Honghu Sun, Jumei Zeng, Song Li, Pengkuan Liang, Chao Zheng, Yong Liu, Tao Luo, Nalin Rastogi, Qun Sun

The interaction between different drug-resistant mutations is important to the development of drug resistance and its evolution. In this study, we aimed to reveal the potential relationships between mutations conferring resistance to two important antituberculosis drugs streptomycin (STR) and fluoroquinolones (FLQ). We used an in vitro competitive fitness assay to reveal the interactions between different mutations ofrpsLandgyrAin drug-resistantMycobacterium smegmatis, followed by the analysis of the frequency ofrpsLandgyrAmutation combinations in 213 STR-FLQ dual-resistant clinicalMycobacterium tuberculosisisolates from Sichuan region, which was also investigated by the whole genome data from 3,056 global clinicalM. tuberculosisisolates. The strains with K43R and K88R mutation inrpsLshowed no difference in relative fitness compared with their susceptible ancestor, while K43N, K43M, K43T, and K88E exhibited a significantly lower relative fitness (P<0.05). For the FLQ-resistant mutants, all mutation types showed no difference in their relative fitness. Among STR-FLQ dual-resistantM. smegmatisstrains, a lower fitness was detected in those with K43N/M/T and K88E instead of K43R and K88R mutations inrpsL. AmongM. tuberculosisisolates harboringrpsLandgyrAdual mutations, the most two frequent combinatorial mutation types were K43R/D94G (n=37) and K43R/A90V (n=24), with the former being the most frequent one by both in vitro tests and clinical survey. Our results suggest that the interaction betweenrpsLandgyrAmutations affects the fitness cost in STR-FLQ dual-resistantM. smegmatisand also the predilection of mutation combinations in clinicalM. tuberculosisisolates.