Abstract
Bacterial biofilms are critical to pathogenesis and infection. They are associated with rising rates of antimicrobial resistance. Biofilms are correlated with worse clinical outcomes, making them important to infectious diseases research. There is a gap in knowledge surrounding biofilm kinetics and dynamics which makes biofilm research difficult to translate from bench to bedside. To address this gap, this work employs a well-characterized crystal violet biomass accrual and planktonic cell density assay across a clinically relevant time course and expands statistical analysis to include kinetic information in a protocol termed the TMBL (Temporal Mapping of the Biofilm Lifecycle) assay. TMBL's statistical framework quantitatively compares biofilm communities across time, species, and media conditions in a 96-well format. Measurements from TMBL can reliably be condensed into response features that inform the time-dependent behavior of adherent biomass and planktonic cell populations. Staphylococcus aureus and Pseudomonas aeruginosa biofilms were grown in conditions of metal starvation in nutrient-variable media to demonstrate the rigor and translational potential of this strategy. Significant differences in single-species biofilm formation are seen in metal-deplete conditions as compared to their controls which is consistent with the consensus literature on nutritional immunity that metal availability drives transcriptomic and metabolomic changes in numerous pathogens. Taken together, these results suggest that kinetic analysis of biofilm by TMBL represents a statistically and biologically rigorous approach to studying the biofilm lifecycle as a time-dependent process. In addition to current methods to study the impact of microbe and environmental factors on the biofilm lifecycle, this kinetic assay can inform biological discovery in biofilm formation and maintenance.