Human Microbiota and Ophthalmic Disease

Abstract

The human ocular surface, consisting of the cornea and conjunctiva, is colonized by an expansive, diverse microbial community. Molecular-based methods, such as 16S rRNA† sequencing, has allowed for more comprehensive and precise identification of the species composition of the ocular surface microbiota compared to traditional culture-based methods. Evidence suggests that the normal microbiota plays a protective immunological role in preventing the proliferation of pathogenic species and thus, alterations in the homeostatic microbiome may be linked to ophthalmic pathologies. Further investigation of the ocular surface microbiome, as well as the microbiome of other areas of the body such as the oral mucosa and gut, and their role in the pathophysiology of diseases is a significant, emerging field of research, and may someday enable the development of novel probiotic approaches for the treatment and prevention of ophthalmic diseases.

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Copyright © 2016
Human Microbiota and Ophthalmic Disease
Louise J. Lu, Ji Liu*
Department of Ophthalmology and Visual Science, Yale School of Medicine, New Haven, CT
INTRODUCTION
The Human Microbiome Project, launched in 2008
by the National Institutes of Health, has revealed a re-
markably abundant and diverse community of microbial
species that inhabit the human body [1]. Understanding
the role of the trillions of bacteria, viruses, and fungi that
comprise the human microbiota is critical to enhance our
understanding of a variety of human diseases and their
pathophysiologies. Advances in the technology of next-
generation sequencing and bioinformatics tools have fa-
cilitated the characterization of the human microbiome
[2]. Discovery of various aspects of the human micro-
biota and its role in physiology and pathogenesis has rev-
olutionized our approach in studying certain diseases and
developing novel treatment modalities. The intestinal mi-
crobiota has been implicated in a variety of autoimmune
and inflammatory diseases, including inflammatory
bowel disease, rheumatoid arthritis, multiple sclerosis,
and type 1 diabetes [3-5].
While the Human Microbiome Project initially stud-
ied five main body areas – the gastrointestinal tract, the
skin, the urogenital tract, the oral mucosa, and the nasal
mucosa [6,11] – an emerging area of research is focusing
on the eye and the microbiota of the ocular surface [7,14].
The fundamental question to explore is whether the ocu-
lar surface harbors a resident microbiota and if so, what
species of micoorganisms comprise it. Subsequently, we
must examine how the microbial community is influ-
enced by host and environmental factors. Finally, we can
provide further clinical significance by investigating the
potential associations between alterations of the micro-
biome and the pathogenesis of ophthalmic diseases. This
study aims to systematically review and summarize evi-
dence regarding the most up-to-date understanding of mi-
crobiota colonizing the ocular surface, as well as
investigate the potential role of the human microbiota in
ophthalmic disease.
THE EYE AND THE OCULAR SURFACE
MICROBIOTA
Structurally, the eye is composed of an internal com-
partment – which consists of the anterior and posterior
chambers, the iris, the lens, the vitreous cavity, the retina,
the ciliary body, the choiroid, and intrinsic ocular mus-
cles – and an external compartment – which consists of
the conjunctiva, the cornea, the sclera, and the tear film
(Figure 1). The internal compartment of the eye main-
tains a sterile environment, and is physically separated
*To whom all correspondence should be addressed: Ji Liu, MD, 40 Temple St., Department of Ophthalmology and Visual Science,
Yale School of Medicine, New Haven, CT 06510; Tele: 203-785-2020; Fax: 203-785-7090; Email: liu.ji@yale.edu.
†Abbreviations: rRNA, Ribosomal RNA; PCR, Polymerase chain reaction; DNA, Deoxyribonucleic acid; IgA, Immunoglobulin A;
CFU, Colony-forming unit; IVT, intravitreal; TMP-SMX, trimethoprim-sulfamethoxazole; CIE, corneal infiltrative events; BDNF, brain-
derived neurotrophic factor.
