Assessing the Genotypic Differences between Strains of <i>Corynebacterium pseudotuberculosis</i> biovar <i>equi</i> through Comparative Genomics

Seven genomes of Corynebacterium pseudotuberculosis biovar equi were sequenced on the Ion Torrent PGM platform, generating high-quality scaffolds over 2.35 Mbp. This bacterium is the causative agent of disease known as “pigeon fever” which commonly affects horses worldwide. The pangenome of biovar equi was calculated and two phylogenomic approaches were used to identify clustering patterns within Corynebacterium genus. Furthermore, other comparative analyses were performed including the prediction of genomic islands and prophages, and SNP-based phylogeny. In the phylogenomic tree, C. pseudotuberculosis was divided into two distinct clades, one formed by nitrate non-reducing species (biovar ovis) and another formed by nitrate-reducing species (biovar equi). In the latter group, the strains isolated from California were more related to each other, while the strains CIP 52.97 and 1/06-A formed the outermost clade of the biovar equi. A total of 1,355 core genes were identified, corresponding to 42.5% of the pangenome. This pangenome has one of the smallest core genomes described in the literature, suggesting a high genetic variability of biovar equi of C. pseudotuberculosis. The analysis of the similarity between the resistance islands identified a higher proximity between the strains that caused more severe infectious conditions (infection in the internal organs). Pathogenicity islands were largely conserved between strains. Several genes that modulate the pathogenicity of C. pseudotuberculosis were described including peptidases, recombination enzymes, micoside synthesis enzymes, bacteriocins with antimicrobial activity and several others. Finally, no genotypic differences were observed between the strains that caused the three different types of infection (external abscess formation, infection with abscess formation in the internal organs, and ulcerative lymphangitis). Instead, it was noted that there is a higher phenetic correlation between strains isolated at California compared to the other strains. Additionally, high variability of resistance islands suggests gene acquisition through several events of horizontal gene transfer.本研究基于离子激流PGM（Ion Torrent PGM）测序平台，对7株假结核棒状杆菌马生物型（Corynebacterium pseudotuberculosis biovar equi）进行全基因组测序，获得了总长超过2.35兆碱基对（Mbp）的高质量测序支架（scaffolds）。该细菌是“鸽热症（pigeon fever）”的致病原，该病害在全球范围内普遍感染马属动物。本研究对该生物型的泛基因组（pangenome）进行了分析，并采用两种系统基因组学方法解析棒状杆菌属（Corynebacterium genus）内的聚类模式。此外，还开展了多项比较基因组学分析，包括基因组岛（genomic islands）与前噬菌体（prophages）的预测、基于单核苷酸多态性（SNP）的系统发育分析。系统发育树结果显示，假结核棒状杆菌可划分为两个独立的进化枝：其一为不还原硝酸盐的羊生物型（biovar ovis）类群，其二为还原硝酸盐的马生物型类群。在马生物型类群中，加利福尼亚分离株彼此亲缘关系更近，而菌株CIP 52.97与1/06-A则构成了马生物型最外围的进化枝。本研究共鉴定得到1355个核心基因（core genes），占泛基因组的42.5%。该泛基因组的核心基因组占比在已报道的同类研究中处于较低水平，提示假结核棒状杆菌马生物型具有较高的遗传变异性。对耐药基因组岛的相似性分析表明，引发更严重感染症状（内脏感染）的菌株之间亲缘关系更为接近。不同菌株间的致病岛（pathogenicity islands）整体较为保守。本研究还鉴定出多种调控假结核棒状杆菌致病性的基因，包括肽酶（peptidases）、重组酶（recombination enzymes）、米科糖苷合成酶（micoside synthesis enzymes）、具有抗菌活性的细菌素（bacteriocins）等多种因子。最后，本研究未发现引发三种不同感染类型（外脓肿形成、内脏脓肿感染、溃疡性淋巴管炎）的菌株之间存在基因型差异；相反，相较于其他菌株，加利福尼亚分离株间的表型相关性更高。此外，耐药基因组岛的高度变异性提示，该类群可通过多次水平基因转移（horizontal gene transfer）事件获取外源基因。