Date Published: April 3, 2019
Publisher: Public Library of Science
Author(s): Erin M. Scott, Carolyn Arnold, Samantha Dowell, Jan S. Suchodolski, Christopher Staley.
Next generation sequencing (NGS) studies have demonstrated a rich and diverse ocular surface-associated microbiota in people that was previously undetected by traditional culture-based methods. The ocular surface microbiome of horses has yet to be investigated using NGS techniques. This study aimed to determine the bacterial composition of the ocular surface microbiome in healthy horses, and to identify whether there are microbial community changes over time and following topical antibiotic use. One eye of 12 horses was treated 3 times daily for 1 week with neomycin-polymyxin-bacitracin ophthalmic ointment. Contralateral eyes served as untreated controls. The inferior conjunctival fornix of both eyes was sampled at baseline prior to initiating treatment (day 0), after 1 week of treatment (day 7), and 4 weeks after concluding treatment (day 35). Genomic DNA was extracted from ocular surface swabs and sequenced using primers that target the V4 region of bacterial 16S rRNA. At baseline, the most abundant phyla identified were Proteobacteria (46.1%), Firmicutes (24.6%), Actinobacteria (12.6%), and Bacteroidetes (11.2%). The most abundant families included Pasteurellaceae (13.7%), Sphingomonadaceae (7.9%), an unclassified Order of Cardiobacteriales (7.7%), and Moraxellaceae (4.8%). Alpha and beta diversity measurements were unchanged in both treatment and control eyes over time. Overall, the major bacterial taxa on the equine ocular surface remained stable over time and following topical antibiotic therapy.
The ocular surface microbiota refers to the resident microorganisms that colonize the cornea, conjunctiva, and tear film. The equine ocular surface is prone to developing serious, vision-threatening ocular diseases such as infectious ulcerative keratitis, and is often treated with topical broad-spectrum antibiotics such as neomycin-polymyxin-bacitracin [1–4]. Evidence suggests that ocular surface microbiota play a protective role in preventing the proliferation of pathogenic species and thus, changes in the microbiome may be linked to ocular diseases [5,6]. Additionally, external factors, such as short or long-term use of topical antibiotics may influence the composition and stability of microbial communities .
All horses had a complete ophthalmic examination performed by a board-certified veterinary ophthalmologist (EMS). This included evaluation of the anterior segment of the eye by slit-lamp biomicroscopy (SL-17, Kowa Optimed Inc., Torrance, CA), and the posterior segment of the eye by indirect ophthalmoscopy (Vantage Plus Wireless Headset, Keeler Instruments Inc., Malvern, PA). A routine minimal ophthalmic database was also performed. This included Schirmer tear test measurements (Intervet Inc., Summit, NJ), fluorescein staining (Amcon Laboratories Inc., St. Louis, MO), and tonometry (Tono-Pen, Dan Scott and Associates, Inc., Westerville, OH). Any horse with an abnormal ophthalmic exam or minimal database result was excluded from the study.
The present study demonstrates the equine ocular surface contains a more diverse bacterial community than previously detected using standard culture techniques. A total of 5 bacterial phyla and 10 families were present in all 24 eyes at >1% relative abundance (Table 3). The most common phyla and their relative proportions colonizing the equine ocular surface, Proteobacteria (46.1%), Firmicutes (24.6%), Actinobacteria (12.6%), and Bacteroidetes (11.2%), are comparable to investigations of the human ocular surface [14,15,17]. Preliminary studies describing the ocular surface microbiome of companion animals utilizing NGS identified the same four phyla at different proportions, with Firmicutes most abundant across all canine and feline samples (34.9% and 43%, respectively) [18,19]. Direct comparison of microbiome studies; however, should be interpreted with caution as there exist variations in methodologies for DNA extraction, sequencing, analysis, and clustering strategies.
This is the first report to investigate the bacterial community of the healthy equine ocular surface using molecular-based techniques. This is also the first report to examine the temporal stability of the equine ocular surface microbiome both over time and following topical antibiotic therapy. Richer and more diverse microbial communities inhabit the ocular surface of the equine eye than previously detected with conventional culture techniques. A stable core bacterial microbiome was identified and discovered to remain consistent over time and with short-term topical broad-spectrum antibiotic use. Investigations comparing the equine ocular surface microbiome of healthy and diseased eyes are currently underway to determine if alterations to ocular microbial communities are associated with disease.