Phylogeography of Francisella tularensis subsp. holarctica, Europe

Francisella tularensis subsp. holarctica isolates from Austria, Germany, Hungary, Italy, and Romania were placed into an existing phylogeographic framework. Isolates from Italy were assigned to phylogenetic group B.FTNF002–00; the other isolates, to group B.13. Most F. tularensis subsp. holarctica isolates from Europe belong to these 2 geographically segregated groups.

F rancisella tularensis is the etiologic agent of tularemia and a highly virulent category A biothreat agent (1,2). The most widely distributed subspecies is F. tularensis subsp. holarctica, which is found throughout much of the Northern Hemisphere and is the only subspecies found in Europe (3). Despite its wide geographic distribution, F. tularensis subsp. holarctica contains low genetic diversity, which indicates recent emergence (4). A recent global phylogeographic analysis (5), and several subsequent analyses (6)(7)(8)(9), assigned most isolates from Europe to 2 phylogenetic groups: B.FTNF002-00 and B.13 (includes multiple subclades descended from branch B.13 [5,6,8]; branch and subclade nomenclature from [5] has been shortened by removing Br and extra 0s from individual branch and subclade names). These groups appear to be geographically segregated: only isolates from B.FTNF002-00 have been reported from the western European countries of Spain, France, and Switzerland, whereas B.13 is the only or dominant type reported from the Czech Republic, Finland, Georgia, Russia, Slovakia, and Ukraine (5)(6)(7)(8)(9). We provide additional information about the geographic distribution of these 2 groups using existing phylogenetic signatures (5,8)  1305-TA1.htm) into the existing global phylogeographic framework.

The Study
All of the isolates were assigned to group B.FTNF002-00 or to group B.13. All 3 isolates from Italy were assigned to group B.FTNF002-00 ( Figure 1, panel A). Although the sample size was small, these isolates were obtained in 3 different years (online Appendix Table), which suggests that this group is ecologically established in Italy. These results increase the known geographic distribution of this group, which appears to be the dominant clone in western Europe ( Figure 2, panel A, purple shading). All 42 isolates from Austria, Germany, Hungary, and Romania were assigned to group B.13 ( Figure 1 We identifi ed new genomic signatures to provide increased genetic resolution within subclade B.20/21. Next-generation sequencing technology (Illumina Inc., San Diego, CA, USA) was used to sequence the genome of an isolate from Hungary (Tul07/2007, GenBank accession no. SRX025133) assigned to subclade B.20/21. Putative single nucleotide polymorphisms (SNPs) were identifi ed in the resulting sequence and the genomes of 4 other strains previously assigned to group B.13 (LVS, AM233362.1; FSC 200, AASP00000000; RC503, SRX000104; Georgia F0673, SRX025885) by using an existing bioinformatics pipeline (5). The more distantly related strain OSU18 (CP000437.1) genome was also included as an outgroup. A maximum-parsimony tree was constructed by using the resulting ≈700 putative SNPs and PAUP 4. and B.FTNF002-00, respectively, in this and previous studies (5)(6)(7)(8)(9). The country of Georgia, which also contains isolates from group B.13 but is not depicted in the map, is indicated by red text and a red arrow pointing toward its location. Isolates assigned to other phylogenetic groups within F. tularensis subsp. holarctica have been reported from some of these countries (5,8), but most isolates from these countries are from groups B.13 and B.FTNF002-00. B) Single nucleotide polymorphism-based phylogeny of previously (5,6,8)  including 20 putative SNPs specifi c to the branch leading to the strain from Hungary (Figure 1, panel B). Consistent with previous analyses (Figure 1, panel A), the strain from Hungary clustered as a sister taxon to strain FSC 200 ( Figure 1, panel B).
To show additional phylogenetic structure within subclade B.20/21, we designed genotyping assays targeting the 20 putative SNPs along the branch leading to the strain from Hungary (Figure 1, panel B) and screened them across 64 isolates assigned to subclade B.20/21. This analysis included the 41 isolates from Austria, Germany, Hungary, and Romania, as well as 23 additional isolates from central Europe, the Czech Republic, Finland, Russia, and Sweden that were previously assigned to this subclade (6,8) (online Appendix Table). The assays were constructed and performed as described (5) Table). Information about assays targeting canonical SNPs for the branches leading to the 6 new subclades are presented in the Table.
This study and previous studies have increased understanding of F. tularensis subsp. holarctica in Europe by placing isolates from multiple countries into the existing global phylogeographic framework. As a result, the genetic background is becoming defi ned for each country (i.e., the specifi c subtypes reported from each country). This information can be useful for identifying intentional (e.g., bioterrorism) or unintentional movement of F. tularensis subsp. holarctica between countries. For example, the isolate from Romania examined in this study was actually isolated in Italy from an infected hare that was shipped from Romania for hunting. Genotyping results are consistent with a Romanian origin for this isolate because it was assigned to the B.13 group that is widespread in central and eastern Europe (Figure 2, panel A) and not to the B.FTNF002-00 group, to which the isolates from Italy were assigned (Figure 1, panel A).
Understanding global phylogeographic patterns is possible only if isolates from multiple geographic locations are placed within the same framework (i.e., examined with the same genomic signatures). Because F. tularensis is genetically monomorphic and highly clonal, SNPs are preferred signatures for determining phylogenetic structure within this species (3). Vogler et al. (5) conducted the fi rst SNP-based global phylogeographic analysis of F. tularensis. Subsequent studies (6)(7)(8)   signatures described by Vogler et al. (5) and new SNPs discovered from new whole-genome sequences or multiple sequence typing data to further refi ne phylogeographic patterns within F. tularensis, particularly F. tularensis subsp. holarctica. These new signatures, when screened across diverse isolate collections, have identifi ed new subclades within preexisting subclades. This pattern will continue as whole-genome sequencing becomes less expensive and more widely available. As a result, the nomenclature of phylogenetic groups within F. tularensis and the particular subclade to which a given isolate is assigned are constantly changing and will continue to change, which makes comparison of results and fi ndings across different studies diffi cult. To address this problem, we have included all known F. tularensis subsp. holarctica SNP-based phylogenetic groups within our phylogenetic trees ( Figure 1, panel A; Figure 2, panel B), including those discovered by other researchers. In addition, for the isolates analyzed in this study (online Appendix Table), where applicable, we have listed the phylogenetic groups to which they were assigned in previous studies.