Resolving taxonomic boundaries and revealing genetic diversity in Enterococcus casseliflavus and closely related taxa

Abstract

Enterococcus casseliflavus is a commensal bacterium that can occasionally cause human infections. A concern is that all strains of this species carry the vanC operon on their chromosomes, which reduces susceptibility to vancomycin. Aside from that, the classification of E. casseliflavus remains challenging because it shares up to 99% 16S rRNA sequence identity with other enterococci species. Here, we reassess the taxonomy and genomic diversity of E. casseliflavus and closely related taxa by comprehensively analyzing publicly available genomes to clarify their evolutionary relationships, ecological distributions, and clinical relevance. We retrieved 156 genomes of clinical and non-clinical isolates that showed ANI >90% with E. casseliflavus from public databases. Using ANI, core genome-based phylogeny, and pangenome reconstruction, we consistently identified three well-defined clusters corresponding to E. casseliflavus, E. entomosocium, and E. innesii. Our results indicate that a higher ANI threshold (>96.6%) is required to accurately discriminate between species within this group, as many strains showed more than 95% ANI with multiple species. Our results also support that E. flavescens and E. casseliflavus represent a single species and should be treated as synonyms. Genes related to biocide resistance, metal tolerance, and virulence were identified across the three species. E. casseliflavus exhibited the greatest diversity of resistance determinants. All species harbored the vanC operon associated with intrinsic vancomycin resistance, while two genomes additionally carried the vanD operon linked to high-level glycopeptide resistance. Overall, our findings refine species boundaries and highlight the importance of genome-based approaches for accurate classification and surveillance within the Enterococcus genus.