Biketov S, Mukamolova G V, Potapov V, Gilenkov E, Vostroknutova G, Kell D B, Young M, Kaprelyants A S
State Scientific Centre for Applied Microbiology, Moscow Region, Russia.
FEMS Immunol Med Microbiol. 2000 Dec;29(4):233-40. doi: 10.1111/j.1574-695X.2000.tb01528.x.
Very little is known about the culturability and viability of mycobacteria following their phagocytosis by macrophages. We therefore studied populations of the avirulent 'Academia' strain of Mycobacterium tuberculosis isolated from murine peritoneal macrophage lysates several days post-infection in vivo. The resulting bacterial suspensions contained a range of morphological types including rods, ovoid forms and coccoid forms. Bacterial viability measured using the MPN method (dilution to extinction in liquid medium) was often much higher than that measured by CFU (plating on solid medium). Viability in the MPN assay was further enhanced when the Micrococcus luteus protein, Rpf, was incorporated into the liquid culture medium at picomolar concentrations. Rpf is an example of a family of autocrine growth factors found throughout the high G+C cohort of Gram-positive bacteria including M. tuberculosis. M. tuberculosis cells obtained from macrophages had altered surface properties, as compared with bacteria grown in vitro. This was indicated by loss of the ability to adsorb bacteriophage DS6A, a reduced tendency to form clumps, acquisition of ethidium bromide stainability following heat treatment, and loss of Rpf-mediated resuscitation following freezing and thawing. These results indicate that a proportion of 'unculturable' M. tuberculosis cells obtained from macrophages is either injured or dormant and that these cells may be recovered or resuscitated using Rpf in liquid medium.
关于分枝杆菌被巨噬细胞吞噬后的可培养性和生存能力，人们了解甚少。因此，我们研究了从体内感染数天后的小鼠腹腔巨噬细胞裂解物中分离出的无毒力结核分枝杆菌“学术型”菌株群体。所得细菌悬液包含一系列形态类型，包括杆菌、卵形菌和球菌。使用MPN法（在液体培养基中稀释至灭绝）测量的细菌生存能力通常远高于通过CFU（在固体培养基上平板培养）测量的结果。当将微球菌属的鲁氏微球菌蛋白Rpf以皮摩尔浓度加入液体培养基时，MPN测定中的生存能力进一步增强。Rpf是在包括结核分枝杆菌在内的革兰氏阳性菌的高G+C菌群中发现的一类自分泌生长因子的一个例子。与体外培养的细菌相比，从巨噬细胞获得的结核分枝杆菌细胞具有改变的表面特性。这表现为吸附噬菌体DS6A的能力丧失、形成团块的倾向降低、热处理后获得溴化乙锭染色能力以及冻融后丧失Rpf介导的复苏能力。这些结果表明，从巨噬细胞获得的一部分“不可培养”结核分枝杆菌细胞要么受到损伤，要么处于休眠状态，并且这些细胞可以在液体培养基中使用Rpf进行恢复或复苏。
