Localization and characterization of the Clostridium difficile biofilm

艰难梭菌生物膜的定位和表征

基本信息

批准号：
8418693
负责人：
Adam Driks
金额：
$ 22.43万
依托单位：
LOYOLA UNIVERSITY CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-02-15 至 2016-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8418693
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

项目摘要

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.

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，医院的医疗保健费用占32亿美元。患者在症状出现前不久就会获得艰难梭菌，如果抗生素治疗耗尽了正常的微生物群，这些生物会持续存在于宿主中，从而创造了有利于艰难梭菌增殖，毒素产生和疾病的疾病。在接下来的30天内，至少有20％的成功治疗CDI症状的患者复发。许多细菌感染的关键早期一步是附着在特定的宿主组织上，该组织可以防止宿主防御能力，这些防御可能会在病原体引起疾病之前消除或杀死病原体。艰难梭菌如何在肠道或逃避宿主防御措施中建立自己是未知的。重要的是，艰难梭菌是否生物膜是一种无处不在的细菌防御机制。生物膜不仅可以促进粘膜附着，而且还排除关键的宿主防御分子，例如抗菌肽和抗体，并避免免疫系统识别。如果艰难梭菌生物膜可以作为孢子的储层，这是一种对抗菌药物抗性的高度健壮，休眠的细胞类型，也可以促进持久性和复发（具有相同菌株的复发）。了解艰难梭菌如何与宿主组织相互作用，并逃避宿主的防御和治疗疗法，将极大地促进新的治疗方法的发展。我们的中心假设是，艰难梭菌在宿主胃肠道（GI）中形成生物膜，此外，生物膜的形成促进了感染，并防止宿主防御和抗生素治疗。作为解决这一假设的第一步，我们确定了实验室培养中艰难梭菌生物膜形成的条件，并表明，六天后，生物膜携带着含有生长和垂死的营养细胞的宏collosies，以及高浓度的休眠孢子。使用激光扫描共聚焦显微镜，我们发现基质中存在多糖和核酸。通过蛋白质印迹分析，我们发现了基质中存在的毒素，通过质谱法，我们确定了9种丰富的基质蛋白，所有这些蛋白都是代谢酶。此外，我们使用荧光原位杂交来表明，艰难梭菌在带有CDI的小鼠的小鼠粘膜和结肠上形成了群落，表明艰难梭菌在宿主中形成了生物膜。艰难梭菌生物膜可以通过促进艰难梭菌附着在结肠中的适当位置来促进CDI；通过抵抗宿主防御和抗菌药物；通过累积毒素并将其引导到宿主组织；并带有一个可以促进复发疾病的休眠孢子库。在这里，我们提出了两个目标。在AIM 1中，我们将在CDI的两个动物模型中定位并表征艰难梭菌生物膜内的艰难梭菌生物膜，我们将确定宿主是否对生物膜产生免疫反应。在AIM 2中，我们将研究艰难梭菌生物膜可能有助于CDI的可能机制。