Characterization of Tularemia Cases in Slovenia with Multiple-Locus Variable-Number Tandem Repeat Analysis

Abstract

Francisella tularensis is the etiologic agent of tularemia, a bacterial zoonotic disease. The genome of F. tularensis shows a recent evolutionary change, especially in reservoirs. Variable number of tandem repeats (VNTR) is described as a high-speed molecular clock and can thus be used as a high-resolution typing system. The main objective of our study was to investigate the molecular diversity of F. tularensis strains and reveal possible sources of infection. Using real-time PCR targeting the ISFtu2 region, we successfully amplified targeted DNA in 13/31 Slovenian patients with a clinical diagnosis of tularemia, and with PCR targeting the fopA gene, we obtained 11/13 PCR products. Sequencing revealed that all samples were identified as F. tularensis subsp. holarctica. We successfully obtained one F. tularensis isolate from a lymph node aspirate by culture on chocolate agar. Our isolate was clustered into major clade B12 (subclade B43). We optimized VNTR typing to be used directly on clinical samples. Multiple-locus VNTR analysis (MLVA) revealed five unique MLVA types; 45.5% samples had the same MLVA type, another 27.3% shared a different MLVA type, and each of the remaining had a unique MLVA type. Most samples differed at only two VNTR markers (Ft-M03 and Ft-M06). Additionally, we investigated samples from small mammals (n = 532) and Ixodes ricinus ticks (n = 232) captured in the same geographical area in which patients with tularemia were found. No F. tularensis DNA was detected in samples of small mammals or I. ricinus ticks. The diversity of MLVA types in Slovenia was high, despite the small region, but most of the samples from the same region shared the same MLVA type. Our results suggest that MLVA is a useful tool for quick molecular characterization of F. tularensis directly from patient samples, especially when investigating geographically localized outbreaks.

Keywords: MLVA; Slovenia; WGS; clinical cases; culture; tularemia.

FIG. 1.

Clusters of human tularemia cases in Slovenia based on patients' place of residence. Color images are available online.

FIG. 2.

Distribution of MLVA types in Slovenia. (A) Colors correspond to tularemia cases according to MLVA type. (B) Minimum spanning tree based on MLVA; the unweighted pair group method with the arithmetic mean phylogram is based on multiple-locus variable-number tandem repeats. Bootstrap values >80% are given at the respective nodes and calculated using 10,000 iterations. The number on the connecting bars indicates genetic distance. MLVA, multiple-locus variable number of tandem repeat analysis. Color images are available online.