Methylene Blue-Photodynamic Therapy Effectively Kills Antibiotic-Resistant Bacteria from Pediatric Patients with Perforated Appendicitis

Abstract Objectives Perforated appendicitis (PA) is the most common cause of intra-abdominal abscess in children and is associated with higher costs, extended hospitalizations, and worse postoperative outcomes compared to uncomplicated appendicitis. Photodynamic therapy (PDT) utilizes a photosensitizer and light to generate cytotoxic reactive species that are efficacious against bacteria. We sought to determine whether PDT could effectively eliminate bacteria isolated from the peritoneal cavities of pediatric patients with perforated appendicitis. Hypothesis In monoculture planktonic samples, PDT achieves a minimum of 99.9% bacterial cell kill (3 log 10 growth reduction per mL). Methods After IRB approval, bacterial subcultures from 30 pediatric surgical patients with PA were developed as planktonic monocultures. Samples were derived from standard clinical collection of peritoneal fluid in patients undergoing surgery for PA. Cultures were incubated with the photosensitizer methylene blue (MB) and exposed to a 665 nm laser to perform PDT. Control conditions were MB alone, laser alone, or no MB and no laser. Log reductions in bacterial growth between groups were compared using two-way ANOVA. Results Bacteria were isolated from 28 (93.3%) specimens, and 26 (86.7%) were polymicrobial. The most prevalent organisms included Escherichia coli (76.7%), the Streptococcus anginosus group (56.7%), Bacteroides fragilis (46.6%), and Pseudomonas aeruginosa (26.7%), with antibiotic resistance common among E. coli isolates. For E. coli , MB-PDT caused a 5.86 ± 0.39 log 10 reduction compared to the no-treatment control (p<0.001). For S. anginosus and P. aeruginosa , MB-PDT resulted in reductions of 5.91 ± 0.88 log 10 and 2.23 ± 0.64 log 10 , respectively (all p<0.001). PDT showed comparable efficacy in isolates with multiple antibiotic resistance relative to those that were antibiotic pan-susceptible for E. coli (p=0.760), S. anginosus group (p>0.999), and P. aeruginosa (p=0.991). Conclusions PDT application to bacterial isolates from patients with perforated appendicitis achieved >99.9% bacterial kill in all isolated strains other than P. aeruginosa , regardless of antibiotic susceptibility, suggesting that PDT may be a viable adjunct to in vivo antimicrobial therapy.