Localization and characterization of the Clostridium difficile biofilm

艰难梭菌生物膜的定位和表征

• 批准号: 8228571
• 负责人: Adam Driks
• 金额: $ 18.69万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-15 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8228571
• 关键词: Accounting; Address; Affect; Alcian Blue; Animal Model; Animals; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Antibodies; Antibody Formation; Antimicrobial Resistance; Bacterial Infections; Cecum; Cells; Clostridium difficile; Colon; Communities; Defense Mechanisms; Development; Disease; Drug resistance; Electron Microscopy; Enzymes; Fluorescent in Situ Hybridization; Foundations; Gastrointestinal tract structure; Germination; Hamsters; Health Care Costs; Hospitals; Host Defense; Immune response; Immune system; Immunofluorescence Microscopy; In Vitro; Infection; Knowledge; Laboratories; Laboratory culture; Laser Scanning Confocal Microscopy; Learning; Location; Mass Spectrum Analysis; Metabolic; Methodology; Metronidazole; Microbial Biofilms; Morbidity - disease rate; Mucous Membrane; Mus; Nucleic Acids; Organism; Pathogenesis; Patients; Peptide antibodies; Pharmacotherapy; Physiological; Play; Polysaccharides; Predisposition; Probiotics; Production; Property; Proteins; Recurrence; Recurrent disease; Relapse; Reproduction spores; Resistance; Role; Serum; Staining method; Stains; Structure; Symptoms; Testing; Therapeutic; Tissues; Toxin; Transmission Electron Microscopy; Vaccines; Vancomycin; Variant; Western Blotting; Work; antimicrobial drug; antimicrobial peptide; cell type; in vivo; killings; light microscopy; mortality; novel; novel therapeutics; pathogen

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. PUBLIC HEALTH RELEVANCE: Clostridium difficile infection (CDI) is a leading cause of morbidity and mortality in hospitals around the world. We will test the hypothesis that to cause CDI, C. difficile attaches in the gut to form a resistant, adherent community called a biofilm. We will also analyze C. difficile biofilms generated in the laboratory, to learn whether they have specialized properties, such as resistance to antibiotics, which could promote disease and affect treatment.

描述（由申请方提供）：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。患者获得C.如果抗生素治疗耗尽了正常的微生物群，这些微生物在宿主中持续存在，为C.艰难梭菌增殖、毒素产生和疾病。至少20%的成功治疗CDI症状的患者在接下来的30天内复发。许多细菌感染的一个关键早期步骤是附着在特定的宿主组织上，这可以防止宿主防御，从而在病原体引起疾病之前将其清除或杀死。不知道C。艰难梭菌在肠道中建立自己或逃避宿主防御。重要的是，它也不知道是否C。difficile制造生物膜，一种无处不在的细菌防御机制。生物膜不仅可以促进附着在粘膜上，还可以排除关键的宿主防御分子，如抗菌肽和抗体，并避免被免疫系统识别。梭艰难梭菌生物膜也可以促进持久性和复发（同一菌株的复发），如果它作为孢子的水库，一个高度强大的，休眠的细胞类型，是抗微生物药物。了解C。艰难梭菌与宿主组织相互作用并逃避宿主防御和治疗，将极大地促进新治疗的开发。 我们的中心假设是C。艰难梭菌在宿主胃肠道（GI）中形成生物膜，并且进一步地，生物膜形成促进感染，并保护免受宿主防御和抗生素治疗。作为解决这一假设的第一步，我们确定了C的条件。艰难梭菌生物膜形成实验室培养，并显示六天后，生物膜窝藏含有生长和死亡的营养细胞的大菌落，以及高浓度的休眠孢子。使用激光扫描共聚焦显微镜，我们发现，多糖和核酸存在于基质中。通过蛋白质印迹分析，我们发现毒素存在于基质中，通过质谱分析，我们鉴定了9种丰富的基质蛋白，它们都是代谢酶。此外，我们还利用荧光原位杂交技术证实了C。艰难梭菌在CDI小鼠盲肠和结肠粘膜形成群落，表明C.艰难梭菌在宿主体内形成生物膜。 C.艰难梭菌生物膜可通过促进C.通过抵抗宿主防御和抗微生物药物;通过积累毒素并将其引导到宿主组织;以及通过窝藏可能促进疾病复发的休眠孢子库，来使细菌难以到达结肠中的适当位置。在此，我们提出两个目标。在目标1中，我们将定位和表征C。我们将在两种CDI动物模型中观察胃肠道内的艰难梭菌生物膜，并确定宿主是否对生物膜产生免疫应答。在目标2中，我们将研究C.艰难的生物膜可能有助于CDI。 公共卫生相关性：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。我们将测试的假设，导致CDI，C。艰难梭菌附着在肠道中，形成一种称为生物膜的抗性粘附群落。我们还将分析C。艰难的生物膜在实验室中产生，以了解他们是否有专门的属性，如耐药性抗生素，这可能会促进疾病和影响治疗。