Integrative genomic, virulence, and transcriptomic analysis of emergent Streptococcus dysgalactiae subspecies equisimilis (SDSE) emm type stG62647 isolates causing human infections

Streptococcus dysgalactiae subspecies equisimilis (SDSE) is a Gram-positive bacterial pathogen that infects humans and is closely related to group A streptococcus (GAS). Compared to GAS, far less is known about SDSE pathobiology. Increased rates of invasive SDSE infections have recently been reported in many countries. One SDSE emm type, stG62647, is known to cause severe diseases, including necrotizing soft-tissue infections, endocarditis, and osteoarticular infections. To increase our understanding of the molecular pathogenesis of stG62647 SDSE isolates causing human infections, we sequenced to closure the genomes of 120 stG62647 SDSE isolates. The genomes varied in size from 2.1 to 2.24 megabase pairs. Consistent with previous data, the great majority of stG62647 isolates had IS1548 integrated into the silB gene, thereby inactivating it. Regions of difference in gene content, including putative mobile genetic elements, were the largest source of genomic diversity. All 120 stG62647 isolates were assayed for virulence using a well-established mouse model of necrotizing myositis. An unexpectedly wide range of virulence was identified (20% to 95%), as assessed by near-mortality data. To explore the molecular mechanisms underlying virulence differences, we analyzed RNAseq transcriptome profiles for 38 stG62647 isolates (comprising the 19 least and most virulent) grown in vitro. Genetic polymorphisms were identified from whole-genome sequence data. Collectively, the results suggest that these SDSE isolates use multiple genetic pathways to achieve an altered virulence phenotype. Our study integrates genomic, mouse virulence, and RNAseq data to advance our understanding of SDSE pathobiology and its molecular pathogenesis. 120 stG62647 SDSE isolates identified in a comprehensive sample of 499 isolates cultured from humans and recovered between 2010 and 2018 in French Brittany were tested for virulence using a mouse model of necrotizing myositis. Of these 120, 19 isolates with near_mortality rates ranging between 20% to 30% (classified as low-virulence isolates), and 19 isolates with near_mortality rates ranging between 70% and 95% (high-virulence isolates), were grown in triplicate, in THY broth in vitro, and RNA-seq cDNA libraries were generated at the mid-exponential phase of growth (ME, OD600=1).

停乳链球菌似马亚种（Streptococcus dysgalactiae subspecies equisimilis, SDSE）是一种可感染人类的革兰氏阳性细菌病原体，与A群链球菌（group A streptococcus, GAS）亲缘关系密切。相较于GAS，学界对SDSE致病生物学的认知仍相对有限。近年来，全球多国均报道了侵袭性SDSE感染的发病率上升趋势。其中一种SDSE的emm分型菌株stG62647，可引发坏死性软组织感染、心内膜炎及骨关节感染等重症疾病。

为深入解析引发人类感染的stG62647型SDSE分离株的分子致病机制，本研究对120株stG62647型SDSE分离株完成全基因组闭合测序。这些菌株的基因组大小介于2.1至2.24兆碱基对之间。与既往研究结果一致，绝大多数stG62647分离株的silB基因中均整合有IS1548插入序列，进而导致该基因失活。基因含量差异区域（包括推定移动遗传元件）是基因组多样性的主要来源。

本研究采用已成熟的坏死性肌炎小鼠模型，对全部120株stG62647分离株开展毒力评价。通过近似死亡率数据评估发现，菌株的毒力分布范围远超预期（20%至95%）。为探究毒力差异背后的分子机制，我们对38株stG62647分离株（分别为毒力最低和最高的各19株）的体外培养样本进行了RNA-seq转录组分析。同时基于全基因组测序数据鉴定了遗传多态性位点。

综合以上结果表明，这些SDSE分离株可通过多种遗传途径实现毒力表型的改变。本研究整合基因组学、小鼠毒力实验及RNA-seq数据，推动了学界对SDSE致病生物学及其分子致病机制的认知。

本研究涉及的120株stG62647型SDSE分离株，采自2010至2018年间法国布列塔尼地区从人类患者体内分离培养的499株菌株的综合性样本库，并通过坏死性肌炎小鼠模型完成毒力测试。在这120株菌株中，分别选取近似死亡率介于20%至30%的19株（归类为低毒力分离株），以及近似死亡率介于70%至95%的19株（高毒力分离株），将其在THY培养基（THY broth）中进行三次重复体外培养，并在指数生长中期（ME，OD600=1）构建RNA-seq cDNA文库。