Genetic Diversity Of Francisella Tularensis Subsp. Holarctica In Kazakhstan

Tularemia is a highly dangerous zoonotic infection due to the bacteria Francisella tularensis. Low genetic diversity promoted the use of polymorphic tandem repeats (MLVA) as first-line assay for genetic description. Whole genome sequencing (WGS) is becoming increasingly accessible, opening the perspective of a time when WGS might become the universal genotyping assay. The main goal of this study was to describe F. tularensis strains circulating in Kazakhstan based on WGS data and develop a MLVA assay compatible with in vitro and in silico analysis. In vitro MLVA genotyping and WGS were performed for the vaccine strain and for 38 strains isolated in Kazakhstan from natural water bodies, ticks, rodents, carnivores, and from one migratory bird, an Isabellina wheatear captured in a rodent burrow. The two genotyping approaches were congruent and allowed to attribute all strains to two F. tularensis holarctica lineages, B.4 and B.12. The seven tandem repeats polymorphic in the investigated strain collection could be typed in a single multiplex PCR assay. Identical MLVA genotypes were produced by in vitro and in silico analysis, demonstrating full compatibility between the two approaches. The strains from Kazakhstan were compared to all publicly available WGS data of worldwide origin by whole genome SNP (wgSNP) analysis. Genotypes differing at a single SNP position were collected within a time interval of more than fifty years, from locations separated from each other by more than one thousand kilometers, supporting a role for migratory birds in the worldwide spread of the bacteria.Author summaryGenotyping of Francisella tularensis has become a routine practice in epidemiology. Despite rapidly accumulating knowledge, the phylogeography of the pathogen is still poorly understood and discussions about geographic and temporal origins continue. One important reason is the poor characterization of the pathogen in many tularemia-endemic countries. This article describes the genetic diversity of Francisella tularensis subsp holartica in Kazakhstan using tandem repeat polymorphisms as well as whole genome sequencing. Thirty-nine strains were analyzed and two lineages were identified, namely B.4 and B.12. The study demonstrates a wider distribution of genotype B.4 in Asia, and identified a more basal branching point in this subclade. The obtained data support the Asian origin hypothesis for F. tularensis. The finding of identical genotypes in strains separated in time by decades and a thousand-kilometers geographic distance, confirms the ability of the bacteria for long-term preservation and fast long distances spread. The isolation of F. tularensis subsp holartica from the bird species Isabellina wheatear allows speculating about a major contribution of birds to the phylogeography of the pathogen. A genotyping protocol was developed utilizing seven polymorphic tandem repeats, two of which were identified within the framework of this work. The in vitro and in silico results are identical when using sequencing reads of 300 base-pairs or more.