Abstract
Antimicrobial resistance is mostly studied by means of phenotypic growth inhibition determinations, in combination with PCR confirmations or further characterization by means of whole genome sequencing (WGS). However, the actual proteins that cause resistance such as enzymes and a lack of porins cannot be detected by these methods. Improvements in liquid chromatography (LC) and mass spectrometry (MS) enabled easier and more comprehensive proteome analysis. In the current study, susceptibility testing, WGS and MS are combined into a multi-omics approach to analyze resistance against frequently used antibiotics within the beta-lactam, aminoglycoside and fluoroquinolone group in E. coli and K. pneumoniae. Our aim was to study which currently known mechanisms of resistance can be detected at the protein level using liquid chromatography–mass spectrometry (LC–MS/MS) and to assess whether these could explain beta-lactam, aminoglycoside, and fluoroquinolone resistance in the studied isolates. Furthermore, we aimed to identify significant protein to resistance correlations which have not yet been described before and to correlate the abundance of different porins in relation to resistance to different classes of antibiotics. Whole genome sequencing, high-resolution LC–MS/MS and antimicrobial susceptibility testing by broth microdilution were performed for 187 clinical E. coli and K. pneumoniae isolates. Resistance genes and proteins were identified using the Comprehensive Antibiotic Resistance Database (CARD). All proteins were annotated using the NCBI RefSeq database and Prokka. Proteins of small spectrum beta-lactamases, extended spectrum beta-lactamases, AmpC beta-lactamases, carbapenemases, and proteins of 16S ribosomal RNA methyltransferases and aminoglycoside acetyltransferases can be detected in E. coli and K. pneumoniae by LC–MS/MS. The detected mechanisms matched with the phenotype in the majority of isolates. Differences in the abundance and the primary structure of other proteins such as porins also correlated with resistance. LC–MS/MS is a different and complementary method which can be used to characterize antimicrobial resistance in detail as not only the primary resistance causing mechanisms are detected, but also secondary enhancing resistance mechanisms.
Highlights
Antimicrobial resistance (AMR) is widely recognized as a serious threat to global public h ealth[1]
Similar to the achievements made for whole genome sequencing (WGS), liquid chromatography combined with mass spectrometry (LC–MS) has advanced to a level in which proteomes can be comprehensively characterized within a few hours using bottom-up shotgun p roteomics[12]
As new AMR protein detection methods are developed that are based on MS9,15,16, it is important to know which resistance mechanisms can be detected at the protein level and if they can predict phenotypic resistance as well
Summary
Antimicrobial resistance (AMR) is widely recognized as a serious threat to global public h ealth[1]. Antibiotic resistant Escherichia coli and Klebsiella pneumoniae are worrisome Both species belong to the family of Enterobacterales and are clinically relevant as they frequently cause various infections in patients of all ages. Similar to the achievements made for WGS, liquid chromatography combined with mass spectrometry (LC–MS) has advanced to a level in which proteomes can be comprehensively characterized within a few hours using bottom-up shotgun p roteomics[12]. Both Chang et al and Trip et al applied this discovery-based approach to detect beta-lactamases in several Acinetobacter baumannii isolates[13,14]. These methods offer a shorter turnaround time than the currently applied phenotypical susceptibility testing techniques that require overnight incubation which results in a delay in reporting time[17]
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