Rapid Identification of New Delhi Metallo-β-Lactamase (NDM) Using Tryptic Peptides and LC-MS/MS

Abstract

There is significant interest in the development of mass spectrometry (MS) methods for antimicrobial resistance protein detection, given the ability of these methods to confirm protein expression. In this work, we studied the performance of a liquid chromatography, tandem MS multiple-reaction monitoring (LC-MS/MS MRM) method for the direct detection of the New Delhi metallo-β-lactamase (NDM) carbapenemase in clinical isolates. Using a genoproteomic approach, we selected three unique peptides (SLGNLGDADTEHYAASAR, AFGAAFPK, and ASMIVMSHSAPDSR) specific to NDM that were efficiently ionized and spectrally well-defined. These three peptides were used to build an assay with turnaround time of 90 min. In a blind set, the assay detected 21/24 blaNDM-containing isolates and 76/76 isolates with negative results, corresponding to a sensitivity value of 87.5% (95% confidence interval [CI], 67.6% to 97.3%) and a specificity value of 100% (95% CI, 95.3% to 100%). One of the missed identifications was determined by protein fractionation to be due to low (∼0.1 fm/μg) NDM protein expression (below the assay limit of detection). Parallel disk diffusion susceptibility testing demonstrated this isolate to be meropenem susceptible, consistent with low NDM expression. Total proteomic analysis of the other two missed identifications did not detect NDM peptides but detected other proteins expressed from the blaNDM-containing plasmids, confirming that the plasmids were not lost. The measurement of relative NDM concentrations over the entire isolate test set demonstrated variability spanning 4 orders of magnitude, further confirming the remarkable range that may be seen in levels of NDM expression. This report highlights the sensitivity of LC-MS/MS to variations in NDM protein expression, with implications for how this technology may be used.

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 theory, a future combination of both applications could result in an all-in-one diagnostic tool for the identification of bacteria and determination of resistance mechanisms. While the ability of targeted LC-MS/MS to accurately and rapidly identify several resistance mechanisms in bacterial subcultures has been demonstrated in previous studies ( Charretier et al, 2015a , b ; Hassing et al, 2016 ; Wang et al, 2017 , 2019a , b ; Cecchini et al, 2018 ; Foudraine et al, 2019 ; Strich et al, 2019 ), to our knowledge, no studies have demonstrated its use for the direct detection of resistance mechanisms in positive blood cultures. Furthermore, most studies focused on a limited number of AMR mechanisms, and not on the prediction of AMR in prospectively collected cultures.…”

“…In addition, by detecting conserved peptides, many protein variants of resistance mechanisms can be detected using single targets ( Welker and van Belkum, 2019 ). In several studies, targeted LC-MS/MS assays were developed for the detection of specific resistance mechanisms such as small-spectrum β-lactamases ( Cecchini et al, 2018 ), carbapenemases ( Wang et al, 2017 , 2019b ; Cecchini et al, 2018 ; Foudraine et al, 2019 ; Strich et al, 2019 ), PBP2a and PBP2c ( Charretier et al, 2015a ), AmpC β-lactamases ( Charretier et al, 2015b ), efflux pumps ( Charretier et al, 2015b ; Cecchini et al, 2018 ), alterations in the quinolone resistance determining region (QRDR) of GyrA ( Hassing et al, 2016 ), MCR-1 ( Wang et al, 2019a ), and aminoglycoside resistance mechanisms ( Foudraine et al, 2021a ). However, the potential of targeted LC-MS/MS to detect resistance mechanisms directly in positive blood cultures has to our knowledge not yet been demonstrated.…”

Parallel Reaction Monitoring Mass Spectrometry for Rapid and Accurate Identification of β-Lactamases Produced by Enterobacteriaceae

“…Specific peptides of A. baumannii identified via LC-MS/MS profiling have been used to classify clinical isolates ( Honghui et al, 2016 ). Targeted high-resolution MS assays have been developed for the detection of KPC, OXA-48, NDM, and VIM enzymes ( Wang et al, 2017 , 2019 ; Foudraine et al, 2019 ; Strich et al, 2019 ). However, there are still many β-lactamases that have not been detected by targeted proteomics.…”

Modified ELISA for Ultrasensitive Diagnosis

“…The potential applications of this method depend on the specificity and strength of the antigen–antibody reaction being evaluated. Due to the limitation of ELISAs requiring the use of a purified antigen and an extraordinarily functional antibody, some researchers strongly propose that other methods be used, such as liquid chromatography–mass spectrometry (LC-MS) [ 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 ] and various lab-on-a-chip methods [ 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 ]. A recent trend in LC-MS development for diagnosis is to focus on the detection of drugs and biomarkers, including peptides and steroids, owing to difficulties in preparing suitable antibodies against these small molecules, whereas lab-on-a-chip–based platforms are thought to offer easier sample preparation, chemical manipulation and reaction, and high throughput using various novel detection techniques with, sometimes, label-free detection.…”