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Journal of Clinical Microbiology, November 2000, p. 4180-4185, Vol. 38, No. 11
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Anders Johansson,1,2,3 Ashraf Ibrahim,4 Ingela Göransson,2 Ulla Eriksson,2 D. Gurycova,5 Jill E. Clarridge III,6 and Anders Sjöstedt2,3,*

Infectious Diseases1 and Clinical Bacteriology,3 Department of Clinical Microbiology, Umeå University, and Department of Microbiology, Defence Research Establishment,2 Umeå, Sweden; Department of Biotechnology, Technical University of Denmark, Lyngby, Denmark4; Department of Epidemiology, Komensky University, Bratislava, Slovak Republic5; and Microbiology, Serology and Molecular Biology Section, PALMS, Veterans Affairs Medical Center, Houston, Texas6

Received 10 April 2000/Returned for modification 12 July 2000/Accepted 23 August 2000

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.

* Corresponding author. Mailing address: Department of Clinical Microbiology, Clinical Bacteriology, Umeå University, SE-901 85 Umeå, Sweden. Phone: 46 90 7851120. Fax: 46 90 7852225. E-mail: anders.sjostedt{at}climi.umu.se.

Journal of Clinical Microbiology, November 2000, p. 4180-4185, Vol. 38, No. 11
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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