Thierry Simon, Tourterel Christophe, Le Flèche Philippe, Derzelle Sylviane, Dekhil Neira, Mendy Christiane, Colaneri Cécile, Vergnaud Gilles, Madani Nora
University Paris-Est, Anses, Animal Health Laboratory, Bacterial Zoonosis Unit, Maisons-Alfort, France.
Univ Paris-Sud, Institut de Génétique et Microbiologie, UMR 8621, Orsay, France; CNRS, Orsay, France.
PLoS One. 2014 Jun 5;9(6):e95131. doi: 10.1371/journal.pone.0095131. eCollection 2014.
Bacillus anthracis is known to have low genetic variability. In spite of this lack of diversity, multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) and single nucleotide polymorphisms (SNPs) including the canonical SNPs assay (canSNP) have proved to be highly effective to differentiate strains. Five different MLVA schemes based on a collection of 31 VNTR loci (MLVA8, MLVA15, MLVA20, MLVA25 and MLVA31) with increased resolving power have been described.
MLVA31 was applied to characterize the French National Reference Laboratory collection. The total collection of 130 strains is resolved in 35 genotypes. The 119 veterinary and environmental strains collection in France were resolved into 26 genotypes belonging to three canSNP lineages and four MLVA clonal complexes (CCs) with particular geographical clustering. A subset of seven loci (MLVA7) is proposed to constitute a first line assay. The loci are compatible with moderate resolution equipment such as agarose gel electrophoresis and show a good congruence value with MLVA31. The associated MLVA and SNP data was imported together with published genotyping data by taking advantage of major enhancements to the MLVAbank software and web site.
The present report provides a wide coverage of the genetic diversity of naturally occurring B. anthracis strains in France as can be revealed by MLVA. The data obtained suggests that once such coverage is achieved, it becomes possible to devise optimized first-line MLVA assays comprising a sufficiently low number of loci to be typed either in one multiplex PCR or on agarose gels. Such a selection of seven loci is proposed here, and future similar investigations in additional countries will indicate to which extend the same selection can be used worldwide as a common minimum set. It is hoped that this approach will contribute to an efficient and low-cost routine surveillance of important pathogens for biosecurity such as B. anthracis.
已知炭疽芽孢杆菌的遗传变异性较低。尽管缺乏多样性，但多位点可变数目串联重复序列（VNTR）分析（MLVA）和单核苷酸多态性（SNP），包括标准SNP检测（canSNP），已被证明在区分菌株方面非常有效。已经描述了基于31个VNTR位点集合（MLVA8、MLVA15、MLVA20、MLVA25和MLVA31）的五种不同MLVA方案，其分辨能力有所提高。
MLVA31被用于对法国国家参考实验室的菌株集合进行特征分析。130株菌株的总集合被分为35种基因型。法国的119株兽医和环境菌株集合被分为26种基因型，属于三个canSNP谱系和四个MLVA克隆复合体（CCs），具有特定的地理聚类。提出了一个由七个位点组成的子集（MLVA7）作为一线检测方法。这些位点与中等分辨率设备如琼脂糖凝胶电泳兼容，并与MLVA31显示出良好的一致性值。利用MLVAbank软件和网站的重大改进，将相关的MLVA和SNP数据与已发表的基因分型数据一起导入。
本报告提供了法国自然发生的炭疽芽孢杆菌菌株遗传多样性的广泛覆盖情况，这可以通过MLVA揭示。获得的数据表明，一旦实现这种覆盖，就有可能设计出优化的一线MLVA检测方法，其包含的位点数量足够少，可以在一次多重PCR或琼脂糖凝胶上进行分型。这里提出了这样一个由七个位点组成的选择，未来在其他国家进行的类似调查将表明这种选择在全球范围内可以作为一个共同的最小集合使用的程度。希望这种方法将有助于对炭疽芽孢杆菌等生物安全重要病原体进行高效、低成本的常规监测。
