings, and bone health. It is increasingly important that clinicians and allied healthcare providers have a basic understanding of these microbes and how they might influence clinical outcomes (1). When you treat a patient, you are treating his or her entire ecosystem.

The Human Microbiome Project was launched in 2008 with the goal of characterizing the microbiome of several different body sites. Initial seminal studies of the gut microbiome were quickly followed by subsequent investigations that described a core microbiome of the skin, urinary tract, oral mucosa, and nasal mucosa (2). The microbes in the eye, or “ocular” microbiome, were not included in these initial studies, but they are gaining significant traction in the scientific community (3, 4). In this article, we’ll discuss what we do and don’t know about the ocular microbiome and how it might influence ocular health and disease. First though, a quick primer on microbiome sample collection and analysis.

Analyzing Eye Microbes

To characterize the ocular microbiome, researchers first sample the ocular tissues using moistened cotton swabs. The cotton swabs are transferred to a sterile tube, which can then undergo several different types of downstream processes. Regrettably, much of the data from the ocular microbiome is difficult to sort through because of discrepancies in results from different methods.

Culture-Dependent Methods

Like other body sites, much of the early ocular microbiome data was based on culture-dependent methods. In this method, samples are streaked onto petri dishes containing bacterial growth media. These cultures are incubated for a period of time, and bacterial growth is quantified by identifying the number of colony-forming units (CFUs). They can also identify bacteria by their phenotype (what they look like) and their biochemical characteristics (5).

Unfortunately, there is no standardized culture method. Various different growth mediums, incubation types, and incubation times are used, making results hard to rationalize (6). Identification can also be difficult, as certain strains of bacteria do not conform to the properties that are characteristic of their genus or species.

The first description of the ocular microbiota from the conjunctiva was published by Dr. Robert Keilty in 1930 (7). Using culture-based methods, he reported that 43 percent of conjunctival cultures from normal subjects were “absolutely sterile.” In 1975, Perkins et al. used more modern culture techniques to characterize normal and infected conjunctiva (8). Bacterial isolates were found in 87 of 96 control eyes, with the primary organisms being Staphylococcus species and Propionibacterium acnes.

Culture-Independent Methods

Culture-independent methods are now the gold standard for microbiome analysis across body sites. Researchers isolate the DNA from a swab sample using commercially available kits and then typically use one of two methods of identification (9). The first option is to sequence all of the bacterial genomes present in the sample (a method called metagenomics) (10). This is the most accurate, but also the most expensive, method. Alternatively, they can selectively amplify (make copies of) a gene that is found in all bacteria but differs slightly from one microbe to the next. The gene most commonly used encodes the 16S subunit of the bacterial ribosome and is thus called 16S sequencing. By matching up the 16S sequences from the sample to known bacterial 16S sequences in a database, scientists can determine what bacteria are present in their sample of interest and in what relative abundance.

In contrast to culture-dependent methods, which occasionally return no bacterial growth, 16S sequencing consistently returns multiple genera of bacteria from ocular surface swabs (11). However, it does not tell you if the bacteria are viable. For instance, if dead bacteria from the skin were to fall onto the ocular surface, these bacteria would not grow in culture, but their DNA would still be isolated and sequenced (12).

Does wearing contacts affect your eyes’ microbiome?

Characterizing the Healthy Ocular Microbiome

Given these various limitations, we need to consider the results of experiments using both methodologies to get an accurate picture of the ocular microbiome. While 16S sequencing and metagenomics capture more microbes than traditional culture-based methods, we cannot know whether these microbes are alive and whether they are true residents of the ocular surface.

Using cultures, the genus most frequently identified from the conjunctival surfaces in healthy individuals is Staphyloccocus, which is consistently isolated from about 20 to 80 percent of conjunctival samples and about 30 to 100 percent of lid margin samples. Less-present genera include the gram-positive Propionibacterium, Corynebacterium, Streptococcus, Micrococcus, Bacillus, and Lactobacillus. Gram negatives are less frequently identified on the healthy ocular surface and include genera like Enterobacter, Proteus, and Acinetobacter, and species like P. aeruginosa and E. coli (3).

In contrast, the first 16S sequencing survey of the healthy human conjunctiva found 12 genera that were present in most samples. These included Pseudomonas, Propionibacterium, Bradyrhizobium, Corynebacteria, Acinetobacter, Brevundimonas, Staphylococcus, Aquabacterium, Sphingomonas, and Streptococcus (13). The microbial diversity of the conjunctiva was found to be higher than that of the lid skin (14).

