Evaluation of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Rapid Detection of β-Lactam Resistance in Enterobacteriaceae Derived from Blood Cultures

Jung, Jette; Popp, Christina; Sparbier, Katrin; Lange, Christoph; Kostrzewa, Markus; Schubert, S.

doi:10.1128/jcm.02691-13

Cited by 116 publications

(92 citation statements)

References 26 publications

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“…One approach to shorten the time to result for antibiotic susceptibility testing (AST) is the use of matrix-assisted laser desorption ionizationtime of flight mass spectrometry (MALDI-TOF MS) for identification and resistance testing. The most common approach using mass spectrometry is the detection of ␤-lactamases with a functional assay that measures their hydrolysis products (4)(5)(6)(7)(8)(9). This test provides results within 1 to 3 h, but, by its very nature, it is limited to the detection of ␤-lactamase-mediated resistance.…”

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Evaluation of a Semiquantitative Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Method for Rapid Antimicrobial Susceptibility Testing of Positive Blood Cultures

et al. 2016

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bWith the increasing prevalence of multidrug-resistant Gram-negative bacteria, rapid identification of the pathogen and its individual antibiotic resistance is crucial to ensure adequate antiinfective treatment at the earliest time point. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for the identification of bacteria directly from the blood culture bottle has been widely established; however, there is still an urgent need for new methods that permit rapid resistance testing. Recently, a semiquantitative MALDI-TOF mass spectrometry-based method for the prediction of antibiotic resistance was described. We evaluated this method for detecting nonsusceptibility against two ␤-lactam and two non-␤-lactam antibiotics. A collection of 30 spiked blood cultures was tested for nonsusceptibility against gentamicin and ciprofloxacin. Furthermore, 99 patient-derived blood cultures were tested for nonsusceptibility against cefotaxime, piperacillin-tazobactam, and ciprofloxacin in parallel with MALDI-TOF mass spectrometry identification from the blood culture fluid. The assay correctly classified all isolates tested for nonsusceptibility against gentamicin and cefotaxime. One misclassification for ciprofloxacin nonsusceptibility and five misclassifications for piperacillin-tazobactam nonsusceptibility occurred. Identification of the bacterium and prediction of nonsusceptibility was possible within approximately 4 h. The early initiation of adequate antibiotic therapy is known to be an important parameter for the outcome of septic patients (1-3). Therefore, empirical treatment must be administered as soon as sepsis is diagnosed; however, with the growing burden of antimicrobial resistance, affecting the sepsis-causing pathogen with an empirical therapy is becoming increasingly difficult. Prediction of antibiotic resistance at the earliest time point is necessary to ensure the best available treatment for the patient and to avoid the extensive use of broad-spectrum therapy. One approach to shorten the time to result for antibiotic susceptibility testing (AST) is the use of matrix-assisted laser desorption ionizationtime of flight mass spectrometry (MALDI-TOF MS) for identification and resistance testing. The most common approach using mass spectrometry is the detection of ␤-lactamases with a functional assay that measures their hydrolysis products (4-9). This test provides results within 1 to 3 h, but, by its very nature, it is limited to the detection of ␤-lactamase-mediated resistance. Recently, Lange et al. (10) described a MALDI-TOF MS method that allows for resistance testing independent of the underlying mechanism. The MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA) relies on a semiquantitative measurement of bacterial proteins. Bacterial cells are incubated in culture medium with or without antibiotics. Subsequently, the cells are lysed and spiked with an internal standard. Mass spectra from the two setups are acquired and normalized, and their peak intensit...

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Evaluation of a Semiquantitative Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Method for Rapid Antimicrobial Susceptibility Testing of Positive Blood Cultures

et al. 2016

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“…Informatics tools for automatic spectrum interpretation, such as STAR-BL prototype software (Bruker Daltonics GmbH), might be helpful for nonexperienced users to facilitate interpretation of the spectrum. Furthermore, they allow the calculation of the hydrolysis ratio of the antibiotics which might a useful tool for future studies (22).…”

