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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 bla NDM-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 bla NDM-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.

Authors+Show Affiliations

Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.Proteomics Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.Department of Laboratory Medicine, Clinical Center, Microbiology Service, National Institutes of Health, Bethesda, Maryland, USA.Kidney Disease Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA. Department of Pathology, Johns Hopkins University, Baltimore, Maryland, USA.Proteomics Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.Department of Laboratory Medicine, Clinical Center, Microbiology Service, National Institutes of Health, Bethesda, Maryland, USA. Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA asuffredini@cc.nih.gov.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, N.I.H., Intramural

Language

eng

PubMed ID

31307990

Citation

Wang, Honghui, et al. "Rapid Identification of New Delhi Metallo-β-Lactamase (NDM) Using Tryptic Peptides and LC-MS/MS." Antimicrobial Agents and Chemotherapy, vol. 63, no. 9, 2019.
Wang H, Strich JR, Drake SK, et al. Rapid Identification of New Delhi Metallo-β-Lactamase (NDM) Using Tryptic Peptides and LC-MS/MS. Antimicrob Agents Chemother. 2019;63(9).
Wang, H., Strich, J. R., Drake, S. K., Chen, Y., Youn, J. H., Rosenberg, A. Z., ... Suffredini, A. F. (2019). Rapid Identification of New Delhi Metallo-β-Lactamase (NDM) Using Tryptic Peptides and LC-MS/MS. Antimicrobial Agents and Chemotherapy, 63(9), doi:10.1128/AAC.00461-19.
Wang H, et al. Rapid Identification of New Delhi Metallo-β-Lactamase (NDM) Using Tryptic Peptides and LC-MS/MS. Antimicrob Agents Chemother. 2019;63(9) PubMed PMID: 31307990.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Rapid Identification of New Delhi Metallo-β-Lactamase (NDM) Using Tryptic Peptides and LC-MS/MS. AU - Wang,Honghui, AU - Strich,Jeffrey R, AU - Drake,Steven K, AU - Chen,Yong, AU - Youn,Jung-Ho, AU - Rosenberg,Avi Z, AU - Gucek,Marjan, AU - Dekker,John P, AU - Suffredini,Anthony F, Y1 - 2019/08/23/ PY - 2019/03/12/received PY - 2019/07/03/accepted PY - 2020/02/23/pmc-release PY - 2019/7/17/pubmed PY - 2019/7/17/medline PY - 2019/7/17/entrez KW - New Delhi metallo-β-lactamase KW - mass spectrometry KW - multiple-reaction monitoring KW - tryptic peptide JF - Antimicrobial agents and chemotherapy JO - Antimicrob. Agents Chemother. VL - 63 IS - 9 N2 - 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 bla NDM-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 bla NDM-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. SN - 1098-6596 UR - https://www.unboundmedicine.com/medline/citation/31307990/Rapid_Identification_of_New_Delhi_Metallo-β-Lactamase_(NDM)_Using_Tryptic_Peptides_and_LC-MS/MS L2 - http://aac.asm.org/cgi/pmidlookup?view=long&pmid=31307990 DB - PRIME DP - Unbound Medicine ER -