Does a True Ocular Microbiome Exist?

Discrepancies in data made scientists debate the existence of an ocular microbiome for a long time. Do bacteria actively colonize the ocular surfaces and replicate there? Or are they only transiently introduced, perhaps falling onto the ocular surface from the surrounding skin of the eyelid or from the touching of the eyes?

To answer this question, Doan et al. collected samples from 107 healthy volunteers and analyzed the ocular microbiome using both culture methods and 16S sequencing in parallel. They concluded that the ocular surface supports a relatively stable, resident microbiome that is distinct from facial skin community, buccal mucosa, and environmental control samples. Corynebacteria, Propionibacteria, and Staphylococci were the predominant bacterial genera identified on the ocular surface using both techniques (15).

Ocular Microbial Diversity

Interestingly, the ocular surface harbors relatively few commensal bacteria and has a low degree of microbial diversity compared to other body sites (15). This is surprising, given that the ocular surface is constantly exposed to the surrounding environment.

Scientists believe that this can be explained by innate immune defenses at the ocular surface. Small proteins called antimicrobial peptides are secreted in tears (16). These help to prevent certain bacteria from colonizing the ocular surface and reduce overall bacterial load. Antimicrobial peptides play a similar role in the small intestine (17), which is also largely devoid of microbes (most of the gut microbiota resides in the large intestine).

Beyond Bacteria: Viruses and Fungi

When you hear the word “microbiome,” you might automatically think “bacteria.” But while bacterial communities are perhaps the most well characterized, the microbiome encompasses a wide range of microorganisms, including archaea, eukaryotes, and viruses. This holds true for all body sites, including the ocular surface.

Using methods similar to 16S that specifically target viruses, researchers have shown that herpes and hepatitis B virus can be detected in the tears of healthy volunteers (18, 19). Doan et al. found that torque teno virus (TTV) was present in 65 percent of conjunctiva samples (15), while researchers at Baylor College found fungi on the ocular surface of children and adolescents (20). These studies suggest that fungi and viruses are also normal residents on the ocular surface.

The Eye Microbiome in Ophthalmic Disease

An understanding of the ocular surface microbiome has countless implications for clinical ophthalmology. In a review of the ocular microbiome, Dartmouth and University of Washington ophthalmologists write:

“A number of ocular surface disorders—including dry eye syndrome, episcleritis, chronic follicular conjunctivitis, pterygium, and Thygeson’s disease, to name only a few—remain essentially idiopathic. All have inflammatory components. By analogy to the situation in the intestine, it is conceivable that dysregulation of an ocular surface microbiotic community (even a very small one, by release of specific toxins or triggering of a large immune response) could trigger or contribute to any or all of these conditions.” (12)

In other words, the specific cause of several diseases of the eye is unknown, but they all share a common theme: inflammation. Microbes are known to play a significant role in shaping immunity. Mice raised in a sterile “germ-free” environment have been shown to have lower levels of secretory IgA in their tear fluid (21). Secretory IgA is the first line of defense against pathogens and toxins at mucosal surfaces. Dysbiosis of the ocular surface could trigger inflammatory events that subsequently lead to disease states.

Associations between ocular microbes and disease are abundant. In one study, patients with dry eye were shown to have significantly more colony-forming units than patients without dry eye (22). Graham et al. reported an increased presence of gram-negative Staphylococcus, Corynebacterium, and Propionibacterium in non-autoimmune dry eye disease (23). Another study found more extensive bacterial loads in patients with Sjögren’s syndrome than in healthy controls (24). Much like the microbiota of other body sites, the ocular microbiota appears to change with age (14) and may also play a role in age-related ophthalmic diseases.

Ocular infection by pathogenic microorganisms underlies non-idiopathic conditions like post-operative endophthalmitis, blebitis, and microbial keratitis. Patients with keratitis have significantly decreased microbial diversity, with overgrowth of Pseudomonas strains (25). These changes typically occur well before diagnosis, suggesting that the ocular microbiome could be a predictor of future eye disease.

Pathogen or Commensal?

While it might seem easy to divide microbes into “good” and “bad,” it’s not quite that simple. Known pathogens have been cultured from people with and without dry eye (23), suggesting that some species that contribute to disease in one person are harmless commensals in another person. These so-called “opportunistic pathogens” are a feature of the microbiome at all body sites. For example, E. coli is well known for causing foodborne illness, but it is also a normal resident of the gut microbiota (26). Likewise, Candida albicans is present at almost all mucosal surfaces in small amounts, but when overgrown, it can cause serious problems (27).

