While most people have heard of the gut microbiome and the many ways it affects our health, they may be unfamiliar with a much smaller and, as of now, more mysterious one: the ocular surface microbiome. Though doctors and researchers know little about it, they suspect the ocular surface microbiome could play a role in several conditions that currently have no cure, from dry eye disease to ocular rosacea to corneal ulcerations.

Consisting of all the microbes naturally living on and inside the body, including bacteria, fungi, viruses and their genes, a microbiome is more than the sum of its parts because of the delicate interactions among these microorganisms. Unravelling those relationships to discover when a microbiome is in balance could help develop treatments that restore a former state of health, in contrast to current treatments such as antibiotics that kill microorganisms in an untargeted way and often lead to further imbalance.

The potential to discover information locked away in the ocular surface microbiome spurred several researchers at the Wilmer Eye Institute, Johns Hopkins Medicine, to apply for a rare consortium grant (which they received) from the National Eye Institute to characterize the resident microbiome of a healthy ocular surface. The highly multidisciplinary team brings together expertise in clinical practice, diagnostic methods, genomic analysis, pathology, biostatistics, proteomics and more.

The three principal investigators — Sezen Karakus, M.D., assistant professor of ophthalmology; Steven Salzberg, Ph.D., Bloomberg Distinguished Professor of Biomedical Engineering, Computer Science and Biostatistics; and Laura Ensign, Ph.D., Marcella E. Woll Professor of Ophthalmology— will work with numerous collaborators on this project.

The team includes Nakul Shekhawat, M.D., M.P.H., Stephen F Raab and Mariellen Brickley-Raab Rising Professor of Ophthalmology; Patricia Simner, Ph.D., M.Sc.; Charles Eberhart, M.D., Ph.D., Charlotte A. Wilson and Margaret K. Whitener Professor of Ophthalmology; Xiangrong Kong, Ph.D.; and Jotham Suez, Ph.D., M.Sc.

“Our general understanding of microbiomes on all surfaces is that when you’re healthy, it’s not that they’re doing nothing. They’re either doing positive things or at the bare minimum, they’re keeping the bad things out,” says Ensign. Because she focuses on developing drugs and other therapeutics, Ensign wonders, “Are there things that we can do to prevent pathogens from being able to take hold that aren’t the use of antibiotics? Could we interrupt the disease process by manipulating the microbiome?”

To pursue that line of research, however, “First we have to understand: What does healthy look like?” Ensign says. “The whole purpose of the consortium is to characterize the resident microbiome of the healthy ocular surface.”

Characterizing the ocular surface microbiome means identifying “which microorganisms are living there, in what number, and their theater of activity,” says Karakus.

“The ocular surface microbiome is the toughest to study because of its low biomass,” Karakus says, “which makes it even more crucial what methods we use, because a little error would make the whole analysis look different.”

Low biomass means a small number of microorganisms live in the microbiome, both because of the much smaller surface area of the eye and the unique environmental characteristics, such as tears, that the ocular surface encounters.

The process to decode a microbiome has three steps: gathering a sample, performing whole-genome sequencing on the sample — which creates a DNA “fingerprint” of each microorganism — and then comparing those DNA sequences to a database of known sequences to identify which microorganisms are present. At each step, contamination can creep in and cause results to be inaccurate. The consortium’s goals are both to standardize and to optimize the methodology.

First, a standardized method to collect samples from the ocular surface needs to be developed, explains Karakus. The researchers will need to decide which location to swab on the eye, which swab to use, how much pressure to apply and how to prepare the patient for collecting the sample.

Performing the genome sequencing involves numerous steps, each of which must be optimized to minimize opportunities for contamination and maximize the yield of the sample. The researchers will test different liquids to preserve samples, different extraction and sequencing platforms and different ways to conduct the sequencing.

Once accurate sequences have been gleaned from a good sample, the next step is to “read” the DNA to determine which organisms are there. To do this, the researchers will run the sequences through open source software, which will compare each sequence against a custom-built database containing the genomes of tens of thousands of bacteria, viruses and other microbes collected from public genome databases. The result will be a list of types of microorganisms present and their respective numbers.

Johns Hopkins is home to a world-renowned expert in this field, says Ensign.

“One of the reasons why I think we were funded by NEI (National Eye Institute) for this consortium is that we have Steven Salzberg, who helped develop the open source software we will use,” Ensign says. Salzberg’s lab also continuously annotates the genomes of microorganisms regularly uploaded by researchers around the world — increasing the software’s ability to identify a growing number of microorganisms.

“The goal is that by the end of a three-year period, we will have agreed upon ways of standardizing not only what types of specimens to collect, but what collection methods to use, what transfer fluid to use, how to store it, how to process it, how to do the sequence analysis,” says Ensign — all to achieve an accurate sample that the software can read to produce a census of what microorganisms are present in a healthy ocular surface microbiome.

“Once we know that our methods are sound and accurate, and standardized across researchers, then we’ll use this information to understand the pathogenesis — how different diseases arise and progress on the ocular surface,” says Karakus. “We need these specific answers to develop targeted treatments.”