The physiology and pathogenicity of Mycobacterium tuberculosis grown under controlled conditions in a defined medium

Abstract

A chemically‐defined culture medium was developed which supported batch growth of Mycobacterium tuberculosis, strain H37Rv, at a minimum doubling time of 14·7 h. This medium also facilitated chemostat culture of M. tuberculosis at a constant doubling time of 24 h. Chemostat growth was optimized at a dissolved oxygen tension of 20% (v/v) and 0·2% (v/v) Tween‐80. Chemostat cultures were dispersed suspensions of single bacilli (1·5–3 µm long), or small aggregates, at a mean density of log10 8·3 cfu ml−1. A limited number of amino acids was utilized (alanine, asparagine, aspartate and serine were depleted by >50%; glycine, arginine, isoleucine, leucine and phenylalanine, by approximately 40%). Chemostat‐grown cells were pathogenic in aerosol‐infected guinea pigs, producing disseminated infection similar to that caused by plate‐grown cells. Cells from chemostat culture were significantly more invasive for J774A.1 mouse macrophages than agar‐ or batch‐grown cells. This study demonstrates the suitability of chemostat culture for the growth of pathogenic mycobacteria in a defined physiological state with potential applications for the controlled production of mycobacterial components for therapeutic and vaccine applications.

Compiling a Molecular Inventory for Mycobacterium bovis BCG at Two Growth Rates: Evidence for Growth Rate-Mediated Regulation of Ribosome Biosynthesis and Lipid Metabolism

“…The first goal of this study was to develop and optimize conditions for growing BCG continuously in a fermentor. James and colleagues have successfully cultivated M. tuberculosis by using a complex nutrient-rich medium containing several carbon sources and an almost full complement of amino acids, but this medium is unsuitable for physiological studies due to difficulties in identifying the growthlimiting substrate (3,27). For physiological studies with chemostat cultures, it is desirable to use defined media containing ingredients that can be easily monitored throughout the experiment.…”

“…As a result, only very few projects have utilized chemostats to grow this group of organisms (3,27,30,35,36). A continuous culture system has been described for M. tuberculosis and has recently been successfully employed to investigate the effects of 1% oxygen on the transcriptome of M. tuberculosis (3,27). This study allowed the effect of oxygen limitation on the transcriptome of M. tuberculosis to be investigated at a constant growth rate and independently of any other environmental parameters (3).…”

Association between spoligotype-VNTR types and virulence ofMycobacterium bovisin cattle

“…Culture conditions may also alter the virulence. 20 While in an experimental infection, animals and pathogenic agents are controlled and uniform, in a field evaluation, the infection occurs by a natural route without any specific control. Thus, more animals have to be evaluated in field studies.…”

Comparative transcriptomics reveals key gene expression differences between the human and bovine pathogens of the Mycobacterium tuberculosis complex

“…For the continuous culture experiments, M. tuberculosis H37Rv and M. bovis AF2122 were grown at 37 uC in defined minimal medium, CAMR Mycobacteria Media (CMM) Mod6 (pH 7.0), at a dissolved oxygen tension of 10 % (50 % air saturation), using the approach described previously for M. tuberculosis H37Rn (Bacon et al, 2004). CMM Mod6 was a modified version of CMM (James et al, 2000) in which all amino acids were removed except for L-asparagine. Glycerol and glucose were omitted and Tween 80 (0.2 %, v/v) was included as sole carbon source.…”