AFM characterization of early P. aeruginosa aggregates highlights emergent mechanical properties.

Pseudomonas aeruginosa (Pa) is a leading cause of chronic lung infections in people with cystic fibrosis (pwCF), where its ability to form resilient, multicellular communities contributes to antibiotic tolerance and long-term persistence. While much of our understanding of Pa biofilms comes from surface-attached models, recent studies have emphasized the clinical relevance of suspended bacterial aggregates-dense, three-dimensional clusters that form early during infection and exhibit key biofilm-like properties. However, the physical characteristics of these aggregates remain poorly defined. Here, we apply atomic force microscopy (AFM) to visualize and quantify the structural and mechanical properties of Pa aggregates formed in synthetic cystic fibrosis sputum medium (SCFM2). Compared to planktonic cultures grown without mucin, aggregates formed in SCFM2 exhibited complex architecture and increased resistance to deformation, as measured by force spectroscopy. These differences emerged despite the absence of mature extracellular matrix components, suggesting that environmental cues and spatial organization alone may be sufficient to enhance aggregate mechanical resilience. Our results demonstrate that AFM provides a powerful, high-resolution approach for studying early-stage bacterial aggregates under physiologically relevant conditions. By resolving structural features and quantifying localized mechanical strength, this method offers new insight into how aggregate architecture contributes to persistence during chronic infection. These findings lay the groundwork for future studies targeting the physical robustness of bacterial communities as an early vulnerability in the pathogenesis of Pa both in CF and in other infection settings.IMPORTANCEChronic infections in people with cystic fibrosis are notoriously difficult to treat, in part due to the ability of Pseudomonas aeruginosa (Pa) to form protective communities known as aggregates. These suspended, multicellular clusters are not well captured by traditional surface-attached biofilm models but are now recognized as an important feature of persistent infection. Understanding how these aggregates resist physical and antimicrobial disruption is essential for developing better therapies. This study uses atomic force microscopy (AFM) to examine Pa aggregates at nanometer resolution in a laboratory model that mimics cystic fibrosis lung secretions. AFM not only visualizes individual aggregates but also measures how strongly they resist being physically deformed. Our findings show that aggregates formed in this environment are structurally robust, compared to single cells. These results highlight the importance of early physical organization in bacterial persistence and suggest new directions for therapies aimed at disrupting bacterial communities before they become established.