Photodynamic Therapy Effectively Kills Bacteria in Perforated Appendicitis: An In Vitro Pilot Study

Author(s):
Korry Wirth, MD ; Laurel Baglia, PhD ; Matthew Byrne, MD; Sadia Afrin, MS; Martin Pavelka; Marjorie Arca, MD; Timothy Baran, PhD; Nicole Wilson, PhD, MD

Background:

Perforated appendicitis is the most common cause of intraabdominal abscess in children. Photodynamic therapy utilizes a photosensitizer and laser light to generate cytotoxic reactive species that have been shown to have efficacy against bacteria. We sought to determine whether PDT would have a significant effect against bacteria isolated from the peritoneal cavities of pediatric patients with perforated appendicitis.

Hypothesis:

We hypothesize that, for monoculture planktonic samples, PDT will achieve at least 99.9% cell kill (3 log10
reduction).

Methods:

In our practice, abscess fluid is collected from all patients with perforated appendicitis and sent to the clinical microbiology laboratory for identification and antibiotic susceptibility testing. IRB approval was obtained to take individual bacterial subcultures from the fluid and develop them in planktonic monocultures. Cultures were incubated with methylene blue (MB) photosensitizer and treated with a 665 nm laser. Controls were isolates treated with MB alone and laser alone. Log reduction in growth between groups was compared using paired t-tests.

Results:

Bacteria were isolated from 28 (93.3%) specimens and 26 (86.7%) were polymicrobial. The most common organisms Escherichia coli (76.7%), Streptococcus anginosus (56.7%), Bacteroides fragilis (46.7%), and Pseudomonas aeruginosa (26.7%), with antibiotic resistance found in E.coli, S. anginosus, and P. Aeruginosa isolates. Treatment with MB alone reduced the growth of E. coli, S. anginosus, and  P. aeruginosa. Treatment with laser alone did not affect bacterial growth of all species. Compared to control conditions, MB-PDT resulted in a 5.90±0.47 log₁₀ reduction (Figure 1B, p<0.0001) in E. coli samples, 5.00±1.84 log₁₀reduction (Figure 1D, p<0.01) in S. anginosus, and 2.57±1.06 (Figure 1F, p < 0.001) reduction in P. Aeruginosa samples. There was a 1.30±0.51 reduction in E. coli samples not treated with light (Figure 1B, p<0.0001). E. coli and S. anginosus samples from different subjects demonstrated high susceptibility to PDT (p>0.95). PDT showed similar effects in E .coli isolates with multiple antibiotic resistance.

Conclusions:

PDT application to bacterial isolates from patients with perforated appendicitis achieves >99.9% bacterial kill, suggesting that PDT may be a viable adjunct to in vivo antimicrobial therapy. Additional in vivo studies using a rabbit model of perforated appendicitis are underway to demonstrate the efficacy of PDT in the peritoneal cavity.