Biotechnol Bioeng. 2025 Nov 13. doi: 10.1002/bit.70098. Online ahead of print.
ABSTRACT
Opportunistic colonization and recurring infection by Pseudomonas aeruginosa are substantial risks to lung functionality for people with underlying respiratory diseases such as cystic fibrosis and chronic obstructive pulmonary disease. The complex metabolic and phenotypic adaptations P. aeruginosa exhibits in response to its environmental conditions make relevant in vitro models of pathogenic populations crucial for identifying and evaluating effective antimicrobial targets. However, an extracellular component that is rarely integrated into these experimental platforms is a spatially extensive, semisolid gel medium representative of biological respiratory mucus layers that P. aeruginosa propagates through via active motility. In this investigation, we examine the applicability of swim plate assays, a qualitative methodology for measuring flagellar swimming motility, as an in vitro platform to study the spatiotemporal development of P. aeruginosa strain PA14. The propagation behavior of PA14 was tracked through time-lapse microscopy and studied under different agar gel compositions incorporating methylcellulose as well as native MUC5AC mucin. To aid quantitative characterization of PA14 population expansion, we paired this experimental workflow with a continuum model that would fit density profile fluctuations to changes in PA14 swimming motility and growth kinetics. We observed higher extracellular concentration and production of the phenazine pyocyanin when PA14 populations were grown in swim plate assays, supporting the emergence of heterogeneous growth environments within the microbial population. PA14 swim plates exhibited a significantly lower spreading velocity in gels containing 0.30% w/v MUC5AC, which model-to-experiment fitting results determined to be driven by reductions in PA14 swimming motility. Continuum model parameters additionally portrayed PA14 expansion in mucin gels, having cell growth outcompeting cell motility, which aligned with experimental assay observations of macrocolonies rapidly developing to high biomass density states. In contrast, PA14 did not show spreading velocity differences in gels containing 0.30% methylcellulose, and fitted parameters did not identify major growth and motility differences when compared to agar-only gels. Combined with the resource accessibility of this experimental platform, the swim plate assay as an in vitro model is well suited to investigations of pathogenic community dynamics in gel conditions over more extensive spatial and time scales.
PMID:41230780 | DOI:10.1002/bit.70098