DOI: 10.1128/mbio.01234-26 ISSN: 2150-7511
Novelty, diversity, and genetic dark matter in enterococci of invertebrates
Bruna F. Sgardioli, Matthew C. Phillips, Arjun Miklos, Kailey Fleiszig-Evans, Abigail L. Manson, Suelen Scarpa de Mello, Terrance Shea, Aysun Urhan, Ryan Whipple, Rauf Salamzade, Jasper Sanders, Paulo A. V. Borges, Maria de Lurdes Nunes Enes Dapkevicius, Ashlee M. Earl, Michael S. Gilmore ABSTRACT
Enterococci appear to have originated in the guts of early terrestrializing arthropods and invertebrates over 425 million years ago—hosts that are now highly diverse and widespread in nature today. Yet most knowledge of the genus comes from human infection-associated lineages with genomes swollen by the recent accretion of foreign DNA conveyed by mobile elements. Because invertebrates dominate terrestrial animal diversity and biomass, they would be predicted to constitute a major but little-explored reservoir of enterococcal diversity. We therefore systematically examined
Enterococcus
association and species diversification in invertebrate hosts of the comparatively natural, isolated, but well-characterized environment of the Azorean island of Terceira. Over 100 invertebrate specimens were examined for associated enterococci, which were taxonomically classified by whole-genome sequencing. Supporting the existence of a large pool of uncharacterized enterococci and
Enterococcus
-adapted genes, 40% (eight of 20) of the
Enterococcus
species identified were either undescribed, including four candidate new species described here, or very recently discovered. In contrast, control isolates from vertebrates were exclusively of known species typical of sampling elsewhere, discounting geographic isolation as a main driver of the novelty observed. Further, because of the abundance of
E. casseliflavus
and
E. flavescens
in this collection, we obtained the resolution necessary to quantify the divergence and decipher the drivers of speciation in the controversial division between these naturally vancomycin-resistant species. These findings provide robust support for the existence of a large pool of new species and unexplored adaptive traits in invertebrate-associated enterococci—diverse environmental survival traits optimized for expression in an enterococcal background, and well positioned for transmission into human-associated enterococcal strains.
IMPORTANCE
Enterococci are auxotrophic gut-associated bacteria that co-evolved with their terrestrial hosts over many eons. In the last 75 years—the “antibiotic era”
—E. faecalis
and
E. faecium
gained genes for antibiotic resistance and enhanced virulence, emerging as leading causes of multidrug-resistant infection. Little is known about the source of those genes or the pathway by which they entered human-associated strains. A recent global survey suggested a potentially large repository of uncharacterized genetic diversity in the enterococci of invertebrates. We directly tested this prospect by examining enterococci of invertebrate hosts in a largely natural and pastoral environment. Our findings provide clear evidence that invertebrates naturally harbor vast unexplored enterococcal diversity. Moreover, associations are likely driven by intrinsic host selection factors rather than geographic isolation. This expands our knowledge of
Enterococcus
biodiversity, including the identification of four novel species, identifying a vast reservoir of enterococcal genes available to species that colonize and infect humans.