Allura Red–Mediated Photodynamic Therapy: A Novel Multi-Assay Evaluation Against Pseudomonas aeruginosa

<ns3:p> Background Pseudomonas aeruginosa is a multidrug-resistant and biofilm-forming bacterium that poses serious challenges to conventional antibiotic therapy. In this study, a dose-dependent antimicrobial photodynamic therapy (aPDT) approach was developed using Allura Red in combination with a 630 nm red laser. Four complementary assays were integrated into a unified evaluation platform: AMDA for singlet oxygen, XTT for metabolic activity, CFU enumeration for bacterial viability, and Crystal Violet (CV) staining for biofilm biomass. Results: The Allura Red–mediated PDT demonstrated dose-dependent effects, with 15 J/cm <ns3:sup>2</ns3:sup> yielding ~87% CFU reduction and &gt;75% biofilm inhibition. Conclusion: This study introduces, for the first time, a novel Allura Red–based, multi-assay PDT framework against P. aeruginosa, providing a comprehensive and translational evaluation method. Background <ns3:italic>Pseudomonas aeruginosa</ns3:italic> is a multidrug-resistant and biofilm-forming bacterium that represents a major challenge to conventional antibiotics. Methods In this study, a novel dose-dependent antimicrobial photodynamic therapy (aPDT) approach was applied using Allura Red combined with red laser light (630 nm) at fluences of 5, 10, and 15 J/cm <ns3:sup>2</ns3:sup> . Four complementary assays were integrated into a single protocol: aminophenyl methylene diphosphonate (AMDA) and XTT for reactive oxygen species (ROS) and singlet oxygen detection, colony-forming unit (CFU) counts for bacterial viability, and Crystal Violet (CV) staining for biofilm biomass. All experiments were performed in triplicate with appropriate controls, and statistical significance was determined using one-way ANOVA (p &lt; 0.05). Results Treatment with 15 J/cm <ns3:sup>2</ns3:sup> produced the highest ROS generation and bacterial killing, with CFU counts reduced by approximately 87%, compared to 71% at 10 J/cm <ns3:sup>2</ns3:sup> and 36% at 5 J/cm <ns3:sup>2</ns3:sup> . Biofilm inhibition exceeded 76% at the highest dose. Statistical analysis confirmed significant dose-dependent effects across all assays. Conclusion The integrated, multidimensional protocol provides a reliable method for assessing PDT efficacy and offers valuable insight into the relationship between oxidative stress, bacterial inactivation, and biofilm disruption. This strategy represents a promising approach against biofilm-associated and drug-resistant pathogens. </ns3:p>