Para-Aminosalicylic acid (PAS) is a group 4 antituberculosis agent. It targets folate metabolism as shown in Fig. S1 in the supplemental material, which also summarizes the known mechanisms of resistance to this prodrug. Recently, we reported a multidrug-resistant (MDR) Mycobacterium tuberculosis Beijing strain harboring a deletion of both dfrA and thyA from Australia (Fig. 1A and Table S1). Since then, we have found deletions affecting both genes in five further MDR Beijing strains (two isolated in Australia and three from Peru) and one extensively drug-resistant (XDR) Beijing strain from China. The Australian MDR strains were recovered from three patients with no apparent epidemiological links who were likely infected in their country of origin (Table S1). The three Peruvian isolates were closely related and consequently shared the same deletion, whereas the remaining strains were distantly related and had deletions that differed in size (Fig. 1A). Consequently, these five distinct deletions were acquired independently, which can be a signal for positive selection of resistance mechanisms. In line with this hypothesis, the strains from Australia and China were found to be PAS resistant when tested with the Bactec MGIT 960 system and on Löwenstein-Jensen medium, respectively (see Supplemental methods). Two out of the three Peruvian deletion mutants were also found to be PAS resistant on 7H10 medium at 8 μg/ml, whereas the two closely related ancestral wild-type strains were found to be susceptible (Fig. 1B). We were unable to retest the strains at 2 μg/ml, the critical concentration recommended by the Clinical and Laboratory Standards Institute and the World Health Organization, which would have clarified whether the result for the third deletion mutant as susceptible was an artifact...