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

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.

Evaluation of PCR-based methods for discrimination of Francisella species and subspecies and development of a specific PCR that distinguishes the two major subspecies of Francisella tularensis.

Previous studies have demonstrated that the four subspecies of the human pathogen Francisella tularensis, despite showing marked variations in their virulence for mammals and originating from different regions in the Northern Hemisphere, display a very close phylogenetic relationship. This property has hampered the development of generally applicable typing methods. To overcome this problem, we evaluated the use of PCR for discrimination of the subspecies using various forms of long arbitrary primers or primers specific for repetitive extragenic palindromic sequences (REP) or enterobacterial repetitive intragenic consensus (ERIC) sequences. Patterns generated by use of REP, ERIC, or long arbitrary primers allowed differentiation at the species level and of the four subspecies of F. tularensis. With each of these three methods, similar or identical clustering of strains was found, and groups of strains of different geographical origins or differing in virulence showed distinct patterns. The discriminatory indices of the methods varied from 0.57 to 0.65; thus, the patterns were not sufficiently discriminatory to distinguish individual strains. The sequence of a fragment generated by amplification with an arbitrary primer was determined, and a region showing interstrain heterogeneity was identified. Specific primers were designed, and a PCR was developed that distinguished strains of F. tularensis subsp. holarctica from strains of other F. tularensis subspecies, including strains of the highly virulent F. tularensis subsp. tularensis. Notably, one European isolate showed the genetic pattern typical of the highly virulent F. tularensis subsp. tularensis, generally believed to exist only in North America. It is proposed that a combination of the specific PCR together with one method generating subspecies-specific patterns is suitable as a rapid and relatively simple strategy for discrimination of Francisella species and subspecies.

Comparative analysis of PCR versus culture for diagnosis of ulceroglandular tularemia.

PCR and culture were comparatively evaluated for their abilities to demonstrate Francisella tularensis in wound specimens from tularemia patients during an outbreak in Sweden in 1998. For transport of the specimens used for PCR, a buffer solution containing a nuclease inhibitor was used, and for transport of the specimens used for culture, a commercial transport system was selected after experimental comparison of various systems. Of 40 patients with culture- and/or serology-verified ulceroglandular tularemia, PCR detected F. tularensis DNA in 30 (75%) patients, whereas culture detected bacterial growth in 25 (62%) patients. Compared to data from a previous study, the present inclusion of a nuclease inhibitor in the transport medium did not improve the sensitivity of the PCR, whereas the sensitivity of the culture procedure was significantly increased by selection of the system used for transport. Among eight patients with clinically suspected tularemia but with negative serology and culture, specimens from four patients showed detectable DNA. In three of these patients the diagnosis was verified by the demonstration of an F. tularensis-specific T-cell response in vitro. In conclusion, PCR was more sensitive than culture for demonstration of F. tularensis in wound specimens. Besides, we showed that tularemia may proceed without development of serum antibodies, and in these patients, PCR may be of special importance for verification of the diagnosis.

Genotypic and phenotypic characterization of two Swedish isolates and two prototypic strains of Coxiella burnetii.

Two Swedish isolates of Coxiella burnetii and the two prototype strains of the species, Nine Mile and Priscilla, were characterized with regard to their multiplication and cytopathic effect on BGM cells and by PCR-based amplification of repetitive element DNA and the C. burnetii-specific plasmids QpH1 and QpRS. Moreover, 1330 bp of each 16S rRNA gene were sequence-determined. All four strains multiplied at virtually the same rate and displayed the same type of vacuoles in the BGM cells. Genetic homogeneity was observed inasmuch as the 16S rDNA sequences were identical and the strains showed identical PCR amplification patterns using primers specific to enterobacterial repetitive intragenic consensus DNA sequences. The two Swedish strains and the Priscilla strain also showed identical patterns after PCR amplification of repetitive extragenic palindromic DNA sequences, whereas the Nine Mile strain demonstrated a similar, but not identical pattern. Thus, the investigated strains demonstrated very similar phenotypic and genotypic characteristics. This finding is discussed in view of the very rare occurrence of domestic Q fever in Sweden.