Jong-Min Park (Presenter)
Yonsei University
Bio: Jong-Min Park is a Ph.D candidate student in the Department of Materials Science and Engineering at Yonsei University. He received his BS degree in Metallurgical Engineering in 2013 from Yonsei University. His research interests include clinical diagnosis for small biomolecule using MALDI-TOF mass spectroscopy and bacterial susceptibility test.
Authorship: Jong-Min Park (1), Dongeun Yong (2), Jae-Chul Pyun(1)
(1) Department of Material Science and Engineering, Collage of Engineering, Yonsei University (2) Department of Laboratory Medicine and Research Institute of Bacterial Resistance, College of Medicine, Yonsei University
| Short Abstract Carbapenem is the strongest β-lactam antibiotics and acts as inhibitors of the enzymes that catalyze formation of peptidoglycan in the cell wall of bacteria. Recently, the emergence of carbapenem-resistant bacteria seriously threatens this class of lifesaving drugs. Therefore, rapid detection of carbapenemase-producing enterobacteriaceae (CPE) is very important to prevent spread of these strains. Carbapenemase is an important enzyme that are produced by (CPE) and catalyze the hydrolysis of carbapenem. Typically, MALDI-TOF MS is not appropriate for small molecule analysis because organic matrices make a lot of noise at low m/z range. Parylene-matrix chip was developed for reduce matrix noise, and used to quantitate carbapenem successfully. Finally, MALDI-TOF MS based carbapenem susceptibility test was carried out with different 60 isolates using Parylene-matrix chip. |
Long Abstract
1. Introduction
o Carbapenem is a β-lactam antibiotics and acts as inhibitors of the enzymes that catalyze formation of peptidoglycan in the cell wall of bacteria. Of the many different β-lactam antibiotics, carbapenems possess the broadest spectrum of activity and greatest potency against bacteria. Therefore, carbapenems are used as antibiotics of last resort when patients are suspected of infected antibiotics resistant bacteria. However, the emergence of multidrug-resistant or carbapenemase-producing enterobacteriaceae (CPE) seriously threatens this class of lifesaving drugs recently[1].
o Rapid detection of CPE is very important to prevent spread of these strains. Carbapenemase has been detected using a number of techniques, including modified Hodge test (MHT), 3-dimensional extract (3-D) bioassay, and DNA-based test. But these detection methods require overnight incubation and can be difficult to interpret.
o Carbapenemase is an important enzyme that are produced by CPE and catalyze the hydrolysis of carbapenem. Carbapenems are hydrolyzed by carbapenemase and molecular weight is increased as 18 Da. Therefore, hydrolysis of carbapenem can be detected using mass spectrometry.
o Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has been widely applied for the analysis of biomolecules with high molecular weight, because of its advantages on easy sample preparation and short analysis time. Therefore, these advantages can be applied to solve the problems of conventional assays. However, when organic matrices are evaporated and ionized during the MALDI process, mass peaks of the resulting fragmented matrix molecules are observed at low mass ranges. These disordered noise peaks of MALDI made identification and quantitation difficult.
o To improve the analysis of small molecule with MALDI-TOF, the Parylene-matrix chip (P-M chip) was developed. This chip consists of a thin Parylene (poly-p-xylylene) film coated onto a dry matrix. During the MALDI process, evaporated organic matrix cannot pass through Parylene film. Therefore, small molecule can be detected quantitatively without matrix noise peaks using the P-M chip.
o Recently, the P-M chip has been used for analysis of penicillin-resistant bacteria[2]. This assay measured the hydrolysis of penicillin into penicilloic acid. β-lactamase-based E.coli antibiotic susceptibility test was carried using a conventional colorimetric assay and MALDI-TOF assay. The penicillin-susceptible and penicillin-resistant E.coli strains showed different mass signal ratios for MALDI-TOF assay. Only 1000 cells are the minimum number required for the MALDI-TOF based penicillin susceptibility test while conventional assay required at least 2 x 10^6 cells.
o In this study, the P-M chip was used in the carbapenemase assay. The assay measured the hydrolysis of 4 carbapenems such as doripenem, ertapenem, imipenem, and meropenem into their hydrolyzed form. Finally, 60 kinds of isolates include carbapenem-producing isolates (R+), non-carbapenem-producing isolates resistant the carbapenems (R-), and non-carbapenem-producing isolates susceptible the carbapenems (S-) were used to determine the feasibility of the MALDI-TOF MS/P-M chip-based carbapenem susceptibility test.
