Assessment of the reversibility of resistance in the absence of antibiotics and its relationship with the resistance gene's fitness cost: a genetic study with mcr-1.

ABSTRACT

BACKGROUND: The intensive use of antibiotics has resulted in strong natural selection for the evolution of antimicrobial resistance (AMR), but whether, and under what circumstances, the removal of antibiotics would result in a rapid reduction in AMR has been insufficiently explored. We aimed to test the hypothesis that in the simple, yet common, case of AMR conferred by a single gene, removing antibiotics would quickly reduce the prevalence of resistance if the AMR gene imposes a high fitness cost and costless resistance is extremely rare among its proximal mutants. METHODS: In this genetic study, to test our hypothesis, we used the mcr-1 gene in Escherichia coli, which confers resistance to the last-resort antibiotic colistin, as a model. A high-throughput reverse genetics approach was used to evaluate mcr-1 variants for their fitness cost and resistance levels relative to a non-functional construct, by measuring relative growth rates in colistin-free media and at 2 µg/mL and 4 µg/mL colistin. We identified costless resistant mcr-1 mutants, and examined their properties within the context of the sequential organisation of mcr-1's functional domains as well as the evolutionary accessibility of these mutations. Finally, a simple population genetic model incorporating the measured fitness cost was constructed and tested against previously published real-world data of mcr-1 prevalence in colonised inpatients in China since the 2017 colistin ban in fodder additives. FINDINGS: We estimated the relative growth rates of 14 742 mcr-1 E coli variants (including the wild type), 3449 of which were single-nucleotide mutants. E coli showed 73·8% less growth per 24 h when carrying wild-type mcr-1 compared with the non-functional construct. 6252 (42·4%) of 14 741 mcr-1 mutants showed colistin resistance accompanied by significant fitness costs, when grown under 4 µg/mL colistin selection. 43 (0·3%) mcr-1 mutants exhibited costless resistance, most of which contained multiple mutations. Among the 3449 single mutants of mcr-1, 3433 (99·5%) had a fitness cost when grown in colistin-free media, with a mean relative growth of 0·305 (SD 0·193) compared with the non-functional variant. 3059 (88·7%) and 1833 (53·1%) of 3449 single mutants outgrew the non-functional mcr-1 in the presence of 2 µg/mL and 4 µg/mL colistin, respectively. Single mutations that gave rise to costless mutants were rare in all three domains of mcr-1 (transmembrane domain, flexible linker, and catalytic domain), but the linker domain was enriched with cost-reducing and resistance-enhancing mutations and depleted with cost-increasing mutations. The population genetics model based on the experimental data accurately predicts the rapid decline in mcr-1 prevalence in real-world data. INTERPRETATION: Many identified costless resistant variants that consist of multiple mutations are unlikely to evolve easily in nature. These findings for colistin and mcr-1 might be applicable to other cases in which AMR entails a substantial fitness cost that cannot be mitigated in proximal mutants. FUNDING: National Natural Science Foundation of China, and National Key Research and Development Program of China.