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

描述（由申请人提供）：在人类和动物治疗中不受控制地大量使用抗生素导致病原菌对几乎所有市售抗生素具有耐药性，这一问题需要新的药物或策略来有效对抗多重耐药（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多药耐药革兰氏阴性病原体是铜绿假单胞菌、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-内酰胺联合使P. aeruginosa对2-内酰胺的抗性增敏（变为4- 64倍敏感），而与多粘菌素B联合诱导对多粘菌素B的抗性（变为4- 8倍耐药）。多胺作用于2-内酰胺和多粘菌素B的分子机制尚不清楚。多胺是所有生物体合成的带正电的化合物。细胞内多胺的浓度受到生物合成、降解和运输的严格调节。多胺的作用主要是促进细胞生长，但在细菌中也有许多其他的作用。主要研究者假设：1)外源性多胺改变细胞内多胺稳态；2)这种改变引起细胞反应以维持细胞内多胺的浓度，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假单胞菌耐多药临床分离株2-内酰胺和多粘菌素B敏感性的影响。最低抑菌浓度、时间杀伤和棋盘试验的标准方法将用于阐明多胺对铜绿假单胞菌、肺炎克雷伯菌和鲍曼假单胞菌耐多药临床分离株2-内酰胺和多粘菌素B敏感性的影响。特异性目的2：确定多胺摄取系统和细胞内多胺浓度在多胺对2-内酰胺和多粘菌素B敏感性的影响中的作用。铜绿假单胞菌PAO1突变体缺乏多胺摄取和生物合成，通过基因替代技术删除两个主要的多胺摄取系统（potABCD/spuABCDEFGH）和多胺生物合成基因（speA/speC）而产生。这些菌株将用于确定多胺摄取和细胞内多胺浓度在多胺对2-内酰胺和多粘菌素B敏感性的影响中的作用。本研究的长期目标是了解多胺对细菌病原体2-内酰胺和多粘菌素B易感性的影响的分子细节，为设计新的治疗耐多药感染的药物或策略提供依据。

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.