VCOM Louisiana Research Day Program

Biomedical Research

Cyrill Slezak, PhD; Karaleen Anderson, MD; Tyson Hillock, OMS-II; Mariel Miller, BS; Peter Dungel, PhD; Olga Kopp, PhD; Katja Sterflinger, PhD; Paul Slezak, MD Utah Valley University, Orem, Utah; Ludwig Boltzmann Institute for Traumatology, Austrian Cluster for Tissue Regeneration, University of Natural Resources and Life Sciences, Vienna, Austria 05 SHOCKWAVES INCREASE IN VITRO RESILIENCE OF RHIZOPUS ORYZAE BIOFILM UNDER AMPHOTERICIN B TREATMENT

LFUS, radial pressure waves (RPW), or SW on R. oryzae biofilms. Methods: LFUS biofilms were grown and treated in 96 well plates, whereas all RPW and SW were grown and treated in 24 well plates. SW was also grown and treated in 15mL falcon tubes with a fibrin glue base to mimic tissue biofilm growth. Falcon tube treatment was conducted in a water bath to mimic energy transfer through tissue. LFUS biofilms were treated with a serial dilution of Amp B (16-0.03 μg/mL) in four main conditions: LFUS only, LFUS Amp B, Amp B only, and an untreated control. The LFUS parameters were 550 kHz for 10 min duration. SW was treated with either electrohydraulic (EH) or electromagnetic (EM) applicators. RPW and SW were treated with a predetermined Amp B MIC50 of 8 μg/mL in four main conditions: SW or RPW only, SW or RPW Amp B, Amp B only, and an untreated control. EH parameters were 0.19 mJ/mm2 at 3Hz with 300 pulses. EM parameters were 0.12 mJ/ mm2 at 3Hz with 300 pulses. RPW parameters were 300 pulses with 1 or 3 Barr. EM water bath parameters were 0.55 mJ/mm2 at 3Hz with 300 pulses. XTT and BrdU labeling assays were used to measure cellular respiration and proliferation,

Background: Extracorporeal shockwave (SW) treatment is used to treat chronic soft tissue wounds, including burns and diabetic ulcers. While previous reports suggest that SW are effective at damaging bacterial biofilms, the effect of shockwaves on fungal biofilms has been largely ignored. Fungi have shown a commensal interaction with Staphylococcus aureus , promoting structural and chemical drug resistance. Many studies have shown that low frequency ultrasound (LFUS) treatments disrupt both bacterial and fungal biofilms, specifically with S. aureus . It is therefore likely that LFUS treatment of polymicrobial biofilms would effectively neutralize both fungal and bacterial biofilms and facilitate drug delivery and efficacy. R. oryzae is a leading cause of Mucormycosis and is associated with increased infection in uncontrolled diabetes, trauma, and burn patients. Amphotericin B (Amp B) has been the first line of treatment for Mucormycosis since the 1950s. However, it can have severe side effects including fatal hepato and nephrotoxicity. Despite the growing clinical significance of this disease, novel antifungal susceptibility testing on this species has remained largely uninvestigated. Objective: The aim of this work is to test for improved drug efficacy through synergistic mechanical biofilm disruption when applying

respectively. Confocal and light microscopy with either calcofluor or crystal violet staining observed biofilm structural integrity. Results: LFUS significantly enhanced Amp B effects at concentrations of < 1 μg/mL when compared to Amp B alone (p > 0.0001). Similarly, RPW showed a synergistic effect on Amp B efficacy (p > 0.001). However, both EH and EM increased cellular respiration with Amp B treatment (p > 0.0001 and p > 0.05, respectively). EM treatment in the water bath further showed increased cellular metabolism regardless of receiving Amp B treatment (p > 0.05). Proliferation surprisingly showed a decrease in combined EH and Amp B therapy when compared to Amp B alone (p > 0.0001). Confocal and light microscopy suggested increased biofilm integrity after synergistic treatment. Conclusions: This study provides evidence of LFUS and RPW enhancing the effectiveness of Amphotericin B through synergistic mechanical agitation when treating R. oryzae biofilm. In contrast, the application of SW resulted in a strong biological response of the biofilm, significantly increasing cellular viability, thus lowering the efficacy of Amp B. These findings further support the regenerative potential of SW by illustrating that this effect exists even in fungal biofilms.

17 2023 Via Research Recognition Day