Biofilm comes back: Controlling regrowth by mitigating the cell-matrix interaction

Biofilms wield a notorious impact on biological infections and environmental sanitation in daily life. Tremendous efforts have been made to develop antimicrobial agents that effectively kill bacterial cells, but preventing biofilm reformation remains unsolved. For instance, a hydrogen peroxide (H₂O₂) and peracetic acid (PAA) mixture can inactivate almost 100 % of Pseudomonas aeruginosa cells in a biofilm within 10 min, but why do cells regrow and recover the biofilm within a day? We hypothesize that interaction between bacterial cells and extracellular polymeric substances (EPS) remaining during biofilm treatment is responsible for biofilm regrowth. This hypothesis is examined by quantifying the number of cells associated with a unit volume of EPS (Manders coefficient) and the mass of EPS associated with a cell in the P. aeruginosa biofilm by processing immunostained biofilm images with the BiofilmQ software. Interestingly, the H₂O₂ + PAA mixture increases the EPS mass associated with a single cell compared with untreated conditions while minimally affecting the Manders coefficient. To resolve this challenge, we devise a sequential strategy in which self-locomotive MnO₂-doped diatoms are initially added to reduce the EPS volume, making bacterial cells more susceptible to the subsequently added H₂O₂ + PAA mixture. This strategy significantly reduces the mass of EPS associated with cells, thereby inhibiting biofilm regrowth over 2 months. Overall, the results of these studies provide valuable insights for developing advanced antibacterial strategies that can significantly improve an ability to control infection and biofouling across a diverse spectrum of household, healthcare, and industrial applications.