Abstract Rationale Secondary bacterial pneumonia is a frequent and deadly complication of acute respiratory distress syndrome (ARDS) and acute lung injury (ALI). While immune and bacterial virulence mechanisms are recognized drivers of pneumonia, the ecological context that enables secondary infection within the injured lung remains poorly understood. We sought to identify alterations in the alveolar metabolic microenvironment induced by ALI that promote the growth of pneumonia-associated pathogens. To do this, we developed a novel ex vivo approach using bronchoalveolar lavage fluid (BALF) as a growth medium to test how injury-associated alveolar metabolites influence pathogen growth. Methods C57BL/6 mice were exposed to three days of hyperoxia (95% O2) to induce ALI, followed by BALF collection. Alveolar leak and inflammation were quantified by IgM ELISA (enzyme-linked immunosorbent assay) and protein assay. Cell-free BALF from hyperoxic (ALI) and normoxic (healthy) mice served as the sole growth medium in a novel ex vivo assay measuring Pseudomonas aeruginosa growth by OD666 and quantitative plating. Metabolomic profiling (¹H NMR) compared BALF metabolite composition between groups, and β-hydroxybutyrate (βHB)—detected only in ALI BALF—was added to healthy BALF to test its effect on P. aeruginosa growth. Results The murine hyperoxia model produced ALI characterized by alveolar leak and inflammation. In our novel ex vivo assay, P. aeruginosa growth was significantly enhanced in BALF collected from mice with ALI. Metabolomic profiling identified multiple metabolites enriched in BALF from ALI mice, including β-hydroxybutyrate (βHB), which was absent from healthy controls. To investigate βHB’s role as a nutrient source for P. aeruginosa during ALI, βHB was added back to BALF from healthy mice in our ex vivo growth assay. Adding βHB to healthy BALF enhanced P. aeruginosa growth to levels comparable to ALI BALF, and a biologically relevant concentration of βHB (250 μM) augmented bacterial growth beyond the ALI reference group, supporting its role as a mediator of pathogen growth. Conclusions Acute lung injury remodels the alveolar metabolic environment in a manner that promotes pathogen growth. A single host-derived metabolite present in alveolar leak, β-hydroxybutyrate, is sufficient to promote P. aeruginosa growth in an ex vivo assay. These findings demonstrate that ALI reshapes nutrient availability within the lungs, creating nutrient-rich ecological niches that favor secondary bacterial pneumonia. This abstract is funded by: None
Heinzinger et al. (2026) studied this question.