Exploring the genomic landscape: identification of aminoglycoside fitness-genes in resistant Escherichia coli

Aminoglycosides resistance is common worldwide in pathogenic bacteria, and helper-drugs are urgently needed for this class of antimicrobials. The objective of this study was to identify genes that are advantageous for growth of E. coli in the presence of aminoglycosides, as these genes may represent targets for helper-drugs. We generated three highly saturated transposon mutant libraries in E. coli MG1655 containing streptomycin (aph(3"")-Ib/aph(6)-Id), gentamicin (aac(3)-IV), or neomycin (aph(3"")-Ia) resistance gene(s). Transposon Directed Insertion-site Sequencing (TraDIS) revealed 106 genes, where transposon insertions caused reduced fitness in the presence of one or more of the antibiotics. Fifty-six, 39 and 32 genes were identified as fitness-genes under streptomycin, gentamicin and neomycin stress (Fig. 1 A). ATP-synthesis, cell division, and stress response genes dominated among streptomycin and gentamicin fitness-genes, while the neomycin fitness-genes were associated with enterobacterial common antigen biosynthesis or magnesium sensing/transport (Fig 1 B). To confirm the importance of the fitness-genes, the genes/gene clusters minCDE, hflCK, clsA and cpxR were deleted among the streptomycin and gentamicin fitness-genes, and phoPQ, wecA, lpp and pal among the neomycin resistance ones. Phenotypic characterization including growth curves, MIC testing and a novel sequencing-based competition approach revealed that all deletion mutants exhibited an attenuated growth in the presence of the corresponding aminoglycoside(s). In summary, we report a set of aminoglycoside fitness-genes which may be putative targets for helper-drugs. Moreover, our study has expanded our understanding of changes that occur in aminoglycoside-resistant E. coli in response to antibiotic exposure.

Figure 1: Aminoglycoside fitness-genes (a) and their functional classification (b) in E. coli MG1655 in the presence of STREP, GEN or NEO.

This INNOTARGETS project has received funding from the European Union"s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement number 956154.