Prevention and cure of murine C. difficile infection by a Lachnospiraceae strain

The dramatically increased risk of symptomatic C. difficile infection (CDI) following use of antibiotics, as well as the ability of fecal microbiota transplant (FMT) prevent disease recurrence, highlights the role of the commensal microbiota in keeping this pathogen in-check. Yet, how microbiota impede C. difficile (C. diff) colonization is unclear, in part due to lack of reductionist in vivo models. Antibiotic-based models of C. diff are influenced by microbiota composition, which can be quite heterogenous both basally and in response to antibiotics making them particularly difficult for mechanistic studies. Germfree (GF) mice circumvent this complexity but their abnormal gut physiology and immunology call into question their physiological relevance. Hence, we utilized mice harboring the 8-species of bacteria referred to as the Altered Schaedler Flora (ASF), which correct many of the abnormalities of GF mice. We observed that, like GF mice, ASF mice were highly prone to rapid lethal CDI (without antibiotics) even when given relatively low doses of this pathogen, although very low doses resulted in most ASF mice surviving the challenge and developing chronic symptomatic CDI. Administering such chronic CDI mice an undefined preparation of Clostridia lowered C. diff levels by several logs and restored health. Such resolution of CDI was associated with colonization of Lachnospiraceae. Fractionation of the Clostridia prep prior to its administration led to appreciation that its ability to impede CDI strongly associated with a specific Lachnospiraceae stain, namely UBA3401. UBA3401 was recalcitrant to being propagated as a pure culture, both in vitro and in monoassociated mice, but could be maintained in ASF mice, wherein it comprised up to 50% of the gut microbiome, which was sufficient to generate high-quality genomic sequence of this bacteria. Sequence analysis and ex vivo study of UBA3401 indicated it had direct ability to secrete substances that impeded C. diff growth. In vivo, administration of UBA3401/ASF feces provided strong protection to a high-dose C. diff challenge that was otherwise uniformly lethal. Thus, UBA3401 may serve as a CDI therapeutic and a tractable platform for study of microbiota-mediated CDI resistance.

抗生素使用后，症状性艰难梭菌感染（C. difficile infection, CDI）的发病风险显著升高；而粪便菌群移植（fecal microbiota transplant, FMT）可预防疾病复发，这两点共同凸显了共生菌群（commensal microbiota）对该病原体的制衡作用。然而，菌群如何阻碍艰难梭菌（C. diff）定植的机制尚不明确，部分原因在于缺乏还原论体内模型（reductionist in vivo models）。基于抗生素的艰难梭菌模型受菌群组成影响，而菌群组成在基础状态及抗生素干预后均存在显著异质性，这使得该模型难以开展机制研究。无菌（Germfree, GF）小鼠可规避这一复杂性，但其肠道生理与免疫学特征异常，使其生理相关性受到质疑。因此，本研究采用了携带8种细菌的小鼠模型，该菌群被称为改变的舍尔德菌群（Altered Schaedler Flora, ASF），其可纠正无菌小鼠的诸多异常表型。研究观察到，与无菌小鼠类似，即使给予相对低剂量的艰难梭菌，无抗生素干预的ASF小鼠仍极易发生快速致死性CDI；但当病原体剂量极低时，多数ASF小鼠可耐受定植并发展为慢性症状性CDI。向这类慢性CDI小鼠输注未明确组分的梭菌（Clostridia）制剂，可使艰难梭菌载量降低数个数量级，并恢复小鼠健康状态。这种CDI的缓解与毛螺菌科（Lachnospiraceae）的定植密切相关。对该梭菌制剂进行分级分离后发现，其阻碍CDI的能力与特定毛螺菌科菌株UBA3401显著相关。UBA3401无论是在体外（in vitro）还是单定植小鼠（monoassociated mice）体内，均难以实现纯培养，但可在ASF小鼠体内稳定定植，其丰度最高可占肠道微生物组（gut microbiome）的50%，这足以获取该菌株的高质量基因组序列。对UBA3401的序列分析与离体实验（ex vivo study）表明，该菌株可直接分泌抑制艰难梭菌生长的物质。体内实验显示，输注携带UBA3401的ASF小鼠粪便，可对高剂量艰难梭菌攻击提供极强的保护作用——否则该攻击将导致小鼠全部死亡。综上，UBA3401有望成为CDI的治疗手段，同时也可作为研究菌群介导CDI抗性的可操作研究平台。