A total of 80 strains, including 60 pneumococcal strains, 8 S. pseudopneumoniae strains, and 12 S. mitis strains, were molecularly characterized and obtained from the National Reference Center for Pneumococci for the creation of the database. Each strain was tested a minimum of 20 and a maximum of 24 times using MALDI-TOF MS. MALDI-TOF MS acquisition and analysis. Mass spectra were acquired using a Microflex LT instrument (Bruker Daltonics). The standard parameters of the CE-IVD method recommended by the manufacturer were used. This instrument was equipped with an N2 laser (377 nm) using the following parameters: mass range, 2,000 to 20,000 Da; ion source 1, 20 kV; ion source 2, 18.15 kV; lens, 6 kV; pulsed ion extraction, 150 ns; laser frequency, 20 Hz. A manual external calibration standard (Bacterial Test Standard; Bruker Daltonics) was used for calibration. Data acquisition was performed using FlexControl (version 3.0; Bruker Daltonics). Eight deposits were performed for each isolate. The dried spots were coated with 1 µl of α-cyano-4-hydroxycinnamic acid (a-HCCA) in 50% acetonitrile-2.5% trifluoroacetic acid and each spot was analysed three times by MALDI-TOF MS. A total of 1890 spectra were produced : S. mitis (n=264), S.pneumoniae (n= 1434), S. pseudopneumoniae (n=192) Fore more details: please contact alexandre.godmer@aphp.fr