Mtp 96 maldi tof target plate

Using the 18 culture conditions of the culturomics concept, the fecal sample was cultivated, and the obtained colonies were identified by MALDI-TOF as described below [5] (link). Proteomic analysis of our strain was carried out with MALDI-TOF as previously described [10] (link), [11] (link). A Microflex spectrometer (Bruker Daltonics, Leipzig, Germany) was used with a MTP 96 MALDI-TOF target plate (Bruker) on which 12 individual colonies were deposited. Twelve spectra were thus obtained, imported into MALDI BioTyper 2.0 software (Bruker) and analysed by standard pattern matching (with default parameter settings) against the main spectra of 7567 bacteria. Comparison with the BioTyper database spectra enabled the identification and discrimination of the analysed species from those in the database in accordance with the obtained score: a score >2 with a validated species enabled the identification at the species level, and a score <1.7 did not enable any identification. After a failed identification of the colony with a clean spectrum, it was identified by sequencing the 16S ribosomal RNA as previously described [18] (link). A threshold of 98.7% similarity level was determined to define a new species without performing DNA-DNA hybridization [19] .

The bacterial diversity of the stool sample was characterized using the 18 culture conditions of standardized culturomics [14] (link). For each culture condition, a liquid preincubation of the sample was performed, and tenfold serial dilutions of this culture were seeded on 5% sheep’s blood–enriched Colombia agar every 3 days for 30 days (bioMérieux, Marcy l’Étoile, France). Colonies were purified and identified using MALDI-TOF MS as previously described [15] (link), [16] (link). The MALDI-TOF MS analysis was carried out using a Microflex Spectrometer (Bruker Daltonics, Leipzig, Germany) with a MTP 96 MALDI-TOF target plate (Bruker). Each colony was tested in duplicate, and the obtained spectra were imported into MALDI BioTyper 2.0 software (Bruker). The spectra were then compared by standard pattern matching (with default parameter settings) to the 7567 references contained in our database, which consists of the Bruker database incremented with data from species that were not already present in the database. A strain is considered identified at the species level for an identification score of ≥1.9. Between identification scores of 1.7 to 1.9, the strain is identified at the genus level. A score of <1.7 does not allow any identification. The 16S rRNA gene was sequenced in order to obtain an identification as previously described [17] (link).