Tissue samples were homogenized and seeded on solid and liquid culture media. All samples were incubated for 14 days. Bacteria were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF MS) using a Microflex LT mass spectrometer and BioTyper software (Bruker Daltonik, Bremen, Germany). Antibiotic susceptibility testing followed guidelines from the European Committee on Antimicrobial Susceptibility Testing (EUCAST).
Biotyper software
Manufactured by Bruker
Sourced in Germany
The Biotyper software is a microbial identification system used for the rapid and reliable identification of microorganisms. It utilizes matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology to generate specific protein profiles, which are then compared to a comprehensive reference database for identification.
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Lab products found in correlation
Microflex lt mass spectrometer, by Bruker (4 mentions)
Hcca matrix solution, by Bruker (2 mentions)
Bacterial test standard, by Bruker (2 mentions)
Ultraflextreme instrument, by Bruker (1 mentions)
Maldi tof ms, by Bruker (1 mentions)
Ppp master mix, by Top-Bio (1 mentions)
Microflex lt maldi tof ms system, by Bruker (1 mentions)
Microflex lt maldi tof instrument, by Bruker (1 mentions)
Microflex platform, by Bruker (1 mentions)
Flexcontrol software, by Bruker (1 mentions)
α cyano 4 hydroxycinnamic acid matrix solution, by Bruker (1 mentions)
Maldi biotyper, by Bruker (1 mentions)
Prepman ultra, by Thermo Fisher Scientific (1 mentions)
Chromas lite 2, by Technelysium (1 mentions)
E z n a cycle pure kit, by Omega Bio-Tek (1 mentions)
Brain heart infusion broth, by Thermo Fisher Scientific (1 mentions)
Microflex lt sh instrument, by Bruker (1 mentions)
Microflex lt, by Bruker (1 mentions)
Microflex lt maldi tof ms, by Bruker (1 mentions)
25 protocols using biotyper software
1
Bacterial Identification and Antibiotic Susceptibility
2
MALDI-TOF MS for Bacterial Identification
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Isolates with negative oxidase and catalase tests were further identified using matrix-assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF MS) following the procedure described by Dušková et al. [12 (link)]. The samples for MALDI-TOF MS analysis were prepared by protein extraction (ethanol/formic acid) according to a standard protocol [13 (link)]. Mass spectrometry measurements were performed using an Ultraflextreme instrument (Bruker Daltonik, Bremen, Germany) operated in the linear positive ion mode using FlexControl 3.4 software. Mass spectra were processed using BioTyper software (version 3.0; Bruker Daltonik). The identification results were expressed by BioTyper log(scores) indicating the similarity of the unknown MALDI-TOF MS profile to Biotyper database entries (version 10.0; 9607 entries). A BioTyper log(score) exceeding 2.0 indicates a highly confident identification at the species level. A BioTyper log(score) between 1.7 and 2.0 means identification at the species level with lower confidence. Only isolates with a log(score) over 1.7 were taken into account.
3
Identification of MCR-positive UTI Isolates
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The mcr-positive isolates obtained from the UTI Clarity agar supplemented with CT were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) with the use of Biotyper software (version 3.1, Bruker Daltonics GmbH, Germany).
4
MALDI-TOF MS Microbiological Analysis
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Microbiological analysis of samples was carried out by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics, Bremen, Germany) based on protein fingerprints. Single colonies of fresh overnight cultures were used for the ethanol-formic acid extraction. Each sample spot was covered with 2 µL of matrix solution (saturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile with 2.5% trifluoroacetic acid; Bruker Daltonics) and air-dried for 15 min. Raw data of protein spectrum of each isolate was imported into the Biotyper software, version 2.0 (Bruker Daltonics) and analyzed [97 (link)].
5
Multiplex PCR for Campylobacter Identification
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Suspected C. jejuni and C. coli colonies cultivated on mCCDA were plated on blood agar at 42 °C under a microaerobic atmosphere for 44 ± 4 h, and bacterial DNA was extracted by boiling. Multiplex PCR using PPP master mix (Top-Bio, Vestec, Czech Republic) was used to identify the genus Campylobacter and 2 species, C. jejuni and C. coli. The primers used (Generi-Biotech, Hradec Kralove, Czech Republic) [24 (link),25 (link),28 (link)] are shown in Table S1 . Amplification was carried out with the following PCR conditions: 95 °C for 5 min, 35 cycles at 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min, and 72 °C for 7 min.
Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) was used for confirmation of Campylobacter species. In detail, when pure bacterial cultures were obtained, MALDI-TOF/MS with ethanol and formic acid extraction was used, and isolates were identified after comparing the bacterial spectrum in a Bruker database MBT 8468 by Biotyper software (version 3.1, Bruker Daltonics GmbH, Bremen, Germany).
Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) was used for confirmation of Campylobacter species. In detail, when pure bacterial cultures were obtained, MALDI-TOF/MS with ethanol and formic acid extraction was used, and isolates were identified after comparing the bacterial spectrum in a Bruker database MBT 8468 by Biotyper software (version 3.1, Bruker Daltonics GmbH, Bremen, Germany).
6
MALDI-TOF Mass Spectrometry for Brucella melitensis Identification
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At both institutes, samples were independently prepared for mass spectrometry by ethanol-formic acid extraction according to manufacturer’s instructions before being spotted on a 96-spot steel plate target and covered with alpha-cyano-4-hydroxy-cinnamic acid (HCCA) matrix solution (Bruker Daltonik GmbH, Bremen, Germany). Mass spectra were measured using a microflex LT MALDI-TOF MS system (Bruker Daltonik) operated by the Biotyper automation software flexControl (v3.4.135.0, Bruker Daltonik). To increase data robustness, twelve technical replicate spectra were acquired from four different target spots using the recommended instrument settings for bacterial identification (linear positive ion detection mode, 60 Hz laser frequency, 20 kV acceleration voltage, 18.1–18.2 kV IS2 voltage). Spectra were initially analyzed at BfR using the Bruker Biotyper software (v3.1) with MSP library version MBT_7311 (7311 entries), the Security-Relevant (SR) Database (104 entries) and a customized in-house database to confirm identification as B. melitensis.
7
Microbial Species Identification Using MALDI-ToF
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The identity of all microbial species was verified using the Microflex LT MALDI-ToF instrument (Bruker Daltonics, Coventry, UK) as previously described23 (link). In brief, single colonies were placed onto a 24-spot steel plate and embedded in an acidic solution of α-cyano-4-hydroxycinnamic acid as a UV-adsorbent matrix. Samples were analysed after drying following the manufacturer’s instructions. Data were processed using the Bruker Biotyper software (version 3.0) and library (version 5.0) for taxonomic classification of the isolates. The identity of the 135 P. aeruginosa isolates were confirmed.
8
Rapid Microbial Identification using MALDI-ToF
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A rapid identification of the microorganisms based upon protein profiles was performed on the MALDI Biotyper system with MALDI-ToF Mass Spectrometry (Infectio MALDI, microflexLT-MS, Bruker, Billerica, MA, USA). Selected bacterial colonies were directly placed on the MALDI-ToF target (MSP 96 target polish steel BC, microscout Target), followed by addition of CHCA matrix solution (1 µL α-cyano-4-hydroxycinnaminic acid 50% acetonitrile–2.5% trifluoro acetic acid). It was then dried at room temperature to allow co-crystallisation. The plates were then subjected to MALDI-ToF MS measurement. The mass spectra obtained from each sample were imported into the Bio-Typer software (Bruker daltonics) and analyzed by standard pattern matching. Results of the pattern-matching were expressed with scores ranging from 0 to 3. Scores below 1.7 were regarded as unreliable identification; a score of ≥1.7 was regarded as identification of genus, and a score of ≥2 indicated species identification as recommended by the manufacturer.
9
Identification of Helicobacter spp. Using MALDI-TOF MS
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Colonies were subjected to a Bruker Daltonics biotyper device using the Microflex platform (Bruker Daltonics, Bremen, Germany) as previously described (van Veen et al., 2010 (link)). Single colonies were transferred onto a target plate and overlaid with matrix solution. FlexControl software (version 3.4) was used for measurements; this version includes eight Helicobacter species (Helicobacter canadensis, Helicobacter canis, Helicobacter cholecystus, H. cinaedi, Helicobacter fenneliae, Helicobacter mustelae, Helicobacter pullorum, and H. pylori with 1–9 spectra for each species). The spectra were analyzed using BioTyper software (version 3.1.66; Bruker) and MBT-Compass and MBT-RUO databases, and extracted profiles were compared to that of H. pylori.
10
Bacterial Identification by Biotyper
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The identification was carried out by the Bruker Biotyper software by comparison of spectra within database.22 (link) The bacterial profile matching is expressed using log scale from 0.00–3.00 for interpretation of results.
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