Microflex maldi tof ms spectrometer

Stools obtained from eight French patients as part of a culturomics project aiming at isolating as many distinct human-associated bacterial species from the gut, were included in the study from 2017 to 2020. All the methods used in this study were carried out in accordance with relevant guidelines and regulations conformed to the Declaration of Helsinki. Informed and oral consent was obtained from the stool donors. Approximately 1 g of each feces specimen was suspended in 2 ml of phosphate-buffered-saline (Life, Technologies, Carisbad, CA, USA). Then, 100 µl of each stool suspension was tenfold diluted up to 10^–10. After that, 50 µl was inoculated on 5% sheep blood-enriched Columbia agar (BioMérieux, Marcy l’Etoile, France) and incubated at 37 °C in anaerobic atmosphere generated by AnaeroGen generator (bioMérieux). After 72 h of incubation, single colonies were selected and subcultured on the same medium in order to obtain pure isolates. Strains were identified using a Microflex MALDI-TOF MS spectrometer (Bruker, Daltonics, Leipzig, Germany) as previously described^{31 (link)}.

Briefly, one isolated bacterial colony was picked from chapman culture plate using a pipette tip and spread it as a thin film on a MTP 96 MALDI‐TOF target plate for identification with a Microflex MALDI‐TOF MS spectrometer (Bruker Daltonics, Leipzig, Germany). In total, 12 distinct deposits for strain Marseille‐P2481were done from 12 individual colonies in duplicate. After air‐drying, 2‐μl matrix solution was applied per spot, as previously reported (Lagier, Khelaifia, et al., 2015). All spectra were recorded in positive linear mode for the mass range of 2,000–20,000 Da (parameter settings: ion source 1 (ISI), 20 kV; IS2, 18.5 kV; lens, 7 kV). The obtained protein spectra were compared with those of 2,480 spectra in the Bruker database enriched with our own database (Lagier, Hugon, et al., 2015). The strain was identified at the species level if the MALDI‐TOF MS score was greater than 1.9. If the score was lower than this threshold, the identification was not considered as reliable and the 16S rRNA gene was sequenced.