Nontuberculous mycobacteria (NTM) are opportunistic pathogens that cause chronic pulmonary infections that are difficult to treat requiring prolonged multidrug therapy. Mycobacterium avium is the most common cause of pulmonary NTM disease in the United States, and infection rates are rising. Clinical persistence of M. avium is thought to reflect heterogeneous bacterial physiological states, yet scalable approaches to quantify clinically relevant phenotypes across diverse isolates remain limited. To address this gap, we assembled a genetically diverse collection of M. avium patient isolates obtained through ARUP, a national clinical reference laboratory. Using this collection, we developed and optimized a high-throughput in vitro biofilm assay to quantitatively assess biofilm formation across clinical isolates. Biofilm formation was assessed using a standardized visual scoring approach, and assay conditions were systematically optimized to improve reproducibility, sensitivity, and dynamic range. This enabled robust and repeatable phenotypic stratification of isolates. Application of this platform revealed substantial heterogeneity in biofilm formation among clinical M. avium isolates, indicating that biofilm capacity is not a uniform trait within this species. Leveraging this phenotypic diversity, we conducted a preliminary genome-wide association analysis to identify bacterial genetic features associated with biofilm formation. We identified an association between biofilm phenotype and the transcriptional regulator fadR, with most fadR-positive isolates exhibiting reduced biofilm formation, suggesting that lipid metabolic regulation and mycomembrane composition may influence biofilm development in M. avium. Together, these findings establish a scalable experimental framework for systematic biofilm phenotyping of clinically relevant M. avium isolates and highlight marked isolate-to-isolate variability in biofilm formation. This approach provides a foundation for future studies investigating how bacterial physiological heterogeneity contributes to persistence and treatment response in NTM lung disease. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
James et al. (Fri,) studied this question.