Effect and mechanism of oxyhydrogen machine in antifungal adjuvant therapy
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摘要:目的
基于体外试验研究氢氧机单独应用或联用抗真菌药对真菌的作用效果及作用机制。
方法选择临床分离获得的12株病原真菌作为试验菌株, 使用氢氧机按固定的流速和档位(气流量3 L/min, 雾化档5档)处理菌悬液，以通气0 min时(未通气时)和通气处理菌悬液5、10 min时为3个观察时点，各时点分别取菌悬液100 μL涂布于沙氏葡萄糖琼脂培养基，于37 ℃温箱内培养2 d后进行菌落计数，计算存活率和杀伤率。采用微量液基稀释法测定4种抗真菌药伏立康唑(VOR)、泊沙康唑(POS)、氟康唑(FLU)、伊曲康唑(ITR)的最低抑菌浓度(MIC)。应用流式细胞仪测定细胞内活性氧(ROS)活性变化。
结果氢氧机5 min通气处理对12株菌株的杀伤率与通气0 min比较，差异均有统计学意义(P < 0.05), 整体杀伤率范围为6.3%~76.2%; 与5 min通气处理相比，氢氧机10 min通气处理对12株菌株的杀伤率增高，差异均有统计学意义(P < 0.05), 整体杀伤率范围为19.6%~93.5%。菌悬液体外药敏试验结果显示，氢氧机通气处理10 min后, ITR、POS、VOR对菌株的MIC值下降了1~4倍, FLU对菌株的MIC值降低了1~5倍; 高浓度氢气处理后，抗真菌药对其中11株菌株的MIC值明显降低。对经过不同时间通气处理的ATCC00279菌株进行细胞内ROS检测发现，与通气0 min比较，通气10 min后菌株对ITR、VOR的ROS百分比增加，差异有统计学意义(P < 0.05)。
结论单独氢氧机通气处理即可实现高达93.5%的菌株杀伤效果，将其与抗真菌药联用还可使菌株对药物的敏感性增强，进一步降低抗真菌药对真菌的MIC，其机制可能与氢氧机能增强真菌细胞内ROS活性有关。
Abstract:ObjectiveTo explore antifungal effect and mechanism of oxyhydrogen machine alone or combined with antifungal agents based on in vitro study.
MethodsTwelve 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.
ResultsThe 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).
ConclusionDirectly 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.
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图 2 各菌株经不同时间通气处理后的残余孢子数量比较
两者比较, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000 1, ns为P>0.05。
表 1 通气不同时间后各菌株的孢子存活率
% 菌株 菌种 0 min 5 min 10 min 382 耳念珠菌 100.0 79.1 50.3 387 耳念珠菌 100.0 80.6 76.1 389 耳念珠菌 100.0 93.7 70.5 Z1 新生隐球菌 100.0 23.8 6.5 Z2 新生隐球菌 100.0 89.7 81.0 Z3 新生隐球菌 100.0 89.1 75.5 SC5314 白念珠菌 100.0 50.4 46.6 ATCC00279 克柔念珠菌 100.0 27.5 17.4 ATCC22019 近平滑念珠菌 100.0 62.2 53.9 R9 耐氟康唑白念珠菌 100.0 91.0 76.9 R14 耐氟康唑白念珠菌 100.0 98.6 97.3 R15 耐氟康唑白念珠菌 100.0 83.6 81.3 
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表 2 4种抗真菌药对12株真菌的体外药敏试验最低抑菌浓度结果
μg/mL 菌株编号 种属 ITR VOR POS FLU 0 min 5 min 10 min 0 min 5 min 10 min 0 min 5 min 10 min 0 min 5 min 10 min AR387 耳念珠菌 0.125 0.125 0.125 0.250 0.250 0.250 0.063 0.063 0.063 64.000 64.000 64.000 AR389 耳念珠菌 2.000 1.000 1.000 4.000 2.000 2.000 1.000 0.500 0.250 4.000 2.000 2.000 R9 白念珠菌 1.000 0.250 0.250 0.500 0.250 0.250 0.063 0.063 0.063 4.000 2.000 1.000 R14 白念珠菌 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.250 0.250 4.000 2.000 2.000 R15 白念珠菌 1.000 1.000 1.000 1.000 1.000 1.000 0.500 0.250 0.125 8.000 4.000 4.000 Z1 新生隐球菌 0.250 0.250 0.250 0.031 0.031 0.031 0.250 0.250 0.250 2.000 1.000 1.000 Z2 新生隐球菌 0.125 0.063 0.063 0.031 0.031 0.031 0.250 0.250 0.250 2.000 0.500 0.500 Z3 新生隐球菌 0.250 0.250 0.250 0.063 0.063 0.063 0.500 0.250 0.250 4.000 4.000 2.000 SC5314 白念珠菌 0.250 0.125 0.125 0.125 0.125 0.125 0.125 0.063 0.063 0.500 0.125 0.125 ATCC00279 克柔念珠菌 0.250 0.125 0.063 0.125 0.125 0.063 0.500 0.250 0.031 0.250 0.250 0.063 ATCC22019 近平滑念珠菌 0.250 0.250 0.063 0.125 0.125 0.063 0.500 0.250 0.250 2.000 1.000 0.063 AR382 耳念珠菌 4.000 2.000 1.000 4.000 1.000 0.250 4.000 2.000 0.500 64.000 64.000 64.000 ITR: 伊曲康唑; VOR: 伏立康唑; POS: 泊沙康唑; FLU: 氟康唑。
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