Francisella tularensis Strain Typing Using Multiple-Locus, Variable-Number Tandem Repeat Analysis

Francisella tularensis, the etiological agent of tularemia, is found throughout the Northern hemisphere. After analyzing the F. tularensis genomic sequence for potential variable-number tandem repeats (VNTRs), we developed a multilocus VNTR analysis (MLVA) typing system for this pathogen. Variation was detected at six VNTR loci in a set of 56 isolates from California, Oklahoma, Arizona, and Oregon and the F. tularensis live vaccine strain. PCR assays revealed diversity at these loci with total allele numbers ranging from 2 to 20, and Nei's diversity index values ranging from 0.36 to 0.93. Cluster analysis identified two genetically distinct groups consistent with the current biovar classification system of F. tularensis. These findings suggest that these VNTR markers are useful for identifying F. tularensis isolates at this taxonomic level. In this study, biovar B isolates were less diverse than those in biovar A, possibly reflecting the history of tularemia in North America. Seven isolates from a recent epizootic in Maricopa County, Ariz., were identical at all VNTR marker loci. Their identity, even at a hypervariable VNTR locus, indicates a common source of infection. This demonstrates the applicability of MLVA for rapid characterization and identification of outbreak isolates. Future construction of reference databases will allow faster outbreak tracking as well as providing a foundation for deciphering global genetic relationships.

土拉弗朗西斯菌（Francisella tularensis）是土拉菌病的病原菌，广泛分布于北半球。本研究针对土拉弗朗西斯菌的基因组序列开展潜在可变数目串联重复序列（variable-number tandem repeats, VNTRs）筛查，进而为该病原菌构建了一套多位点可变数目串联重复序列分析（multilocus VNTR analysis, MLVA）分型系统。针对采自美国加利福尼亚州、俄克拉荷马州、亚利桑那州、俄勒冈州的56株分离株，以及土拉弗朗西斯菌活疫苗株的分析显示，6个VNTR位点均存在序列变异。聚合酶链式反应（PCR）检测结果表明，上述位点存在显著多态性：等位基因数目范围为2至20，奈氏多样性指数（Nei's diversity index）取值介于0.36至0.93之间。聚类分析结果显示，可将供试菌株划分为2个遗传差异显著的类群，该划分结果与土拉弗朗西斯菌现行的生物型分类系统一致。上述研究结果表明，本研究所筛选的VNTR分子标记可有效用于该分类层级下土拉弗朗西斯菌分离株的鉴定分型。本研究中，生物型B的分离株多态性低于生物型A，该现象或可反映北美地区土拉菌病的流行历史。采自美国亚利桑那州马里科帕县近期动物疫情的7株分离株，在所有VNTR标记位点上均呈现完全一致的基因型；即便在高变VNTR位点上，这些菌株的基因型仍完全一致，表明其感染来源具有同源性。该结果证实了MLVA分型系统可快速完成暴发分离株的特征鉴定与分型。未来参考数据库的构建，不仅可实现疫情暴发的快速溯源追踪，同时也为解析全球范围内土拉弗朗西斯菌的遗传进化关系提供了研究基础。