Keywords: microbiota, microbiome, ophthalmic disease, ocular surface, genomics, infection, ophthalmology
REVIEW
The human ocular surface, consisting of the cornea and conjunctiva, is colonized by an expansive, diverse
microbial community. Molecular-based methods, such as 16S rRNA†sequencing, has allowed for more
comprehensive and precise identification of the species composition of the ocular surface microbiota com-
pared to traditional culture-based methods. Evidence suggests that the normal microbiota plays a protective
immunological role in preventing the proliferation of pathogenic species and thus, alterations in the homeo-
static microbiome may be linked to ophthalmic pathologies. Further investigation of the ocular surface mi-
crobiome, as well as the microbiome of other areas of the body such as the oral mucosa and gut, and their
role in the pathophysiology of diseases is a significant, emerging field of research, and may someday en-
able the development of novel probiotic approaches for the treatment and prevention of ophthalmic dis-
eases.
YALE JOURNAL OF BIOLOGY AND MEDICINE 89 (2016), pp.325-330.

from the immune system by the blood-retinal barrier. In
contrast, the external compartment of the eye is exposed
to microorganisms in the environment [8].
The ocular surface is comprised of the cornea and its
overlying tissue, the conjunctiva. Thus, the ocular surface
microbiota refers to the resident microorganisms that col-
onize the conjunctiva and the cornea and importantly, ex-
cludes the eyelid (microbes found in the eyelid are
considered part of the skin microbiota, which is included
in the five main research areas in the Human Microbiome
Project). Direct exposure to the external environment
means that the ocular surface is susceptible to a gamut of
antigens and pathogens. In addition to serving as a phys-
ical barrier against the external environment, the ocular
surface has a critical role in innate immunity [9]. The mu-
cosal immune system protects the conjunctival and
corneal epithelia via innate and adaptive defense mecha-
nisms present in the tissue and tear film [10]. While the
ocular surface epithelium is constantly in contact with
commensal bacteria, the epithelial cells of the cornea and
conjunctiva in a healthy individual do not undergo an in-
flammatory response. Studies have shown that ocular sur-
face epithelial cells recognize and selectively respond to
microbial components of ocular pathogenic bacteria by
producing pro-inflammatory cytokines [9]. The lack of an
inflammatory response to non-pathogenic bacteria sug-
gests a unique innate immune response of the ocular sur-
face epithelium that supports the colonization of a resident
microbiota.
While a substantial amount of evidence strongly sup-
ports the existence of an ocular surface microbiota, it is
worthwhile to note that analysis of the ocular microbiome
is currently in its early stages. Willcox et. al. argues that
determination of the ocular microbiome with molecular
techniques has lagged in relation to analysis of the micro-
biome of other areas of the body, and a greater number of
rigorous cross-sectional and longitudinal studies examin-
ing changes in the microbiota are necessary to maintain
current hypotheses [7]. Turnbaugh et al., in 2007, ques-
tioned the existence of a stable core microbiome, propos-
ing that there exist only transiently present organisms on
the ocular surface, as opposed to a resident microbiota
[11]. In recent years however, investigation of the ocular
microbiome using molecular techniques such as 16S
rRNA gene analysis have identified, with greater preci-
sion, a wider range of homeostatic species that colonize
the ocular surface, reinforcing the concept of a stable,
unique ocular surface microbiota.
CHARACTERIZATION OF THE OCULAR
MICROBIOTA
Previous analyses of the ocular surface microbiota,
performed using microbiological culture techniques, re-
ported a significantly different and less diverse profile
than what has most recently been discovered using mo-
lecular techniques. The characterization of the ocular sur-
face microbiota using culture-based methods was
purported to be dominated by Gram-positive species, es-
pecially Staphylococcus, Streptococcus, Corynebac-
terium, and Propionibacterium [12]. In addition, some
Gram negative species, such as Haemophilus and Neisse-
ria, as well as fungal isolates were cultured from the ocu-
lar surface of healthy human subjects [12,13]. A major
disadvantage of culture-based techniques that may ac-
count for its inaccuracy in microbiome characterization is
that species detection is significantly biased towards fast-
growing microorganisms that can successfully be culti-
vated on standard media [14-16].
