Exploratory Study on the Ocular Surface Microbiome and Correlation to Clinical Parameters

The ocular surface is the first line of defense of the eye, it is therefore where external threats are sensed, and potential insults neutralised. Over the course of evolution, various microbes, especially bacteriae, have come to colonise the ocular surface as commensals. The commensals have a role to maintain the homeostasis of the ocular surface. 1 The innate immunity of the ocular surface is very active, and consists of active mechanisms to suppress inflammation 2. For example, there exist macrophages, dendritic cells, suppressor cells, regulatory cells, B cells, IgA, lysozyme, anti-microbial peptides and barriers against external agents. The normal commensals of the ocular surface maintain a basal level of activation of innate defence by stimulating the pattern recognition receptors on ocular surface epithelial cells. This normal composition of microbes is important since inflammation and infection will result if there is introduction of a pathogenic strain that overcomes the flora, or if a dominant strain secretes excessively immunogenic products, such as the exotoxin A of Staphylococcus which triggers marginal keratitis, a form of type IV hypersensitivity. The flora load of microbiome could also influence tear function as a higher flora load was found to be associated with increased mucin degradation 3 and reduced globet cell densitiy 4. Previous studies [I'm not sure which studies these are] at SERI/SNEC also point to the importance of microbes. For example, in dry eye patients, there is increased lysophospholipids in the tear, and this may contribute to inflammatory mediators such as arachidonic acid and other metabolites. The lysophospholipids are formed by phospholipase A2 reactions, and the latter may be microbial in origin. Since dry eye is a known inflammatory disease of the ocular surface, this is one way that microbes can contribute to the pathology.

The microbiome is the sum total of the microbe found in a body location. In the eye the microbiome can be investigated non-invasively because of the accessible location of the ocular surface. Despite the importance of the ocular surface microbiome, understanding of this topic is very superficial and largely based on culture methods5-10. This can be misleading because some of the microbes are not so amenable to routine cell cultures and their presence may be undetected or their representation under-estimated 11. Recently developed metagenomics sequencing, compared to cultures, are advantageous to get an un-biased view of microbiome11.

Three prior studies have evaluated the microbiome profiles of the ocular surface with metagenomics sequencing of 16s rRNA 4, 12 13. Differences in microbiome were observed between subjects with dry eyes or blepharitis, and healthy subjects. Moreover, a much greater diversity of ocular flora was discovered compared to results based on bacterial culture.

The school of chemical and environmental life science at Nanyang Technological University has evaluated the microbiomes using such techniques. The basis of this is that prokaryotic organisms have unique DNA signatures which can typically characterise the species of origin, and the relative prevalence of the organisms of different species can be deduced from the relative abundance of the different DNA detected. The global human microbiome project seeks to understand the microbiome in different parts of the human body. This is best understood in the intestine, but publications on the ocular surface have yet to emerge.

Recently our collaborators have been found that 3 bacteria dominated the ocular surface of the eye. These are Klebsiella pneumoniae, Pseudoalteromonas agarivorans and Lactobacillus rhamnosus. This dominance of 3 types of bacteria was unexpected, since the dominant view of ocular surface commensals is that coagulase negative Staphylococcus dominates, followed by Staphylococcus aureus and Streptococcus, with less gram negative bacteria, and some Propionibacter spp 11. Among the 3 species, Klebsiella pneumonia is a known gram negative pathogen that can cause infectious keratitis, and in some cases endophthalmitis. The roles of Pseudoalteromonas and Lactobacillus are entirely unknown. The former is a non-pathogenic free living gram negative bacteria known to be present in marine settings. Surprisingly the high levels of the 3 predominant bacteria are relatively constant among the 3 human participants, suggesting a certain amount of uniformity in the colonisation of the ocular surface by major bacteria microbes in non-diseased individuals.