Abstract
To develop effective and accurate typing of strains of Francisella tularensis, a potent human pathogen and a putative bioterrorist agent, we combined analysis of insertion-deletion (indel) markers with multiple-locus variable-number tandem repeat analysis (MLVA). From 5 representative F. tularensis genome sequences, 38 indel markers with canonical properties, i.e., capable of sorting strains into major genetic groups, were selected. To avoid markers with a propensity for homoplasy, we used only those indels with 2 allelic variants and devoid of substantial sequence repeats. MLVA included sequences with much diversity in copy number of tandem repeats. The combined procedure allowed subspecies division, delineation of clades A.I and A.II of subspecies tularensis, differentiation of Japanese strains from other strains of subspecies holarctica, and high-resolution strain typing. The procedure uses limited amounts of killed bacterial preparations and, because only 1 single analytic method is needed, is time- and cost-effective.
Highlights
To develop effective and accurate typing of strains of Francisella tularensis, a potent human pathogen and a putative bioterrorist agent, we combined analysis of insertiondeletion markers with multiple-locus variable-number tandem repeat analysis (MLVA)
Human disease caused by F. tularensis subsp. tularensis may be fulminate or even lethal, whereas disease caused by other subspecies is less severe, often incapacitating and protracted [4]
pulsed-field gel electrophoresis (PFGE) typing is, far from ideal for the purpose. It involves making concentration-adjusted suspensions of live bacteria, which has the potential for creating infectious aerosols, is time-consuming, produces complex banding pattern data, and has a restrictive discriminatory capacity when applied to F. tularensis [7,14,15,16,17]
Summary
To develop effective and accurate typing of strains of Francisella tularensis, a potent human pathogen and a putative bioterrorist agent, we combined analysis of insertiondeletion (indel) markers with multiple-locus variable-number tandem repeat analysis (MLVA). Based on virulence tests and biochemical assays, F. tularensis is divided into 4 subspecies, a division that has recently been corroborated by genetic typing [1,2]. Subspecies determination of F. tularensis typically involves biochemical fermentations Such analyses are labor-intensive, hampered by the fastidious growth characteristics of the organism on artificial media, and associated with a substantial risk for laboratory-acquired infections [2,9]. It involves making concentration-adjusted suspensions of live bacteria, which has the potential for creating infectious aerosols, is time-consuming, produces complex banding pattern data, and has a restrictive discriminatory capacity when applied to F. tularensis [7,14,15,16,17]
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