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Reinfection dynamics of mycobacterium tuberculosis
Author(s)
Mitchell, Joni
Date Issued
2007
Type
Thesis
Publisher
Cape Peninsula University of Technology
Abstract
Reinfection is an important mechanism leading to recurrent tuberculosis. Recently, molecular epidemiological studies have shown that in high incidence settings, recurrent tuberculosis may occur through reinfection. Animal model experiments have shown that a reinfecting mycobacterial strain is specifically targeted to existing granulomas and that these structures are more dynamic than was previously thought. In this study we hypothesised that primary infection with M. tuberculosis may reprogramme human macrophages thereby preventing or facilitating reinfection with a secondary mycobacterial strain.
Two antibiotic-resistant M. tuberculosis H37Rv variants were generated by electrotransformation of marked plasmids, designated KanRand HygR . A THP1 human macrophage cell line was infected and reinfected with different combinations of these marked strains as well as a hypervirulent M. tuberculosis Beijing strain. Mycobacterial growth has been assessed by colony forming unit enumeration and confirmed with polymerase chain reaction (PCR) analysis.
In vitro growth curves of wild-type and differentially marked M. tuberculosis H37Rv Kan Rand HygR strains were compared in the BACTECTM mycobacterial growth indicator tube (MGITTM) system in parallel with conventional liquid culturing. In vitro liquid culture growth curves of hypervirulent clinical Beijing strain isolates were also compared to M. tuberculosis H37Rv growth curves. Through this it was established that there was no fitness cost as result of plasmid integration and that these strains of varying virulence had similar growth curves. Competitive dynamics within THP1 human macrophage cells were then assessed and have shown that there were no significant differences in growth patterns between primary and secondary infecting strains during THP1 cell reinfection.
The findings of this study answered fundamental questions regarding reinfection of mycobacterial strains. It was established here that human macrophages can indeed be reinfected with a second virulent mycobacterial strain.
Two antibiotic-resistant M. tuberculosis H37Rv variants were generated by electrotransformation of marked plasmids, designated KanRand HygR . A THP1 human macrophage cell line was infected and reinfected with different combinations of these marked strains as well as a hypervirulent M. tuberculosis Beijing strain. Mycobacterial growth has been assessed by colony forming unit enumeration and confirmed with polymerase chain reaction (PCR) analysis.
In vitro growth curves of wild-type and differentially marked M. tuberculosis H37Rv Kan Rand HygR strains were compared in the BACTECTM mycobacterial growth indicator tube (MGITTM) system in parallel with conventional liquid culturing. In vitro liquid culture growth curves of hypervirulent clinical Beijing strain isolates were also compared to M. tuberculosis H37Rv growth curves. Through this it was established that there was no fitness cost as result of plasmid integration and that these strains of varying virulence had similar growth curves. Competitive dynamics within THP1 human macrophage cells were then assessed and have shown that there were no significant differences in growth patterns between primary and secondary infecting strains during THP1 cell reinfection.
The findings of this study answered fundamental questions regarding reinfection of mycobacterial strains. It was established here that human macrophages can indeed be reinfected with a second virulent mycobacterial strain.
Additional information
Thesis (MTech (Biomedical Technology))--Cape Peninsula University of Technology, 2007
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