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ABSTRACTFrancisella tularensisis a potent pathogen and a possible bioterrorism agent. Little is known, however, to explain the molecular basis for its virulence and the distinct differences in virulence found between the four recognized subspecies, F. tularensissubsp. tularensis, F. tularensissubsp. mediasiatica, F. tularensissubsp. holarctica, and F. tularensissubsp. novicida. We developed a DNA microarray based on 1,832 clones from a shotgun library used for sequencing of the highly virulent strain F. tularensissubsp. tularensisSchu S4. This allowed a genome-wide analysis of 27 strains representing all four subspecies. Overall, the microarray analysis confirmed a limited genetic variation within the species F. tularensis, and when the strains were compared, at most 3.7% of the probes showed differential hybridization. Cluster analysis of the hybridization data revealed that the causative agents of type A and type B tularemia, i.e., F. tularensissubsp. tularensisand F. tularensissubsp. holarctica, respectively, formed distinct clusters. Despite marked differences in their virulence and geographical origin, a high degree of genomic similarity between strains of F. tularensissubsp. tularensisand F. tularensissubsp. mediasiaticawas apparent. Strains from Japan clustered separately, as did strains of F. tularensissubsp. novicida. Eight regions of difference (RD) 0.6 to 11.5 kb in size, altogether comprising 21 open reading frames, were identified that distinguished strains of the moderately virulent subspecies F. tularensissubsp. holarcticaand the highly virulent subspecies F. tularensissubsp. tularensis. One of these regions, RD1, allowed for the first time the development of an F. tularensis-specific PCR assay that discriminates each of the four subspecies.

ABSTRACTMembers of the genus Francisellaand the speciesF. tularensisappear to be genetically very similar despite pronounced differences in virulence and geographic localization, and currently used typing methods do not allow discrimination of individual strains. Here we show that a number of short-sequence tandem repeat (SSTR) loci are present in F. tularensisgenomes and that two of these loci, SSTR9 and SSTR16, are together highly discriminatory. Labeled PCR amplification products from the loci were identified by an automated DNA sequencer for size determination, and each allelic variant was sequenced. Simpson's index of diversity was 0.97 based on an analysis of 39 nonrelated F. tularensisisolates. The locus showing the highest discrimination, SSTR9, gave an index of diversity of 0.95. Thirty-two strains isolated from humans during five outbreaks of tularemia showed much less variation. For example, 11 of 12 strains isolated in the Ljusdal area, Sweden in 1995 and 1998 had identical allelic variants. Phenotypic variants of strains and extensively cultured replicates within strains did not differ, and, for example, the same allelic combination was present in 55 isolates of the live-vaccine strain of F. tularensisand another one was present in all 13 isolates of a strain passaged in animals. The analysis of short-sequence repeats of F. tularensisstrains appears to be a powerful tool for discrimination of individual strains and may be useful for a detailed analysis of the epidemiology of this potent pathogen.