(521) Understanding how nutrient availability impacts biofilm formation of nontuberculous mycobacterium (NTM)

Background: The incidence of NTM infections is steadily increasing each year among the global Cystic Fibrosis (CF) population. Treatment of NTM infections is challenging due to both inherent drug resistance and physiological tolerance. Mycobacterium abscessus is particularly problematic due to its resistance to most antibiotics and its correlation with rapid lung function decline. Biofilm formation, which enhances M. abscessus antibiotic resistance in vitro, is affected by carbon and nitrogen availability in some NTM species. It is currently unknown if M. abscessus aggregation is similarly controlled by carbon and nitrogen availability, and the mechanisms and regulatory nodes through which the nutritional environment regulate M. abscessus aggregation are likewise unclear. Finally, the spatial arrangement of M. abscessus biofilms and its relevance to CF airway colonization have not been explored. Therefore, this study aims to achieve three goals: 1) Determine how carbon and nitrogen availability influences M. abscessus biofilm formation, 2) identify how the CF sputum environment impacts the evolution of M. abscessus biofilm and colony morphology phenotypes, and 3) define the in situ spatial arrangement of M. abscessus biofilms in CF sputum.

Methods: We used an in vitro aggregation assay to identify how carbon and nitrogen availability in synthetic cystic fibrosis medium (SCFM), a medium designed to mimic CF sputum nutrient environment, impacts M. abscessus aggregation dynamics. We also experimentally evolved an M. abscessus strain in SCFM to elucidate the evolutionary effects of adaptation in the CF sputum environment. We characterized the colony morphology and aggregation dynamics of the evolved populations and performed whole-genome sequencing (WGS) to identify mutations that emerged during the evolution experiment. Finally, we utilized MiPACT-HCR, a novel tissue-fixing and molecular imaging technique, to preserve and characterize the in situ environment of multiple NTM-positive CF sputum samples. We characterized the composition, distribution, and 3D structure of M. abscessus biofilms in sputum.

Results: Aggregation assay shows that M. abscessus aggregation is predominantly regulated by nitrogen availability, rather than carbon. Increased nitrogen content correlates with dispersal of aggregates and vice versa. Evolution of M. abscessus in SCFM generated new variants with distinct colony morphology and a prolonged aggregation phenotype compared to the wild-type ancestor. WGS analysis identified new mutations with relevance to metabolism and metabolite transport. Finally, CF sputum imaging shows that M. abscessus can exist as both single-species intracellular aggregates or large polymicrobial extracellular biofilms in situ.

Conclusions: The study is currently ongoing. Our future goals will focus on understanding the molecular mechanisms and regulatory pathways that mediate M. abscessus aggregation in response to nutritional flux. Molecular genetics and targeted metabolomics will be used to determine the role of flux-dependent metabolites in regulating aggregation. Additionally, using the information gained from in situ characterization, we will generate a laboratory model that can simulate M. abscessus biofilms as we have observed them in CF sputum.

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