Abstract
The ability of all pathogens to survive within the host is key to their success in establishing disease. Environmental conditions that affect the growth of a pathogen in the host include nutrient status, environmental pH, oxygen availability, and host defences. Studying the response of Mycobacterium tuberculosis (M. tuberculosis) exposed to these relevant host conditions in vitro will further increase our understanding of how these environments have an impact on the molecular mechanisms M. tuberculosis adopts to combat the effects of external influences such as antimycobacterials. The methods presented here are used to investigate the effect of environmental factors on the development of drug-resistant M. tuberculosis. Cultures grown under controlled conditions in continuous culture are sampled and the frequency with which resistant mutants develop are determined. These studies provide data that aid our understanding of the complex interaction between the host environment and invading bacterium that allow resistant strains to develop and continue to cause disease.
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References
Bacon J, Marsh PD (2007) Transcriptional responses of Mycobacterium tuberculosis exposed to adverse conditions in vitro. Curr Mol Med 7(3):277–86, Review
Gillespie SH (2002) Evolution of drug resistance in Mycobacterium tuberculosis: clinical and molecular perspective. Antimicrob Agents Chemother 46:267–274
Warner DF, Mizrahi V (2006) Tuberculosis chemotherapy: the influence of bacillary stress and damage response pathways on drug efficacy. Clin Microbiol Rev 19:558–570
Elliot AM, Berning SE, Iseman MD, Peloquin CA (1995) Failure of drug penetration and acquisition of drug resistance in chronic tuberculous empyema. Tuber Lung Dis 76:463–467
Clemens DL, Horwitz MA (1995) Characterisation of Mycobacterium tuberculosis phagosome and evidence that phagosome maturation is inhibited. J Exp Med 181:257–270
Sturgill-Koszycki S, Schlesinger PH, Chakraborty P, Haddix PL, Collins HL, Fok AK, Allen RD, Gluck SL, Heuser J, Russell DG (1994) Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science 263(5147):678–681
Hackman DJ, Rotsein OD, Zhang W, Gruenheid S, Gros P, Grinstein S (1998) Host resistance to intracellular infection: mutation of natural resistance associated macrophage protein 1 (nramp 1) impairs phagosomal acidification. J Exp Med 188:351–364
Bacon J, James BW, Wernisch L, Williams A, Morley KA, Hatch GJ, Mangan JA, Hinds J, Stoker NG, Butcher PD, Marsh PD (2004) The influence of reduced oxygen availability on pathogenicity and gene expression in Mycobacterium tuberculosis. Tuberculosis 84:205–217
Bacon J, Dover LG, Hatch KA, Zhang Y, Gomes JM, Kendall SL, Wernisch L, Stoker NG, Butcher PD, Besra GS, Marsh PD (2007) The lipid composition and transcriptional response of Mycobacterium tuberculosis grown under iron-limitation in continuous culture: identification of a novel wax ester. Microbiology 153(5):1435–1444
Golby P, Hatch KA, Bacon J, Cooney R, Riley P, Allnutt J, Hinds J, Nunez J, Marsh PD, Hewinson RG, Gordon SV (2007) Comparative transcriptomics reveals key gene expression differences between the human and bovine pathogens of the Mycobacterium tuberculosis complex. Microbiology 153(10):3323–36
Billington OJ (1999) Physiological cost of rifampicin resistance induced in vitro in Mycobacterium tuberculosis. Antimicrob Agents Chemother 43(8):1866–1869
Davies AP (2000) Comparison of fitness of two isolates of Mycobacterium tuberculosis, one of which had developed multi-drug resistance during the course of treatment. J Infect 41:184–187
Gillespie SH (2001) Antibiotic resistance in the absence of selective pressure. Int J Antimicrob Agents 17:171–176
Arnold C, Westland L, Mowat G, Underwood A, Magee J, Gharbia S (2004) Single-nucleotide polymorphism-based differentiation and drug resistance detection in Mycobacterium tuberculosis from isolates or directly from sputum. Eur J Clin Microbiol Infect Dis 11:122–130
Acknowledgments
This work was funded by the Department of Health and the Health Protection Agency, UK. The views expressed in this chapter are those of the authors and not necessarily those of the Department of Health or Health Protection Agency. The authors acknowledge Dr Brian James for the huge contribution he has made to the development of chemostat models for the growth of M. tuberculosis, to Dr Alpana Bose and Dr Claire Jenkins for their technical help and to Professor Philip Marsh for his constructive comments.
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Bacon, J., Hatch, K.A., Allnutt, J. (2010). Application of Continuous Culture for Measuring the Effect of Environmental Stress on Mutation Frequency in Mycobacterium tuberculosis . In: Gillespie, S., McHugh, T. (eds) Antibiotic Resistance Protocols. Methods in Molecular Biology, vol 642. Humana Press. https://doi.org/10.1007/978-1-60327-279-7_10
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DOI: https://doi.org/10.1007/978-1-60327-279-7_10
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