Effect of serial subculturing on the genetic composition and cytotoxicactivity of Mycobacterium tuberculosis

Continuous subculture has been observed to produce changes in the virulenceof micro-organisms, e.g. rabies virus, poliovirus and Mycobacterium bovis BCG. The latter has been used as a vaccine for tuberculosis for thelast 100 years; however, in some instances its efficacy has been observedto be very low. In order to determine whether similar changes can be producedin Mycobacterium tuberculosis , we selected four isolates, M.tuberculosis H37Rv, a Beijing strain (DR-689), and two moreisolates 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-IS 6110 analysis to determine whether genomic changes were produced. We alsochecked their genomic composition by microarray analysis. Changes in virulencewere studied by measuring the cytotoxic effect of parental and subculturedisolates on a THP-1 macrophage monolayer. The most visible change was thechange of position of an IS 6110 band of ∼1400 bp to ∼1600 bpin the Beijing isolate subcultured in the ox bile medium. Analysis by microarrayand PCR confirmation did not reveal any genomic changes. Cytotoxic activitywas decreased in the isolates at levels close to that of BCG, and more consistentlyin those subcultured in the presence of ox bile.