Francisella tularensis Molecular Typing Using Differential Insertion Sequence Amplification

Abstract

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.

Occurrence and Control of Tularemia in Drinking Water

“…holarctica (type B), and F. tularensis subsp. mediasiatica (Larson et al 2011). F. tularensis subsp.…”

“…F. novicida is another member of this genus, but there is contention over whether it should be considered as a separate species or transferred to the rank of subspecies (Johansson et al 2010). F. tularensis is genetically monomorphic with a >99% sequence similarity between subspecies tularensis and holarctica (Larson et al 2011).…”

Francisella tularensisBacteria Associated with Feline Tularemia in the United States

“…For genotyping, we used the PCR-based differential insertion sequence amplification (DISA) method with the CR10 C+L+S primer set and pulsed-field gel electrophoresis (PFGE) of Pme I-digested Francisella spp. DNA, as previously described ( 9 ).…”

Francisella tularensis Subtype A.II Genomic Plasticity in Comparison with Subtype A.I

“…This isolate was identified as F . tularensis WY-00W4114 (hereafter referred to as W4114) and further characterized by pulsed-field gel electrophoresis (PFGE) and differential insertion sequence amplification (DISA) [ 17 ]. Even though both the PFGE clustering algorithm and the PCR-based DISA assay classified this isolate as an A.II strain, considerable chromosomal diversity existed in W4114 when compared to other strains within this subtype; in addition, the A.II subpopulation possessed greater diversity in PFGE typing patterns than the A.I clade [ 17 ].…”