The Use of Polyamines in Treatment of Multidrug-Resistant Gram-Negative Pathogens

描述(申请人提供)：人类和动物治疗中大量抗生素的无节制使用导致了对几乎所有商业抗生素具有耐药性的病原菌的选择，这个问题需要新的药物或策略来有效地对抗多重耐药(MDR)病原菌。目前，与医疗保健感染和医院暴发相关的最有问题的耐多药革兰氏阴性病原菌是铜绿假单胞菌、肺炎克雷伯菌和鲍曼不动杆菌。PI发现，外源性多胺(精胺、亚精胺和腐胺)与2-内酰胺类抗生素联合使用可使铜绿假单胞菌对2-内酰胺类抗生素敏感(对敏感4倍)，而与多粘菌素B联合使用可诱导铜绿假单胞菌对多粘菌素B产生4~8倍耐药性。多胺对2-内酰胺和多粘菌素B的作用分子机制尚不清楚。多胺是在所有生物体内合成的带正电荷的化合物。细胞内多胺的浓度受到生物合成、降解和运输的严格调控。多胺的作用主要是为了细胞生长，但据报道，在细菌中也有其他各种作用。主要研究者假设：1)外源性多胺改变了细胞内多胺的稳态；2)这种改变诱导了细胞对维持细胞内多胺浓度的反应；3)细胞反应包括多胺对2-内酰胺和多粘菌素B敏感性的影响。为了验证这一假设，提出了以下具体目标：具体目标1：阐明多胺对铜绿假单胞菌、肺炎克雷伯菌和鲍曼不动杆菌耐药临床分离株2-内酰胺类和多粘菌素B敏感性的影响。标准的最低抑菌浓度法、时间杀伤法和棋盘试验将被用来阐明多胺对铜绿假单胞菌、肺炎克雷伯菌和鲍曼不动杆菌多药耐药临床分离株2-内酰胺类和多粘菌素B敏感性的影响。具体目标2：确定多胺摄取系统和胞内多胺浓度在多胺对2-内酰胺和多粘菌素B敏感性影响中的作用。利用基因置换技术，通过缺失两个主要的多胺摄取系统(potABCD/spuABCDEFGH)和多胺生物合成基因(spEA/Spec)，将产生缺乏多胺吸收和生物合成的突变株PAO1。这些菌株将被用来确定多胺摄取和胞内多胺浓度在多胺对2-内酰胺和多粘菌素B敏感性影响中的作用。本研究的长期目标是了解多胺对细菌病原体中2-内酰胺类和多粘菌素B敏感性影响的分子细节，为设计新的治疗药物或治疗MDR感染的策略提供依据。 公共卫生相关性：多药耐药(MDR)病原体引起的传染病是主要的公共卫生问题，因为抗击这些疾病的治疗选择有限。多胺已被认为是对2-内酰胺类耐药病原体的敏感剂。本研究旨在阐明多胺类药物在使用现有的2-内酰胺类抗生素治疗MDR病原体方面的适用性，并确定与多胺效应相关的遗传决定因素。

DESCRIPTION (provided by applicant): The uncontrolled use of large amounts of antibiotics for human and animal therapy has resulted in the selection of pathogenic bacteria resistant to almost all commercially available antibiotics and this problem requires new agents or strategies active against the multidrug resistant (MDR) pathogenic bacteria. Currently, the most problematic MDR Gram-negative pathogens associated healthcare-infections and nosocomial outbreaks are Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii. PI discovered that exogenous polyamines (spermine, spermidine, and putrescine) in combination with 2-lactams sensitized 2- lactam resistance (become 4- to 64-fold sensitive) but in combination with polymyxin B induced resistance to polymyxin B (become 4- to 8-fold resistant) in P. aeruginosa. Molecular mechanism for the polyamine effect on 2-lactam and polymyxin B was unknown. Polyamines are positively charged compounds synthesized in all living organisms. Intracellular concentration of polyamines is strictly regulated by biosynthesis, degradation, and transport. The role of polyamines is primarily for cellular growth, but a variety of other roles has been reported in bacteria. The principal investigator hypothesizes that 1) exogenous polyamines alter the polyamine homeostasis in cells; 2) this alteration induces a cellular response to maintain intracellular concentration of polyamines, and 3) the cellular response includes the polyamine effect on 2-lactam and polymyxin B susceptibility. To test the hypothesis following specific aims are proposed: Specific Aim 1: Elucidate the polyamine effect on 2-lactam and polymyxin B susceptibility in MDR clinical isolates of P. aeruginosa, K. pneumoniae, and A. baumannii. Standard methods of minimal inhibitory concentration, time-killing, and checkerboard assays will be employed to elucidate the polyamine effect on 2-lactam and polymyxin B susceptibility for MDR clinical isolates of P. aeruginosa, K. pneumoniae, and A. baumannii. Specific Aim 2: Determine role of polyamine uptake systems and intracellular polyamine concentration in the polyamine effect on 2-lactam and polymyxin B susceptibility. Mutant P. aeruginosa PAO1 lacking polyamine uptake and biosynthesis will be produced by deletion of two major polyamine uptake systems (potABCD/spuABCDEFGH) and the genes for polyamine biosynthesis (speA/speC) employing gene- replacement techniques. These strains will be used to determine role of polyamine uptake and intracellular polyamine concentration in the polyamine effect on 2-lactam and polymyxin B susceptibility. Long-term goal of this study is to understand molecular details of the polyamine effect on 2-lactam and polymyxin B susceptibility in bacterial pathogens, which will provide the basis for designing a new therapeutic agent or strategy to treat MDR infections. PUBLIC HEALTH RELEVANCE: Infectious diseases caused by multidrug resistant (MDR) pathogens are major public health problems since the limited therapeutic options to combat them. Polyamines have been known as sensitizing 2- lactam resistant pathogens. This study is to elucidate the applicability of the polyamines to treat the MDR pathogens using currently existing 2-lactam antibiotics and to identify genetic determinant associated with the polyamine effect.