Table_5_Pathogenomes of Atypical Non-shigatoxigenic Escherichia coli NSF/SF O157:H7/NM: Comprehensive Phylogenomic Analysis Using Closed Genomes.XLSX

The toxigenic conversion of Escherichia coli strains by Shiga toxin-converting (Stx) bacteriophages were prominent and recurring events in the stepwise evolution of enterohemorrhagic E. coli (EHEC) O157:H7 from an enteropathogenic (EPEC) O55:H7 ancestor. Atypical, attenuated isolates have been described for both non-sorbitol fermenting (NSF) O157:H7 and SF O157:NM serotypes, which are distinguished by the absence of Stx, the characteristic virulence hallmark of Stx-producing E. coli (STEC). Such atypical isolates either never acquired Stx-phages or may have secondarily lost stx during the course of infection, isolation, or routine subculture; the latter are commonly referred to as LST (Lost Shiga Toxin)-isolates. In this study we analyzed the genomes of 15 NSF O157:H7 and SF O157:NM strains from North America, Europe, and Asia that are characterized by the absence of stx, the virulence hallmark of STEC. The individual genomic basis of the Stx (−) phenotype has remained largely undetermined as the majority of STEC genomes in public genome repositories were generated using short read technology and are in draft stage, posing a major obstacle for the high-resolution whole genome sequence typing (WGST). The application of LRT (long-read technology) sequencing provided us with closed genomes, which proved critical to put the atypical non-shigatoxigenic NSF O157:H7 and SF O157:NM strains into the phylogenomic context of the stepwise evolutionary model. Availability of closed chromosomes for representative Stx (−) NSF O157:H7 and SF O157:NM strains allowed to describe the genomic basis and individual evolutionary trajectories underlying the absence of Stx at high accuracy and resolution. The ability of LRT to recover and accurately assemble plasmids revealed a strong correlation between the strains’ featured plasmid genotype and chromosomally inferred clade, which suggests the coevolution of the chromosome and accessory plasmids. The identified ancestral traits in the pSFO157 plasmid of NSF O157:H7 strain LSU-61 provided additional evidence for its intermediate status. Taken together, these observations highlight the utility of LRTs for advancing our understanding of EHEC O157:H7/NM pathogenome evolution. Insights into the genomic and phenotypic plasticity of STEC on a lineage- and genome-wide scale are foundational to improve and inform risk assessment, biosurveillance, and prevention strategies.

志贺毒素转化型（Stx）噬菌体介导的大肠杆菌菌株产毒转化，是肠出血性大肠杆菌（EHEC）O157:H7从肠致病性大肠杆菌（EPEC）O55:H7祖先逐步演化过程中显著且反复发生的事件。目前已有研究报道了不发酵山梨醇（NSF）O157:H7与发酵山梨醇（SF）O157:NM血清型的非典型减毒分离株，这类菌株缺失产志贺毒素大肠杆菌（STEC）的标志性毒力因子——志贺毒素（Stx）。此类非典型菌株要么从未获得Stx噬菌体，要么在感染、分离或常规传代过程中次生丢失了stx基因；后者通常被称为志贺毒素丢失（LST）分离株。本研究对来自北美、欧洲及亚洲的15株缺失STEC标志性毒力因子stx的NSF O157:H7与SF O157:NM菌株进行了基因组分析。此前，由于公共基因组库中的绝大多数产志贺毒素大肠杆菌（STEC）基因组均通过短读长测序技术（short read technology）获得且处于草图阶段（draft stage），极大阻碍了高分辨率全基因组序列分型（WGST）的开展，导致Stx（-）表型的个体基因组基础长期未得到明确解析。长读长测序技术（LRT）的应用使我们获得了闭合基因组，这对于将非典型非产志贺毒素NSF O157:H7与SF O157:NM菌株置于逐步演化模型的系统基因组学背景中进行分析至关重要。通过对代表性Stx（-）NSF O157:H7与SF O157:NM菌株的闭合染色体进行分析，我们得以高精度、高分辨率地解析Stx缺失的基因组基础与个体演化轨迹。长读长测序技术能够回收并精准组装质粒，研究发现菌株的特征质粒基因型与染色体推断的进化枝之间存在显著相关性，这提示染色体与附属质粒存在共演化现象。在NSF O157:H7菌株LSU-61的pSFO157质粒中鉴定到的祖先性状，进一步为其处于演化中间状态提供了证据。综上，上述结果凸显了长读长测序技术（LRT）在加深我们对EHEC O157:H7/NM致病组演化认知方面的应用价值。在谱系及全基因组层面解析产志贺毒素大肠杆菌（STEC）的基因组与表型可塑性，是优化风险评估、生物监测及预防策略的基础。