Genomics-based detection and identification of mi-
crobial species has exposed a significantly expanded di-
versity of ocular surface microbiota than what had been
previously uncovered by culture-based methods. The first
gene sequencing-based survey of the bacterial species
found on the ocular surface, using 16S rRNA PCR, was
conducted in 2011 by researchers at the Bascom Palmer
Eye Institute. Deep sequencing of conjunctival DNA re-
vealed an average of 221 species of bacteria per subject
[14]. The bacteria were classified into five phyla and 59
distinct genera, with twelve genera being ubiquitous
among all subjects in the analyzed cohort (Table 1). Of
the five bacterial phyla, Proteobacteria, Actinobacteria,
326 Lu and Liu: Microbiota and ophthalmic disease
Figure 1. Anatomy of the eye
(© 2016 American Academy of
Ophthalmology)

and Firmicutes accounted for more than 87 percent of all
sequences; the remaining two phyla, Cyanobacteria and
Bacteroides, were present in contamination-level quanti-
ties and were excluded from further analysis. The twelve
common genera – Pseudomonas, Propionibacterium,
Bradyrhizobium, Corynebacterium, Acinetobacter, Bre-
vundimonas, Staphylococci, Aquabacterium, Sphin-
gomonas, Streptococcus, Streptophyta, and
Methylobacterium – comprised more than 96 percent of
the classified microbiome [14]. The five most abundant
genera out of the twelve ubiquitous genera identified by
Dong et al. were Pseudomonas, Bradyrhizobium, Propi-
onibacterium, Acinetobacter, and Corynebacterium, fol-
lowed by Brevundimonas, Staphylococcus,
Aquabacterium, Sphyngomonas, and Streptococcus. As
these twelve genera accounted for more than 96 percent of
the total classified bacterial DNA sequences, the data
strongly suggests that the healthy conjunctiva is colonized
by a resident homeostatic microbiota. To date, the find-
ings from this study remain the most comprehensive DNA
sequencing-based characterization of bacterial diversity at
the ocular surface.
Consistent with findings that genus composition of
the microbiota varies at different layers in other areas of
the body such as the human epidermis, the microbiota of
the ocular surface appears to have vertical stratification of
species composition [17]. Swabbing the ocular surface
with light pressure yielded sequences of opportunistic and
environmental species, such as Rothia, Herbaspirillum,
Leptothrichia, and Rhizobium. These bacteria captured
from the superficial layer likely represent transient species
on the ocular surface. In contrast, using a “deep” swab, in
which dry cotton was applied with greater pressure,
yielded an abundance of Staphylococci, Cornyebacteriae,
and Proteobacteria species, which localize to the mucosal
layer and conjunctival epithelium [14]. Deep swabbing is
thus necessary to obtain an accurate and comprehensive
characterization of the diversity of the ocular surface mi-
crobiota.
HOST AND ENVIRONMENTAL FACTORS
THAT MAY ALTER THE OCULAR SURFACE
MICROBIOTA
The ocular surface microbiota can become altered by
host factors, environmental insults, and disease states [18].
Disruption of the ocular surface may breach the innate im-
mune system at the corneal and conjunctival epithelia and
allow microbial ligands to trigger ocular inflammation
[12,19]. Alterations in the microbiota of the ocular sur-
face have been associated with conditions such as dry eye
syndrome, contact lens wear, keratoprosthesis, antibiotics,
and infection [12].
The tear film, which lubricates the ocular surface ep-
ithelia, contains antimicrobial compounds such as
lysozyme, lactoferrin, immunoglobulin A (IgA), lipocalin,
and complement [41]. Given the immunological role of
tears in the defense against potential pathogens, it is rea-
sonable to conjecture that certain situations that affect the
tear film, such as dry eye syndrome and contact lens wear,
may alter the ocular surface microbiota.
