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

描述（由申请人提供）：人类和动物治疗中大量抗生素的不受控制的使用导致选择对几乎所有市售抗生素具有耐药性的病原菌，该问题需要对多药耐药（MDR）病原菌具有活性的新试剂或策略。目前，最有问题的MDR革兰氏阴性病原体相关的医疗保健感染和医院爆发是铜绿假单胞菌，肺炎克雷伯菌，鲍曼不动杆菌。PI发现，外源性多胺（精胺、亚精胺和腐胺）与2-内酰胺类药物联合可敏化2-内酰胺类药物耐药性（敏感性增加4- 64倍），但与多粘菌素B联合可诱导铜绿假单胞菌对多粘菌素B产生耐药性（耐药性增加4- 8倍）。多胺对2-内酰胺和多粘菌素B作用的分子机制尚不清楚。多胺是在所有生物体中合成的带正电荷的化合物。多胺的细胞内浓度受到生物合成、降解和运输的严格调节。多胺的作用主要是细胞生长，但在细菌中也有各种其他作用。主要研究者假设：1）外源性多胺改变了细胞中的多胺稳态; 2）这种改变诱导细胞应答以维持细胞内多胺浓度; 3）细胞应答包括多胺对2-内酰胺和多粘菌素B敏感性的影响。具体目标1：阐明多胺对铜绿假单胞菌、克雷伯菌、铜绿假单胞菌、铜绿假单胞菌、铜绿假单pneumoniae和A.鲍曼不动杆菌。将采用最小抑菌浓度、时间杀灭和棋盘测定的标准方法来阐明多胺对铜绿假单胞菌、克雷伯氏菌和铜绿假单胞菌MDR临床分离株2-内酰胺和多粘菌素B敏感性的影响。pneumoniae和A.鲍曼不动杆菌。具体目标二：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敏感性影响中的作用。缺乏多胺摄取和生物合成的突变体铜绿假单胞菌PAO 1将通过使用基因置换技术缺失两个主要的多胺摄取系统（potABCD/potABCDEFGH）和多胺生物合成的基因（speA/speC）来产生。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敏感性影响的分子细节，这将为设计新的治疗药物或治疗MDR感染的策略提供基础。 公共卫生相关性：由多药耐药（MDR）病原体引起的传染病是主要的公共卫生问题，因为有限的治疗选择来对抗它们。已知多胺可使2-内酰胺抗性病原体致敏。本研究旨在阐明多胺对现有2-内酰胺类抗生素治疗MDR病原体的适用性，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.