Project description:We used transposon insertion sequencing (Tn-Seq) to identify the genes that are required for in vitro growth and intramacrophage growth of the live vaccine strain of F. tularensis (LVS).
Project description: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.
Project description:We analyzed 10 isolates of Francisella tularensis subspecies holarctica from China and assigned them to known clades by using canonical single-nucleotide polymorphisms. We found 4 diverse subtypes, including 3 from the most basal lineage, biovar japonica. This result indicates unprecedented levels of diversity from a single region and suggests new models for emergence.
Project description: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.
Project description:In vitro experimental evolution has complemented clinical studies as an excellent tool to identify genetic changes responsible for the de novo evolution of antimicrobial resistance. However, the in vivo context for adaptation contributes to the success of particular evolutionary trajectories, especially in intracellular niches where the adaptive landscape of virulence and resistance are strongly coupled. In this work, we designed an ex vivo evolution approach to identify evolutionary trajectories responsible for antibiotic resistance in the Live Vaccine Strain (LVS) of Francisella tularensis subsp. holarctica while being passaged to increasing ciprofloxacin (CIP) and doxycycline (DOX) concentrations within macrophages. Overall, adaptation within macrophages advanced much slower when compared to previous in vitro evolution studies reflecting a limiting capacity for the expansion of adaptive mutations within the macrophage. Longitudinal genomic analysis identified resistance conferring gyrase mutations outside the Quinolone Resistance Determining Region. Strikingly, FupA/B mutations that are uniquely associated with in vitro CIP resistance in Francisella were not observed ex vivo, reflecting the coupling of intracellular survival and resistance during intracellular adaptation. To our knowledge, this is the first experimental study demonstrating the ability to conduct experimental evolution to antimicrobial resistance within macrophages. The results provide evidence of differences in mutational profiles of populations adapted to the same antibiotic in different environments/cellular compartments and underscore the significance of host mediated stress during resistance evolution.