Mortality from multi-drug resistant (MDR) bacterial infections is projected to cause 10 million deaths per year worldwide by 2050, making antibiotic resistance a vital threat to society. Fueling this crisis even further is the increased use of antibiotics among 45-97% of COVID-19 patients, which will likely boost selective pressure for MDR phenotypes. Unfortunately, with the misuse and overuse of these chemical compounds, pathogens have evolved several mechanisms to resist all currently used anti-infective agents. The World Health Organization recently deemed carbapenem resistant Pseudomonas aeruginosa as one of the most difficult infections to treat, so future management of this species and other Gram-negative pathogens will require novel, yet undiscovered, antibiotics. As a bacterial group, environemtnal Pseudomonas strains (env-Ps) are well known for their extensive genomic content and diversity. Owed to their genetic complexity is the production of an assorted repertoire of secondary metabolites that have been shown to prevent the growth of pathogenic fungi, breakdown complex recalcitrant compounds, exhibit anti-tumor activity, and inhibit a wide range of bacterial pathogens including methicillin-resistant Staphylococcus aureus and Mycobacterium tuberculosis. Moreover, soil and freshwater environments are dominated worldwide by pseudomonads, whose global abundance suggest the expression of certain traits that are advantageous to ecological survival. In contrast, P. aeruginosa is observed infrequently in ecological settings. One trait that is likely to contribute to such fitness effects of env-Ps is the ability to antagonize nearby competitors through production of antimicrobial compounds. Previous work showed that water-derived env-Ps were able to inhibit cystic fibrosis (CF) derived pathogens including P. aeruginosa, Burkholderia, Achromobacter, and Stenotrophomonas species, and subsequently identified gene clusters involved in antagonistic activity within the environmental strains. As a continuation of this project, env-Ps from nutrient-rich water systems are hypothesized to be sources of potent antimicrobial activity. Indeed, preliminary data shows that env-Ps from a polluted river exhibited the remarkable ability to inhibit CF-derived extensively drug resistant (XDR) pathogens, including carbapenem resistant P. aeruginosa. In this study, an innovation approach using culturable bacteria will be utilized to link antagonistic activity (phenotype) to diverse biosynthetic gene clusters (genotype) involved in antimicrobial activity. By investigating direct competitive interactions between env-Ps and XDR pathogens, novel antagonistic factors are expected to be identified. This will be achieved by (i) isolating and determining the antimicrobial activity of env-Ps from polluted water columns; (ii) identification of biosynthetic gene clusters involved in the activity; and (iii) initial characterization of encoded undiscovered compounds. Combined re...