Rapid and Accurate Detection of Aminoglycoside-Modifying Enzymes and 16S rRNA Methyltransferases by Targeted Liquid Chromatography-Tandem Mass Spectrometry

Abstract

New and rapid diagnostic methods are needed for the detection of antimicrobial resistance to aid in the curbing of drug-resistant infections. Targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a method that could serve this purpose, as it can detect specific peptides of antimicrobial resistance mechanisms with high accuracy. In the current study, we developed an accurate and rapid targeted LC-MS/MS assay based on parallel reaction monitoring for detection of the most prevalent aminoglycoside modifying enzymes and 16S ribosomal RNA methyltransferases in E. coli and K. pneumoniae that confer resistance to aminoglycosides. Specific tryptic peptides needed for detection were selected and validated for AAC(3)-Ia, AAC(3)-II, AAC(3)-IV, AAC(3)-VI, AAC(6’)-Ib, AAC(6’)-Ib-cr, ANT(2”)-I, APH(3’)-VI, ArmA, RmtB, RmtC and RmtF. In total, 205 isolates containing different aminoglycoside resistance mechanisms that consisted mostly of E. coli and K. pneumoniae were selected for assay development and evaluation. Mass spectrometry results were automatically analyzed and were compared to whole genome sequencing results. Of the 2460 isolate and resistance mechanism combinations tested, 2416 combinations matched. Discrepancies were further analyzed by repeating LC-MS/MS analysis and performing additional PCRs. Mass spectrometry results were also used to predict resistance and susceptibility to gentamicin, tobramycin and amikacin in only the E. coli and K. pneumoniae isolates (n=191). The category interpretations were correctly predicted for gentamicin in 97.4% of the isolates, for tobramycin in 97.4% of the isolates, and for amikacin in 82.7% of the isolates. Targeted LC-MS/MS can be applied for accurate and rapid detection of aminoglycoside resistance mechanisms.

Liquid Chromatography-Tandem Mass Spectrometry Analysis Demonstrates a Decrease in Porins and Increase in CMY-2 β-Lactamases in Escherichia coli Exposed to Increasing Concentrations of Meropenem

“…When peptides for other resistance mechanisms, such as ESBLs, carbapenemases ( Foudraine et al, 2019 ), fluoroquinolone resistance mechanisms ( Hassing et al, 2016 ), and aminoglycoside resistance mechanisms ( Foudraine et al, 2021a ) would be added to the current panel of CMY, E. coli cAmpC, OmpC and OmpF, a single multiplex LC-MS/MS assay would be able to detect several prevalent mechanisms that confer resistance to beta-lactams, fluoroquinolones and aminoglycosides in for instance E. coli . While such an assay could be used to confirm or rule out the presence of specific resistance mechanisms after routine susceptibility testing, it may be more beneficial to use it as a rapid screening test for important liquid cultures such as blood cultures.…”

“…Tryptic peptides from 6 to 20 amino acids without a cysteine and preferably without a methionine or a double lysine/arginine motif at the N- or C-terminus (ragged end) were selected as candidate peptides ( Supplementary Table 1 ). In addition to porin and β-lactamase peptides, internal quality control peptides were included from a previous study for verification of adequate sample pre-treatment ( Foudraine et al, 2021a ). General peptide uniqueness of all candidate peptides was assessed by performing BLASTp searches in the NCBI non-redundant protein sequence database.…”

“…In contrast, targeted liquid chromatography-tandem mass spectrometry (LS-MS/MS) offers increased sensitivity and specificity by separating and filtering peptides prior to detection. Therefore, targeted LC-MS/MS using a triple quadrupole or an Orbitrap mass spectrometer can be used for the accurate detection and quantification of many antimicrobial resistance mechanisms ( Charretier et al, 2015a , b ; Charretier and Schrenzel, 2016 ; Hassing et al, 2016 ; Wang et al, 2017 , 2019 ; Cecchini et al, 2018 ; Foudraine et al, 2019 , 2021a ).…”

Using Targeted Liquid Chromatography-Tandem Mass Spectrometry to Rapidly Detect β-Lactam, Aminoglycoside, and Fluoroquinolone Resistance Mechanisms in Blood Cultures Growing E. coli or K. pneumoniae

“…In previous studies, we demonstrated the possibility to detect different carbapenemase enzymes and aminoglycoside resistance mechanisms with high accuracy using LC-MS/MS (Foudraine et al, 2019(Foudraine et al, , 2021a. In these studies, resistance mechanisms were identified by detection of selected specific peptides, and SIL peptides were used to automate the analysis.…”

“…+As previously reported (Foudraine et al, 2019).# As reported (Foudraine et al, 2022). * As previously reported (Foudraine et al, 2021a). "Anonymous" variants were unique β-lactamase variants similar to the reference sequence which were not (yet) annotated at the time of peptide selection.…”