Maldi biotyper microflex lt

Morphology of colonies was visually analyzed, and all morphologically different colonies were identified by MALDI-TOF MS in a MALDI Biotyper Microflex LT mass spectrometer (Bruker Daltonik, Germany); see Figure 1B.
A colony was directly spotted on the MALDI plate, overlaid with 1 μL of saturated α-cyano-4-hydroxycinnamic acid and air-dried. The loaded plate was then placed in the instrument according to the manufacturer’s instructions. The mass spectra were acquired within 10 min. The spectra were imported into the integrated MALDI Biotyper software (version 3.0) and analyzed by standard pattern matching with a default setting. A score ≥ 2.00 indicated identification at the species level and a score from 1.70 to 1.99 indicated identification at the genus level, whereas any score under 1.70 meant no significant similarity of the obtained spectrum with any database entry.

For each product, the colonies grown on solid media were carefully examined for morphology and all phenotypically different colonies were subjected to identification by MALDI-TOF MS in a MALDI Biotyper Microflex LT mass spectrometer (Bruker Daltonik, Bremen, Germany). When a colony was directly spotted on the MALDI plate, it was overlaid with 1 μL of saturated formic acid, and air-dried. Subsequently, 1 μL of acetonitrile-matrix solution was added to each spot, and air-dried. The loaded plate was placed in the instrument according to the manufacturer’s instructions. The mass spectra were acquired, imported into the integrated MALDI Biotyper software (version 3.0), and analyzed using standard pattern matching with a default setting. A score of ≥2.00 indicated identification at the species level, while a score from 1.70 to 1.99 indicated identification at the genus level. Any score under 1.70 meant no significant similarity of the obtained spectrum with any database entry.

One microliter of each extract was spotted on a MALDI-Biotyper steel target plate, allowed to dry and, subsequently, overlaid with 1 µL of alpha-cyano-4-hydroxycinnamic acid (HCCA) matrix (5 mg/mL HCCA in 50% acetonitrile, 2.5% TFA, 47.5% water) and left to dry completely again. Mass spectra of each spot were obtained using the MALDI- Biotyper Microflex LT instrument (MALDI Biotyper, Bruker Daltonics Inc., Billerica, MA, USA) and the software FlexControl version 3.3 by scanning from 720 to 5000 m/z in MS-positive ion linear mode. The laser frequency was set to 30 Hz and the final spectrum was an average of 1000 laser shots (10 groups of 100 shots collected in a spiral pattern).

Without a detailed extraction step, bacteria from single colonies were used for MALDI-TOF analysis in a MALDI Biotyper Microflex LT (Bruker Daltonik, Bremen, Germany). For the acquisition of the mass spectra the authors followed the manufacturer’s recommendations. The mass spectra were acquired within less than 5 minutes including the sample preparation. The BioTyper 3.0 software compared the obtained spectra with a reference database containing 3740 reference spectra (representing 319 genera and 1,946 species) and expressed the resulting similarity value as a log score. A score of ≥ 2.000 indicated identification on the species level, a score of ≥ 1.700 indicated identification on the genus level whereas any score under 1.700 meant no significant similarity of the obtained spectrum with any database entry. If the results were questionable, the procedure was repeated.

From each agar plate showing microbial growth, a representative of each morphotype was subcultured and controlled for purity. A colony of each pure culture was suspended in 300 µL ultrapure water and stored at −80 °C until further processing. Samples were extracted and identified using a MALDI Biotyper system (MALDI Biotyper Microflex LT, Bruker Daltonics, Bremen, Germany) following the protocol for ethanol-formic acid extraction [14 ]. The volumes of formic acid and acetonitrile (both Carl Roth) were adapted as specified in the protocol for single, small colonies. The obtained protein spectral profiles were matched against the MALDI Biotyper reference database (software version 4.1.90, 8936 entries) and expressed as score values ranging from 0 to 3.0. According to the manufacturer, scores >1.7 indicate a reliable genus identification, scores >2.0 a reliable genus and probable species identification, and scores >2.3 a highly probable species identification. Detailed germ numbers and MALDI Biotyping results are provided in the Supplementary Table S1.

The fungal strains were cultured on Sabouraud dextrose agar with chloramphenicol and gentamicin (Becton Dickinson GmbH, Heidelberg, Germany) and incubated at 30 °C for up to one week, until sufficient growth was observed. The isolates were subjected to MALDI-TOF MS analysis using a Bruker MALDI Biotyper microflex LT (Bruker Daltonics, Bremen, Germany).

The clinical isolates tested in this study were collected in routine clinical workflow from specimens submitted to the Pisa University Hospital, Italy, over a two-year period (S1 Table). Multiple isolates from the same patient and body site were excluded. Cultures were processed per standard laboratory practices and, once pure culture was obtained on blood agar plates, strains were identified according to the operating procedures of our laboratory. This included microscopy of Gram-stained preparations and biochemical analysis using the API 50 CHB test kit according to the manufacturer’s instructions and the ATBPlus software (bioMérieux, Marcy l'Etoile, France). In parallel, bacteria from single colonies were used for MALDI-TOF MS analysis in a MALDI Biotyper Microflex LT mass spectrometer (Bruker Daltonik, Bremen, Germany). Failure to identify the organism, or any discrepancies between MALDI-TOF MS and biochemical identification, prompted 16S rRNA gene sequencing of the isolate.
The study was approved by the Ethical Committee Area Vasta Nord-Ovest, University of Pisa, and conducted in full accordance with the principles of the Declaration of Helsinki. Samples were taken as part of the standard patient care and used anonymously. For this type of study, no written informed consent was necessary.