Whole-Genome Relationships among Francisella Bacteria of Diverse Origins Define New Species and Provide Specific Regions for Detection

Francisella tularensis is a highly virulent zoonotic pathogen that causes tularemia and, because of weaponization efforts in past world wars, is considered a tier 1 biothreat agent. Detection and surveillance of F. tularensis may be confounded by the presence of uncharacterized, closely related organisms. Through DNA-based diagnostics and environmental surveys, novel clinical and environmental Francisella isolates have been obtained in recent years. Here we present 7 new Francisella genomes and a comparison of their characteristics to each other and to 24 publicly available genomes as well as a comparative analysis of 16S rRNA and sdhA genes from over 90 Francisella strains. Delineation of new species in bacteria is challenging, especially when isolates having very close genomic characteristics exhibit different physiological features-for example, when some are virulent pathogens in humans and animals while others are nonpathogenic or are opportunistic pathogens. Species resolution within Francisella varies with analyses of single genes, multiple gene or protein sets, or whole-genome comparisons of nucleic acid and amino acid sequences. Analyses focusing on single genes (16S rRNA, sdhA), multiple gene sets (virulence genes, lipopolysaccharide [LPS] biosynthesis genes, pathogenicity island), and whole-genome comparisons (nucleotide and protein) gave congruent results, but with different levels of discrimination confidence. We designate four new species within the genus; Francisella opportunistica sp. nov. (MA06-7296), Francisella salina sp. nov. (TX07-7308), Francisella uliginis sp. nov. (TX07-7310), and Francisella frigiditurris sp. nov. (CA97-1460). This study provides a robust comparative framework to discern species and virulence features of newly detected Francisella bacteria. IMPORTANCE: DNA-based detection and sequencing methods have identified thousands of new bacteria in the human body and the environment. In most cases, there are no cultured isolates that correspond to these sequences. While DNA-based approaches are highly sensitive, accurately assigning species is difficult without known near relatives for comparison. This ambiguity poses challenges for clinical cases, disease epidemics, and environmental surveillance, for which response times must be short. Many new Francisella isolates have been identified globally. However, their species designations and potential for causing human disease remain ambiguous. Through detailed genome comparisons, we identified features that differentiate F. tularensis from clinical and environmental Francisella isolates and provide a knowledge base for future comparison of Francisella organisms identified in clinical samples or environmental surveys.