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.

Presence of Francisella tularensis subsp. holarctica DNA in the Aquatic Environment in France

“…PCR-based methods seem more suitable for detecting Francisella in environmental samples. However, a wide diversity of Francisella species is present in the environment, including many water species (e.g., F. ulginis , F. salminarina , F. salina ), fish- and mollusc-associated species (e.g., F. noatunensis , F. orientalis , F. halioticida , F. marina ), and endocytobionts of marine ciliates (e.g., F. endociliophora and F. adeliensis ) [ 8 ]. Consequently, the choice of PCR targets is critical for the nature of Francisella species identified.…”

“…holarctica , and 6–18 copies for F. novicida ) and thus improves the sensibility of the qPCR. This DNA target is present in other Francisella species (i.e., there are 1–2 copies of IS Ftu2 in the F. philomiragia genome) [ 8 , 22 ]. Consequently, qPCR results from the PCR targeting the IS Ftu2 sequence should be interpreted cautiously because it could detect a wide diversity of Francisella species.…”

“…This was observed with our ISFtu2-qPCR controls, which demonstrated both detection of F. philomiragia and F. noatunensis . Öhrman et al, by in silico approaches, demonstrated that ISFtu2-qPCR can detect F. novicida and F. philomiragia but does not detect other Francisella species such as F. opportunistica and Francisella endocytobionts [ 8 ]. Consequently, the ISFtu2-qPCR is a Francisella sp.…”

“…novicida, is an aquatic bacterium with low virulence in humans [ 5 ] and can also be considered a different species, F. novicida [ 6 , 7 ]. Based on the most recent phylogenetic studies, we choose in the article to considered F. novicida as a species [ 8 ]. Other Francisella species are occasionally associated with human infections, like the aquatic bacterium F. philomiragia [ 5 ].…”

“…Other Francisella species are occasionally associated with human infections, like the aquatic bacterium F. philomiragia [ 5 ]. In the past twenty years, multiple new environmental Francisella species were described, all of them isolated from water or from aquatic fauna [ 8 ].…”