Muscillo M, La Rosa G, Sali M, De Carolis E, Adone R, Ciuchini F, Fasanella A
Department of Environment and Primary Prevention, Istituto Superiore Sanità Roma, Rome.
J Clin Microbiol. 2005 Sep;43(9):4758-65. doi: 10.1128/JCM.43.9.4758-4765.2005.
Several circulating Bacillus anthracis strains isolated in Italy and belonging to the A1.a cluster, genotype 3 (A1.a-3) are genotypically indistinguishable from Carbosap, a live attenuated vaccine strain, containing both pXO1 and pXO2 plasmids. The genotype was assessed by using eight-locus multilocus variable-number tandem repeat analysis. We describe here the use of a ninth locus able to explore variability among strains that have the same genotype. It is important to be able to genotype the wild isolate of B. anthracis strains from outbreaks of anthrax in areas where Carbosap vaccination of cattle and sheep is common practice. A total of 27 representative field strains isolated in Italy and four vaccinal strains, namely, Carbosap, Sterne, Pasteur I, and Pasteur II, were characterized by a ninth marker, called pXO2-A. Twenty-three field strains were genotype 3 and therefore identical to Carbosap. The marker was in the pXO2 plasmid and is based on the polymorphism of the already-known VX2-3 locus. Detection was obtained by PCR with fluorescence-labeled forward primers in order to produce appropriate fragments for capillary electrophoresis with an ABI 310 genetic analyzer. Genetic relationships showed heterogeneity in all of the examined samples. Interestingly, with respect to genotype 3, samples grouped into eight different subtypes, A to H, and the subtype G, had only two samples indistinguishable from Carbosap. The results of the present study confirm the validity of a hierarchical progressive protocol for discrimination among closely related isolates.
在意大利分离出的几种属于A1.a簇、基因型3（A1.a - 3）的循环炭疽芽孢杆菌菌株，在基因上与含有pXO1和pXO2质粒的减毒活疫苗菌株Carbosap无法区分。通过八基因座多位点可变数目串联重复分析评估基因型。我们在此描述使用第九个基因座来探索具有相同基因型的菌株之间的变异性。能够对炭疽病爆发地区的炭疽芽孢杆菌野生分离株进行基因分型很重要，在这些地区，对牛羊进行Carbosap疫苗接种是常见做法。总共27株在意大利分离的代表性野外菌株和4株疫苗菌株，即Carbosap、Sterne、巴斯德I和巴斯德II，用一个名为pXO2 - A的第九个标记进行了表征。23株野外菌株是基因型3，因此与Carbosap相同。该标记位于pXO2质粒中，基于已知的VX2 - 3基因座的多态性。通过使用荧光标记的正向引物进行PCR检测，以便产生适合用ABI 310遗传分析仪进行毛细管电泳的片段。遗传关系表明所有检测样本均存在异质性。有趣的是，对于基因型3，样本分为八个不同的亚型，A至H，其中亚型G只有两个样本与Carbosap无法区分。本研究结果证实了用于区分密切相关分离株的分层递进方案的有效性。