Dry eye syndrome has consistently been associated
with inflammatory ocular surface conditions such as an-
terior blepharitis, keratitis, and ocular rosacea [20]. Gra-
327Lu and Liu: Microbiota and ophthalmic disease
Phylum
Genus
Proteobacteria
Acetinobacteria
Firmicutes
Unclassifiedb
Pseudomonas
Bradyrhizobium
Propionibacterium
Acinetobacter
Corynebacterium
Brevundimonas
Staphylococcus
Aquabacterium
Sphyngomonas
Streptococcus
Other
Unclassifiedb
Percentage of all sequencesa(%)
64%
19.6%
3.9%
12.5%
18%
12%
11%
9%
8%
4%
2%
2%
0.5%
0.5%
2%
31%
Table 1. Composition of the ocular surface microbiota by phylum and genus, determined according to rel-
ative abundance of classified 16S rRNA gene reads [14].
aDong et al. analyzed 115,003 sequences in total.
bThe Ribosomal Database Project-II software was unable to classify 12.5% and 31% of sequences to the phyum and genus level,
respectively. Unclassified bacteria are designated as novel phylotypes.

ham et al. used both conventional culture and 16S rDNA
PCR to compare the bacterial population of the ocular sur-
face of normal and dry eye subjects, collecting conjuncti-
val swab specimens from a cohort of patients with dry
eyes as well as healthy control subjects over a three-month
period. They found that certain bacteria species were
found in samples from dry eye subjects only, including
Bacillus sp. and Klebsiella oxytoca, in addition to an as-
sociation between elevated bacterial count (CFU/swab)
and the incidence of blepharitis [21]. Increasing bacterial
count was correlated with a decrease in goblet cells among
the subgroup of 27 subjects, consistent with previous stud-
ies that have demonstrated a depletion of goblet cells in
other areas of the body after colonization by bacteria [22].
A reduction in goblet cells, which produce the mucins
found on the ocular surface, results in a thinned tear film
and diminishes the barrier to infiltration and colonization
of the ocular surface by external pathogens [22]. The pro-
duction of mucins on the ocular surface may pose resem-
blance to the production of fucosylated and sialylated
glycoproteins in the intestinal tract [40]. Perhaps the
human host secretes glycans and polysaccharides to pro-
mote the growth of certain microbial species in the ocular
surface in an analogous manner to findings in the intes-
tinal tract.
Alterations of the ocular surface microbiota in con-
tact lens wear has also been studied. Larkin et al. exam-
ined the microbial colonization of the conjunctiva in
contact lens wearers and compared them to control sub-
jects. Their results included the finding that the ocular sur-
face of contact lens wearers yielded higher bacterial
counts than that of control subjects. However, the authors
found no qualitative variation in the species of bacteria
identified between the lens-wearing and control groups
[23].
Studies of the bacterial microbiota colonizing the oc-
ular surface of patients with Boston type 1 keratoprosthe-
ses (K-Pros) have found no quantitative difference in
positive cultures from conjunctival swabs of K-Pro and
control patients [24,25]. Qualitatively however, re-
searchers found that samples from K-Pro patients grew
not only coagulase-negative Staphylococcus, which was
the singular species of bacteria to grow in the control
group samples, but also a variety of other Gram-positive
bacteria [24].
Ophthalmic antibiotics are used to treat and prevent a
variety of infectious and inflammatory ocular conditions.
