Polyphasic discrimination of from closely related species and a whole-genome multilocus (wgMLST) scheme for the evaluation of diversity within this clade.

is an environmentally ubiquitous genus with significant roles in bioelectrochemical applications and human infections. However, identification problems involving , , and have been reported, potentially hindering research progress in these areas. In this study, we explored how to discriminate between these species. By comparing the genomes of spp. available in public databases with that of the newly sequenced strain DSM9451, we showed that this strain is a member of the species . Of the eight public genomes associated with this species, only two were correctly identified in public databases. Phenotypic analysis revealed distinct features of with respect to and . However, only differences in the intensity of biochemical reactions were observed between and . To discriminate between these closely related species and explore their diversity, a whole-genome multilocus sequence typing scheme was developed. The scheme distinguished related species and revealed significant diversity among and isolates, suggesting that both species may harbor isolates with significantly different metabolic properties. These differences are so wide within the clade that they may explain why these two species are difficult to distinguish. The identification of exclusive genes of each species allowed the design of a simple molecular method to differentiate from closely related species, which will help in clarifying its role in human infections and environmental processes.IMPORTANCEMisidentification within the genus, particularly between , , and , has been reported and misperceives scientific research on spp. in diverse fields, including both biotechnological applications and human infections. Near-complete 16S rRNA gene sequencing fails to correctly classify many species, and MALDI-TOF systems used in clinical microbiology laboratories are suboptimal for species-level identification. In this study, we identified phenotypic characteristics that can guide differentiation and classification, and building upon the identification of species-specific genes, we suggest an accurate and cost-effective molecular test as an alternative to genome sequencing. The proposed whole-genome multilocus sequence typing scheme allows the exploration of species and strain diversity, highlighting the limitations of generalizing results from studies of a single strain. As an emergent pathogen and biotechnological candidate, the proper identification by a single molecular test will enhance the insights about these species towards biotechnology development and public health safety.