Reviewer response for version 1

Abstract

Enterococcus raffinosus is an understudied member of its genus possessing a characteristic megaplasmid contributing to a significantly large genome size. Although less commonly associated with human infection compared to other enterococci, this species can cause disease and persist in diverse niches such as gut, urinary tract, blood, and environment. Few complete genome assemblies have been published to date for E. raffinosus. In this study, we report the complete assembly of the first clinical urinary E. raffinosus strain, Er676, isolated from a postmenopausal woman with history of recurrent urinary tract infection (rUTI). We additionally completed the assembly of clinical type strain ATCC49464. Comparative genomic analyses reveal inter-species diversity driven by large accessory genomes. The presence of a conserved megaplasmid indicates it is a ubiquitous and vital genetic feature of E. raffinosus. We find that the E. raffinosus chromosome is enriched for DNA replication and protein biosynthesis genes while the megaplasmid is enriched for transcription and carbohydrate metabolism genes. Prophage analysis suggests that diversity in the chromosome and megaplasmid sequences arises, in part, from horizontal gene transfer. Er676 demonstrated the largest genome size reported to date of E. raffinosus and the highest probability of human pathogenicity. Er676 also possesses multiple antimicrobial resistance genes (ARGs), all of which are encoded on the chromosome, and has the most complete prophage sequences. Complete assembly and comparative analyses of the Er676 and ATCC49464 genomes provide important insight into the inter-species diversity of E. raffinosus that enables its ability to colonize and persist in the human body. Investigating genetic factors that contribute to this species pathogenicity will lend valuable tools to combat diseases caused by this opportunistic pathogen.