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Francisella tularensis molecular typing using differential insertion sequence amplification.

Marilynn A Larson1, Paul D Fey, Amanda M Bartling

  • 1University of Nebraska Medical Center, Department of Pathology and Microbiology, 985900 Nebraska Medical Center, Omaha, NE 68198-5900, USA. malarson@unmc.edu

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|May 27, 2011
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Summary

A new assay using insertion sequence (IS) elements effectively subtypes Francisella tularensis, the bacterium causing tularemia. This rapid method aids in distinguishing virulent strains and supports epidemiological investigations.

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Area of Science:

  • Microbiology
  • Genetics
  • Epidemiology

Background:

  • Tularemia is a severe zoonotic disease caused by Francisella tularensis.
  • Francisella tularensis genomes exhibit plasticity due to insertion sequence (IS) elements.
  • Current genotyping methods for F. tularensis are labor-intensive and time-consuming.

Purpose of the Study:

  • To investigate insertion sequence (IS) elements as a tool for subtyping Francisella tularensis.
  • To develop a rapid and efficient molecular assay for differentiating F. tularensis strains.
  • To assess the assay's utility in epidemiological and environmental studies.

Main Methods:

  • Development of a differential IS amplification (DISA) assay targeting specific IS elements.
  • Analysis of amplicon sizes and sequences from various F. tularensis subspecies and related Francisella species.
  • Comparison of DISA results with pulsed-field gel electrophoresis (PFGE) genotyping.
  • Application of the DISA assay to identify tick-borne F. tularensis isolates.

Main Results:

  • The DISA assay successfully differentiated virulent F. tularensis subsp. tularensis (subtypes A.I and A.II) and subsp. holarctica (type B) from other Francisella species.
  • Heterogeneity in amplicon sizes and sequences was observed within subtype A.I and A.II isolates, attributed to a 312-bp fragment from ISFtu1.
  • DISA results correlated well with PFGE genotyping, offering faster results and requiring less sample processing.
  • The assay accurately identified and differentiated tick-borne F. tularensis, demonstrating its environmental applicability.

Conclusions:

  • Insertion sequence (IS) elements provide a valuable basis for developing rapid molecular subtyping methods for Francisella tularensis.
  • The differential IS amplification (DISA) assay offers a practical and efficient approach for strain differentiation and epidemiological surveillance of tularemia.
  • This IS-targeting strategy enhances capabilities for investigating tularemia outbreaks and characterizing sources.