How does a microbe turn from “good” to “bad”? Scientists have uncovered an interesting feature of bacteria called quorum sensing (28). Once enough of a certain type of bacteria are present (a quorum), the bacteria can sense this through cell-to-cell signaling. They can then change aspects of their physiology to become more virulent and overcome the innate immune response of the host. This epigenetic change occurs in all microbiotas. In the ocular microbiota, quorum sensing has been demonstrated to occur in microbes like S. aureus and P. aeruginosa (29, 30).

Wearing Contacts Changes the Physiology of the Eye

In 2014, an estimated 40.9 million people reported using contact lenses (31), an increase from 38 million people in 2004 (32). Contact lenses come in many different types of polymers and can be worn as either daily disposables, daily wear, or extended wear. Common lens-related irritation and redness affect an estimated one-third of contact lens wearers, and much rarer keratitis infections can cause blindness (31). A recent study found that most contact wearers are not compliant with recommended hygienic practices (33).

Researchers believe contact lenses give bacteria something to adhere to, making it easier for pathogens to colonize the surface of the eye. Like in the gut, bacteria on the ocular surface consume host-produced glycoproteins called mucins (34). Mucin decreases bacterial adherence to the cornea (35). Researchers have hypothesized that altered chemistry of tears under a contact lens results in changes to this mucin structure and that the combined shear stress and chemical stress increases bacterial adherence (36).

How Does This Influence the Ocular Microbiome?

Extended wear of contact lenses is associated with an increased number of pathogenic organisms in the conjunctival tissues (37). Extended use contact wearers show lower levels of secretory IgA antibodies in their tears against P. aeruginosa, the opportunistic pathogen responsible for many cases of microbial keratitis (38). Shin et al. found that contact lens wearers had relatively higher abundance of Methylobacterium, Lactobacillus, Acinetobacter, and Pseudomonas and relatively lower abundance of Corynebacterium, Staphylococcus, Streptococcus, and Haemophilus, compared to non-wearers. Overall, contact lens wear shifted the ocular microbiota towards a more skin-like microbial community (39).

The effect of contact lens wear on the microbiota will likely vary by type of contact lenses, duration of their wear, and the age of the wearer. A study published by Zhang et al. early this year found that people who wore orthokeratology lenses, a type of rigid gas-permeable contact lens, had decreased abundance of the genera Bacillus, Tatumella, and Lactobacillus. These lenses subject the eye to increased pressure and hypoxia, factors that likely influence the microbiota. In contrast, wearers of soft contact lenses had significantly decreased Delftia and increased Elizabethkingia but had a microbiota that looked more like non-wearers (40). Preclinical trials on antimicrobial lenses are underway, and it will be very interesting to see how these affect the ocular microbiome (41).

Conclusion

If you’ve been following my work for a while, you know that this is usually the point of a microbiome-related article where I would say: Probiotics! Prebiotics! Fermented foods! Unfortunately, our current understanding of the ocular microbiota, what factors affect it, and what this means for disease states is fairly limited. (One preliminary open-label study did find that one month of probiotic Lactobacillus acidophilus eye drops improves signs and symptoms of vernal keratoconjunctivitis (42), but large-scale double-blind controlled trials will need to confirm.)

Research on the ocular microbiota lags far behind other body sites like the gut, skin, and oral cavity. Still, if this microbial community is influenced by similar factors as communities at other body sites, we can infer a few simple practices that might keep it healthy. Recommend to your patients that they:

• Avoid harmful chemicals in and around the eyes. Think: harsh shampoos, face wash, etc.

• Wear contacts sparingly and practice good hygiene: If they choose to wear contacts, advise that they wash their hands before inserting or removing them and take them out at night (43). If they experience any kind of irritation, they should consider avoiding contact lens use for a while.

• Get enough shut-eye: Preliminary studies suggest that sleep may be important for cultivating a healthy ocular microbiota (44).

• Cultivate a healthy whole-body ecosystem: While the microbiotas of different body sites are distinct, they do share one thing: the host. The host immune system is constantly talking with microbes. If a patient has dysbiosis at one site, it could shape the immune system and indirectly influence the microbes that colonize other sites. For example, changes in the oral microbiome have been associated with neurodegeneration in glaucoma (45).