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.

Tularaemia

“…Recently, however, interstrain variation in short tandem nucleotide repeats has been identified [19,20]. By use of PCR-based techniques, isolates derived from patients at different periods of time or from various regions within the Nordic countries can now be differentiated [20].…”

“…A test amenable for rapid and simple use on suspect colonies is the agglutination of suspended bacteria by specific antiserum. Due to the recent development of molecular methods, polymerase chain reaction (PCR)-based assays can now be performed for a rapid final confirmation of bacterial isolates [18][19][20].…”

“…The original bases for the differentiation were biochemical properties, epizootology, and virulence assay in rabbits [21]. More recently, subtle differences in nucleic acid sequences have been established [18][19][20].…”

Genetic tools for highly pathogenic Francisella tularensis subsp. tularensis

“…There are four subspecies of F. tularensis, each differing in their relative degree of pathogenesis in the human host, ranging from the weakly pathogenic subsp. novicida, the moderately pathogenic subspecies holarctica and mediasiatica, to the highly pathogenic subspecies tularensis (Garcia Del Blanco et al, 2002;Hollis et al, 1989;Johansson et al, 2001Johansson et al, , 2004Titball et al, 2003). The high mortality of serious forms of tularaemia, coupled with the very infectious nature of F. tularensis aerosols, prompted the past development of F. tularensis subsp.…”

Identification of the Source of <i>Francisella tularensis</i> Infection by Multiple-Locus Variable-Number Tandem Repeat Analysis

“…(4)(5)(6)(7)(8). It has been shown to be highly effective in the discrimination of individual strains.…”