Extensive allelic variation among Francisella tularensis strains in a short-sequence tandem repeat region

Abstract

Members of the genus Francisella and the species F. tularensis appear 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. tularensis genomes 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. tularensis isolates. 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. tularensis and another one was present in all 13 isolates of a strain passaged in animals. The analysis of short-sequence repeats of F. tularensis strains 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.

FIG. 1

DNA sequence of the SSTR9 locus. The locus is located in an ORF of the Schu S4 strain. The putative start and stop codons are boxed. Locations of PCR primers are shaded. The 9-bp tandem repeats are indicated by arrows; broken lines indicate sequence heterogeneity of the 9-bp SSTR.

FIG. 2

Sequence of the SSTR16 region in the Schu S4 strain. The 16-bp direct repeats are marked by arrows and are present in 18 copies. Primer positions are shaded. The direct repeats are located inside an IS element; terminal inverted repeats of the IS element are shown in boldface. Thirty-five copies of the IS elements have been identified in the current assembly of the Schu S4 genome, each containing 2 to 18 copies of the 16-bp tandem repeat.

FIG. 3

Representative electrophoretic analysis of PCR fragments of the SSTR9 loci from different isolates of F. tularensis. Fragments were detected by fluorescence on an ABI 377XL DNA sequencer. Strains of each of the four subspecies are represented; F. tularensis subsp. tularensis in lanes 7 (FSC198), 9 (FSC199), and 17 (FSC054); F. tularensis subsp. holarctica nonrelated European and North American isolates in lanes 1 (FSC176), 3 (FSC180), 4 (FSC247), 5 (FSC188), 6 (FSC012), 14 (FSC025), 16 (FSC032), 20 (FSC150), 21 (FSC076), 22 (FSC155), 23 (FSC080), 24 (FSC157), 25 (FSC089), 26 (FSC161), and 27 (FSC097), isolates from Ljusdal, Sweden, 1995 and 1998 in lanes 2 (FSC245), 11 (FSC200), 13 (FSC201), 15 (FSC202), and 29 (FSC102), and nonrelated Japanese isolates in lanes 8 (FSC017), 10 (FSC021), 12 (FSC022), and 19 (FSC075); F. tularensis subsp. mediasiatica in lane 18 (FSC149); and F. tularensis subsp. novicida in lane 28 (FSC040). Allele sizes (in base pairs) are indicated to the left.