Enzymatic and mechanical disruption before successive photodynamic therapy targets the extracellular matrix of Candida albicans

To evaluate whether pretreatment strategies targeting the extracellular matrix (ECM), such as DNase I and low-frequency ultrasound, enhance the efficacy of successive antimicrobial photodynamic therapy (aPDT) against Candida albicans biofilms and to assess the effects on biofilm components. Forty-eight-hour C. albicans (ATCC 90028) biofilms were treated under four conditions: (I) aPDT [Photodithazine (PDZ) (25 mg/L) for 20 min + Light-Emitting Diode (LED) (660 nm, 18 J/cm²)], (II) DNase+aPDT [5 min with 20 U/mL DNase I before aPDT], (III) sonication+aPDT [7 W, 170-190 J before aPDT], (IV) Dn+So+aPDT. Ten successive aPDT applications were performed. Colony-forming units per milliliter (CFU/mL) was assessed after each application. After applications 1, 5, and 10, total and insoluble dry weight, soluble/insoluble proteins, water-soluble polysaccharides (WSP), alkali-soluble polysaccharides (ASP), and extracellular DNA (eDNA) were quantified. Biofilm architecture was analyzed by scanning electron microscopy (SEM). All groups showed significant CFU/mL reduction after ten aPDT applications compared to untreated control: aPDT (5.7 log₁₀), Dn+aPDT (5.5 log₁₀), So+aPDT (5.3 log₁₀), Dn+So+aPDT (5.3 log₁₀) (p<0.05). Insoluble proteins decreased after the first application (46%), and soluble proteins after the fifth and tenth (45%; p≤0.032). WSP and eDNA were significantly reduced (p≤0.047). No significant differences were observed for total/insoluble dry weight, proteins, and ASP. SEM revealed wall deformities and fewer clusters. Successive aPDT applications reduced CFU/mL, WSP, and eDNA in C. albicans biofilms regardless of pretreatment strategy.