Clinical Significance of Clostridium difficile Toxin Variants

描述（由申请人提供）： 艰难梭菌相关性疾病（CDAD）是退伍军人医院和北美其他医院日益严重的问题。最近在加拿大和美国的多医院爆发与一种特定的C。艰难梭菌毒素变体，称为BI/NAP 1/027，这可能至少部分解释了全国CDAD发病率的总体增加。毒素变体BI/NAP 1/027菌株的特征在于毒素A和B（C.艰难梭菌）、另外的毒素（称为二元毒素CDT）的存在以及对氟喹诺酮类抗生素的抗性增加。由于BI/NAP 1/027菌株与这些医院暴发中更严重的疾病相关，因此被称为高毒力菌株。确定各种毒素和其他因素对BI菌株高毒力的贡献，不仅有助于了解这些菌株成为北美主要流行菌株的原因，还有助于阐明CDAD发病机制中的基本分子步骤。历史上，毒素A被认为是C.艰难梭菌，因为其有效的肠毒性活性和毒素A单独在动物模型中产生疾病的能力。然而，对天然存在的毒素A-阴性、B-阳性（A-/B+）变异株的临床观察，以及我们在仓鼠模型中证明A-/B+菌株毒力的工作，以及其他最近的证据表明，毒素B，而不是A，是C的基本毒力因子。很难我们推测毒素B而不是毒素A是C的主要毒力因子。艰难梭菌，包括流行性BI菌株。我们还假设，在我们医院的特定菌株的频率将部分取决于该菌株产生毒素B的水平。本提案的具体目标是：目标1：前瞻性筛查C。艰难临床分离株，以确定特定菌株的频率，将体外毒素产生与频率相关联，并获得用于构建突变体的候选高毒力BI分离株。目的2：构建BI强毒力菌株毒素B和毒素A的独立突变体。目标三：对BI衍生毒素B和毒素A突变体的毒素产生和致病性位点（PaLoc）基因表达进行体外分析。目的4：在仓鼠模型中检测独立的毒素B和毒素A突变体，以确定毒力是否被消除。 公共卫生相关性： 项目叙述对退伍军人医疗保健的潜在影响：CDAD仍然是医院获得性感染性腹泻的主要原因，VA医院受到的影响尤其严重，因为CDAD的风险因素、高龄、住院时间延长和频繁的抗生素暴露是VA医院患者的特征。对CDAD发病机制的进一步了解，应导致新的治疗，诊断和策略，以中断持续流行的CDAD影响VA和非VA住院患者。

DESCRIPTION (provided by applicant): PROJECT SUMMARY/ABSTRACT Clostridium difficile-associated disease (CDAD) is an increasing problem in Veterans Hospitals and other hospitals throughout North America. Recent multi-hospital outbreaks in Canada and in the U.S. have been linked to the emergence of a specific C. difficile toxin variant, referred to as BI/NAP1/027 which may explain, at least in part, the overall increase in national rates of CDAD. The toxin variant BI/NAP1/027 strains are characterized by increased production of toxin A and B (the major known virulence factors of C. difficile), the presence of an additional toxin, referred to as binary toxin CDT, as well as increased resistance to fluoroquinolone antibiotics. Because of their association with more severe disease in these hospital outbreaks, the BI/NAP1/027 strains have been referred to as hypervirulent. Determining the contribution of the various toxins and other factors to hypervirulence of the BI strains will not only improve the understanding of why these strains have become the dominant epidemic strains in North America, but will also help elucidate the fundamental molecular steps in the pathogenesis of CDAD. Historically, toxin A has been implicated as the primary virulence determinant of C. difficile because of its potent enterotoxic activity and the ability of toxin A alone to produce disease in animal models. However, clinical observations on the naturally-occurring toxin A- negative, B-positive (A-/B+) variant strains, and our work demonstrating virulence of A-/B+ strains in the hamster model, in addition to other recent evidence, suggest that toxin B, not A, is the essential virulence factor in C. difficile. We hypothesize that toxin B, not toxin A is the essential virulence factor of C. difficile, including epidemic BI strains. We also hypothesize that the frequency of a particular strain in our hospital will be determined in part by the level of toxin B production by that strain. The specific objectives of this proposal are: Objective 1: To prospectively screen C. difficile clinical isolates to determine the frequency of specific strains, correlate toxin production in vitro with frequency, and to obtain candidate hypervirulent BI isolates for construction of mutants. Objective 2: To construct independent mutants of toxin B and toxin A in a hypervirulent BI strain. Objective 3: To conduct in vitro analysis of the BI-derived toxin B and toxin A mutants for toxin production and for pathogenicity locus (PaLoc) gene expression. Objective 4: To test the independent toxin B and toxin A mutants in the hamster model to determine if virulence is abrogated. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Potential impact on Veterans Health Care: CDAD remains the major cause of hospital-acquired infectious diarrhea and VA hospitals have been particularly affected because the risk factors for CDAD, advanced age, prolonged hospital stay and frequent antibiotic exposure are characteristics of VA Hospital patients. An improved understanding of the pathogenesis of CDAD, should lead to new treatments, diagnosis, and strategies to interrupt the ongoing epidemic of CDAD affecting VA and non-VA hospitalized patients.