Project Summary/Abstract -- DePas The emergence of nontuberculous mycobacteria (NTM) as dangerous, antibiotic resistant pulmonary pathogens is outpacing research into their mechanisms of pathogenesis. Our long-term goal is to characterize NTM in the infection environment and develop new therapeutic approaches aimed at specific in vivo bacterial activities. The objective of this proposal is to directly assess NTM biofilm formation and growth rate in situ and determine how the infection environment impacts these processes. We hypothesize that the spatial and chemical environment of sputum from people with Cystic Fibrosis (CF) sputum supports the formation of antibiotic tolerant, slow-growing NTM biofilms through regulated cellular processes. The rationale for this proposal is that the biofilm state and growth rate of a specific bacterial pathogen can have drastic influences on the efficacy of antibiotics and the host immune response. We will test our central hypothesis with two specific aims: 1) Determine how NTM biofilm formation is regulated by the CF chemical environment and how it impacts antibiotic tolerance and 2) Determine how anoxia-induced dormancy influences physiological tolerance and biofilm formation of NTM. The proposed work will combine three innovative complementary techniques into one coherent strategy for investigating infection-relevant phenotypes such as biofilm formation and dormancy. We will employ a novel tissue clearing/bacterial visualization technique MiPACT-HCR in both aims. In Aim 2, we will also utilize a 3D model of the CF infection environment, the Agar Block Biofilm Assay. We will use a new in vitro aggregation assay that allows us to track and quantify the transition from planktonic cells to biofilms in both Aims. The proposal is significant because it will provide an accurate description of the physiological state of NTM during human infection to inform antibiotic choice and dosage decisions. It is also significant in that it will provide molecular targets for development of anti-biofilm and anti-dormancy strategies against NTM. The expected outcome of this work is a thorough understanding of the structure and prevalence of NTM communities during infection of patients with CF and insight into the mechanistic pathways driving biofilm formation and dormancy. These results will have a positive impact by assisting physicians make more appropriate treatment choices for NTM infections.