The Antibiotic Resistance of Conjunctiva and Nasophar-
ynx Evaluation (ARCANE) study, which aimed to deter-
mine the effects of repeated exposure of topical antibiotics
on resistance patterns of the conjunctival microbiota, con-
cluded that the repeated use of macrolide and fluoro-
quinolone ophthalmic antibiotics leads to a significant
increase in Gram-positive species, particularly Staphylo-
coccus epidermidis, isolated from culture [26]. Fontes et
al. investigated the effect of orally administered trimetho-
prim-sulfamethoxazole (TMP-SMX) administration on
the conjunctival microbiota in patients with HIV infec-
tion. Chronic administration of TMP-SMX was found to
be associated with an altered conjunctival microbiota that
contained a significantly greater percentage of antibiotic-
resistant Staphylococcus species [27]. Yin et al. also dis-
covered a shift in the conjunctival flora as a result of
antibiotic use. Repeated use of a topical antibiotic, such
as moxifloxacin, after intravitreal (IVT) injection was cor-
related with increased antibiotic resistance of the ocular
surface microbiota [28].
Infectious pathologies have also been linked to shifts
in the homeostatic ocular microbiome. An analysis of the
microbial composition of the ocular surface of healthy
eyes and eyes with bacterial keratitis, utilizing DNA se-
quencing, found that bacterial keratitis was associated
with the depletion of homeostatic ocular microorganisms
and the emergence of a pathological microbiome domi-
nated by Pseudomonas aeruginosa [29]. Investigation of
the conjunctival flora in patients with human immunode-
ficiency virus (HIV) infection compared to HIV-negative
patients, however, concluded that there was no significant
difference between the types and proportions of microbial
organisms isolated from HIV-positive and HIV-negative
eyes [30]. Analysis of the conjunctival flora of HIV pa-
tients who received antibiotic treatment with systemic
clarithromycin demonstrated a significant decrease in the
conjunctival flora [30].
ROLE OF THE MICROBIOME IN OCULAR
DISEASE
Many diseases involving the ocular surface, such as
dry eye syndrome, chronic follicular conjunctivitis, and
various inflammatory eye diseases, appear to have idio-
pathic etiologies. Evidence indicating the existence of a
resident ocular surface microbiota suggests that the nor-
mal microbiota plays a protective role in preventing pro-
liferation of pathogenic species, and that ophthalmic
pathologies are linked to alterations in the homeostatic mi-
crobiome.
Ocular Microbiota and Ophthalmic Diseases
Recent studies have investigated the link between al-
terations in the ocular surface microbiota and ophthalmic
disease. Sankaridurg et al. conducted a study exploring
the microbial colonization of soft contact lenses as a risk
factor associated with corneal infiltrative events (CIE) and
found that colonization of lenses with pathogenic bacteria,
especially Gram-negative bacteria such as Serratia
marcescens and Haemophilus influenzae, was signifi-
cantly associated with CIE [31]. Lee et al. conducted DNA
sequencing analysis of ocular surface samples from ble-
pharitis patients and health controls and concluded that
blepharitis may be induced by a change in the microbial
328 Lu and Liu: Microbiota and ophthalmic disease

composition – namely, greater quantities of Streptophyta,
Corynebacterium, and Enhydrobacter species [20].
As suggested by Dong et al., a number of potentially
pathogenic bacteria may reside at the ocular surface [14].
Post-operative infectious endophthalmitis is a serious, vi-
sion-threatening complication of ocular surgery that in-
volves inflammation of the ocular surface, the anterior and
posterior compartments of the eye, as well as adjacent
structures. Acute, post-operative endophthalmitis pre-
sumably occurs when the patient’s own periocular bacte-
ria enter the sterile intraocular compartments of the eye
during surgery and cause diffuse infection and inflamma-
tion. Thus, pre-operative, intra-operative, and post-opera-
tive antibiotic prophylaxis are used to prevent acute
endophthalmitis [32]. Consideration of the ocular surface
microbiota may have two-fold significance in acute en-
dophthalmitis. First, alterations in the microbiota can in-
fluence the species of microorganisms that colonize the
ocular surface and heighten the risk for intraocular infec-
tion by pathogenic bacteria. Second, knowledge of the
composition of both the core and transient microbiota of
the ocular surface can aid in the determination of the most
effective antibiotic prophylaxis to prevent post-operative
acute endophthalmitis in patients [33].
