Evolution of rifampicin resistance due to substandard drugs in E. coli and M. smegmatis .

Poor-quality medicines undermine the treatment of infectious diseases such as tuberculosis, which requires months of treatment with rifampicin and other drugs. Rifampicin resistance is a critical concern for tuberculosis treatment. While sub-therapeutic doses of medicine are known to select for antibiotic resistance, the effect of drug degradation products on the evolution of resistance is unknown. Here, we demonstrate that substandard drugs that contain degraded active pharmaceutical ingredients select for gene alterations that confer resistance to standard drugs. We generated drug resistant E. coli and M. smegmatis strains by serially culturing bacteria in the presence of the degradation product of rifampicin, rifampicin quinone. We conducted Sanger sequencing to identify mutations in rifampicin resistant populations. Strains resistant to rifampicin quinone developed cross-resistance to the standard drug rifampicin, with some populations showing no growth inhibition at maximum concentrations of rifampicin. Sequencing of the rifampicin quinone treated strains indicated that they acquired mutations in the DNA-dependent RNA polymerase B subunit. These mutations were localized in the rifampicin resistance determining region (RRDR), consistent with other reports of rifampicin resistant E. coli and Mycobacteria. Rifampicin quinone treated mycobacteria also had cross-resistance to other rifamycin class drugs: rifabutin and rifapentine. Our results strongly suggest that substandard drugs not only hinder individual patient outcomes, but also restrict future treatment options by actively contributing to the development of resistance to standard medicines.