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Rapid Detection of OXA-48-Producing Enterobacteriaceae by Matrix-Assisted Laser Desorption Ionization−Time of Flight Mass Spectrometry

et al. 2016

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A rapid and sensitive (100%) matrix-assisted laser desorption ionization؊time of flight mass spectrometry (MALDI-TOF MS) assay was developed to detect OXA-48-type producers, using 161 previously characterized clinical isolates. Ertapenem was monitored to detect carbapenem resistance, and temocillin was included in the assay as a marker for OXA-48-producers. Structural analysis of temocillin is described. Data are obtained within 60 min. Carbapenemase-producing Enterobacteriaceae (CPE) are increasingly being isolated worldwide. As these bacteria are difficult to detect and control, their spread is considered a public health concern (1, 2). Dissemination of the group D carbapenemases, particularly, bla OXA-48 isolates, is of major concern (3-5). These isolates are characterized by low carbapenem MICs and often go undetected by most phenotypic methods.The development of mass spectrometry techniques, such as matrix-assisted laser desorption ionizationϪtime of flight mass spectrometry (MALDI-TOF MS), has yielded sensitivity with faster turnaround times (6-10). The aim of this study was to determine whether a MALDI-TOF MS ertapenem assay can be used to identify and classify CPE producers, with particular emphasis on OXA-48-type isolates. We propose the use of temocillin as a resistance marker for OXA-48-type enzymes (11), as isolates harboring these types of carbapenemases show very high levels of resistance to temocillin (12, 13), and there is no specific inhibitor for group D carbapenemases. Phenylboronic acid (PBA) and dipicolinic acid (DPA) are used as inhibitors of, respectively, group A and B carbapenemases.A total of 161 characterized nonrepeat Enterobacteriaceae isolates, 117 of which carried a carbapenemase enzyme, were tested (Table 1). Seventy-two of the 161 isolates were genotypically characterized by the Antibiotic Resistance Surveillance Program of the Spanish National Center of Microbiology (14) All isolates were analyzed by the disk diffusion method (KPC/ MBL and OXA-48 confirm kit; Rosco Diagnostica A/S, Taastrup, Denmark) with disks containing meropenem (10 g), meropenem plus PBA, meropenem plus DPA, meropenem plus cloxacillin (CL), and temocillin (30 g). A 0.5-McFarland suspension of the isolates on Mueller-Hinton II agar plates (Becton Dickinson, USA) was used, followed by an overnight incubation of the plates at 37°C. The susceptibility cutoff used for temocillin was Ͻ12 mm (12).For the MALDI-TOF MS assay, isolates were previously grown overnight at 37°C in Trypticase soy agar with 5% sheep blood (TSA II) (Becton Dickinson). The hydrolysis of ertapenem (0.5 mg/ml Invaz [Merck & Co., Inc.], 0.001% SDS, 50 mM NH 4 HCO 3 [pH 7.0]) (6, 9) and temocillin (1.5 mg/ml Negaban [Eumedica], 0,001% SDS, 50 mM NH 4 HCO 3 [pH 7.0]) (9) was evaluated after resuspension of the bacteria that filled a 1-l inoculation loop in 10 l of the antibiotic solution in an Eppendorf tube and incubation of the suspension at 37°C under agitation. When inhibitors were used, bacteria were resuspended in 5 l of ertapenem (1 mg/ml ...

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“…Overnight growth of colonies would still be required to identify organisms not identifiable by this method (e.g., polymicrobial specimens, Gram-positive rods, anaerobes, and yeast) and to perform susceptibility testing. That being said, methods have been developed using pelleted bacteria from positive blood cultures to assess antimicrobial resistance by MALDI-TOF MS (22,23). A valuable future study would be to employ our pellet method in parallel with a method for the rapid detection of antimicrobial resistance.…”

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Rapid Identification of Positive Blood Cultures by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Using Prewarmed Agar Plates

et al. 2014

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bThis study describes an inexpensive and straightforward method for identifying bacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) directly from positive blood cultures using prewarmed agar plates. Different inoculation methods and incubation times were evaluated to determine the optimal conditions. The two methods using pelleted material from positive culture bottles performed best. In particular, the pellet streak method correctly identified 94% of the Gram negatives following 4 h of incubation and 98% of the Gram positives following 6 h of incubation.

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Evaluation of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Rapid Detection of β-Lactam Resistance in Enterobacteriaceae Derived from Blood Cultures

Cited by 116 publications

References 26 publications

Evaluation of a Semiquantitative Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Method for Rapid Antimicrobial Susceptibility Testing of Positive Blood Cultures

Evaluation of a Semiquantitative Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Method for Rapid Antimicrobial Susceptibility Testing of Positive Blood Cultures

Rapid Detection of OXA-48-Producing Enterobacteriaceae by Matrix-Assisted Laser Desorption Ionization−Time of Flight Mass Spectrometry

Rapid Identification of Positive Blood Cultures by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Using Prewarmed Agar Plates

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