All MALDI-TOF mass spectra acquired for this study can be accessed via the Open Science Foundation (https://osf.io/ksz7r/ ). The bacterial isolates were cultured from Microbank™ freezing beads (Pro-lab Diagnostics, Toronto, Canada) onto 5% Sheep Blood agar plates (bioMérieux, Marcy-l’Étoile, France) and subcultured before MALDI-TOF mass spectra acquisition. Strains were incubated under aerobic conditions at 37°C except for strains of the species Bacterioides fragilis, Actinomyces israelii, and Winkia neuii, which were incubated under anaerobic conditions using a whitley A95 anaerobic workstation (Don Whitley Scientific Limited, Bingley, United Kingdom). Strains of the species Streptococcus pneumoniae, Bordetella pertussis, and Bordetella parapertussis were incubated under 5% enriched CO2 conditions. All mass spectra were acquired on reusable steel target plates [MBT Biotarget 96 (Bruker Daltonics, Bremen, Germany) and steel target plates (Industrietechnik mab AG, Basel, Switzerland)].
Mbt biotarget 96
Manufactured by Bruker
Sourced in Germany
The MBT Biotarget 96 is a laboratory equipment designed for the rapid identification of microorganisms. It utilizes matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry technology to analyze protein profiles of microbial samples. The core function of the MBT Biotarget 96 is to enable efficient and reliable microbial identification in clinical, research, and industrial settings.
Automatically generated - may contain errors
Lab products found in correlation
Formic acid, by Thermo Fisher Scientific (6 mentions)
Microflex smart instrument, by Bruker (3 mentions)
2 5 dihydroxybenzoic acid matrix, by Merck Group (2 mentions)
Flexanalysis software v 3, by Bruker (2 mentions)
Acetonitrile, by Carl Roth (2 mentions)
Sheep blood agar plates, by bioMérieux (1 mentions)
Whitley a95 anaerobic workstation, by Don Whitley Scientific (1 mentions)
Msp 96 polished steel target, by Bruker (1 mentions)
Filter paper, by GE Healthcare (1 mentions)
5 protocols using mbt biotarget 96
1
MALDI-TOF Mass Spectrometry Protocol
2
Rapid Lipid A Extraction from E. coli
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Lipid A from E. coli was extracted using the MBT Lipid Xtract™ Kit, following the manufacturer’s instructions. Briefly, bacteria were grown overnight in the Mueller–Hinton agar, the equivalent of a 1-μL inoculation loop placed into a 1.5-mL low-binding microtube, and mixed in 50 μL of MBT Lipid Xtract Hydrolysis buffer. A total of 44 μL of the cell suspension was discarded, and the remaining 6 μL was submitted with the lid of the tube closed to a heating process at 90 °C for 10 min. The tubes were left for 2 min with the lid open to completely evaporate the buffer. The dried pellets were washed with 50 μL of MBT Lipid Xtract washing buffer for a few seconds without dissolving the pellet. The total volume of the washing buffer was discarded by pipetting. Finally, 5 μL of the matrix was pipetted up and down for 15–20 s to resuspend the dried pellet, and 2 μL of the suspension was spotted onto either an MSP 96 polished steel target (Bruker Daltonics, Part-No. 8280800) or an MBT Biotarget 96 (Bruker Daltonics, Part-No. 1840375).
3
Rapid Antimicrobial Susceptibility Testing
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DOT-MGA was performed as previously described (Idelevich et al., 2018a (link)). Briefly, microdroplets containing 3 μl of antibiotic solution and 3 μl of bacterial suspension (final inoculum approximately 5 × 105 CFU/ml) were pipetted onto an MBT Biotarget 96 (Bruker Daltonik, Bremen, Germany). Sterility and growth controls were spotted on a second target. Targets were incubated for 3 or 4 h at 35 ± 1°C using a plastic transport box (Bruker Daltonik) serving as a humidity chamber in order to prevent the microdroplets from evaporating. After incubation, the remaining liquid was removed from the spots using filter paper, (size 37 × 100 mm, GE Healthcare GmbH, Freiburg im Breisgau, Germany). After overlaying the spots with 1 μl of α-cyano-4-hydroxycinnamic acid matrix including internal standard (MBT MASTeR prototype kit, Bruker Daltonik), MALDI-TOF MS spectra were acquired on a microflex smart instrument (Bruker Daltonik). The method was performed in triplicate.
4
MALDI-TOF MS Analysis of Bacterial Proteins
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The bacterial suspensions were set to 0.5 McFarland standard turbidity in deionized water, pelleted (13,800 × g for 5 min at room temperature [rt]), and resuspended in 300 μL of deionized water. Then, 900 μL of 99.8% (vol/vol) ethanol (Carl Roth, Karlsruhe, Germany) was added. Following two centrifugation steps (9,600 × g for 2 min at rt) and air drying (4 to 6 min), the pellets were resuspended in 20 μL of 70% (vol/vol) formic acid (Thermo Fisher Scientific, Waltham, MA, USA). The suspensions were mixed with 20 μL of acetonitrile (Carl Roth, Karlsruhe, Germany) and centrifuged (9,600 × g for 2 min at rt). Next, 1 μL of supernatants was spotted on a MALDI target plate (MBT Biotarget 96, Bruker Daltonik, Bremen, Germany) and left to dry at rt, and 1 μL of 130 μM 2,5-dihydroxybenzoic acid matrix (Sigma-Aldrich, St. Louis, MO, USA) was added onto each protein-containing spot. The extracted membrane proteins were analyzed by MALDI-TOF MS using a Microflex smart instrument (mass range of 2 to 40 kDa, laser intensity of 70%, number and frequency of shots of 200; Bruker Daltonik, Bremen, Germany). The analysis of spectra was performed using flexAnalysis software v.3.3 (Bruker Daltonik, Bremen, Germany).
5
MALDI-TOF MS Analysis of Bacterial Proteins
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The bacterial suspensions were set to 0.5 McFarland standard turbidity in deionized water, pelleted (13,800 × g for 5 min at room temperature [rt]), and resuspended in 300 μL of deionized water. Then, 900 μL of 99.8% (vol/vol) ethanol (Carl Roth, Karlsruhe, Germany) was added. Following two centrifugation steps (9,600 × g for 2 min at rt) and air drying (4 to 6 min), the pellets were resuspended in 20 μL of 70% (vol/vol) formic acid (Thermo Fisher Scientific, Waltham, MA, USA). The suspensions were mixed with 20 μL of acetonitrile (Carl Roth, Karlsruhe, Germany) and centrifuged (9,600 × g for 2 min at rt). Next, 1 μL of supernatants was spotted on a MALDI target plate (MBT Biotarget 96, Bruker Daltonik, Bremen, Germany) and left to dry at rt, and 1 μL of 130 μM 2,5-dihydroxybenzoic acid matrix (Sigma-Aldrich, St. Louis, MO, USA) was added onto each protein-containing spot. The extracted membrane proteins were analyzed by MALDI-TOF MS using a Microflex smart instrument (mass range of 2 to 40 kDa, laser intensity of 70%, number and frequency of shots of 200; Bruker Daltonik, Bremen, Germany). The analysis of spectra was performed using flexAnalysis software v.3.3 (Bruker Daltonik, Bremen, Germany).
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