Localization and characterization of the Clostridium difficile biofilm

描述（由申请人提供）：艰难梭菌感染（CDI）是世界各地医院发病率和死亡率的主要原因。在美国，它每年在医院的医疗保健费用中占32亿美元以上。患者在症状出现前不久感染艰难梭菌，如果抗生素治疗耗尽了正常的微生物群，这些微生物就会在宿主体内持续存在，为艰难梭菌的增殖、毒素产生和疾病创造有利条件。成功治疗CDI症状的患者中至少有20%在接下来的30天内复发。许多细菌感染的关键早期步骤是附着在特定的宿主组织上，这可以防止宿主的防御，在病原体引起疾病之前将其清除或杀死。目前尚不清楚艰难梭菌如何在肠道内建立自身或逃避宿主的防御。重要的是，尚不清楚艰难梭菌是否会形成生物膜，这是一种普遍存在的细菌防御机制。生物膜不仅可以促进与粘膜的附着，而且可以排除关键的宿主防御分子，如抗菌肽和抗体，避免被免疫系统识别。艰难梭菌生物膜也可能促进持久性和复发（同一菌株的复发），如果它作为孢子的储存库，孢子是一种高度健壮的休眠细胞类型，对抗微生物药物具有抗性。了解艰难梭菌如何与宿主组织相互作用，并逃避宿主防御和治疗，将极大地促进新疗法的发展。

DESCRIPTION (provided by applicant): Clostridium difficile infection (CDI) is a leading cause of morbidity and mortality in hospitals around the world. In the US, it accounts for over $3.2 billion/year in health care costs in hospitals. Patients acquire C. difficile shortly before symptoms appear and these organisms persist in the host if antibiotic treatment had depleted the normal microbiota, creating conditions favorable for C. difficile proliferation, toxin production and disease. At least 20% of patients successfully treated for CDI symptoms have a recurrence within the next 30 days. A critical early step in many bacterial infections is attachment to specific host tissues, which guards against host defenses that may remove or kill the pathogen before it can cause disease. It is unknown how C. difficile establishes itself in the gut or evades host defenses. Importantly, it is also not known whether C. difficile makes a biofilm, a ubiquitous bacterial defense mechanism. A biofilm could not only facilitate attachment to the mucosa but also exclude critical host-defense molecules such as antimicrobial peptides and antibodies, and avoid recognition by the immune system. A C. difficile biofilm could also promote persistence and relapse (a recurrence with the same strain) if it serves as a reservoir for spores, a highly robust, dormant cell type that is resistant to antimicrobial drugs. Understanding how C. difficile interacts with host tissues, and evades host defenses and therapeutics, would greatly facilitate development of novel treatments. Our central hypothesis is that C. difficile forms a biofilm in the host gastrointestinal (GI) tract and, further, that biofilm formation facilitates infection, and protects against host defenses and antibiotic treatment. As a first step in addressing this hypothesis, we identified conditions for C. difficile biofilm formation in laboratory culture and showed that after six days, the biofilm harbors macrocolonies containing both growing and dying vegetative cells, as well as high concentrations of dormant spores. Using laser-scanning confocal microscopy, we found that polysaccharides and nucleic acids are present in the matrix. By western blot analysis, we found toxin present in the matrix, and by mass spectrometry, we identified 9 abundant matrix proteins, all of which are metabolic enzymes. Further, we used fluorescence in-situ hybridization to show that C. difficile forms communities at the mucosa of the cecum and colon in mice with CDI, indicating that C. difficile forms a biofilm in the host. C. difficile biofilms could contribute to CDI by facilitating attachment of C. difficile to appropriate locations in the colon; by resisting host defenses and antimicrobial drugs; by accumulating toxin and directing it to host tissues; and by harboring a depot of dormant spores that could facilitate relapsing disease. Here, we propose two Aims. In Aim 1, we will localize and characterize C. difficile biofilms within the GI tract in two animal models of CDI, and we will determine if the host generates an immune response to the biofilm. In Aim 2, we will examine possible mechanisms by which C. difficile biofilms may contribute to CDI.