Rapid detection of carbapenemase-producing Enterobacteriaceae using MALDI-TOF MS

Knox, James R.; Jadhav, Snehal; Sevior, Danielle; Agyekum, Alex; Whipp, Margaret; Waring, Lynette; Iredell, Jonathan R.; Palombo, Enzo A.

doi:10.1016/j.pathol.2015.12.138

Cited by 2 publications

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“…However, the Carba NP test displayed a lower sensitivity than was initially reported (76% in this study versus 100%) (5,6), mainly due to false-negative results obtained with OXA-48-type producers. This finding is in agreement with previous studies reporting the limited ability of Carba NP test to detect OXA-48-type-producing isolates and some weak carbapenemases of class A, like GES-5 and SME-1 (33,34). For the MALDI-TOF MS assay, the limitation in detecting OXA-48-type producers was overtaken by addition of NH 4 HCO 3 to the reaction buffer.…”

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confidence: 92%

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Meropenem Hydrolysis Assay with NH ₄ HCO ₃ , a Reliable Tool for Direct Detection of Carbapenemase Activity

et al. 2015

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A comparison of a matrix-assisted laser desorption ionization-time of flight mass spectrometric (MALDI-TOF MS) meropenem hydrolysis assay with the Carba NP test showed that both methods exhibited low sensitivity (approximately 76%), mainly due to the false-negative results obtained with OXA-48-type producers. The addition of NH 4 HCO 3 to the reaction buffer for the MALDI-TOF MS assay dramatically improved its sensitivity (98%). Automatic interpretation of the MALDI-TOF MS assay, using the MBT STAR-BL software, generally agreed with the results obtained after manual analysis. For the Carba NP test, spectrophotometric analysis found six additional carbapenemase producers. Nosocomial infections caused by carbapenem-resistant Enterobacteriaceae and Pseudomonas spp. are now emerging worldwide and are difficult to treat (1). Previous data have shown that strict epidemiological intervention based on the rapid detection of carbapenemase production can prevent the spread of those bacteria (2). Therefore, the introduction of rapid and sensitive methodologies for the detection of carbapenemase-producing bacteria is of utmost importance. Recently, two new highly sensitive and rapid methods for the direct detection of carbapenemase activity were developed. In 2011, two research groups demonstrated that matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can detect carbapenemase activity, based on mass spectral profiles obtained from carbapenem molecules (3, 4). The second method, the Carba NP test, is a biochemical method used in the detection of carbapenemase activity in Enterobacteriaceae, Pseudomonas spp., and Acinetobacter species (5-7). This test is based on a decrease in pH resulting from the hydrolysis of the ␤-lactam ring of carbapenem molecules, which is detected using phenol red as a pH indicator.The aim of this study was to validate the efficiency of a MALDI-TOF MS meropenem hydrolysis assay and the Carba NP test for the detection of carbapenemase producers. The performances of both methods were compared with that of a recently published modification of the MALDI-TOF MS assay (MALDI-TOF BIC) in an effort to increase the reliability of detecting OXA-48 producers (8). Furthermore, the automatic interpretation of all methods was evaluated.The methods were tested against a group of 124 Enterobacteriaceae and 37 Pseudomonas aeruginosa isolates from collections of the Faculty of Medicine and University Hospital in Plzen (Czech Republic), the National Medicines Institute in Warsaw (Poland), the Robert Koch Institute in Wernigerode (Germany), and the University Hospital in Larissa (Greece). The isolates were previously characterized, as described below. All isolates were tested for extended-spectrum ␤-lactamase (ESBL) and AmpC expression by the ESBL double-disk synergy test (DDST) with cefotaxime, ceftazidime, aztreonam, and amoxicillin-clavulanate disks in the absence and presence of cloxacillin (250 g/ml) (9). Susceptibility to carbapenems was determined by using imipenem, merope...

