Bacteriophage-based probiotic preparations and their effect on the gut microflora

描述(申请人提供)：人类胃肠道(Gl)是由丰富和多样化的微生物区系定居。多种因素(例如，抗生素治疗、饮食等)可能会改变肠道中正常和有益的细菌菌群，这可能会导致各种慢性和退行性疾病，这些疾病在幼儿和老年人中尤其严重。用于缓解这些疾病的方法之一是摄取益生菌；即各种非致病细菌(例如乳杆菌和双歧杆菌)，当摄取足够数量时，这些细菌可能会改善健康。然而，一个潜在有用的益生菌干预措施并没有得到太多的关注，那就是使用噬菌体来靶向人类胃肠道中的“问题”细菌物种。噬菌体(或称噬菌体)是感染细菌的病毒。它们是地球上最丰富的微生物，在维持众多生态系统中的细菌种群平衡方面发挥着关键作用。裂解噬菌体具有显著的抗菌活性；然而，与抗生素相比，它们是非常特异的；即，它们只裂解其目标细菌种类的菌株。本申请中提出的研究解决了我们的假设，即由于其高度特异性，噬菌体可能有助于有利地操纵哺乳动物肠道的微生物群(通过靶向Gl道中特定的“问题”细菌病原体)，并且基于噬菌体的益生菌不会对肠道有益的微生物群产生有害影响。我们将使用单核细胞增多性李斯特菌和L单核细胞增多症特异性噬菌体制剂(LMP-102)作为模型系统来评估我们的假设。我们认为，将L的单核细胞增多性噬菌体制剂作为常规饮食的一部分，可以显著降低摄入的单核细胞增多性李氏杆菌定植于Gl道并增殖到足够水平，穿过肠粘膜，通过肠系膜淋巴结和血流传播的风险。具体目标如下：SA#1：开发基因工具来鉴定和监测单核细胞增生性乳杆菌噬菌体制剂(LMP-102)的单个单噬菌体成分，在体内外；SA#2：测定LMP-102单噬菌体在小鼠胃肠道中的持久性(在存在和不存在目标单核细胞增多菌的情况下)，并确定它们对单核细胞增多性乳杆菌在实验感染小鼠体内定植的影响；SA#3：比较LMP-102和氨苄西林对健康小鼠和实验感染L单核细胞基因小鼠肠道菌群的影响。本申请中的研究旨在提供有关噬菌体-细菌在胃肠道中相互作用的关键初步数据，并可能导致开发新的益生菌制剂，用于预防和/或治疗由各种细菌病原体引起的疾病，这些细菌具有口腔入口，需要在胃肠道短期或长期定植才能致病。

DESCRIPTION (provided by applicant): The human gastrointestinal (Gl) tract is colonized by an abundant and diverse microbiota. Numerous factors (e.g., antibiotic treatment, diet, etc.) may alter the normal and beneficial bacterial flora of the gut, which may lead to various chronic and degenerative diseases that are particularly problematic in young children and the elderly. One of the approaches used to alleviate those disorders is the ingestion of probiotic bacteria; i.e., various nonpathogenic bacteria (e.g., lactobacilli and bifidobacteria) that may improve health when ingested in sufficient quantities. However, a potentially useful probiotic intervention that has not received much attention is to use bacteriophages to target "problem" bacterial species in the human Gl tract. Bacteriophages (or phages) are viruses that infect bacteria. They are the most abundant microorganisms on earth, and they play a key role in maintaining balanced bacterial populations in numerous ecosystems. Lytic phages have remarkable antibacterial activity; however, in contrast to antibiotics, they are very specific; i.e., they only lyse strains of their targeted bacterial species. The studies proposed in this application address our hypothesis that, because of their high specificity, bacteriophages may be useful in favorably manipulating the microflora of the mammalian intestinal tract (by targeting specific "problem" bacterial pathogens in the Gl tract), and that phage-based probiotics will have no deleterious effect on the beneficial microflora of the gut. We will begin to evaluate our hypothesis by using L. monocytogens and a L monocytogenes-specific phage preparation (LMP-102) as a model system. We propose that making a L monocytogenes-specific phage preparation part of a routine diet may significantly reduce the risk of ingested L. monocytogenes colonizing the Gl tract and proliferating to sufficient levels to cross the intestinal mucosa and disseminate via the mesenteric lymph nodes and bloodstream. The following three specific aims will be addressed: SA #1: Develop genetic tools to identify and monitor individual monophage components of L. monocytogenes-spectflc phage preparation (LMP-102) in vitro and in vivo; SA #2: Determine the persistence of LMP-102 monophages in the Gl tract of mice (in the presence and absence of the targeted L. monocytogenes strain) and determine their effect on L. monocytogenes colonization in experimentally-infected mice; SA #3: Compare the in vivo effects of LMP-102 and ampicillin on the intestinal microflora of healthy mice and mice experimentally-infected with L monocytogenes. The studies in this application are designed to yield critical preliminary data concerning bacteriophage-bacterial interactions in the Gl tract, and they may lead to the development of novel probiotic preparations for the prophylaxis and/or treatment of illnesses caused by various bacterial pathogens that have an oral portal of entry and require short- or long-term colonization in the Gl tract in order to cause disease.