Effect and mechanism of oxyhydrogen machine in antifungal adjuvant therapy

Objective To explore antifungal effect and mechanism of oxyhydrogen machine alone or combined with antifungal agents based on in vitro study.

Methods Twelve strains of clinically isolated pathogenic fungi were selected as study strains, and the bacterial suspension was treated with a hydrogen-oxygen machine at a fixed flow rate and gear (gas flow rate of 3 L/min at the fifth atomization gear position). 100 μL of bacterial suspension was respectively coated on Sarge glucose AGAR medium at 0 min of ventilation (without ventilation) and 5 and 10 min of ventilation for bacterial suspension, and colony counts were performed after the medium was cultured at 35℃ for 2 days. Survival rate and kill rate were calculated. The minimal inhibitory concentrations (MICs) were evaluated before or after oxyhydrogen treatment for four antifungal drugs: voriconazole (VOR), posaconazole (POS), fluconazole (FLU), itraconazole (ITR). The intracellular reactive oxygen species (ROS) activity was measured by flow cytometry.

Results The killing rate of 12 strains after 5 min ventilation by hydrogen oxygen machine showed significant difference compared with that at 0 min of ventilation (P < 0.05). The overall killing rate ranged from 6.3% to 76.2%. The killing rate of 12 strains at 10 min of ventilation by hydrogen oxygen machine increased compared with 5 min of ventilation(P < 0.05), with the overall killing rate of 19.6% to 93.5%. The results of external drug sensitivity test of bacterial suspension liquid showed that the MIC values of ITR, VOR, and POS against the strains after 10 min treatment were decreased 1 time to 4 times, and the MIC value of FLU against the strains was reduced 1 time to 5 times. After treatment with high concentration of hydrogen, the MICs of antifungal agents against 11 strains were significantly decreased. The intracellular ROS detection of ATCC00279 strain after different time of ventilation showed that the percentage of ROS in strains by ITR and VOR increased after 10 min of ventilation compared with 0 min of ventilation (P < 0.05).

Conclusion Directly ventilation by oxyhydrogen machine enables to obtain direct killing of up to 93.5% of the pathogenic fungi. Oxyhydrogen treatment combined with antifungal drugs can increase the susceptibility of the strain to the drugs to further decrease MIC of antifungal agents against fungi. The mechanisms might be associated with the increased intracellular ROS activity of fungal cells by oxyhydrogen machine.