The incidence of nontuberculous mycobacterial (NTM) infections in cystic fibrosis (CF) patients is increasing, with some CF clinics in the US reporting NTM prevalence upwards of 25%. In addition, the current treatment regimen for NTM involves long courses of antibiotic cocktails that demonstrate limited efficacy and cause frequent and serious side effects. Mycobacterium abscessus, in particular, is difficult to treat and correlates with a more rapid decline in lung function compared to Mycobacterium avium complex. Studies with zebrafish and human cell cultures have demonstrated that M. abscessus is more virulent when aggregated into cord-like biofilms, in part because of the decreased ability of phagocytes to efficiently engulf and kill corded M. abscessus compared to diffuse M. abscessus cells. Translating these findings into useful clinical strategies for treating NTM infections will be greatly aided by 1.) A thorough understanding of the environmental conditions and genetic networks that control NTM aggregation, and 2.) Information about the in vivo aggregation state of NTM during infection. To address item 1, we developed an in vitro aggregation assay in which NTM such as M. abscessus and the model strain Mycobacterium smegmatis aggregate and disperse regularly in liquid culture. We found that M. smegmatis aggregation was dependent on carbon source type and availability. In particular, glycerol catabolism induces aggregation while pyruvate or amino acid catabolism leads to growth as dispersed cells. In contrast, oxygen availability does not induce changes in aggregation state. Currently, we are performing experiments in order to elucidate the genetic regulators that trigger aggregation in response to glycerol catabolism.