Genomics, proteomics and phenotypic analysis in 190 strains of E. coli (70) and K. pneumoniae (120) for investigating antimicrobial resistance.. A systematic exploration of antimicrobial resistance to beta-lactams, aminoglycosides and fluoroquinolones in Escherichia coli and Klebsiella pneumoniae using genoproteomics

AbstractBackground: Antimicrobial resistance is generally studied using a combination of growth inhibition and DNA detection methods. However, continuous improvements in mass spectrometry allow to examine antimicrobial resistance at the intermediate protein level as well. In the current study, these three modalities are combined into a multi-omics approach to analyse resistance against important antibiotics within the beta-lactam, aminoglycoside and fluoroquinolone group in E. coli and K. pneumoniae.Objectives: First, to analyse which antimicrobial resistance genes are expressed at the protein level and if these can be detected using untargeted liquid chromatography-mass spectrometry. Second, to assess which of these mechanisms contribute to beta-lactam, aminoglycoside, and fluoroquinolone resistance in E. coli and K. pneumoniae. Third, to correlate porin abundance and porin alterations to resistance. Fourth, to identify other proteins which correlate to resistance.Methods: Whole genome sequencing, high-resolution bottom up LC-MS/MS and antimicrobial susceptibility testing by broth microdilution were performed for 190 clinical E. coli and K. pneumoniae isolates. Resistance genes and proteins were identified using the Comprehensive Antibiotic Resistance Database. All proteins were extensively annotated using both Prokka and the NCBI refSeq database.Results & Conclusion: Proteins of small spectrum beta-lactamases, extended spectrum beta-lactamases, carbapanemases, AmpC beta-lactamases, and proteins of 16S-RMTases and aminoglycoside acetyltransferases are expressed in E. coli and K. pneumoniae without applying antibiotic pressure and can be detected by untargeted LC-MS/MS. These mechanisms could explain phenotypic resistance in most of the studied isolates. Differences in abundance and structure of other proteins such as porins also correlated with resistance and can sometimes explain additional differences in MICs. The findings and generated data in this study may guide future studies aiming to decipher antimicrobial resistance or search for new resistance markers.