Hospital acquired Clostridioides (Clostridium) difficile infection is exacerbated by the continued evolution of C. difficile strains, a phenomenon studied by multiple laboratories using stock cultures specific to each laboratory. Intralaboratory evolution of strains contributes to interlaboratory variation in experimental results adding to the challenges of scientific rigor and reproducibility. To explore how microevolution of C. difficile within laboratories influences the metabolic capacity of an organism, three different laboratory stock isolates of the C. difficile 630 reference strain were whole-genome sequenced and profiled in over 180 nutrient environments using phenotypic microarrays. The results identified differences in growth dynamics for 32 carbon sources including trehalose, fructose, and mannose. An updated genome-scale model for C. difficile 630 was constructed and used to contextualize the 28 unique mutations observed between the stock cultures. The integration of phenotypic screens with model predictions identified pathways enabling catabolism of ethanolamine, salicin, arbutin, and N-acetyl-galactosamine that differentiated individual C. difficile 630 laboratory isolates. The reconstruction was used as a framework to analyze the core-genome of 415 publicly available C. difficile genomes and identify areas of metabolism prone to evolution within the species. Genes encoding enzymes and transporters involved in starch metabolism and iron acquisition were more variable while C. difficile distinct metabolic functions like Stickland fermentation were more consistent. A substitution in the trehalose PTS system was identified with potential implications in strain virulence. Thus, pairing genome-scale models with large-scale physiological and genomic data enables a mechanistic framework for studying the evolution of pathogens within microenvironments and will lead to predictive modeling to combat pathogen emergence.

艰难梭菌菌株的持续进化加剧了医院获得的艰难梭菌( Clostridium )感染，多个实验室使用每个实验室特有的原种培养物研究了这一现象。菌株的实验室内进化导致实验结果的实验​​室间变异，增加了科学严谨性和可重复性的挑战。为了探索实验室内艰难梭菌的微进化如何影响生物体的代谢能力，三种不同的实验室储备分离株艰难梭菌使用表型微阵列在 180 多种营养环境中对 630 个参考菌株进行了全基因组测序和分析。结果确定了 32 种碳源的生长动态差异，包括海藻糖、果糖和甘露糖。构建了一个更新的艰难梭菌630基因组规模模型，并用于将在原种培养物之间观察到的 28 个独特突变置于上下文中。表型筛选与模型预测的整合确定了使乙醇胺、水杨苷、熊果苷和 N-乙酰半乳糖胺分解代谢的途径，从而区分了单个艰难梭菌630 实验室分离株。重建被用作分析 415 个可公开获得的C. difficile的核心基因组的框架基因组并确定物种内易于进化的代谢区域。编码参与淀粉代谢和铁获取的酶和转运蛋白的基因更具可变性，而艰难梭菌不同的代谢功能（如 Stickland 发酵）则更加一致。海藻糖 PTS 系统中的一个替代物被确定为对菌株毒力有潜在影响。因此，将基因组规模模型与大规模生理和基因组数据配对，为研究病原体在微环境中的进化提供了一个机制框架，并将导致预测建模以对抗病原体的出现。