In Situ Hybridization Visualizes Bacterial Clusters on Cells of the Human Conjunctival Epithelium

Abstract

Purpose : The ocular surface is continually cleansed by a tear film containing potent anti-microbial proteins. However, evidence from culture and microbiome studies suggests that the conjunctiva normally harbors a small but distinctive bacterial population. A key question is where these bacteria are located and how this might relate to their role in ocular health and disease.

Methods : Microbes and epithelial cells of inferior conjunctiva were collected from 17 healthy adult subjects using eye drop-wetted nylon microfiber swabs. Cells were released into PBS, fixed and transferred by filtration to 0.2 micron pore polycarbonate membranes. Bacteria were visualized by in situ hybridization with fluorescent Alexa 594 16S rRNA-specific complementary DNA oligonucleotides. Random sequence Alexa 488 fluorescent oligos provided controls. Epithelial cell and bacterial numbers were measured in purified DNA by quantitative PCR with pan-bacterial 16S primers and Alu repeat-specific primers. Illumina MiSeq 16S high-throughput metagenomic analysis of bacterial species in sample and control DNA preps used pre-amplification of 16S rRNA genes to improve performance and decrease reagent-related microbial background.

Results : Illumina 16S gene sequencing identified Staphylococcus epidermidis as the most abundant species, at 49%, followed by the genera Corynebacterium, Propionibacter, and Streptococcus. In situ hybridization of swab samples indicated that few of the bacteria were free-floating and that nearly all were tightly associated with about 3% of the conjunctival epithelial cells. The 16S rRNA-containing bacteria were not associated with lectin-stained goblet cells or lymphocytes. Similar morphology in these clusters of 10 or more bacteria suggest that they may represent living micro-colonies of a single cell type. We are testing this hypothesis with genus-specific in situ hybridization probes.

Conclusions : Bacterial cells recovered from the human ocular surface are tightly bound in clusters to a minor fraction of nucleated conjunctival epithelial cells. Few are associated with cells involved in clearing away microbes, such as lymphocytes or goblet cells. These bacteria may occupy a protected niche located below the surface layer of the conjunctiva, where they could serve as a stem population that maintains the ocular microbiome.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

In situ hybridization detects bacteria (red) on a conjunctival cell.