Francisella tularensis strain typing using multiple-locus, variable-number tandem repeat analysis

Abstract

Francisella tularensis, the etiological agent of tularemia, is found throughout the Northern hemisphere. After analyzing the F. tularensis genomic sequence for potential variable-number tandem repeats (VNTRs), we developed a multilocus VNTR analysis (MLVA) typing system for this pathogen. Variation was detected at six VNTR loci in a set of 56 isolates from California, Oklahoma, Arizona, and Oregon and the F. tularensis live vaccine strain. PCR assays revealed diversity at these loci with total allele numbers ranging from 2 to 20, and Nei's diversity index values ranging from 0.36 to 0.93. Cluster analysis identified two genetically distinct groups consistent with the current biovar classification system of F. tularensis. These findings suggest that these VNTR markers are useful for identifying F. tularensis isolates at this taxonomic level. In this study, biovar B isolates were less diverse than those in biovar A, possibly reflecting the history of tularemia in North America. Seven isolates from a recent epizootic in Maricopa County, Ariz., were identical at all VNTR marker loci. Their identity, even at a hypervariable VNTR locus, indicates a common source of infection. This demonstrates the applicability of MLVA for rapid characterization and identification of outbreak isolates. Future construction of reference databases will allow faster outbreak tracking as well as providing a foundation for deciphering global genetic relationships.

FIG. 1

Dot plot homology at individual marker loci. Dot plot homology analysis was performed using self comparison of each VNTR locus's nucleotide sequence. The panels represent the entire amplicon generated with primers from Table 1. All analyses used a 100% similarity requirement as indicated in each panel. The allele sizes presented in this figure are 321, 248, 183, 479, 159, and 191 bp (Ft-V1 through Ft-V6, respectively).

FIG. 2

Dendrogram based upon six MLVA markers. This dendrogram was generated using UPGMA analysis based upon allele differences among isolates. Letters to the right of each branch correspond to geographical origin: California isolates are identified by a county-specific three letter code, Arizona county isolates are designated by AZ, and Oklahoma isolates are listed as OK. Numbers associated with branch lengths represent bootstrap values using 1,000 simulations. An asterisk designates strains of known F. tularensis biovar B classification.