# Development of standardized resources for characterization of the resident ocular surface microbiome.

> **NIH NIH U24** · JOHNS HOPKINS UNIVERSITY · 2024 · $145,027

## Abstract

PROJECT SUMMARY
The exposed epithelial surfaces of the body, including the skin, gut, female reproductive tract, and airways, are
colonized by microorganisms that contribute to homeostasis and disease. While the resident microbiota has
been extensively characterized in many of these sites, the ocular surface microbiome is relatively
understudied. The use of metagenomic sequencing approaches has facilitated moving beyond targeted
culturing approaches to more fully characterize the breadth of organisms present in a specimen. However, the
relative microbial biomass at the ocular surface is much lower than other mucosal surfaces, such as the gut.
Metagenomic characterization of low microbial biomass specimens presents numerous challenges, as sources
of contamination not only arise during the sampling procedure and from the environment itself, but even from
laboratory processing methods. As a result, the lack of protocol standardization and omission of key controls
for sources of contamination limits the interpretation and potential for comparison across studies. To address
these challenges, we have assembled a large multidisciplinary team of experts in topics including (i)
development of standardized protocols and clinical validation of diagnostic tests utilizing metagenomic
sequencing for low biomass biospecimens, (ii) development of open source metagenome analysis tools, (iii)
clinical assessment of ocular surface and external eye findings among a large, diverse patient population, and
(iv) wet lab characterization of microbes under strict cleanliness guidelines. We previously described the use of
metagenomic sequencing to detect the presence of pathogens in biopsies from the brain, paraffin embedded
ocular tissue specimens, and cerebral spinal fluid (CSF). By comparing a range of specimen pre-treatment and
processing approaches and sophisticated software tools, we were able to optimize the methods to maximize
organism detection and minimize or remove contamination. We validated our metagenomic sequencing and
analytical approaches to the rigor required for use as a diagnostic test. Here, we hypothesize that following
similar approaches with low microbial biomass ocular specimens will facilitate the characterization of the
healthy ocular surface microbiome. In Aim 1, we will compare specimen processing approaches and validate
our analytical methods. In Aim 2, we will compare specimen collection approaches, including collection
materials and procedures. We will then use our optimized specimen collection and processing approaches to
collect ocular specimens longitudinally for characterization of organism persistence. In Aim 3, we will identify a
subset of participants with persistent and/or unique organisms to collect additional specimens for validation of
composition and characterization of viability. Upon study completion, we will have contributed to the
characterization and understanding of the healthy ocular surface microbiome, and developed proto...

## Key facts

- **NIH application ID:** 11002858
- **Project number:** 3U24EY035078-01S1
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Laura Ensign
- **Activity code:** U24 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $145,027
- **Award type:** 3
- **Project period:** 2023-07-01 → 2026-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11002858

## Citation

> US National Institutes of Health, RePORTER application 11002858, Development of standardized resources for characterization of the resident ocular surface microbiome. (3U24EY035078-01S1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/11002858. Licensed CC0.

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