Ocular Microbiome…. Dead on Arrival?
Darlene Miller, Kara Cavuoto, Anat Galor
Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Department of Ophthalmology, Miami, Florida
Background: DNA signatures from viable, injured and dead microorganisms are ubiquitous in all natural and anthropological environments. They are pervasive in foods, water, molecular reagents, kits and our human genomic databases. Recent reports have documented the presence of this undetected microbial dark matter and or contaminating nucleic acid (DNA) signatures in low biomass microbiomes (lungs, placenta, blood) rendering the results inaccurate and invalid.
Purpose: Our purpose was to determine the presence and diversity of contaminating DNA signatures (microbial dark matter) in published ocular microbiomes.
Methods: We examined the three most referenced ocular microbiomes for the presence and diversity of reported bacterial members (genera) of the “metagenomic contaminome” associated with moderate and low biomass niches. We also confirmed the presence of this group in parallel cultures (n=3) and among unpublished ocular surface culture surveys from our laboratory (2011-2021).
Results: We documented 183 different genera that have been reported as possible contaminating DNA signatures in reagents, kits, molecular grade water, blank negative controls, laboratory environments, sequencing plate cross contamination and as “contaminants or microbial dark matter” in whole genome sequence databases. Signatures were from 9 phyla, dominated by Pseudomonadota (Proteobacteria) , 41.5%, n=76, Bacillota (Firmicutes) , 30.1%, n=55, and Actinomycetota (Actinobacteria) , 15.9%, n=29. The remaining 5 phyla, included Bacteroidota (Bacteroidetes) , 8.7%, n=16, Acidobacteriota, 1.09%, n=1, Deinococcota, 1.09%, n=2 and Fusobacteriota, 1.09%, n=2 and unclassified, 1.09%, n=2. Seventy-six or 41.5% of the “contaminants” were recovered from 2 or more sources. Contaminating bacterial DNA signatures were found in all 3 published microbiomes and constituted greater than 60% (65.5%, 70.7% and 72.9%) of each of the reported ocular surface microbiomes. Less than 10% of these isolates were recovered in the 3 parallel culture dependent microbiomes. Greater than 90% (92%, n=12/13, 100%, n=9/9, 100%, n=4/4) of the 3 parallel culture dependent microbiomes were classified as contaminants. Sixty-nine percent of conjunctiva (42 genera), lids (25 genera, 68%), lacrimal sac (33 genera, 69.7%) and cornea (47 genera, 59.6%) at our Institute documented the growth of “contaminants”.
Conclusions: The presence and diversity of live and dead contaminating DNA signatures (microbial dark matter) may overlap and obscure the true resident ocular microbiota leading to inaccurate, incomplete and distorted profiles that compromise the validity of current and future ocular metagenomic studies.