结核分枝杆菌能量代谢对吡嗪酰胺抗结核作用的影响

目的研究结核分枝杆菌能量代谢对吡嗪酰胺抗结核作用的机制和作用靶点。方法将饥饿3、5和10d的结核分枝杆菌及未经过营养饥饿的结核分枝杆菌分别加入100μg／ml吡嗪酰胺和脂肪酸、苯甲酸和水杨酸进行处理，观察营养饥饿处理过程对于吡嗪酰胺抗结核分枝杆菌作用的影响，以及酸性物质对吡嗪酰胺抗菌活性的影响。同时用流式细胞仪检测结核分枝杆菌膜电位和平均荧光强度，以探讨吡嗪酰胺的作用机制。结果未经过营养饥饿的结核分枝杆菌和饥饿3、5和10d的结核分枝杆菌经吡嗪酰胺处理后，菌落形成单位（CFU）分别减少23．08％、37．75％、82．32％和81．03％；营养饥饿5d后，结核分枝杆菌的膜电位明显降低，经吡嗪酰胺处理后的膜电位则大幅降低，加入能量物质葡萄糖后，因吡嗪酰胺作用而降低的膜电位可得到恢复。脂肪酸、苯甲酸和水杨酸对于正常生长的和营养饥饿后的结核分枝杆菌均有促进吡嗪酰胺抗菌效果的作用，对营养饥饿后的结核分枝杆菌作用更明显。结论饥饿状态和弱酸能增强吡嗪酰胺对结核分枝杆菌的抗菌作用，吡嗪酰胺可能是通过于扰结核分枝杆菌的膜电位及其能量代谢而产生作用。

Nutrient starved incubation conditions enhance pyrazinamide activity against Mycobacterium tuberculosis

OBJECTIVE: To investigate the mechanism of the action and the target of pyrazinamide (PZA), by examining the effect of nutrient-starved conditions on PZA activity. METHODS: M. tuberculosis starved in PBS for 3, 5 or 10 days were treated with PZA, and with benzoic acid, salicylic acid or fatty acid in combination with PZA. The activity of PZA against M.tuberculosis was determined by the number of colony-forming unit (CFU). The membrane potential of M.tuberculosis was determined by flow-cytometry with Rhodamine to investigate the mechanism of PZA activity. RESULTS: The CFU of M. tuberculosis treated with PZA for 0, 3, 5, 10 days nutrient starvation compared with their respective controls, decreased by 23.08%, 37.75%, 82.32%, 81.03%, respectively. The membrane potential of M. tuberculosis in nutrient-starved cultures declined rapidly over the first 5 days' starvation. The starved bacilli treated with PZA had significantly lower level of membrane potential compared with the control. The effect of PZA on lowering the membrane potential was antagonized by addition of glucose, which provided energy for the bacilli. Fatty acids, benzoic acid and salicylic acid-enhanced PZA activity in both normal and starved bacilli, especially in starved bacilli. CONCLUSION: Nutrient starved incubation conditions and weak acids could enhance pyrazinamide activity against Mycobacterium tuberculosis. PZA can impair Mycobacterium tuberculosis cell viability by disrupting the energy supply system and reducing membrane potential of the cells.