Biology subjects

Hawryluk, D. S.

Publications and source records attributed to Hawryluk, D. S..

3 recordsLinked to original sources

bioRxiv2025

Intra-Abdominal Photodynamic Therapy in a Rabbit Model of Perforated Appendicitis

ImportancePerforated appendicitis is a common and morbid pediatric surgical condition frequently requiring prolonged antibiotic therapy. Adjunctive intraoperative strategies for local antimicrobial therapy are limited, and photodynamic therapy (PDT) may offer a targeted, resistance-independent approach. ObjectiveTo evaluate the feasibility, safety, and preliminary efficacy of laparoscopic methylene blue photodynamic therapy (MB-PDT) for intra-abdominal disinfection in a rabbit model of perforated appendicitis. DesignExperimental preclinical animal study conducted between 2023 and 2025. SettingSingle-center laboratory-based animal research facility affiliated with an academic medical center. ParticipantsNineteen New Zealand White rabbits underwent surgically induced perforated appendicitis with peritonitis via appendiceal ligation and electrocautery perforation. InterventionsRabbits received laparoscopic MB-PDT (n=9) or control conditions (methylene blue only, no light; n=10) 24 - 40 hours after appendiceal perforation. MB-PDT involved peritoneal lavage with 300 {micro}g/mL methylene blue, followed by 665 nm laser illumination to 25 J/cm2. Peritoneal aspirates were collected before and 24h after intervention. Bacterial burden was quantified, and isolated bacteria were submitted to in vitro PDT. Tissue samples from intraperitoneal organs were collected 24 hours post-intervention. Main Outcomes and MeasuresPrimary outcomes included feasibility of PDT delivery, safety (histologic evidence of off-target injury), and preliminary in vivo efficacy (change in bacterial burden pre-vs. post-intervention). Secondary outcomes included clinical parameter changes and in vitro MB-PDT efficacy. ResultsOf the 14 rabbits that developed peritonitis, 10 (age 5-6 months, weight 3.1-3.8 kg) completed follow-up. Laparoscopic MB-PDT delivery was feasible and caused no off-target histologic injury. Bacterial burden increased in MB-PDT animals relative to controls, though not significantly (p=0.18). Post-intervention, body temperature showed a trend toward reduction in MB-PDT animals (-0.37 {+/-} 1.32{degrees}C) vs controls (0.26 {+/-} 0.76{degrees}C; p=0.27). In vitro MB-PDT significantly reduced bacterial burden for all tested species (p<0.001), with greater efficacy in Gram-positive bacteria. Conclusions and RelevanceLaparoscopic MB-PDT was feasible and safe in a rabbit model of perforated appendicitis, with high in vitro antimicrobial efficacy. Although in vivo bacterial reduction was not demonstrated, these results support further investigation of MB-PDT as a novel intraoperative adjunct for treating intra-abdominal infection.

microbiology↗

bioRxiv2025

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

ObjectivesPerforated 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. HypothesisIn monoculture planktonic samples, PDT achieves a minimum of 99.9% bacterial cell kill (3 log10 growth reduction per mL). MethodsAfter 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. ResultsBacteria 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 log10 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 log10 and 2.23 {+/-} 0.64 log10, 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). ConclusionsPDT 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.

microbiology↗

bioRxiv2025

Susceptibility of bacterial species commonly found in abdominal abscesses to low-dose photodynamic therapy: Effects of methylene blue concentration, fluence rate, and fluence

ObjectivesThe objective of this study was to determine the effects of methylene blue (MB) concentration, laser fluence rate, and laser fluence on efficacy of in vitro photodynamic therapy (PDT) for four bacteria commonly found in human abscesses. Materials and MethodsPDT experiments were performed with four of the most common bacteria found in abdominal abscesses: Escherichia coli (E. coli), Enterococcus faecalis (E. faecalis), Staphylococcus aureus (S. aureus), and Pseudomonas aeruginosa (P. aeruginosa). MB concentration was varied from 50-300 {micro}g/mL, and laser fluence rate was varied from 1-4 mW/cm2 at a fluence of 7.2 J/cm2. Higher fluence rates and fluences were explored for P. aeruginosa. Primary outcomes were reduction in colony forming units (CFU) following PDT, and measured MB uptake following drug incubation. ResultsGram-positive bacteria (E. faecalis and S. aureus) were eradicated at all MB concentrations and laser fluence rates tested. Efficacy was reduced for E. coli, but still resulted in >6 log10 reduction in CFU when MB concentration was at least 100 {micro}g/mL. P. aeruginosa required higher fluence (28.8 J/cm2) to achieve comparable efficacy, while increasing fluence rate did not have a significant effect on PDT efficacy. MB uptake was reduced in Gram-negative species compared to Gram-positive, particularly P. aeruginosa, although uptake was not significantly correlated with CFU reduction. ConclusionsGram-positive bacteria can be eradicated in vitro with low levels of MB (50 {micro}g/mL), laser fluence (7.2 J/cm2), and laser fluence rate (1 mW/cm2). E. coli showed substantial cell killing (>6 log10 CFU reduction) with these same parameters. Low MB uptake and PDT efficacy in P. aeruginosa could be overcome by increasing the laser fluence, while increasing fluence rate did not have an effect.

microbiology↗