Detection of Carbapenemase-Producing Bacteria by Using an Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method

Abstract

cThe emergence of carbapenemase-producing bacteria poses a new challenge in the management of antibiotic therapies for patients. This report describes a new method using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for rapid detection of carbapenemase activity in enterobacteria, Pseudomonas aeruginosa, and Acinetobacter baumannii. In a panel of 78 isolates, including 41 carbapenemase-producing strains, the ULPC-MS/MS assay showed 100% agreement with molecular characterization, whereas six carbapenemase-producing isolates were not detected by the modified Hodge test.T he emergence and dissemination of resistance to carbapenem in Gram-negative pathogens such as Enterobacteriaceae, Acinetobacter baumannii, and Pseudomonas aeruginosa represent a challenge for optimizing antibiotic therapies and preventing outbreaks, especially regarding carbapenemase-producing bacteria, by comparison to other resistance mechanisms. Using the Ambler classification of beta-lactamases, the principal carbapenemase enzymes detected in infected patients worldwide belong to class B (metallo-beta-lactamases, including IMP, VIM, and NDM), to class A (beta-lactamases, including Klebsiella pneumoniae carbapenemase [KPC]), and to class D (oxacillinases, including OXA-48 and OXA-23). Moreover, clinical infections due to these carbapenemase-producing strains, which are also resistant to most betalactam antibiotics, are associated with higher morbidity and mortality (1). Bacterial resistance to carbapenem is usually screened using susceptibility and phenotypic assays such as the modified Hodge test (MHT) (2). However, these tests have shown poor sensitivity and specificity in detecting carbapenemase-producing isolates, especially in A. baumannii (3). While PCR-based detection of the carbapenemase gene is considered the gold standard method, in view of the high genetic diversity of genes coding for carbapenemase, numerous PCR assays targeting the most prevalent enzymes must be developed in this approach (2, 4). Recently, mass spectrometry (MS) technologies, such as matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), have been shown to be capable of characterizing carbapenemase-producing bacteria (5,6,7,8).This study reports on a rapid UPLC-MS/MS assay to detect carbapenemase-producing Enterobacteriaceae, P. aeruginosa, and A. baumannii isolates based on the ability of bacterial isolates to hydrolyze ertapenem and meropenem in solution.A total of 78 bacterial strains (27 reference strains and 51 strains isolated from clinical samples) were included in the study: 41 carbapenemase-producing strains (including 23 enterobacterial strains, 3 P. aeruginosa strains, and 15 A. baumannii strains) and 37 strains susceptible to carbapenems or harboring decreased susceptibility to carbapenems due to non-carbapenemase-based mechanisms. Escherichia coli ATCC 25922 and Citrobacter freundii NDM-1-positive strain...

Detection of carbapenemases in Enterobacteriaceae: a challenge for diagnostic microbiological laboratories

“…Incubation of the reaction mixture for 4 h increased both the sensitivity and specificity of the method to 100%. Recently, similar results were obtained by Carricajo et al [121], using ultra-performance LC-tandem MS. Unfortunately, the equipment required for LC-MS is not currently available in most diagnostic laboratories, in contrast to MALDI-TOF mass spectrometers.…”

“…Spectrophotometric detection of carbapenem hydrolysis has been proposed as a reference method for confirmation of carbapenemase activity . Although this approach is labour‐intensive, it cannot be used routinely in diagnostic laboratories.…”

Non-molecular detection of carbapenemases in Enterobacteriaceae clinical isolates

“…Other mass spectrometry systems such as liquid chromatography-MS (LC-MS) [137], ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) [140], capillary electrophoresis-electrospray-ionization-tandem-MS (CE-ESI-MS/MS) [141] or PCR-electrospray ionization-MS (PCR/ESI-MS) [142] are being evaluated for its use in the detection of carbapenemases. Despite its optimal performance in the detection of carbapenemases [139e142] its usefulness is limited to the research setting due to the advanced equipment needed.…”

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for the Rapid Detection of Antimicrobial Resistance Mechanisms and Beyond

“…(v) Finally, mass spectrometry analysis can be used to detect antimicrobial resistance mediated by all possible resistance mechanisms. Two main technologies are used: liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MALDI-TOF MS. LC-MS/MS has been developed mainly to detect ␤-lactamases (37)(38)(39)(40). The different procedures consist of either detecting the enzyme directly from bacteria or detecting the hydrolysis of the antibiotic by the ␤-lactamase enzyme.…”