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.