Bacterial biofilms thwart the control of nosocomial infections, such as those resulting from medical device implantation. Elevated levels of one or more quorum sensing (QS) signals have been linked to the formation of bacterial biofilms, which makes eradication of bacteria colonizing the growth-amenable surfaces of implants difficult due to their biofilm-mediated resistance to antibiotics and host defense mechanisms. Therefore, it is desirable to develop ways to control quorum sensing and thus biofilm formation of P. aeruginosa and other Gram-negative pathogens. As with many Gram-negative organisms, N-acyl homoserine lactones (AHL) function as major QS signals for P. aeruginosa, specifically N-butanoyl-l-homoserine lactone (C(4)-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C(12)-HSL). A small number of enzymes have been found that show potential for modulating QS by degrading AHLs, but there development has been limited. We have isolated enzymes from Geobacilli that possess demonstrable activity toward AHLs (functioning as lactonases) and are able to disrupt AHL-dependent processes of test organisms in liquid culture, including P. aeruginosa biofilm formation. Our most active Geobacillus enzyme, a very stable, soluble enzyme that can be readily purified, is already being expressed in a commercially viable system. We propose to further characterize these enzymes, relative to known AHL lactonases, to judge their suitability for our ultimate objective to formulate these enzymes into coatings for medical device products. PUBLIC HEALTH RELEVANCE Preventing medical device-related Pseudomonas aeruginosa (PA) infections by inhibiting quorum sensing (QS) dependent processes such as biofilm formation, allowing host defense mechanisms and antibiotic strategies to be more effective, is a concept that holds great potential. Towards these ends, we discovered enzymes isolated from species of Geobacillus are capable of hydrolyzing the lactone ring of N-acyl-L- homoserine lactones, the molecular trigger of bacterial QS for Gram-negative bacteria. Furthermore, co-culturing purified these Geobacillus enzymes inhibits QS-dependent processes, such as PA biofilm formation. In Phase I, we will thoroughly characterize the lactonase activities of our Geobacillus enzymes. We will also quantitatively compare our Geobacillus enzymes to other emerging lactonase enzymes. [unreadable] [unreadable] [unreadable]