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

Abstract

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.

FIG. 1

PCR amplification of DNA from various Francisella strains using the REP1R-I and REP2-I primers. Samples represent F. tularensis subsp. tularensis strains (FSC138, FSC043, FSC041, and FSC198) (lanes 1 to 4), F. tularensis subsp. holarctica strains (FSC196, FSC155, FSC150, FSC108, and FSC157) (lanes 5 to 9), an F. tularensis subsp. mediaasiatica strain (FSC149) (lane 10), an F. tularensis subsp. holarctica strain from Japan (FSC024) (lane 11), F. tularensis subsp. novicida strains (FSC040 and FSC156) (lanes 12 and 13), and an F. philomiragia strain (FSC 144) (lane 14). Strain designations are indicated in Table 1. Lane N, water used as a negative control. Lane M, molecular markers (sizes in base pairs).

FIG. 2

PCR amplification of DNA from various Francisella strains using the LP-RAPD2 primer. Samples represent F. tularensis subsp. tularensis strains (FSC138, FSC043, FSC041, and FSC198) (lanes 1 to 4), F. tularensis subsp. holarctica strains (FSC196, FSC155, FSC150, FSC108, and FSC157) (lanes 5 to 9), an F. tularensis subsp. mediaasiatica strain (FSC149) (lane 10), an F. tularensis subsp. holarctica strain from Japan (FSC024) (lane 11), F. tularensis subsp. novicida strains (FSC040 and FSC156) (lanes 12 and 13), and an F. philomiragia strain (FSC144) (lane 14). Strain designations are indicated in Table 1. Lane N, water used as a negative control. Lane M, molecular markers (sizes in base pairs).

FIG. 3

Multiplex PCR amplification using F. tularensis-specific primers. Samples represent F. tularensis subsp. tularensis strains (FSC138, FSC043, FSC041, and FSC198) (lanes 1 to 4), F. tularensis subsp. holarctica strains (FSC196, FSC155, FSC150, FSC108, and FSC157) (lanes 5 to 9), an F. tularensis subsp. mediaasiatica strain (FSC149) (lane 10), an F. tularensis subsp. holarctica strain from Japan (FSC024) (lane 11), F. tularensis subsp. novicida strains (FSC040 and FSC156) (lanes 12 and 13), and an F. philomiragia strain (FSC144) (lane 14). Strain designations are indicated in Table 1. Lane N, water used as a negative control. Lane M, molecular markers (sizes in base pairs).