Wayne L G, Hayes L G
Department of Veterans Affairs Medical Center, Long Beach, California 90822, USA.
Infect Immun. 1996 Jun;64(6):2062-9. doi: 10.1128/iai.64.6.2062-2069.1996.
It was demonstrated previously that abrupt transfer of vigorously aerated cultures of Mycobacterium tuberculosis to anaerobic conditions resulted in their rapid death, but gradual depletion of available O2 permitted expression of increased tolerance to anaerobiosis. Those studies used a model based on adaptation of unagitated bacilli as they settled through a self-generated O2 gradient, but the model did not permit examination of homogeneous populations of bacilli during discrete stages in that adaptation. The present report describes a model based on culture of tubercle bacilli in deep liquid medium with very gentle stirring that keeps them in uniform dispersion while controlling the rate at which O2 is depleted. In this model, at least two stages of nonreplicating persistence were seen. The shift into first stage, designated NRP stage 1, occurred abruptly at a point when the declining dissolved O2 level approached 1% saturation. This microaerophilic stage was characterized by a slow rate of increase in turbidity without a corresponding increase in numbers of CFU or synthesis of DNA. However, a high rate of production of glycine dehydrogenase was initiated and sustained while the bacilli were in this state, and a steady ATP concentration was maintained. When the dissolved O2 content of the culture dropped below about 0.06% saturation, the bacilli shifted down abruptly to an anaerobic stage, designated NRP stage 2, in which no further increase in turbidity was seen and the concentration of glycine dehydrogenase declined markedly. The ability of bacilli in NRP stage 2 to survive anaerobically was dependent in part on having spent sufficient transit time in NRP stage 1. The effects of four antimicrobial agents on the bacilli depended on which of the different physiologic stages the bacilli occupied at a given time and reflected the recognized modes of action of these agents. It is suggested that the ability to shift down into one or both of the two nonreplicating stages, corresponding to microaerophilic and anaerobic persistence, is responsible for the ability of tubercle bacilli to lie dormant in the host for long periods of time, with the capacity to revive and activate disease at a later time. The model described here holds promise as a tool to help clarify events at the molecular level that permit the bacilli to persist under adverse conditions and to resume growth when conditions become favorable. The culture model presented here is also useful for screening drugs for the ability to kill tubercle bacilli in their different stages of nonreplicating persistence.
先前的研究表明，将活力旺盛且通气良好的结核分枝杆菌培养物突然转移至厌氧条件下会导致其迅速死亡，但逐步耗尽可用氧气则可使细菌对厌氧环境的耐受性增强。那些研究使用的模型是基于未搅动的杆菌在自身产生的氧气梯度中沉降时的适应性，但该模型无法在适应过程的不同阶段对杆菌的均匀群体进行检测。本报告描述了一种基于在深层液体培养基中培养结核杆菌的模型，通过非常轻柔的搅拌使其保持均匀分散，同时控制氧气耗尽的速率。在这个模型中，观察到了至少两个非复制性持续存在阶段。向第一阶段即非复制性持续存在阶段1的转变在溶解氧水平下降至接近1%饱和度时突然发生。这个微需氧阶段的特征是浊度增加缓慢，而CFU数量或DNA合成没有相应增加。然而，在杆菌处于该状态时，甘氨酸脱氢酶的产生速率很高且持续存在，并且ATP浓度保持稳定。当培养物中的溶解氧含量降至约0.06%饱和度以下时，杆菌突然转变至厌氧阶段，即非复制性持续存在阶段2，在此阶段浊度不再增加，甘氨酸脱氢酶的浓度显著下降。处于非复制性持续存在阶段2的杆菌在厌氧条件下存活的能力部分取决于在非复制性持续存在阶段1中度过了足够的过渡时间。四种抗菌药物对杆菌的作用取决于杆菌在给定时间所处的不同生理阶段，并反映了这些药物公认的作用方式。有人提出，能够转变至两个非复制阶段中的一个或两个阶段，即对应于微需氧和厌氧持续存在阶段，是结核杆菌能够在宿主体内长期潜伏，并在之后恢复活力并引发疾病的原因。这里描述的模型有望成为一种工具，有助于阐明在分子水平上使杆菌在不利条件下持续存在并在条件有利时恢复生长的事件。这里呈现的培养模型也可用于筛选药物在结核杆菌不同非复制性持续存在阶段杀死它们的能力。
