Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
Slovenian Museum of Natural History, Ljubljana, Slovenia.
Vector Borne Zoonotic Dis. 2021 May;21(5):351-357. doi: 10.1089/vbz.2020.2711. Epub 2021 Feb 18.
is the etiologic agent of tularemia, a bacterial zoonotic disease. The genome of 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 strains and reveal possible sources of infection. Using real-time PCR targeting the IS2 region, we successfully amplified targeted DNA in 13/31 Slovenian patients with a clinical diagnosis of tularemia, and with PCR targeting the gene, we obtained 11/13 PCR products. Sequencing revealed that all samples were identified as subsp. . We successfully obtained one 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 ( = 532) and ticks ( = 232) captured in the same geographical area in which patients with tularemia were found. No DNA was detected in samples of small mammals or 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 directly from patient samples, especially when investigating geographically localized outbreaks.
是土拉弗朗西斯菌的病原体，一种细菌性人畜共患病。 的基因组显示出最近的进化变化，特别是在储存库中。可变数目的串联重复（VNTR）被描述为高速分子钟，因此可以用作高分辨率的分型系统。我们研究的主要目的是调查 菌株的分子多样性，并揭示可能的感染源。使用针对 IS2 区域的实时 PCR，我们成功地扩增了 13/31 名斯洛文尼亚临床诊断为土拉弗朗西斯菌病的患者的目标 DNA，并且使用针对 基因的 PCR，我们获得了 11/13 的 PCR 产物。测序表明，所有样本均被鉴定为 亚种。我们通过在巧克力琼脂上培养成功地从淋巴结吸出物中获得了一个 分离株。我们的分离株聚类到主要分支 B12（子分支 B43）。我们成功地优化了 VNTR 分型，以便直接用于临床样本。多位点 VNTR 分析（MLVA）显示了五种独特的 MLVA 类型；45.5%的样本具有相同的 MLVA 类型，另外 27.3%的样本具有不同的 MLVA 类型，其余每个样本都具有独特的 MLVA 类型。大多数样本仅在两个 VNTR 标记（Ft-M03 和 Ft-M06）上存在差异。此外，我们还调查了在发现土拉弗朗西斯菌病患者的同一地理区域捕获的小哺乳动物（ = 532）和 蜱（ = 232）的样本。在小哺乳动物或 蜱样本中均未检测到 DNA。尽管斯洛文尼亚的 MLVA 类型多样性很高，但由于该地区较小，因此来自同一地区的大多数样本都具有相同的 MLVA 类型。我们的研究结果表明，MLVA 是一种从患者样本中快速进行 分子特征分析的有用工具，尤其是在调查地理位置局部爆发时。
