Journal article

Effect of serial subculturing on the genetic composition and cytotoxic activity of Mycobacterium tuberculosis

Continuous subculture has been observed to produce changes in the virulence of micro-organisms, e.g. rabies virus, poliovirus and Mycobacterium bovis BCG. The latter has been used as a vaccine for tuberculosis for the last 100 years; however, in some instances its efficacy has been observed to be very low. In order to determine whether similar changes can be produced in Mycobacterium tuberculosis, we selected four isolates, M. tuberculosis H37Rv, a Beijing strain (DR-689), and two more isolates with deletion of the phospholipase C locus (plcA-plcB-plcC ), and subjected them to serial culturing on Middlebrook 7H9 medium, with or without ox bile. After 100 passages, we performed RFLP-IS6110 analysis to determine whether genomic changes were produced. We also checked their genomic composition by microarray analysis. Changes in virulence were studied by measuring the cytotoxic effect of parental and subcultured isolates on a THP-1 macrophage monolayer. The most visible change was the change of position of an IS6110 band of approximately 1400 bp to approximately 1600 bp in the Beijing isolate subcultured in the ox bile medium. Analysis by microarray and PCR confirmation did not reveal any genomic changes. Cytotoxic activity was decreased in the isolates at levels close to that of BCG, and more consistently in those subcultured in the presence of ox bile.


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