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

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.

A fuzzy distance metric for measuring the dissimilarity of planar chromatographic profiles with application to denaturing gradient gel electrophoresis data from human skin microbes: demonstration of an individual and gender-based fingerprint.

A newly devised fuzzy metric for measuring the dissimilarity between two planar chromatographic profiles is proposed in this paper. It does not require an accurately assigned sample-feature matrix and can cope with slight imprecision of the positional information. This makes it very suitable for 1-D techniques which do not have a second spectroscopic dimension to aid variable assignment. The usefulness of this metric has been demonstrated on a large data set consisting of nearly 400 samples from Denaturing Gradient Gel Electrophoresis (DGGE) analysis of microbes on human skin. The pattern revealed by this dissimilarity metric was compared with the one represented by a sample-feature matrix and highly consistent results were obtained. Several pattern recognition techniques have been applied on the dissimilarity matrix based on this dissimilarity metric. According to rank analysis, within-individual variation is significantly less than between-individual variation, suggesting a unique individual microbial fingerprint. Principal Coordinates Analysis (PCO) suggests that there is a considerable separation between genders. These results suggest that there are specific microbial colonies characteristic of individuals.

Evaluation of detection of Legionella spp. in water samples by fluorescence in situ hybridization, PCR amplification and bacterial culture.

One hundred water samples (32 from clinical units and 68 from private households) were examined for Legionella by culture, fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR). Twenty-four samples were positive by culture (22 L. pneumophila; 2 non-pneumophila species), 36 by FISH (32 L. pneumophila; 4 non-pneumophila species) and 75 by PCR (41 positive for L. pneumophila; 26 positive for L. pneumophila and a non-pneumophila species; 8 positive for non-pneumophila species). PCR and FISH results were compared to bacterial culture as the "gold standard" method by calculating sensitivities and specificities, respectively: PCR assays, 96% and 47%; FISH assays, 67% and 72%, respectively. In comparison with FISH the lower specificity of PCR is probably caused by dead Legionella bacteria and/or free Legionella DNA in potable water, and the higher sensitivity of PCR may be explained by the detection limit of fluorescence microscopy. In conclusion, the relatively high specificity, sensitivity and quickness of the FISH assay offer significant advantages over conventional PCR and culture-based techniques.