D-cycloserine intra patient WGS

This study focuses on understanding the mechanism of resistance of Mycobacterium tuberculosis to D-cycloserine, a poorly understood second-line drug currently used for the treatment of multidrug-resistant tuberculosis. Our analysis includes the use of a unique set of serial clinical isolates, which were collected from a patient over a period of 21 months. The isolates underwent routine drug susceptibly testing at the time of sample collection and were further analysed by whole genome sequencing and RNAseq. Whole genome sequencing revealed that the isolates had multiple mutations conferring resistance to anti-tuberculosis drugs. Of interest to this study, the strain acquired mutations in ald, a gene that has been associated with resistance to D-cycloserine. Transcriptomic analysis revealed upregulation of Rv0576 and Rv0577 in the isolate with ald mutations; Rv0577 is a putative glyoxalase which could be involved in metabolic detoxification. Rv0576 and Rv0577 were cloned and transformed into the reference strain M. tuberculosis H37Rv and a clinical isolate of M. tuberculosis to determine whether over-expression of these two genes could be linked to the mechanism of D-cycloserine resistance. We assessed the susceptibility of parental and transformed strains to a panel of anti-tuberculosis drugs including D-cycloserine and demonstrated that overexpression of Rv0577 led to an increase in the minimum inhibitory concentration of D-cycloserine. These findings provide novel insight into the mechanism of D-cycloserine resistance in M. tuberculosis.