Chemogenomic screen for imipenem resistance in Gram-negative bacteria (E.coli)

Carbapenem-resistant Gram-negative bacteria are considered as a major threat to global health. Imipenem (IMP) is used as a last line of treatment against these pathogens but its efficiency is diminished with the emergence of resistance. We applied a powerful whole genome screen in Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa that were submitted to chemical mutagenesis, selection for IMP resistance and characterized by next generation sequencing. A comparative analysis of 45-50 clones for each species showed that the most mutated genes encoded for proteins that were involved in membrane/cell envelope biogenesis, transcription and signal transduction. The rpoD gene was one of the most mutated gene common to the three species and an E. coli rpoD_mutated knock-in was 4-fold resistant to IMP. E. coli and K. pneumoniae shared many common mutated genes, particularly in genes involved in membrane/cell envelope biogenesis and the contribution in IMP susceptibility was experimentally proven for amidases, transferases, and transglycosidases. P. aeruginosa, differed from the two Enterobacteriaceae with two different resistance mechanisms one involving mutations in the oprD porin or alternatively in two-component systems, the latter also observed in the Enterobacteriaceae. Our chemogenomic screen with the three species has highlighted shared and species-specific responses to IMP