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confidence: 92%

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Meropenem Hydrolysis Assay with NH ₄ HCO ₃ , a Reliable Tool for Direct Detection of Carbapenemase Activity

et al. 2015

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“…Unlike other phenotypic assays, which rely upon the use of various indirect indicators of β-lactamase-mediated hydrolysis of β-lactams, MALDI-TOF MS allows direct visualization of mass-peaks corresponding to intact β-lactam substrates and their degradation products released upon hydrolysis and, therefore, may be considered as a reference method for detecting β-lactamase activity. However, most MALDI-TOF MS-based assays for carbapenemase detection described up to date required preparation of fresh solutions of carbapenems either from chemical substances or therapeutic formulations, which made them less suitable for routine diagnostics (Hrabák et al, 2011;Hoyos-Mallecot et al, 2014;Knox et al, 2014;Papagiannitsis et al, 2015;Ghebremedhin et al, 2016;Sakarikou et al, 2017). Recently, M. Oho et al reported on the development of MALDI-TOF MS assay with imipenem susceptibility disk and zinc sulfate solution for detection of CPE (Oho et al, 2021).…”

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Detection of carbapenemase-producing Enterobacterales by means of matrix-assisted laser desorption ionization time-of-flight mass spectrometry with ertapenem susceptibility-testing disks as source of carbapenem substrate

et al. 2022

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MALDI-TOF mass spectrometry has become widely used in clinical microbiology and has proved highly accurate for detection of carbapenemases in Gram-negative bacteria. However, the use of carbapenem-hydrolysis assays in routine diagnostics is hampered by the need for antibiotic substances and for making their fresh solutions each time an assay is conducted. Here, we evaluated the use of commercial antibiotic susceptibility-testing disks as source of ertapenem substrate in MALDI-TOF MS-based assay for detection of carbapenemase-producing Enterobacterales (CPE). The assay was validated on 48 CPE isolates of 8 different species expressing NDM-, VIM-, KPC- and OXA-48-type carbapenemases and exhibiting various levels of resistance to carbapenems (MIC range: 0.25– > 32 mg/l), as well as on 48 carbapenemase-non-producing isolates. The assay conditions were optimized as follows: 10-μl loopful of bacterial colonies was suspended in 150 μl 0.01 M Na-PBS buffer, pH 7.4, a 10 μg ertapenem susceptibility-testing disk was immersed in the suspension and incubated 3 h at 35°C, after which supernatant was obtained by centrifugation and applied on a target plate with alpha-cyano-4-hydroxycinnamic acid matrix. Mass spectra were analyzed between 440 and 560 m/z. Carbapenemase activity was detected in all tested CPE isolates by the appearance of m/z peaks corresponding to ertapenem hydrolysis products: [Mh + H]+:494.2, [Mh + Na]+:516.2, [Mh + 2Na]+:538.2, [Mh/d + H]+:450.2, [Mh/d + Na]+:472.2, and simultaneous decrease or loss of peaks of intact antibiotic: [M + H]+:476.2, [M + Na]+:498.1, [M + 2Na]+:520.1. No hydrolysis peaks or loss of intact ertapenem peaks were observed for carbapenemase-negative strains. We therefore report the development of a sensitive, specific and cost-effective MALDI-TOF MS-based assay for detection of CPE, which makes use of antibiotic disks readily available in most laboratories.

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Rapid detection of carbapenemase-producing Enterobacteriaceae using MALDI-TOF MS

Cited by 2 publications

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Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Meropenem Hydrolysis Assay with NH ₄ HCO ₃ , a Reliable Tool for Direct Detection of Carbapenemase Activity

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Meropenem Hydrolysis Assay with NH ₄ HCO ₃ , a Reliable Tool for Direct Detection of Carbapenemase Activity

Detection of carbapenemase-producing Enterobacterales by means of matrix-assisted laser desorption ionization time-of-flight mass spectrometry with ertapenem susceptibility-testing disks as source of carbapenem substrate

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Rapid detection of carbapenemase-producing Enterobacteriaceae using MALDI-TOF MS

Cited by 2 publications

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Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Meropenem Hydrolysis Assay with NH 4 HCO 3 , a Reliable Tool for Direct Detection of Carbapenemase Activity

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Meropenem Hydrolysis Assay with NH 4 HCO 3 , a Reliable Tool for Direct Detection of Carbapenemase Activity

Detection of carbapenemase-producing Enterobacterales by means of matrix-assisted laser desorption ionization time-of-flight mass spectrometry with ertapenem susceptibility-testing disks as source of carbapenem substrate

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Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Meropenem Hydrolysis Assay with NH ₄ HCO ₃ , a Reliable Tool for Direct Detection of Carbapenemase Activity

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Meropenem Hydrolysis Assay with NH ₄ HCO ₃ , a Reliable Tool for Direct Detection of Carbapenemase Activity