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

Previous studies have demonstrated that the four subspecies of the human pa thogen Francisella tularensis, despite showing marked variations in their v irulence 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 ove rcome this problem, we evaluated the use of PCR for discrimination of the s ubspecies using various forms of long arbitrary primers or primers specific for repetitive extragenic palindromic sequences (REP) or enterobacterial r epetitive intragenic consensus (ERIC) sequences. Patterns generated by use of REP, ERIC, or long arbitrary primers allowed differentiation at the spec ies level and of the four subspecies of F. tularensis. With each of these t hree methods, similar or identical clustering of strains was found, and gro ups 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 off. tularensis subsp. h olarctica from strains of other F. tularensis subspecies. including strains of the highly virulent F. tularensis subsp. tularensis, Notably, one Europ ean isolate showed the genetic pattern typical of the highly virulent F. tu larensis subsp. tularensis, generally believed to exist only in North Ameri ca. 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 su bspecies.