2. Methods
2.1. Materials
Bacterial samples include R+, R-, and S- isolates were collected by Medicine and research institute of bacterial resistance in Yonsei university college of medicine, Seoul, Korea. All of the isolates have been characterized using MHT, agar microdilution method for determination of minimal inhibitory concentration (MIC), and sequencing of the carbapenemase genes after PCR.
2.2. Preparation of Parylene-matrix chips
To prepare the P-M chips, a matrix solution containing 10 mg/ml 2,5-dihydroxybenzoic acid (DHB) in acetonitrile/water (1:1, v:v) with 0.05% trifluoroacetic acid (TFA) was dropped on each sample spot of stainless steel target plate. After drying the spotted matrix, a Parylene-N film was coated on the target plate using a Parylene coater. The thickness of the Parylene-N film was maintained by adjusting the initial amount of the Parylene-N dimer.
2.3. Measurement of carbapenem using the Parylene-matrix chip
Quantitative analysis of 4 carbapenems was carried with DHB matrix and P-M chip. Each carbapenem solution was dropped on Bruker 96-well target plate and P-M chip and allowed dry. After drying, DHB solution was dropped on 96-well target plate only and allowed dry. MALDI-TOF MS spectra were measured after drying between m/z 200 and 700, using a Microflex LRF mass spectrometer equipped with a nitrogen laser (337 nm). Signal-to-noise (S/N) ratio and limit of detection (LOD) were compared between DHB plate and P-M chip. Then, quantitative analysis of 4 carbapenem mixture was carried with P-M chip.
2.4. Carbapenem hydrolysis assay with different bacterial isolates
The carbapenem hydrolysis assay was performed using P-M chip. Bacterial isolate was grown overnight on Mueller-Hinton agar. Bacterial colony was transferred by 1 µL sized loop into reaction buffer (doripenem, ertapenem, imipenem, and meropenem in Tris-HCl buffer) and well suspended. Bacterial suspension was incubated in 37¡ÆC for 2, 4 hours. After incubation, the reaction mixture was centrifuged, and supernatant was dropped on Bruker 96-well target plate and allowed to dry. Then DHB solution was dropped on dried sample and allowed to dry. For P-M chip, the supernatant was dropped on P-M chip and allowed to dry. MALDI-TOF MS spectra were measured after drying between m/z 200 and 700. The mass signals of carbapenem and hydrolyzed carbapenem were estimated by summing the peak intensities of the appropriate ions.
3. Results
3.1. Measurement of carbapenem with the Parylene-matrix chip
In the CPE susceptibility assay, 4 carbapenems were used as the substrate. Carbapenem analysis with a DHB matrix yielded peaks in the low m/z range that were difficult to discriminate from noise. Four carbapenems were detected well using P-M chip, much less noise was observed and S/N ratios were greatly improved. Because mass peaks of carbapenem were observed as too many adduct ions, 2~3 signature peaks that mainly changed while carbapenemase reaction occurred were selected. The mass signal of carbapenem was then calculated as the sum of the intensities of signature carbapenem ions in each mass spectrum. LOD of carbapenem was estimated to be 10 µM. Moreover, the mass signal of carbapenem increased quantitatively in the range of 10 µM to 1 mM. This results show that carbapenem susceptibility test can be effectively and quantitatively assayed using a MALDI-TOF MS approach based on P-M chip. Furthermore, carbapenem mixture solution was analyzed using MALDI-TOF MS. The mass peaks of each carbapenem in the mixture were obtained using P-M chip with less noise than conventional DHB matrix. Concentration of each carbapenem in the mixture was adjusted quantitatively, the mass peaks of adjusted carbapenem was changed quantitatively. This results show that the carbapenem susceptibility test can be assayed against 4 carbapenems simultaneously for each isolates.
