Effect of aminoacyl-tRNA synthetase mutations on susceptibility to ciprofloxacin in Escherichia coli

Background: Chromosomal mutations that reduce ciprofloxacin susceptibility in Escherichia coli characteristically map to drug target genes (gyrAB and parCE), and genes encoding regulators of the AcrAB-TolC efflux pump. Mutations in RNA polymerase can also reduce susceptibility, by up-regulating the MdtK efflux pump. Objectives: We asked whether mutations in additional chromosomal gene classes could reduce susceptibility to ciprofloxacin. Methods: Experimental evolution, complemented by WGS analysis, was used to select and identify mutations that reduce susceptibility to ciprofloxacin. Transcriptome analysis, genetic reconstructions, susceptibility measurements and competition assays were used to identify significant genes and explore the mechanism of resistance. Results: Mutations in three different aminoacyl-tRNA synthetase genes (leuS, aspS and thrS) were shown to re- duce susceptibility to ciprofloxacin. For two of the genes (leuS and aspS) the mechanism was partially dependent on RelA activity. Two independently selected mutations in leuS (Asp162Asn and Ser496Pro) were studied in most detail, revealing that they induce transcriptome changes similar to a stringent response, including up-regulation of three efflux-associated loci (mdtK, acrZ and ydhJK). Genetic analysis showed that reduced susceptibility depended on the activity of these loci. Broader antimicrobial susceptibility testing showed that the leuS mutations also reduce susceptibility to additional classes of antibiotics chloramphenicol, rifampicin, mecillinam, ampicillin and trimethoprim). Conclusions: The identification of mutations in multiple tRNA synthetase genes that reduce susceptibility to ciprofloxacin and other antibiotics reveals the existence of a large mutational target that could contribute to re- sistance development by up-regulation of an array of efflux pumps