Epistasis between antibiotic resistance mutations drives the evolution of extensively drug-resistant tuberculosis

Background and objectives: Multidrug resistant (MDR) bacteria are a growing threat to global health. Studies focusing on single antibiotics have shown that drug resistance is often associated with a fitness cost in the absence of drug. However, little is known about the fitness cost associated with resistance to multiple antibiotics. Methodology: We used Mycobacterium smegmatis as a model for human tuberculosis (TB) and an in vitro competitive fitness assay to explore the combined fitness effects and interaction between mutations conferring resistance to rifampicin (RIF) and ofloxacin (OFX); two of the most important first- and second-line anti-TB drugs, respectively. Results: We found that 4 out of 17 M. smegmatis mutants (24%) resistant to RIF and OFX showed a statistically significantly higher or lower competitive fitness than expected when assuming a multiplicative model of fitness effects of each individual mutation. Moreover, 6 out of the 17 double drug-resistant mutants (35%) had a significantly higher fitness than at least one of the corresponding single drug-resistant mutants. The particular combinations of resistance mutations associated with no fitness deficit in M. smegmatis were the most frequent among 151 clinical isolates of MDR and extensively drug-resistant (XDR) Mycobacterium tuberculosis from South Africa. Conclusions and implications: Our results suggest that epistasis between drug resistance mutations in mycobacteria can lead to MDR strains with no fitness deficit, and that these strains are positively selected in settings with a high burden of drug-resistant TB. Taken together, our findings support a role for epistasis in the evolution and epidemiology of MDR- and XDR-TB.