Identification of Intestinal Bacteria Protective against C. Difficile Colitis

描述（由申请人提供）：艰难梭菌相关疾病（CDAD）是一个日益严重的公共卫生问题。艰难梭菌在结肠中的生长被认为被共生菌群的正常成分所抑制。因此，艰难梭菌感染的主要触发因素是暴露于抗生素，这改变了正常肠道微生物群的组成。大约20%的CDAD患者使用抗生素进行常规治疗失败并复发。这些患者中有很大一部分终生依赖抗生素，并经常出现胃肠道功能障碍。其他患者发展为暴发性疾病，这与高死亡率有关。正常肠道菌群的保护作用是由粪便移植细菌治疗的临床成功所支持的。在这个过程中，复发性CDAD患者的肠道接种健康供者的粪便。我们最近在一份病例报告中证明，这一过程确实与供体细菌在受体结肠中的建立有关。然而，由于实用和美观的考虑，粪便移植并没有广泛应用。我们的长期目标是开发一种标准化的，易于管理的结肠细菌配方，可以有效地治疗CDAD患者。在这个建议中，我们将使用SSU rRNA基因的高变区进行大规模平行焦磷酸测序，以表征复发性CDAD患者及其供者粪便物质的组成。在这项探索性工作中，我们将获得以下特定目标的关键数据：1)确定细菌治疗前后患者粪便微生物组的组成和多样性，2)确定细菌治疗后粪便微生物组的再定殖率和稳定性，以及3)确定是否可以保存单个供体的粪便样本并随后用于恢复几例CDAD患者的正常肠道功能。从第一个目标中获得的数据将为需要更多患者的这项工作的未来扩展奠定基础。该结果将允许我们进行功率计算，以确定此类研究所需的患者数量，以获得更明确的结果。CDAD患者的细菌治疗代表了一个独特的机会来研究在成人患者中建立新的微生物群。从第二个目标中获得的数据将为进一步研究这种独特临床情况下宿主-微生物相互作用的未来研究奠定基础。最后，第三个目标代表了我们最终目标的第一步，并且有可能大大降低追求健康，保护性结肠微生物群特征的复杂性。

DESCRIPTION (provided by applicant): Clostridium difficile associated disease (CDAD) is a major growing public health problem. The growth of C. difficile bacteria in the colon is thought to be inhibited by normal constituents of the mutualist bacteria flora. Therefore, the major trigger for the C. difficile infection is exposure to antibiotics which alter the composition of the normal intestinal microbiota. About 20% of patients with CDAD fail conventional therapy with antibiotics and develop recurrent disease. A significant fraction of these patients develop life-long dependency on antibiotics and often dysfunction of their gastrointestinal tract. Other patients develop fulminant disease, which is associated with high mortality rate. The protective role of the normal intestinal flora is supported by clinical success of bacteriotherapy by way of fecal transplantation. In this procedure the intestinal tracts of patients with recurrent CDAD are inoculated with fecal material from healthy donors. We had recently demonstrated in a case report that this procedure indeed is associated with establishment of donor bacteria in the recipient's colon. However, fecal transplantation is not widely available because of practical and aesthetic considerations. Our long-term goal is to develop a standardized, easy-to- administer formulation of colonic bacteria that can efficiently treat patients with CDAD. In this proposal we will use massively parallel pyrosequencing of hypervariable regions from SSU rRNA genes to characterize the composition of fecal material from patients with recurrent CDAD and their donors. In this exploratory work we will obtain critical data in pursuit of the following specific aims: 1) Determine the composition and diversity of the patient fecal microbiome before and after bacteriotherapy, 2) Determine the rate of recolonization and stability of the fecal microbiome following bacteriotherapy, and 3) Determine if a fecal sample from a single donor can be preserved and later used to restore normal bowel functioning to several CDAD patients. The data obtained from the first aim will form the foundation for future extension of this work requiring more patients. The results will allow us to perform power calculations to determine the number of patients needed in such studies to obtain more definitive results. Bacteriotherapy of patients with CDAD represents a unique opportunity to study establishment of new microflora in an adult patient. The data obtained from the second aim will lay the foundation into future studies that will be further investigate host- microbial interactions in this unique clinical situation. Finally, the third aim represents the first step toward our ultimate goal and has the potential to significantly reduce the complexity of the pursuit for the signature of healthy, protective colonic microflora. PUBLIC HEALTH RELEVANCE: Clostridium difficile is the major known cause of antibiotic associated disease, and represents a growing problem. We are normally protected against this infection by normal bacteria that live in our intestines. Antibiotics used in medical practice weaken this protection. A significant fraction of patients with this infection cannot be treated by conventional treatments, which also involve antibiotics. Our ultimate goal is to develop a standardized formulation of protective bacteria. In this grant we will make critical initial steps toward this goal by studying composition of intestinal bacteria in patients undergoing fecal transplantation for C. difficile infection refractory to conventional treatment.