Methylene blue photodynamic therapy of bacterial species found in human abscesses: planktonic, biofilm, and 3D silicone models

Abstract

Deep tissue abscesses are inflammatory, purulent lesions encased in a fibrin-rich pseudocapsule that include multiple bacterial and fungal species. We have initiated a Phase 1 clinical trial exploring the safety and feasibility of methylene blue photodynamic therapy (MB-PDT) at the time of abscess drainage. To optimize treatment parameters for future clinical applications, our goal is to generate physically accurate three-dimensional (3D) abscess models upon which bacteria can be grown. Here, we report results of MB-PDT against four representative bacterial species found in human abscesses in planktonic culture, as biofilms on silicone, and pilot results in 3D silicone molds derived from human abscess computed tomography (CT) images. In all cases, MB-PDT was performed with 665 nm light at a fluence rate of 4 mW/cm² for 30 minutes, resulting in a fluence of 7.2 J/cm². In planktonic cultures, MB-PDT was effective against <i>Escherichia coli, Enterococcus faecalis,</i> and methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) (4- to 7-fold log CFU reduction). For <i>Klebsiella pneumoniae,</i> increased fluence was required to achieve comparable efficacy. When bacteria were grown as biofilms on silicone, MB-PDT efficacy was reduced (1- to 2-fold CFU reduction). A 3D silicone model was generated based on pelvic abscess CT images, and MRSA was grown in this model for six days. Crystal violet staining showed abundant growth on the silicone, without penetration into the model. These results motivate exploration of both light and drug dose ranging for biofilm samples. Future experiments will additionally focus on MB-PDT of bacteria grown on 3D silicone surfaces.