Worldwide genetic relationships among Francisella tularensis isolates determined by multiple-locus variable-number tandem repeat analysis

Abstract

The intracellular bacterium Francisella tularensis is the causative agent of tularemia and poses a serious threat as an agent of bioterrorism. We have developed a highly effective molecular subtyping system from 25 variable-number tandem repeat (VNTR) loci. In our study, multiple-locus VNTR analysis (MLVA) was used to analyze genetic relationships and potential population structure within a global collection of 192 F. tularensis isolates, including representatives from each of the four subspecies. The VNTR loci displayed between 2 and 31 alleles with Nei's diversity values between 0.05 and 0.95. Neighbor-joining cluster analysis of VNTR data revealed 120 genotypes among the 192 F. tularensis isolates, including accurate subspecies identification. F. tularensis subsp. tularensis (type A) isolates showed great diversity at VNTR loci, while F. tularensis subsp. holarctica (type B) isolates showed much lower levels despite a much broader geographical prevalence. The resolution of two distinct clades within F. tularensis subsp. tularensis (designated A.I and A.II) revealed a previously unrecognized genetic division within this highly virulent subspecies. F. tularensis subsp. holarctica appears to have recently spread globally across continents from a single origin, while F. tularensis subsp. tularensis has a long and complex evolutionary history almost exclusively in North America. The sole non-North American type A isolates (Slovakian) were closely related to the SCHU S4 strain. Significant linkage disequilibrium was detected among VNTR loci of F. tularensis consistent with a clonal population structure. Overall, this work greatly augments the study of tularemia ecology and epidemiology, while providing a framework for future forensic analysis of F. tularensis isolates.

FIG. 1.

VNTR marker location within the physical map of F. tularensis subsp. tularensis SCHU S4 genome. Positions are given with reference to the predicted origin of replication set at position 0.

FIG. 2.

Number of alleles and allelic diversity detected at 25 VNTR markers. Bars indicate allele number, and filled diamonds indicate Nei's diversity index (D), calculated as 1 − Σ(allele frequency)² analyzed across 120 F. tularensis genotypes and 192 isolates.

FIG. 3.

Genetic relationships among global F. tularensis isolates based on allelic differences at 25 VNTR markers. The NJ dendrogram was rooted with F. tularensis subsp. novicida isolates. Geographical origin, number of isolates (n), number of genotypes (G), and F. tularensis subspecies are indicated. Reference strains from American Type Culture Collection (ATCC) and Gamaleya Institute of Epidemiology and Microbiology (GIEM) are specified in boxes. Major clades containing type A and type B F. tularensis isolates were arbitrarily designated A.I and A.II and B.I to B.V, respectively. Bootstrap values greater than 50% are shown and were calculated using 1,000 iterations. Scale bar represents genetic distance.

FIG. 4.

Isolate identification within individual subgroups of the NJ dendrogram. The origin (including state, province, and country) and year are specified to the right of each branch or clade. Reference strains from the American Type Culture Collection (ATCC) and Gamaleya Institute of Epidemiology and Microbiology (GIEM) are identified in boxes. Branch lengths among clades are not to scale, due to space constraints. Major clades containing type A and type B F. tularensis isolates were arbitrarily designated A.I and A.II and B.I to B.V, respectively. Filled circles indicate isolates from geographically confined tularemia outbreaks.