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:Francisella tularensis is the causative agent of the zoonotic disease tularemia. In Germany, most human infections are caused by contact with infected hares. The aim of this study was to characterize Francisella tularensis subsp. holarctica strains isolated from hares in Germany and to develop bioinformatics tools to analyze their genetic relatedness. In total, 257 German isolates-obtained mainly from hares (n = 233), other vertebrate animals, and ticks, but also from humans (n = 3)-were analyzed within this study. Publically available sequence data from 49 isolates were used to put our isolates into an epidemiological context and to compare isolates from natural foci and humans. Whole-genome sequences were analyzed using core-genome Multi-Locus-Sequence-Typing, canonical Single Nucleotide Polymorphism (SNP) typing and whole-genome SNP typing. An overall conformity of genotype clustering between the typing methods was found, albeit with a lower resolution for canonical single SNP typing. The subclade distribution, both on local and national levels, among strains from humans and hares was similar, suggesting circulation of the same genotypes both in animals and humans. Whilst close to identical isolates of the same subclade were found distributed over large areas, small geographical foci often harbored members of different subclades. In conclusion, although genomic high-resolution typing was shown to be robust, reproducible and allowed the identification of highly closely related strains, genetic profiling alone is not always conclusive for epidemiological linkage of F. tularensis strains.
Project description:In November 2012, a group of 7 persons who participated in a hare hunt in North Rhine-Westphalia, Germany, acquired tularemia. Two F. tularensis subsp. holarctica isolates were cultivated from human and hare biopsy material. Both isolates belonged to the FTN002-00 genetic subclade (derived for single nucleotide polymorphisms B.10 and B.18), thus indicating likely hare-to-human transmission.
Project description:Comparative genome hybridization of the Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica populations have shown that genome content is highly conserved, with relatively few genes in the F. tularensis subsp. tularensis genome being absent in other F. tularensis subspecies. To determine if organization of the genome differs between global populations of F. tularensis subsp. tularensis and F. tularensis subsp. holarctica, we have used paired-end sequence mapping (PESM) to identify regions of the genome where synteny is broken. The PESM approach compares the physical distances between paired-end sequencing reads of a library of a wild-type reference F. tularensis subsp. holarctica strain to the predicted lengths between the reads based on map coordinates of two different F. tularensis genome sequences. A total of 17 different continuous regions were identified in the F. tularensis subsp. holarctica genome (CR(holar)(c)(tica)) which are noncontiguous in the F. tularensis subsp. tularensis genome. Six of the 17 different CR(holarctica) are positioned as adjacent pairs in the F. tularensis subsp. tularensis genome sequence but are translocated in F. tularensis subsp. holarctica, implying that their arrangements are ancestral in F. tularensis subsp. tularensis and derived in F. tularensis subsp. holarctica. PCR analysis of the CR(holarctica) in 88 additional F. tularensis subsp. tularensis and F. tularensis subsp. holarctica isolates showed that the arrangements of the CR(holarctica) are highly conserved, particularly in F. tularensis subsp. holarctica, consistent with the hypothesis that global populations of F. tularensis subsp. holarctica have recently experienced a periodic selection event or they have emerged from a recent clonal expansion. Two unique F. tularensis subsp. tularensis-like strains were also observed which likely are derived from evolutionary intermediates and may represent a new taxonomic unit.
Project description:Francisella tularensis is a tier 1 agent causing the zoonosis tularemia. This highly infectious Gram-negative bacterium is occasionally isolated from human samples (especially blood samples) in routine clinical microbiology laboratories. A rapid and accurate method for identifying this pathogen is needed in order to optimize the infected patient's healthcare management and prevent contamination of the laboratory personnel. MALDI TOF mass spectrometry has become the gold standard for the rapid identification of most human pathogens. However, F. tularensis identification using such technology and commercially available databases is currently considered unreliable. Real-time PCR-based methods for rapid detection and accurate identification of F. tularensis are not available in many laboratories. As a national reference center for tularemia, we developed a MALDI TOF database allowing accurate identification of the species F. tularensis and its differentiation from the closely related neighbor species F. tularensis subsp. novicida and F. philomiragia. The sensitivity and specificity of this database were validated by testing 71 F. tularensis strains and 165 strains from 63 species not belonging to the Francisella genus. We obtained accurate identification at the species level and differentiation of all the tested bacterial strains. In particular, F. tularensis could be accurately differentiated from other small Gram-negative bacilli occasionally isolated from human samples, including species of the HACEK group and Brucella melitensis.
Project description:Francisella tularensis is a highly infectious human intracellular pathogen that is the causative agent of tularemia. It occurs in several major subtypes, including the live vaccine strain holarctica (type B). F. tularensis is classified as category A biodefense agent in part because a relatively small number of organisms can cause severe illness. Three complete genomes of subspecies holarctica have been sequenced and deposited in public archives, of which OSU18 was the first and the only strain for which a scientific publication has appeared. We re-assembled the OSU18 strain using both de novo and comparative assembly techniques, and found that the published sequence has two large inversion mis-assemblies. We generated a corrected assembly of the entire genome along with detailed information on the placement of individual reads within the assembly. This assembly will provide a more accurate basis for future comparative studies of this pathogen.
Project description:In 2018, the incidence of tularemia increased twofold in the west of France, with many pneumonic forms, suggesting environmental sources of infection. We investigated the presence of Francisellatularensis subsp. holarctica and other Francisella species DNA in the natural aquatic environment of this geographic area. Two sampling campaigns, in July 2019 and January 2020, allowed the collection of 87 water samples. Using a combination of real-time PCR assays, we tested the presence of either Francisella sp., F. tularensis/F. novicida, and F. tularensis subsp. holarctica, the latter being the only tularemia agent in Europe. Among 57 water samples of the first campaign, 15 (26.3%) were positive for Francisella sp., nine (15.8%) for F. tularensis and/or F. novicida, and four (7.0%) for F. tularensis subsp. holarctica. Ratios were 25/30 (83.3%), 24/30 (80.0%), and 4/30 (13.3%) for the second campaign. Among the thirty sites sampled during the two campaigns, nine were positive both times for Francisella sp., seven for F. tularensis and/or F. novicida, and one for F. tularensis subsp. holarctica. Altogether, our study reveals a high prevalence of Francisella sp. DNA (including the tularemia agent) in the studied aquatic environment. This aquatic environment could therefore participate in the endemicity of tularemia in the west of France.