Phylogeography of Francisella tularensis subspecies holarctica from the country of Georgia

Namita Nayyar (WF Team)

13 years ago

Phylogeography of Francisella tularensis subspecies holarctica from the country of Georgia

– Reported, January 20, 2012

Francisella tularensis, the causative agent of tularemia, displays subspecies-specific differences in virulence, geographic distribution, and genetic diversity. F. tularensis subsp. holarctica is widely distributed throughout the Northern Hemisphere. In Europe, F. tularensis subsp. holarctica isolates have largely been assigned to two phylogenetic groups that have specific geographic distributions. Most isolates from Western Europe are assigned to the B.Br.FTNF002-00 group, whereas most isolates from Eastern Europe are assigned to numerous lineages within the B.Br.013 group. The eastern geographic extent of the B.Br.013 group is currently unknown due to a lack of phylogenetic knowledge about populations at the European/Asian juncture and in Asia. In this study, we address this knowledge gap by describing the phylogenetic structure of F. tularensis subsp. holarctica isolates from the country of Georgia, and by placing these isolates into a global phylogeographic context.

We identified a new genetic lineage of F. tularensis subsp. holarctica from Georgia that belongs to the B.Br.013 group. This new lineage is genetically and geographically distinct from lineages previously described from the B.Br.013 group from Central-Eastern Europe. Importantly, this new lineage is basal within the B.Br.013 group, indicating the Georgian lineage diverged before the diversification of the other known B.Br.013 lineages. Although two isolates from the Georgian lineage were collected nearby in the Ukrainian region of Crimea, all other global isolates assigned to this lineage were collected in Georgia. This restricted geographic distribution, as well as the high levels of genetic diversity within the lineage, is consistent with a relatively older origin and localized differentiation.

We identified a new lineage of F. tularensis subsp. holarctica from Georgia that appears to have an older origin than any other diversified lineages previously described from the B.Br.013 group. This finding suggests that additional phylogenetic studies of F. tularensis subsp. holarctica populations in Eastern Europe and Asia have the potential to yield important new insights into the evolutionary history and phylogeography of this broadly dispersed F. tularensis subspecies.

Francisella tularensis is a highly clonal, recently-emerged pathogen that causes tularemia, which presents in several main forms: pneumonic (30%-60% mortality), ulceroglandular, and oropharyngeal . The latter two are associated with lower mortality. F. tularensis is currently divided into three subspecies (tularensis, holarctica and mediasiatica), with F. novicida recognized as a very closely related species, or as another subspecies by some authors . These taxa vary in virulence, geographic distribution, overall genetic diversity, and host/vector associations . Human tularemia is a disease at which the clinical severity depends upon the route of infection, subspecies of the infection strain, and timely therapeutic response . Cases in Europe are caused by F. tularensis subsp. holarctica, and in many rural areas of the Balkans and countries further east outbreaks are water-borne, resulting in oropharyngeal tularemia . No known cases by F. tularensis subsp. mediasiatica are known and only a few by F. novicida have been documented . F. tularensis subsp. tularensis is restricted to North America, whereas F. tularensis subsp. holarctica is found throughout the Northern Hemisphere . Despite its wider geographic distribution F. tularensis subsp. holarctica has markedly lower genetic diversity than F. tularensis subsp. tularensis .

Significant gains toward deciphering the evolutionary history of F. tularensis overall and, in particular, F. tularensis subsp. holarctica have been made by using whole genome comparisons for single nucleotide polymorphism (SNP) discovery coupled with subsequent canonical SNP (canSNP) analysis . Numerous new groups were identified within F. tularensis subsp. holarctica , two of which, B.Br.013 (includes subclades B.Br.013/014 and B.Br.LVS ) and B.Br.FTNF002-00, were predominant in Europe but geographically segregated . In the Western European countries of Spain, France, and Switzerland almost all isolates belong to the highly monomorphic B.Br.FTNF002-00 group . In contrast, in large portions of Central and Eastern Europe, from the Czech Republic to Russia, most F. tularensis subsp. holarctica isolates are assigned to various lineages within the B.Br.013 group .

Initial analyses with previously described canSNP assays revealed that all 25 Georgian isolates belong to the B.Br.013 group. One of the Georgian strains (F0673) was sequenced using the Illumina Genome Analyzer II sequencing platform resulting in very high sequence coverage (averaging 1,076X) when aligned to the LVS genome . Subsequent whole genome sequence (WGS) comparisons among three published B.Br.013 group genomes (FSC 200, LVS, and RC503), the genome of strain F0673 generated for this study, and the published OSU18 genome (as an outgroup) revealed 650 putative SNPs. Most of these putative SNPs (n = 470) were phylogenetically located on the branches separating OSU18 from the genomes in the B.Br.013 group . Maximum parsimony analysis of the putative SNPs produced a phylogeny with a very low homoplasy index (0.02), consistent with the highly clonal nature of F. tularensis. The phylogenetic topology of the FSC 200, LVS, and RC503 genomes is consistent with previous publications , and the small number of putative SNPs unique to the Georgian strain is consistent with the low genetic diversity observed among other lineages within F. tularensis subsp. holarctica . The new branch (B.Br.027) leading to the Georgian strain arises from a common ancestor that is basal to the previously described diversified lineages within the B.Br.013 group and is separated from them by only 45 putative SNPs, with 39 of these putative SNPs leading to the Georgian strain (B.Br.027) and the other six putative SNPs along a branch (B.Br.026 defining a monophyletic lineage containing the other sequenced strains from this group.

We describe a new subpopulation in the B.Br.013 group from Georgia that is genetically and geographically distinct from the other B.Br.013 group subpopulations found in Europe. Members of this new lineage are endemic to parts of Eastern Europe and Western Asia, though the complete geographic range remains unknown. The basal positioning of the Georgian lineage and its restricted geographic distribution illustrates the need for studies on additional Asian and East European isolates to gain a better understanding of the clonal expansion of F. tularensis subsp. holarctica.

All 25 Georgian isolates were screened with an 11-marker MLVA system (Additional file 4) [25]. This was done in order to determine the level of genetic diversity within each identified subclade. The MLVA Diversity (D) was calculated for each subclade using the following equation: G/N (G = MLVA genotypes; N = number of isolates). Diversity was not calculated for subclades with a single isolate.

Credits:Gvantsa Chanturia, Dawn N Birdsell,Merab Kekelidze,Ekaterine Zhgenti, George Babuadze, Nikoloz Tsertsvadze, Shota Tsanava,Paata Imnadze,Stephen M Beckstrom-Sternberg, James S Beckstrom-Sternberg, Mia D Champion, Shripad Sinari, Miklos Gyuranecz,Jason Farlow,Amanda H Pettus, Emily L Kaufman, Joseph D Busch, Talima Pearson, Jeffrey T Foster, Amy J Vogler, David M Wagner, and Paul Keim

More Information at:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3224097/?tool=pubmed

– WF Team