An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through two stages of nonreplicating persistence

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.

Oxygen depletion induced dormancy in Mycobacterium smegmatis

“…All experiments were conducted with M. smegmatis mc P 155 [5]. Cultures were carried out in 20U125mm test tubes according to Wayne and Hayes [4] using Dubos Tween-albumin broth (Difco, Detroit, MI) at 37³C. Seventeen ml of culture was used throughout and, therefore, the ratio of head space air volume (8.5 ml) to liquid volume (17 ml) was always 0.5.…”

“…Growth and survival of the bacterial populations were monitored by viable count measurements as described [4]. Oxygen depletion was monitored using the oxygen indicator dye methylene blue as described [4].…”

“…This demonstrated a potential in M. tuberculosis for survival under anaerobic conditions. In pioneering work over the last decades Wayne and coworkers developed an in vitro system for mycobacterial dormancy and con¢rmed that oxygen depletion is a signal that triggers a dormancy response of the bacilli [1,4].…”

Culturability of Mycobacterium tuberculosis cells isolated from murine macrophages: a bacterial growth factor promotes recovery

“…The behaviour of M. tuberculosis following phagocytosis by macrophages is quite di¡erent from that observed using Wayne's model for dormancy, in which M. tuberculosis H37Rv cells maintain high viability when they undergo a metabolic shift-down under micro-aerophilic conditions in vitro [12,13]. This transition is associated with cell wall thickening in both pathogenic M. tuberculosis and non-pathogenic M. bovis BCG strains [44].…”

“…For example, Khomenko obtained ¢lterable forms of a bacillus from TB patients, but it is unclear whether these represent dormant bacteria, or injured, moribund, or even dead cells [10,11]. Wayne used the term dormant' to describe a population of M. tuberculosis cells observed after the transition from an aerobic to an anaer-obic state [12,13]. However, the signi¢cance of this state provoked in vitro for the persistence of mycobacteria in vivo remains to be clari¢ed.…”