Cold atmospheric plasma improves antifungal responsiveness of Aspergillus flavus and Fusarium keratoplasticum conidia and mycelia

The purpose of this study is to evaluate sublethal cold atmospheric plasma
(CAP) treatment of filamentous fungal pathogen susceptibility to commonly
used antifungal drugs in vitro. Response to CAP in combination with
voriconazole, fluconazole, amphotericin B, and caspofungin was evaluated
in Aspergillus flavus and Fusarium keratoplasticum conidia and mycelium;
conidial response to fluconazole was also assessed in three strains of F.
falciforme. Conidial susceptibility to antifungal drugs alone or in
combination with CAP was assessed using a modified CLSI broth
microdilution assay with MIC determination and colony-forming unit (CFU)
enumeration. Mycelial viability and biofilm thickness changes in response
to antifungal drugs alone or in combination with CAP were assessed over 24
hours post treatment. CAP enhanced antifungal drug efficacy against all
fungal species, though effects differed by drug and growth form. CAP
enhanced antifungal drug susceptibility in conidia, with the strongest
effect observed for F. keratoplasticum conidia, where caspofungin MIC
decreased fourfold and sensitivity to fluconazole, which exerted no effect
in absence of CAP, was restored when combined with sublethal CAP
treatment. In A. flavus, CAP lowered the MIC of voriconazole but increased
the MIC of amphotericin B, despite reductions in viable cell counts.
Differential responses to CAP and fluconazole were observed across three
strains of F. falciforme, suggesting variability in CAP response. In
treated biofilms, CAP alone initially reduced mycelial viability and
biofilm thickness, but partial recovery of the fungus was observed over
time in most cases. When combined with antifungal drugs, CAP significantly
enhanced reduction of mycelial viability and thickness beyond antifungal
treatment alone. In F. keratoplasticum biofilms, the combination of CAP
and antifungal drugs produced sustained reductions in mycelial viability
and biofilm thickness, whereas A. flavus biofilms were more resistant to
CAP treatment and exhibited consistent recovery after 24 hours.