Molecular Analysis of Francisella tularensis Subspecies tularensis and holarctica

Fey, Paul D.; Dempsey, Major M.P.; Olson, Michael E.; Chrustowski, M.S.; Engle, Jeniffer L.; Jay, Justin J.; Dobson, Michael E.; Kalasinsky, Kathryn S.; Shea, April A.; Iwen, Peter C.; Wickert, R.C.; Francesconi, Stephen C.; Crawford, R. M. M.; Hinrichs, Steven H.

doi:10.1309/jn3nthk4vvwkjt4a

Cited by 18 publications

(23 citation statements)

References 3 publications

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“…In research laboratories, isolates of F. tularensis have been identified and classified using a variety of molecular typing methods, including amplified fragment length polymorphism (AFLP) analysis [21], pulse-field gel electrophoresis (PFGE) [22], [23], insertion/deletion (INDEL) mutation analysis [24], multi-locus variable number of tandem repeats analysis (MLVA) [25], [26], multi-locus sequence typing (MLST) [2], and whole genome single-nucleotide polymorphism (SNP) analysis [1]. The highest typing resolution has been achieved by MLVA of rapidly mutating tandem repeats, but at a cost sometimes of incorrectly characterizing relationships among distantly related isolates.…”

Section: Introductionmentioning

confidence: 99%

A Real-Time PCR Array for Hierarchical Identification of Francisella Isolates

et al. 2009

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A robust, rapid and flexible real-time PCR assay for hierarchical genetic typing of clinical and environmental isolates of Francisella is presented. Typing markers were found by multiple genome and gene comparisons, from which 23 canonical single nucleotide polymorphisms (canSNPs) and 11 canonical insertion-deletion mutations (canINDELs) were selected to provide phylogenetic guidelines for classification from genus to isolate level. The specificity of the developed assay, which uses 68 wells of a 96-well real-time PCR format with a detection limit of 100 pg DNA, was assessed using 62 Francisella isolates of diverse genetic and geographical origins. It was then successfully used for typing 14 F. tularensis subsp. holarctica isolates obtained from tularemia patients in Sweden in 2008 and five more genetically diverse Francisella isolates of global origins. When applied to human ulcer specimens for direct pathogen detection the results were incomplete due to scarcity of DNA, but sufficient markers were identified to detect fine-resolution differences among F. tularensis subsp. holarctica isolates causing infection in the patients. In contrast to other real-time PCR assays for Francisella, which are typically designed for specific detection of a species, subspecies, or strain, this type of assay can be easily tailored to provide appropriate phylogenetic and/or geographical resolution to meet the objectives of the analysis.

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Section: Introductionmentioning

confidence: 99%

A Real-Time PCR Array for Hierarchical Identification of Francisella Isolates

et al. 2009

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“…analysis (8), pulsed-field gel electrophoresis (12,29), amplified fragment length polymorphism (12,13), canonical insertiondeletions (22), and multilocus variable-number tandem repeat (VNTR) analysis (MLVA) (11,18). These subpopulations are correlated with different host and vector distributions (11,19) and may differ in virulence (29).…”

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confidence: 99%

Phylogeography of Francisella tularensis : Global Expansion of a Highly Fit Clone

et al. 2009

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Francisella tularensis contains several highly pathogenic subspecies, including Francisella tularensis subsp. holarctica, whose distribution is circumpolar in the northern hemisphere. The phylogeography of these subspecies and their subclades was examined using whole-genome single nucleotide polymorphism (SNP) analysis, high-density microarray SNP genotyping, and real-time-PCR-based canonical SNP (canSNP) assays. Almost 30,000 SNPs were identified among 13 whole genomes for phylogenetic analysis. We selected 1,655 SNPs to genotype 95 isolates on a high-density microarray platform. Finally, 23 clade-and subclade-specific canSNPs were identified and used to genotype 496 isolates to establish global geographic genetic patterns. We confirm previous findings concerning the four subspecies and two Francisella tularensis subsp. tularensis subpopulations and identify additional structure within these groups. We identify 11 subclades within F. tularensis subsp. holarctica, including a new, genetically distinct subclade that appears intermediate between Japanese F. tularensis subsp. holarctica isolates and the common F. tularensis subsp. holarctica isolates associated with the radiation event (the B radiation) wherein this subspecies spread throughout the northern hemisphere. Phylogenetic analyses suggest a North American origin for this B-radiation clade and multiple dispersal events between North America and Eurasia. These findings indicate a complex transmission history for F. tularensis subsp. holarctica.

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“…PFGE analysis was shown previously to type and subtype F. tularensis (12,31) and was used as the reference methodology to evaluate a collection of 88 wild-type and 4 well-characterized F. tularensis isolates. PFGE was performed by using two different restriction endonucleases (PmeI and BamHI).…”

Section: Resultsmentioning

confidence: 99%

“…Agarose-embedded chromosomal DNA for PFGE was prepared and digested with the restriction endonucleases PmeI and BamHI (Fermentas, Inc., Glen Burnie, MD) as previously described (12). Agarose gels (1%, wt/vol) contained Francisella DNA digested with PmeI or BamHI and were run for 17.5 h or 16.5 h, respectively, with an initial switch time of 1.8 s and a final switch time of 10.7 s. Migration profiles of the restriction fragments were normalized to SmaI-digested Staphylococcus aureus NCTC 8325 by using Bionumerics software (Applied Maths, Inc., Austin, TX).…”

Section: Methodsmentioning

confidence: 99%

Francisella tularensis Molecular Typing Using Differential Insertion Sequence Amplification

et al. 2011

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Tularemia is a potentially fatal disease that is caused by the highly infectious and zoonotic pathogen Francisella tularensis. Despite the monomorphic nature of sequenced F. tularensis genomes, there is a significant degree of plasticity in the organization of genetic elements. The observed variability in these genomes is due primarily to the transposition of direct repeats and insertion sequence (IS) elements. Since current methods used to genotype F. tularensis are time-consuming and require extensive laboratory resources, IS elements were investigated as a means to subtype this organism. The unique spatial location of specific IS elements provided the basis for the development of a differential IS amplification (DISA) assay to detect and distinguish the more virulent F. tularensis subsp. tularensis (subtypes A.I and A.II) and subsp. holarctica (type B) strains from F. tularensis subsp. novicida and other near neighbors, including Francisella philomiragia and Francisella-like endosymbionts found in ticks. Amplicon sizes and sequences derived from DISA showed heterogeneity within members of the subtype A.I and A.II isolates but not the type B strains. These differences were due to a 312-bp fragment derived from the IS element ISFtu1. Analysis of wild-type F. tularensis isolates by DISA correlated with pulsed-field gel electrophoresis genotyping utilizing two different restriction endonucleases and provided rapid results with minimal sample processing. The applicability of this molecular typing assay for environmental studies was demonstrated by the accurate identification and differentiation of tick-borne F. tularensis. The described approach to IS targeting and amplification provides new capability for epidemiological investigations and characterizations of tularemia source outbreaks.

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Molecular Analysis of Francisella tularensis Subspecies tularensis and holarctica

Cited by 18 publications

References 3 publications

A Real-Time PCR Array for Hierarchical Identification of Francisella Isolates

A Real-Time PCR Array for Hierarchical Identification of Francisella Isolates

Phylogeography of Francisella tularensis : Global Expansion of a Highly Fit Clone

Francisella tularensis Molecular Typing Using Differential Insertion Sequence Amplification

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