Abstract/Project Summary The CDC reported that in 2019, 2.8 million infections due to antibiotic resistant bacteria occurred in the US, causing over 35,000 deaths. Bacterial biofilms are responsible for many of these infections, as biofilms confer resistance to multiple antibiotics via nonspecific factors such as exclusion of the drug and altered bacterial metabolism. Multidrug resistant Pseudomonas aeruginosa, a strong biofilm-forming pathogen, was responsible for 32,000 infections and 2,700 deaths in 2019. Most of these infections are associated with biofilms, and P. aeruginosa displays the nonspecific antibiotic resistance conferred by the complex biofilm it forms. To combat the increasing threat of this pathogen innovative intervention strategies must be designed. One of these strategies is synergy with currently used antibiotics. We employ high throughput screens (HTS) of chemical libraries and screening of complex plat products to identify synergistic compounds and substances that effectively eradicate preformed biofilms. A multistep HTS of over 6,000 synthetic compounds identified two promising candidates, triclosan (TRI) and oxyclozanide (OXY), that synergize with the antibiotic tobramycin (TOB) against established P. aeruginosa biofilms. Triclosan is a well-studied antimicrobial approved by the FDA. However it has not been studied for synergy with TOB. In addition, a screen of plant-derived substances identified Larrea tridentata and a component of Hydrastis canadensis as capable of eradicating preformed P. aeruginosa biofilms. Berberine (BER), a compound made by Hydrastis, inhibited P. aeruginosa biofilms in our assays and has previously been shown to synergize with TOB. We propose to test this synergy in a biofilm wound infection model, which has not been done. We have now shown that extracts of Larrea, which is also known as Creosote (CRE), are highly active against established P. aeruginosa biofilms, and we have shown this extract to be effective in a murine biofilm wound model of P. aeruginosa infection. This model is based on in vivo bioluminescence imaging (BLI), which greatly speeds the process of in vivo testing of biofilm infection in animals. Bioluminescent P. aeruginosa form a biofilm on the underside of a scab over the wound, allowing quantification of the infection in individual animals over time by non-invasive imaging. Here, we propose to develop the treatments we have identified by testing TOB synergy of the compounds and extracts in the BLI wound model. This process will establish novel treatments for P. aeruginosa biofilm infections in a rapid and quantitative manner.