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Supernatant was centrifuged at 15?500?g for 5?min to collect bacteria. The pellet was washed once with deionized water. Then, an ethanol/formic acid extraction procedure was applied: a small amount of bacteria was resuspended in 300?��L of water. Later, 900?��L of absolute ethanol was added and the mixture was centrifuged at 15?500?g for 2?min, and the supernatant was discarded. Fifty microlitres Selleckchem JQ1 of formic acid (70% v/v) was added to the pellet and mixed thoroughly before the addition of 50?��L of acetonitrile. The mixture was centrifuged again at 15?500?g for 2?min. One microlitre of the supernatant was placed onto a spot of the steel card and air-dried at room temperature. The supernatant was overlaid with 1?��L of matrix solution [saturated solution of HCCA (��-cyano-4-hydroxy cinnamic acid) in organic solvent (50% acetonitrile and 2.5% trifluoroacetic acid)] and air-dried. MALDI-TOF MS.? Measurements were performed in an Autoflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Leipzig, Germany) equipped with a 200-Hz smartbeam laser. Spectra were recorded in the linear, positive mode at a laser frequency of 200?Hz within a mass range from 2000�C20?000?Da. The IS1 voltage was 20?kV, the IS2 voltage was maintained at 18.6?kV, lens voltage was 6?kV and the extraction delay time was 40?ns. For each spectrum, 500 laser shots were collected and analyzed (10?��?50 laser shots from different positions of the target spot). The spectra were externally calibrated using the R428 standard calibrant mixture (Escherichia coli extracts including the additional proteins RNase A and myoglobin; Bruker Daltonics). Calibration masses were: RL36, 4364.3?Da; RS22, 5095.8?Da; RL34, 5380.4?Da; RL33meth, 6254.4?Da, RL32, 6315?Da; RL29, 7273.5?Da; RS19, 10299.1?Da; RNase A, 13682.2?Da; myoglobin, 16952.5?Da. Data analysis.? For automated data analysis, raw spectra were processed using the MALDI biotyper, E-64 version 2.0, software (Bruker Daltonics) with default settings. The software performs normalization, smoothing, baseline substraction and peak picking, creating a list of the most significant peaks of the spectrum (m/z values with a given intensity, with the threshold set to a minimum of 1% of the highest peak and a maximum of 100 peaks). To identify unknown bacteria, every peak list generated was matched directly against reference libraries (3290 species) using the integrated patterns matching algorithm of biotyper, version 2.0 (Bruker Daltonics). The unknown spectra were compared with a library of reference spectra based on a pattern recognition algorithm using peak position, peak intensity distributions and peak frequencies. Results scoring.? MALDI-TOF identifications were classified using modified score values proposed by the manufacturer: a score ��2 indicated species identification, a score in the range 1.7�C1.9 indicated genus identification, and a score