A cultivation-based approach to decipher oxidative stress responses in healthy humans gut microbiota

The gut microbiota plays a critical role in maintaining health, and its dysbiosis has been associated with several diseases, such as inflammatory bowel disease. The oxidative environment caused by chronic inflammation has been identified as an important factor driving disease-specific changes in microbiota composition. However, in vivo data are often confounded by high donor variability and the complex host environment. Furthermore, the tolerance of gut anaerobes to specific oxidants remains largely unexplored. This complexity underscores the need to study microbial oxidative stress responses under controlled in vitro conditions. To address this, we developed a cultivation-based model to study the oxidative stress tolerance of representative intestinal strains (n=41) and fecal-derived gut microbiota from healthy adults (n=7) in a high-throughput manner. We evaluated twelve different hydrogen peroxide (H2O2) and oxygen (O2) conditions each. Our experimental setup included successive passages to examine stress responses and recovery patterns. We comprehensively characterized a representative panel of 41 gut microbial species for the potential to grow and recover from H2O2 and O2 stress, providing important information about strain-specific tolerances relative to others. Our comparative assessment of pure strain tolerances confirmed the high sensitivity of Faecalibacterium spp. and the revealed high sensitivity of Fusicatenibacter saccharivorans and Lachnospira eligens to both H2O2 and O2. We also corroborate that several Bacteroides spp. exhibit uniquely high tolerance to both oxidants compared to the rest of the tested species. Furthermore, pure strain tolerances could explain the main taxonomic patterns in complex communities when exposed to selected oxidative stress modeling conditions, i.e., 0.71 mM H2O2 or aerobic incubation. These conditions induced biologically relevant oxidative stress-mediated dysbiosis in fecal-derived microbial communities, including the loss of sensitive anaerobes (e.g., butyrate-producers) and the bloom of facultative anaerobes (e.g., Enterococcus, Streptococcus, Escherichia-Shigella). The proposed H2O2 and O2 conditions can be used to rapidly screen biologically relevant oxidative stress responses in fecal cultures, with the potential to characterize donor-specific shifts in the community or metabolic activities.

肠道菌群（gut microbiota）在维持机体健康中发挥关键作用，其菌群失调与多种疾病（如炎症性肠病）密切相关。慢性炎症所引发的氧化环境，被认为是驱动菌群组成发生疾病特异性改变的重要因素。然而，体内（in vivo）实验数据常受供体间高异质性与复杂宿主环境的干扰。此外，肠道厌氧菌对特定氧化剂的耐受机制目前仍未得到充分探索。这种复杂性凸显了在可控体外（in vitro）环境下研究微生物氧化应激响应的必要性。为解决这一问题，本研究构建了基于培养的模型，以高通量方式探究41株代表性肠道菌株（n=41）与7份健康成人粪便来源肠道菌群（n=7）的氧化应激耐受能力。我们分别设置了12种不同的过氧化氢（H₂O₂）与氧气（O₂）培养条件。本实验设置了连续传代步骤，以考察微生物的应激响应与恢复模式。我们对41株肠道微生物代表菌株进行了全面表征，分析其在过氧化氢与氧气应激下的生长与恢复潜力，从而明确不同菌株间的特异性耐受差异。我们对纯培养菌株耐受能力的比较分析证实，粪杆菌属（Faecalibacterium spp.）对过氧化氢与氧气均具有较高敏感性；同时发现食糖黏杆菌（Fusicatenibacter saccharivorans）与埃利格斯毛螺菌（Lachnospira eligens）同样对两种氧化剂具有高敏感性。此外，我们证实相较于其他受试菌株，拟杆菌属（Bacteroides spp.）的多个物种对两种氧化剂均表现出独特的高耐受能力。此外，当暴露于选定的氧化应激模拟条件（即0.71 mM H₂O₂或有氧培养）时，纯培养菌株的耐受特征可解释复杂菌群中的主要分类学变化模式。上述条件可在粪便来源的微生物群落中诱导具有生物学意义的氧化应激介导的菌群失调，具体表现为敏感厌氧菌（如丁酸产生菌）的消亡与兼性厌氧菌（如肠球菌属、链球菌属、埃希氏菌-志贺氏菌属）的增殖。本研究提出的过氧化氢与氧气培养条件，可用于快速筛选粪便培养物中具有生物学意义的氧化应激响应，有望表征供体特异性的菌群结构或代谢活性变化。