2025-2026

Investigating Biofilm Disruption in Pseudomonas putida by Exogenous Pyruvate Oxidase (POX)

Bacterial biofilms increase resistance to antibiotics, representing one of the major clinical concerns in bacterial infection. The purpose of this project was to investigate the potential of an enzymatic therapeutic approach using exogenous Pyruvate Oxidase (POX) to disrupt Pseudomonas putida biofilms through pyruvate depletion and hydrogen peroxide (H₂O₂) generation. Established P. putida biofilms were assigned to five experimental groups: Control (+Pyruvate), Inactivated POX (+Pyruvate), Starvation (-Pyruvate), Active POX (+Pyruvate), and Active POX + Cat (+Pyruvate and Catalase). All groups were prepared with 0.05 M potassium phosphate buffer (KPi, pH 7.0) and the cofactors thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD), and magnesium ions (Mg²⁺). Biofilm biomass was quantified using Crystal Violet (CV) assays (absorbance at 590 nm) and P. putida viability was assessed by colony-forming unit (CFU) counts. As a result, the One-way ANOVA and Tukey’s multiple comparison test suggested the Active POX (+Pyruvate) group had significantly less biofilm biomass and CFU (P. putida viability) than all other groups. The Active POX + Cat (+Pyruvate) group dispersed biofilms significantly more than the Control (+Pyruvate) and Inactivated POX (+Pyruvate) groups. Adding catalase reduced the killing effect on P. putida, resulting in a higher P. putida viability compared to when only Active POX (+Pyruvate) was used. This implies that H₂O₂ generation is key for antimicrobial activity and breaking up biofilms, while pyruvate depletion may play a role in weakening biofilm structure. Taken together, these mechanisms indicate that using POX could serve as an enzymatic pretreatment to enhance antibiotic effectiveness against biofilm-related infections.