Bacterial taxonomy, which began as a largely intuitive process, has become increasingly objective with the advent of numerical taxonomy and techniques for the measurement of evolutionary divergence in the structure of semantides, i.e., large, information-bearing molecules such as nucleic acids and proteins. These developments have forced the

No abstract

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 O 2 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 O 2 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 O 2 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 O 2 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 O 2 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.

Great progress has been made in the latter half of the twentieth century in the understanding of the immunology of tuberculosis and of strategies for chemotherapeutic management of this disease. Indeed, given the evidence that the dominant, and perhaps sole, ecologic niche of Mycobacterium tuberculosis is the infected human host, it seemed reasonable to hope that the disease could not only be controlled, but eradicated by the end of this century (1). These hopes are dashed by the periodic resurgence of tuberculosis in various populations. Undoubtedly socioeconomic factors have played a major role in the failure to eradicate this disease, but another, neglected, factor is the apparent ability of the tubercle bacillus to remain in a relatively quiescent state in the host, neither replicating and causing progression of disease, nor dying off and disappearing from the host's tissues, in spite of apparently adequate immune responses and aggressive chemotherapy.

There is ample clinical evidence, as well as evidence from animal experiments, that Mycobacterium tuberculosis can persist in tissues for months to decades without replicating, yet with the ability to resume growth and activate disease. Our knowledge of both macrophage physiology and the nature of tuberculous lesions in man and animals suggests that hypoxia is a major factor in inducing nonreplicating persistence (NRP) of tubercle bacilli. In vitro models reinforce this conclusion and provide insights into mechanisms that make NRP possible. There is evidence from in vitro models that the strategies employed by the bacilli to permit hypoxic NRP include restriction of biosynthetic activity to conserve energy, induction of alternative energy pathways, and stabilization of essential cell components to lessen the need for repair or replacement.