Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, The Netherlands.
Microbiology (Reading). 2011 Nov;157(Pt 11):3113-3123. doi: 10.1099/mic.0.051953-0. Epub 2011 Aug 26.
Rapid identification of hypervirulent Clostridium difficile strains is essential for preventing their spread. Recent completion of several full-length C. difficile genomes provided an excellent opportunity to identify potentially unique genes that characterize hypervirulent strains. Based on sequence comparisons between C. difficile strains we describe two gene insertions into the genome of hypervirulent PCR ribotypes 078 and 027. Analysis of these regions, of 1.7 and 4.2 kb, respectively, revealed that they contain several interesting ORFs. The 078 region is inserted intergenically and introduces an enzyme that is involved in the biosynthesis of several antibiotics. The 027 insert disrupts the thymidylate synthetase (thyX) gene and replaces it with an equivalent, catalytically more efficient, thyA gene. Both gene insertions were used to develop ribotype-specific PCRs, which were validated by screening a large strain collection consisting of 68 different PCR ribotypes supplemented with diverse 078 and 027 strains derived from different geographical locations and individual outbreaks. The genetic markers were stably present in the hypervirulent PCR ribotypes 078 and 027, but were also found in several other PCR ribotypes. Comparative analysis of amplified fragment length polymorphisms, PCR ribotype banding patterns and toxin profiles showed that all PCR ribotypes sharing the same insert from phylogenetically coherent clusters. The identified loci are unique to these clusters, to which the hypervirulent ribotypes 078 and 027 belong. This provides valuable information on strains belonging to two distinct lineages within C. difficile that are highly related to hypervirulent strains.
快速鉴定产毒力强的艰难梭菌对于防止其传播至关重要。最近完成的几个艰难梭菌全长基因组为鉴定可能具有特征性的产毒力强菌株的独特基因提供了极好的机会。基于艰难梭菌菌株之间的序列比较，我们描述了两种基因插入产毒力强的 PCR 型 078 和 027 的基因组中。分别对这两个区域，即 1.7 kb 和 4.2 kb 进行分析，结果表明它们包含几个有趣的开放阅读框。078 区插入基因间，引入了一种参与几种抗生素生物合成的酶。027 插入破坏了胸苷酸合酶（thyX）基因，并以催化效率更高的 thyA 基因取代。这两个基因插入物都被用来开发 PCR 型特异性引物，通过对包含 68 种不同 PCR 型的大型菌株集进行筛选来验证这些引物，这些菌株集还补充了来自不同地理位置和个体暴发的各种 078 和 027 菌株。遗传标记在产毒力强的 PCR 型 078 和 027 中稳定存在，但也存在于其他几种 PCR 型中。扩增片段长度多态性、PCR 型带型和毒素谱的比较分析表明，所有携带相同插入物的 PCR 型都来自系统发育一致的聚类。鉴定的基因座是这些聚类所特有的，产毒力强的 PCR 型 078 和 027 就属于这些聚类。这为属于艰难梭菌两个不同谱系的菌株提供了有价值的信息，这些谱系与产毒力强的菌株高度相关。
