Rapid differentiation of Francisella species and subspecies by fluorescent in situ hybridization targeting the 23S rRNA

Abstract

Background: Francisella (F.) tularensis is the causative agent of tularemia. Due to its low infectious dose, ease of dissemination and high case fatality rate, F. tularensis was the subject in diverse biological weapons programs and is among the top six agents with high potential if misused in bioterrorism. Microbiological diagnosis is cumbersome and time-consuming. Methods for the direct detection of the pathogen (immunofluorescence, PCR) have been developed but are restricted to reference laboratories.

Results: The complete 23S rRNA genes of representative strains of F. philomiragia and all subspecies of F. tularensis were sequenced. Single nucleotide polymorphisms on species and subspecies level were confirmed by partial amplification and sequencing of 24 additional strains. Fluorescent In Situ Hybridization (FISH) assays were established using species- and subspecies-specific probes.Different FISH protocols allowed the positive identification of all 4 F. philomiragia strains, and more than 40 F. tularensis strains tested. By combination of different probes, it was possible to differentiate the F. tularensis subspecies holarctica, tularensis, mediasiatica and novicida. No cross reactivity with strains of 71 clinically relevant bacterial species was observed. FISH was also successfully applied to detect different F. tularensis strains in infected cells or tissue samples. In blood culture systems spiked with F. tularensis, bacterial cells of different subspecies could be separated within single samples.

Conclusion: We could show that FISH targeting the 23S rRNA gene is a rapid and versatile method for the identification and differentiation of F. tularensis isolates from both laboratory cultures and clinical samples.

Figure 1

Primer positions for the synthesis of two overlapping 23S rRNA gene fragments covering the complete 23S rRNA gene. Francisella tularensis generally contains three rRNA operons in its entire genome. Analysis of the available whole genomes revealed that theses operons have identical nucleotide sequences.

Figure 2

Phylogentic tree based on nearly complete nucleotide sequences of 23S rRNA gene of F. tularensis and F. philomiragia generated using a neighbor joining analysis. Bootstrap values based on 1000 resamplings are indicated at branch points.

Figure 3

Left: Artificial mixture of F. tularensis subsp. tularensis (Schu S4, red circle) and F. philomiragia (ATCC 25017, green circle), phase contrast microscopy. Right: Fluorescence microscopy after hybridization with probes Bwall1448-Cy3 and Bwphi1448-6-FAM with 50% formamide. The green, pleomorphic cells of F. philomiragia can be easily distinguished from the smaller, coccoid rods of the highly virulent F. tularensis subsp. tularensis strain showing red fluorescence.

Figure 4

Left: Artificial mixture of F. tularensis subsp. tularensis (Schu S4, green circle) and F. tularensis subsp. mediasiatica (FSC 148, red circle), phase contrast microscopy. Right: Fluorescence microscopy after hybridization with probes Bwmed1379-Cy3 and Bwtume168II-6-FAM with 20% formamide. F. tularensis subsp. tularensis cells only bind to probe Bwtume168II-6-FAM (green fluorescence) whereas bacterial cells of F. tularensis subsp. mediasiatica bind to both probes resulting in a yellow-orange fluorescence.

Figure 5

Two-step algorithm for the rapid identification and differentiation of Francisella strains using fluorescence in situ hybridization. After an initial hybridization step with three probes including the "pan-Francisella" probe Bw-all1488, negative samples can directly be reported. Performing internal controls with probe EUB-338 allows recognizing false negative results caused by technical problems. After hybridization with all species- and subspecies-specific probes in parallel, initially positive samples can be further differentiated by following the algorithm depicted in step two allowing unambiguous identification to subspecies level.

Figure 6

Specific detection of F. tularensis subsp. holarctica in a liver tissue sample (mouse) fixed in formalin and embedded in paraffin for more than four years. After deparaffinization and fluorescence in situ hybridization, bacterial cells can be visualized in small granuloma (A: phase contrast microscopy; B: fluorescence microscopy, DAPI staining; C: Specific staining of F. tularensis subsp. holarctica).

Figure 7

Cytospin preparation of infected U 937 cell culture followed by specific detection of the facultative pathogen F. tularensis subsp. novicida (MOI 10:1, 24 h). (A: phase contrast microscopy; B: FISH, probe EUB338-6-FAM; C: FISH, probe Bwnov168-Cy3).

Figure 8

Mixed sample of bacterial cells from F. tularensis tularensis (ATCC 6223) and F. tularensis subsp. holarctica LVS (ratio 100:1). Contamination lower than 1% could be identified using appropriate probe sets. (A: FISH staining with probe EUB338-6-FAM for staining of all bacteria in liquid samples. B: Specific staining of F. tularensis subsp. holarctica).