Maldi biotyper 2

Skin samples were recovered using cotton swabs from the inflamed areas of AD children and matched areas on non-AD controls. Swabs were either maintained at 4°C or frozen at -20°C before seeding. After Gram staining, swabs were quantitatively seeded on non-selective media under aerobic or anaerobic conditions (Trypticase Soy Agar with 5% horse blood (BioMérieux, Marcy L’Etoile, France) and Schaedler with vitamin K1 and 5% Sheep Blood (Becton Dickinson, Le Pont de Claix, France) agar plates) for 2 to 5 days, respectively. Bacterial colonies were identified on agar plates and the number of colonies grown was expressed in CFU/ml. MALDI-TOF-MS identification was carried out on a Microflex mass spectrometer (Bruker Daltonics, Bremen, Germany) using freshly isolated colonies. Briefly, a colony was added to a matrix and applied to a metal plate before ionization with a laser so that the total protein content could be analyzed by measuring their time of flight. Identification of clinical isolates was achieved by comparing the mass spectra obtained to reference spectra in the database, using MALDI Biotyper 2.0 (Bruker Daltonics). The degree of spectral concordance was expressed as a logarithmic identification score (LogScore) according to the manufacturer’s instructions.

All isolates were purified and analysed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) (Microflex LT, MALDI-TOF-MS, Bruker Daltonics, Coventry, UK) in a positive linear mode (2000 to 20000 m/z range). The resulting spectra for each culture were analysed by MALDI-Biotyper 2.0 software (Bruker Daltonics, Coventry, UK). The software evaluates each spectrum compared to a reference spectrum in the Bruker Taxonomy Database identifying the best match from database records. Results were expressed as scores (QI) from 0 to 3, as recommended by the manufacturer. Scores QI ≤ 1.7 were not considered as reliable identification. A score of QI ≥ 1.7 corresponded to “genus” identification. Only scores higher than QI ≥ 2 were considered a reliable identification of species.

The MSP profile showing the highest score was selected for each isolate and was included to construct the dendrogram using the statistical toolbox in MATLAB 7.1 integrated in the MALDI Biotyper 2.0 software (Bruker Daltonics). Based on the principle that identification score reflects the agreement of the spectra with the standard A. baumannii database entry, the MSP profile showing the highest score could mean that the specific spectra represents the most typical aspects of a certain strain from the database. This selection of the highest score marking spectra was necessary, especially when highly similar strains were studied, because several mass spectral features related to limited reproducibility of the method might eclipse mass spectral differences between the strains. Test strain clonality was determined with cut-off values at a distance of 250 [6 (link)].
For PFGE analysis of the 32 isolates, SmaI-digested genomic DNA was prepared according to the manufacturer's instructions (Bio-Rad, Hercules, CA, USA). Fragments were separated for 20 h at 6.0 V/cm at 11°C using a CHEF-DR II System (Bio-Rad) with initial and final pulse times of 0.5 s and 30 s, respectively [14 (link)]. The pattern was analyzed using the Fingerprinting II software (Bio-Rad). The cut-off value of 75 was applied for grouping as it was used previously [15 (link), 16 (link)].

The cultured bacteria were suspended in 300 μl of bi-distilled water and mixed with 900 ml of ethanol (Carl Roth GmbH). The measurement of the sample was then conducted following procedures previously described by El-Ashker et al. [19 (link)]. Measurement was performed with an Ultraflex III TOF/TOF mass spectrometer (Bruker Daltonics) equipped with a 200-Hz smartbeam 1 laser. The parameter setting was as follows: delay, 80 ns; ion source, 1 V, 25 kV; ion source, 2 V, 23.4 kV; lens voltage, 6 kV; and mass range 0–20 137 kDa. The raw spectra was analyzed by MALDI BIOTYPER 2.0 software (Bruker Daltonics, Billerica, MA, USA) using the default settings. The procedure of the MALDI-TOF mass spectrometry measurement was performed automatically without any user intervention. The software generated a list of peaks up to 100. The peaks with a mass-to-charge ratio difference of <250 ppm were considered to be identical. The peak list that was generated was used for matches against the reference library by directly using the integrated pattern-matching algorithms of the software. All parameters were the same regardless of the presumptive bacterial species analyzed. The BIOTYPER 2.0 database was composed using only Campylobacter and related species [20 (link)].