Reorganized Genomic Taxonomy of Francisellaceae Enables Design of Robust Environmental PCR Assays for Detection of Francisella tularensis

Abstract

In recent years, an increasing diversity of species has been recognized within the family Francisellaceae. Unfortunately, novel isolates are sometimes misnamed in initial publications or multiple sources propose different nomenclature for genetically highly similar isolates. Thus, unstructured and occasionally incorrect information can lead to confusion in the scientific community. Historically, detection of Francisella tularensis in environmental samples has been challenging due to the considerable and unknown genetic diversity within the family, which can result in false positive results. We have assembled a comprehensive collection of genome sequences representing most known Francisellaceae species/strains and restructured them according to a taxonomy that is based on phylogenetic structure. From this structured dataset, we identified a small number of genomic regions unique to F. tularensis that are putatively suitable for specific detection of this pathogen in environmental samples. We designed and validated specific PCR assays based on these genetic regions that can be used for the detection of F. tularensis in environmental samples, such as water and air filters.

Keywords: Francisella taxonomy; assay; phylogeny; tularemia.

Figure A1

Phylogenetic tree of family Francisellaceae.

Figure 1

Whole genome maximum likelihood phylogeny of the complete Francisella near neighbor (FNN) dataset calculated with RAxML. Genus Francisella is divided into four separate clades indicated with different colors and numbered 1–4. The four most proximate genera to Francisella are also presented (the complete phylogenetic tree of Francisellaceae is presented in Figure A1).

Figure 2

Clustered heatmap visualizing ANI values between Francisella, Allofrancisella, Parafrancisella, and Pseudofrancisella species.

Figure 3

Phylogenetic tree of F. tularensis visualizing subspecies and major genetic subgroups.

Figure 4

Visualization of genomic regions that are differentially present/absent across the three subspecies of F. tularensis and/or major phylogenetic groups within the subspecies. A phylogeny is presented in the middle of the figure, and the multiple circles around the phylogeny correspond to different genomic regions. The color of each cell in the circles corresponds to the BSR value for each genomic component in each examined whole genome sequence.