Sequential Phage Pretreatment and TiO2–Thyme Essential Oil Photocatalysis: A Synergistic Approach to Pseudomonas aeruginosa Biofilm Inhibition and Control

This work introduces an original sequential bio-inspired strategy combining lytic phage pretreatment with TiO2–thyme essential oil (TEO) photocatalysis, achieving near-complete inhibition of both biofilm initiation and maturation. By simultaneously targeting planktonic cells, mature biofilms, and extracellular DNA (eDNA), this approach addresses key mechanisms involved in biofilm persistence. Pseudomonas aeruginosa ATCC 4114 was selected as the biological model due to its relevance in water distribution systems and its strong biofilm-forming ability. Experimental results showed that phage pretreatment alone inhibited biofilm formation by planktonic cells by up to 99.6% (inactivation rate constant k = 0.034 min−1) and weakened bacterial attachment in mature biofilms by 89.06% (k = 0.011 min−1). To further enhance photocatalytic efficacy, titanium dioxide (TiO2) was combined with TEO at 0.05% (v/v) as a bio-inspired photosensitizer. UV–Vis spectroscopy confirmed TiO2-TEO interactions that extended light absorption into the visible region (400–700 nm), thereby enhancing photocatalytic efficiency. This combination was designed to suppress residual biofilm development and disrupt extracellular DNA (eDNA), a critical component of biofilm structure and stability. The integrated approach involving phage pretreatment followed by TiO2–TEO (0.05%) photocatalysis achieved 99.99% inhibition of both biofilm initiation and maturation phases, with significantly increased kinetic parameters (A = 2.62 for planktonic cells and A = 3.65 for sessile cells; k = 0.076 min−1 and 0.063 min−1, respectively; p < 0.01). This study provides novel insights into water disinfection strategies using photocatalytic treatment, emphasizing the importance of monitoring post-treatment bacterial virulence factor expression.