噬菌体TM4复苏结核休眠菌的初步研究

Preliminary study on resuscitation of Mycobacteriophage TM4 in dormant cells of M. tuberculosis

目的探索噬菌体TM4对结核休眠菌复苏的作用。方法采用密闭培养对数生长期结核菌构建休眠菌模型;采用药敏检测和透射电子显微镜(电镜)观察作为模型检测方法;采用菌落计数和电镜观察作为复苏的检测指标。结果密闭培养180 d休眠菌模型构建成功;培养3 d复苏促进因子(Rpf)E组管底菌量多于空白组和TM4组,培养8 d噬菌体TM4组管底菌量多于空白组,后续观察见TM4组和Rpf E组管底菌量均较前有所增加。混合液培养第1日时,空白组、TM4组和Rpf E组的菌落计数均为0;培养第6日时,菌落计数分别是0、0.7×102和2×104CFU/mL;培养第14日时,菌落计数分别是3.4×10、1.68×107和2.1×109CFU/mL。培养第17日时,电镜观察发现混合物TM4组有大量薄壁结核菌、部分厚壁结核休眠菌和结核菌细胞碎片,Rpf E组满视野薄壁的结核菌,空白组含大量厚壁的结核休眠菌和少数薄壁结核菌。结论噬菌体TM4能够复苏结核休眠菌。

Objective To investigate the potential of mycobacteriophage TM4 in resuscitating dormant cells of M. tuberculosis. Methods Dormant cells of M. tuberculosis were induced by prolonged closed culture of log-phase M. tuberculosis at 37 ℃, which were proven through drug sensitivity test and electron microscope. M. tuberculosis colony count and electron microscope were used to detect whether the dormant cells of M. tuberculosis had been resuscitated by TM4. Results Dormant ceils of M. tuberculosis were obtained successfully by closed culture for 180 d at 37 ~C. The amount of M. tuberculosis at the bottom of the tube in Rpf E group was more than that of the other groups through naked eye after culturing 3 d at 37 ℃. While the amount of M. tuberculosis at the bottom in TM4 group did not increased obviously until culturing for 8 d. M. tuberculosis was still non-cuhurable in the three groups after culturing 1 d. Along with further culture, part of M. tuberculosis turned cuhurable, the concentration of resuscitated M. tuberculosis in control, TM4, and Rpf E groups was 0, 0.7 × 10^2, and 2 × 10^4 CFU/mL, respectively after culturing 6 d. The concentration of resuscitated M. tuberculosis in control, TM4, and Rpf E groups increased to 3.4 × 10, 1.68 × 10^7, and 2. 1× 10^9 CFU/mL, respectively after culturing 14 d. Various shapes of M. tuberculosis for culturing 17 d in the three groups were watched through electron microscope. There were lots of thin-wall M. tuberculosis, a few dormant cells of M. tuberculosis with thick wall, and cell pieces in TM4 group; whereas the field of view of Rpf E group was filled with plentiful thin-wall M. tuberculosis; as we excepted, a lot of thick-wall M. tuberculosis and a few thin wall M. tuberculosis were observed in control group. Conclusion Mycobacteriophage TM4 can resuscitate dormant cells of M. tuberculosis.