Miller et al. evaluated the concept that infectious mi-
croorganisms play a role, perhaps as a cofactor to genetic
and environmental risk factors, in ocular adnexal neo-
plasms. Several microorganisms may have a pathogenic
role in ocular malignancies, including human papilloma
virus in conjunctival papilloma and squamous cell carci-
noma, HIV in conjunctival squamous cell carcinoma, Ka-
posi sarcoma-associated herpes virus in conjunctival
Kaposi sarcoma, and Helicobacter pylori, Chlamydia, and
hepatitis C virus in ocular adnexal mucosa-associated
lymphoid tissue lymphomas [12]. Depletion or alteration
in the commensal microbiota, due to antibiotics or other
factors, may allow for colonization of the ocular surface
by opportunistic pathogens, producing a greater risk for
infection-associated ocular adnexal neoplasms [34].
Non-ocular Microbiota and Ophthalmic Diseases
Finally, we must consider the potential role of the
general, non-ocular microbiome in ophthalmic disease.
The pathophysiology of glaucoma involves local inflam-
matory responses. A study comparing the oral microbiome
of patients with glaucoma and healthy controls found that
patients with glaucoma had a higher quantity of bacterial
organisms compared to controls [35]. The researchers of
the study proposed that an increased bacterial count can
lead to neurodegeneration of the optic nerve via activa-
tion of microglia in the retina and optic nerve; the altered
commensal microbiome induces changes in cytokine sig-
naling and complement activation [35]. Additionally,
other glaucoma researchers have implicated the role of the
human microbiome in modulating levels of brain-derived
neurotrophic factor (BDNF), which has been shown to
have an effect on the survival of retinal ganglion cells in
an animal model [36].
A number of recent studies have proposed a link be-
tween the intestinal microbiome and uveitis [37]. Uveitis,
or intraocular inflammation, has various etiologies which
include infectious pathologies as well as a number of im-
mune-mediated conditions. Animal models of experi-
mental autoimmune uveitis have demonstrated that
administration of oral antibiotics that altered the intestinal
microbiota resulted in a significant attenuation of the
uveitis [38]. Presumably, an altered commensal flora led
to an increase in regulatory T lymphocytes in lymphoid
tissues as well as in the eye, leading to decreased inflam-
mation. Another animal model study of experimental au-
toimmune uveitis found that oral antibiotics or a germ-free
state significantly decreased the severity of uveitis [39].
CONCLUSION
Advances in next-generation sequencing and bioin-
formatics tools have revealed an expansive, diverse mi-
crobial community inhabiting the human cornea and
conjunctiva. The most abundant genera in the microbiota
of the ocular surface, identified using 16S rRNA se-
quencing, were Pseudomonas, Bradyrhizobium, Propi-
onibacterium, Acinetobacter, and Corynebacterium. The
ocular surface microbiota can be altered by a variety of
host factors, environmental influences, and pathological
states, including dry eye syndrome, contact lens wear, ker-
atoprosthesis, antibiotics, and infection. Evidence strongly
suggests that the homeostatic microbiome plays a protec-
tive role in preventing colonization of pathogenic species.
Thus, disruption of the normal ocular surface microbiota
may play a significant role as a cofactor in the pathogen-
esis of ophthalmic diseases, such as contact lens-associ-
ated corneal infiltrative events, blepharitis, and
post-operative infectious endophthalmitis. Futhermore, re-
cent studies suggest that the microbiome of other areas of
the body are involved in the pathophysiology of certain
ophthalmic diseases, such as the oral microbiome and
glaucoma, as well as the intestinal microbiome and
uveitis. Continued investigation of the ocular surface mi-
crobiome is necessary to enhance our understanding of the
role of homeostatic microorganisms in ophthalmic dis-
eases and inspire the development of novel, probiotic-
based therapies for the prevention and treatment of ocular
disease.
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