Tomida Junko, Morita Yuji, Shibayama Keigo, Kikuchi Ken, Sawa Tomohiro, Akaike Takaaki, Kawamura Yoshiaki
Department of Microbiology, School of Pharmacy, Aichi Gakuin University, Nagoya, Japan.
Department of Bacteriology II, National Institute of Infectious Diseases, Tokyo, Japan.
PLoS One. 2017 Oct 13;12(10):e0186241. doi: 10.1371/journal.pone.0186241. eCollection 2017.
Helicobacter cinaedi is associated with nosocomial infections. The CRISPR-Cas system provides adaptive immunity against foreign genetic elements. We investigated the CRISPR-Cas system in H. cinaedi to assess the potential of the CRISPR-based microevolution of H. cinaedi strains. A genotyping method based on CRISPR spacer organization was carried out using 42 H. cinaedi strains. The results of sequence analysis showed that the H. cinaedi strains used in this study had two CRISPR loci (CRISPR1 and CRISPR2). The lengths of the consensus direct repeat sequences in CRISPR1 and CRISPR2 were both 36 bp-long, and 224 spacers were found in the 42 H. cinaedi strains. Analysis of the organization and sequence similarity of the spacers of the H. cinaedi strains showed that CRISPR arrays could be divided into 7 different genotypes. Each genotype had a different ancestral spacer, and spacer acquisition/deletion events occurred while isolates were spreading. Spacer polymorphisms of conserved arrays across the strains were instrumental for differentiating closely-related strains collected from the same hospital. MLST had little variability, while the CRISPR sequences showed remarkable diversity. Our data revealed the structural features of H. cinaedi CRISPR loci for the first time. CRISPR sequences constitute a valuable basis for genotyping, provide insights into the divergence and relatedness between closely-related strains, and reflect the microevolutionary process of H. cinaedi.
嗜人埃希杆菌与医院感染有关。CRISPR-Cas系统可提供针对外来遗传元件的适应性免疫。我们研究了嗜人埃希杆菌中的CRISPR-Cas系统，以评估基于CRISPR的嗜人埃希杆菌菌株微进化的潜力。使用42株嗜人埃希杆菌菌株进行了基于CRISPR间隔区组织的基因分型方法。序列分析结果表明，本研究中使用的嗜人埃希杆菌菌株有两个CRISPR位点（CRISPR1和CRISPR2）。CRISPR1和CRISPR2中共识直接重复序列的长度均为36 bp，在42株嗜人埃希杆菌菌株中发现了224个间隔区。对嗜人埃希杆菌菌株间隔区的组织和序列相似性分析表明，CRISPR阵列可分为7种不同的基因型。每种基因型都有不同的祖先间隔区，并且在菌株传播过程中发生了间隔区的获得/缺失事件。跨菌株保守阵列的间隔区多态性有助于区分从同一家医院收集的密切相关菌株。多位点序列分型（MLST）的变异性很小，而CRISPR序列显示出显著的多样性。我们的数据首次揭示了嗜人埃希杆菌CRISPR位点的结构特征。CRISPR序列构成了基因分型的宝贵基础，可以深入了解密切相关菌株之间的差异和相关性，并反映嗜人埃希杆菌的微进化过程。
