Genetic subtyping of Francisella tularensis and Borrelia burgdorferi using multiple-locus variable-number tandem repeat analysis.

摘要

Technological advances have allowed fine-scale detection of genetic variation within pathogen genomes providing the research community with a valuable means for evaluating the ecology and evolution of microbial pathogens. Identification of genetic motifs undergoing relatively fast mutation allows discrimination among individual strains while providing quantifiable genetic signatures with a wide range of information content. Genetic subtyping systems based on multiple-locus variable-number tandem repeat analysis (MLVA) have shown great capacity for exploiting nucleotide sequence variation within genetically homogenous pathogens such as Bacillus anthracis and Yersina pestis. This dissertation describes the development and application of MLVA typing systems for the microbial pathogens Francisella tularensis and Borrelia burgdorferi.;Genetic diversity of F. tularensis was evaluated on both global and continental scales. Great strain discrimination among a collection of 56 North American F. tularensis isolates was provided by an F. tularensis MLVA typing system comprised of 6 MLVA markers. All isolates were successfully resolved into taxonomically correct Francisella groups. The subsp. tularensis groups showed greater genetic diversity than subsp. holarctica group. Subsequently a 25 MLVA marker subtyping system was developed. A global collection of F. tularensis representing all known subspecies was analyzed to evaluate world-wide genetic relationships. All strains clustered into taxonomically distinct clades with North American isolates displaying greater diversity than those from Eurasia. Within the highly virulent subsp. tularensis , two distinct subdivisions (A.I. and A.II) were identified.;Analysis of genetic diversity within an expanded collection of North American F. tularensis isolates revealed spatial genetic structure within subsp. tularensis and correlated with geographical ranges of known tularemia vectors and hosts. This correlation provides direction for future ecological and evolutionary studies addressing the ecology of tularemia in North America.;Among a group of 41 globally diverse B. burgdorferi, B. afzelii , and B. garinii isolates, a ten marker MLVA system provided great strain discrimination (Chapter 5) identifying 30 unique genotypes. Cluster analysis revealed five distinct genotypic clades wherein B. afzelii, and B. garinii showed greater genetic similarity suggesting these subgroups are close relatives and perhaps recently derived from B. burgdorferi.