3.2. Carbapenemase-based bacterial antibiotic susceptibility test
Bacterial samples include R+, R-, and S- isolates were used to validate the MALDI-TOF MS-based antibiotic susceptibility test. R+ isolates were identified by PCR, and several carbapenemases were detected such as IMP-1, VIM-2, OXA-23, ISAba1-OXA-51, NDM-1, KPC-2, and SIM-1[3]. For the carbapenemase assay, equal number of isolate cells were treated with 0.5 g/L of each carbapenem for 2, 4 hours in 37¡ÆC incubator. Reaction solutions were then centrifuged, and then supernatants were subjected to MALDI-TOF MS with the P-M chip. The signature peaks of hydrolyzed carbapenem were selected as described previously. The mass peaks of carbapenem and hydrolyzed carbapenem were observed simultaneously. The mass signals of carbapenem and hydrolyzed carbapenem were estimated by summing the signature peak intensities of the appropriate ions. The mass signal ratios of hydrolyzed carbapenem to carbapenem were calculated and used to compare the carbapenemase existence of R+ isolates and R-, and S- isolates. For R+ isolates, the mass signals of carbapenem were decreased while the mass signals of hydrolyzed carbapenem were increased according to time (0, 2, 4 hours). For R- and S- isolates, the mass signals of hydrolyzed carbapenem were not increased according to time. R+ and R-, S- isolates exhibited different mass signal ratios at a same assay conditions. Conventional MHT and 3-D bioassay had a lower sensitivity and specificity than the MALDI-TOF MS assay. Moreover, these conventional assays required additional overnight culture process. This results show that the P-M chip enables rapid and sensitive detecting of CPE. Furthermore, the criteria for MALDI-TOF MS based carbapenem susceptibility test are needed because analysis methods include signature peak, mass spectrometry setting, and standard strain are different in each hospital.
4. Conclusions
In this work, the P-M chip was applied to quantitatively analyze the carbapenem susceptibility test. The LOD of carbapenems was found to be around 10 µM and the dynamic detection range was estimated to be 10 µM to 1 mM. Compared with the conventional organic matrix, the P-M chip exhibits improved S/N ratio and LOD. To validate the MALDI-TOF MS-based carbapenem susceptibility test, R+, R-, S- isolates were used. Carbapenem susceptibility was measured using MALDI-TOF more sensitive and selective than conventional assay. And additional overnight culture is unnecessary for MALDI-TOF assay. Therefore, the MALDI-TOF MS-based carbapenem susceptibility test using P-M chip is rapid and has greater sensitivity than other conventional carbapenemase detection methods, such as the MHT, and 3-D bioassay.
References & Acknowledgements:
[1] Papp-Wallace, K.M., et al., Carbapenems: Past, Present, and Future. Antimicrobial Agents and Chemotherapy, 2011. 55(11): p. 4943-4960.
[2] Park, J.-M., et al., Highly sensitive bacterial susceptibility test against penicillin using parylene-matrix chip. Biosensors and Bioelectronics, 2015. 71: p. 306-312.
[3] Lee, W., et al., Comparison of matrix-assisted laser desorption ionization–time-of-flight mass spectrometry assay with conventional methods for detection of IMP-6, VIM-2, NDM-1, SIM-1, KPC-1, OXA-23, and OXA-51 carbapenemase-producing Acinetobacter spp., Pseudomonas aeruginosa, and Klebsiella pneumoniae. Diagnostic Microbiology and Infectious Disease, 2013. 77(3): p. 227-230.
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