Chemogenomic Screen for Imipenem Resistance in Gram-Negative Bacteria

Abstract

Gram-negative carbapenem-resistant bacteria are a major threat to global health. The use of genome-wide screening approaches to probe for genes or mutations enabling resistance can lead to identification of molecular markers for diagnostics applications. We describe an approach called Mut-Seq that couples chemical mutagenesis and next-generation sequencing for studying resistance to imipenem in the Gram-negative bacteria Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The use of this approach highlighted shared and species-specific responses, and the role in resistance of a number of genes involved in membrane biogenesis, transcription, and signal transduction was functionally validated. Interestingly, some of the genes identified were previously considered promising therapeutic targets. Our genome-wide screen has the potential to be extended outside drug resistance studies and expanded to other organisms. ABSTRACT Carbapenem-resistant Gram-negative bacteria are considered a major threat to global health. Imipenem (IMP) is used as a last line of treatment against these pathogens, but its efficacy is diminished by the emergence of resistance. We applied a whole-genome screen in Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa isolates that were submitted to chemical mutagenesis, selected for IMP resistance, and characterized by next-generation sequencing. A comparative analysis of IMP-resistant clones showed that most of the highly mutated genes shared by the three species encoded proteins involved in transcription or signal transduction. Of these, the rpoD gene was one of the most prevalent and an E. coli strain disrupted for rpoD displayed a 4-fold increase in resistance to IMP. E. coli and K. pneumoniae also specifically shared several mutated genes, most 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 isolates with two different resistance mechanisms, with one involving mutations in the oprD porin or, alternatively, in two-component systems. Our chemogenomic screen performed with the three species has highlighted shared and species-specific responses to IMP. IMPORTANCE Gram-negative carbapenem-resistant bacteria are a major threat to global health. The use of genome-wide screening approaches to probe for genes or mutations enabling resistance can lead to identification of molecular markers for diagnostics applications. We describe an approach called Mut-Seq that couples chemical mutagenesis and next-generation sequencing for studying resistance to imipenem in the Gram-negative bacteria Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The use of this approach highlighted shared and species-specific responses, and the role in resistance of a number of genes involved in membrane biogenesis, transcription, and signal transduction was functionally validated. Interestingly, some of the genes identified were previously considered promising therapeutic targets. Our genome-wide screen has the potential to be extended outside drug resistance studies and expanded to other organisms.