Interaction between rpsL and gyrA mutations affects the fitness and dual resistance of Mycobacterium tuberculosis clinical isolates against streptomycin and fluoroquinolones

Abstract

Background:

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).

Materials and methods:

We used an in vitro competitive fitness assay to reveal the interactions between different mutations of rpsL and gyrA in drug-resistant Mycobacterium smegmatis, followed by the analysis of the frequency of rpsL and gyrA mutation combinations in 213 STR–FLQ dual-resistant clinical Mycobacterium tuberculosis isolates from Sichuan region, which was also investigated by the whole genome data from 3,056 global clinical M. tuberculosis isolates.

Results:

The strains with K43R and K88R mutation in rpsL showed 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-resistant M. smegmatis strains, a lower fitness was detected in those with K43N/M/T and K88E instead of K43R and K88R mutations in rpsL. Among M. tuberculosis isolates harboring rpsL and gyrA dual 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.

Conclusion:

Our results suggest that the interaction between rpsL and gyrA mutations affects the fitness cost in STR–FLQ dual-resistant M. smegmatis and also the predilection of mutation combinations in clinical M. tuberculosis isolates.

Keywords::

• Mycobacterium
• fitness
• dual resistance
• epistasis

Acknowledgments

We sincerely thank our colleagues from Public Health Clinical Center of Chengdu for the collection of samples and the performance of drug susceptibility tests. This work was supported by the Science and Technology Department of Sichuan Province (2017TJPT0014 and 2016SZ0068), and the Ministry of Science & Technology of China (2015DFR31060).

Disclosure

The authors report no conflicts of interest in this work.

Supplementary materials

Figure S1 Model of mutations appearing in rpsL and gyrA in this study.

Notes: (A) Structure of Escherichia coli rpsL (Protein Data Bank accession number 5U4I). The position of K43 residue (yellow) and K88 residue (red) screened in this study was highly conserved between E. coli and Mycobacterium tuberculosis. (B) M. tuberculosis gyrA (Protein Data Bank accession number 3IFZ). The position of G88 residue (yellow), A90 residue (red), and D94 residue (cyan) screened in present study is shown. Amino acids were placed in the cartoon structure in dots format using the PyMOL Viewer.

Figure S2 The alignment of rpsL in M. tuberculosis and E. coli.

Table S1 Epistasis (ε) in mutants resistant to STR and FLQ