Evolution of Rifampin Resistance in Escherichia coli and Mycobacterium smegmatis Due to Substandard Drugs

Poor-quality medicines undermine the treatment of infectious diseases, such as tuberculosis, which require months of treatment with rifampin and other drugs. Rifampin resistance is a critical concern for tuberculosis treatment. While subtherapeutic 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 Escherichia coli and Mycobacterium smegmatis strains by serially culturing bacteria in the presence of the rifampin degradation product rifampin quinone. We conducted Sanger sequencing to identify mutations in rifampin-resistant populations. Strains resistant to rifampin quinone developed crossresistance to the standard drug rifampin, with some populations showing no growth inhibition at maximum concentrations of rifampin. Sequencing of the rifampin quinone-treated strains indicated that they acquired mutations in the DNAdependent RNA polymerase B subunit. These mutations were localized in the rifampin resistance-determining region (RRDR), consistent with other reports of rifampinresistant E. coli and mycobacteria. Rifampin quinone-treated mycobacteria also had cross-resistance to other rifamycin class drugs, including 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.