Roberts Darby M, Thomas Jonathan E, Salmon Jacklyn H, Cubeta Marc A, Stapelmann Katharina, Gilger Brian C
Departement of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, United States of America.
Department of Nuclear Engineering, North Carolina State University, Raleigh, North Carolina, United States of America.
PLoS One. 2025 Aug 11;20(8):e0326940. doi: 10.1371/journal.pone.0326940. eCollection 2025.
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 (from 16 to 4-8 μg/mL) 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 (from 0.25 to 0.06-0.125 μg/mL) but increased the MIC of amphotericin B (from 4 to >4 μg/mL), 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.
本研究的目的是评估亚致死剂量冷大气等离子体（CAP）处理对丝状真菌病原体体外常用抗真菌药物敏感性的影响。在黄曲霉和角膜塑形镰刀菌的分生孢子和菌丝体中评估了CAP与伏立康唑、氟康唑、两性霉素B和卡泊芬净联合使用的效果；还在三株镰状镰刀菌中评估了分生孢子对氟康唑的反应。使用改良的CLSI肉汤微量稀释法，通过测定最小抑菌浓度（MIC）和计算菌落形成单位（CFU），评估单独使用抗真菌药物或与CAP联合使用时分生孢子对药物的敏感性。在处理后的24小时内，评估单独使用抗真菌药物或与CAP联合使用时菌丝体活力和生物膜厚度的变化。CAP增强了对所有真菌物种的抗真菌药物疗效，尽管不同药物和生长形式的效果有所不同。CAP增强了分生孢子对抗真菌药物的敏感性，在角膜塑形镰刀菌分生孢子中观察到的效果最强，卡泊芬净的MIC降低了四倍（从16降至4-8μg/mL），而对氟康唑的敏感性（在无CAP时无作用）在与亚致死剂量CAP联合处理时得以恢复。在黄曲霉中，CAP降低了伏立康唑的MIC（从0.25降至0.06-0.125μg/mL），但增加了两性霉素B的MIC（从4增至>4μg/mL），尽管活细胞数有所减少。在三株镰状镰刀菌中观察到对CAP和氟康唑的不同反应，表明CAP反应存在变异性。在处理过的生物膜中，单独的CAP最初降低了菌丝体活力和生物膜厚度，但在大多数情况下，随着时间的推移观察到真菌有部分恢复。当与抗真菌药物联合使用时，CAP显著增强了对菌丝体活力和厚度的降低效果，超过单独使用抗真菌治疗。在角膜塑形镰刀菌生物膜中，CAP与抗真菌药物联合使用使菌丝体活力和生物膜厚度持续降低，而黄曲霉生物膜对CAP治疗更具抗性，在24小时后表现出一致的恢复。
