Ultraflextreme matrix assisted laser desorption ionization time of flight mass spectrometry

Isolates were stored as a frozen stock in trypticasein soy broth (TSB, Biocorp, Poland) with Haemophilus Test Medium Supplement (HTMS, Oxoid, Hampshire, United Kingdom), in the presence of 30% (v/v) glycerol at − 70 ± 2 °C until its use. Bacteria were then re-cultured by applying a frozen bacterial suspension on chocolate agar (BioMerieux, Craponne, France), incubated for 24 h in microaerophilic (5–10% CO₂, 80–90% N₂, 5–10% O₂, Generbag microaer, BioMerieux, Craponne, France) conditions at 35 °C. Haemophilus spp. isolates were identified as previously shown^{13 (link)} by colony morphology, Gram-staining and API NH microtests (BioMerieux, Craponne, France), as well as by using the Ultraflextreme Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (Bruker Daltonics, Bremen, Germany) (MALDI-TOF MS) with MALDI-Biotyper 3.0 software (Bruker Daltonics, Bremen, Germany) according to the procedure described earlier^{29 (link)}.

Isolates were stored as a frozen stock in trypticasein soy broth (TSB, Biocorp, Warsaw, Poland) supplemented with Haemophilus test medium supplement (HTMS, Oxoid, Hampshire, Great Britain) with addition of 30% (v/v) glycerol at −70 ± 2 °C until its use. Bacteria were then re-cultured by applying the frozen stock to a chocolate agar (BioMérieux, Craponne, France) and incubated for 24 h at 35 °C in microaerophilic (5–10% CO₂, 80–90% N₂, 5–10% O₂, Generbag microaer, BioMérieux, Craponne, France) conditions. Haemophilus spp. isolates were then identified by colony morphology, Gram-staining, and identified to the species level by API NH microtests (BioMérieux, Craponne, France) and by the Ultraflextreme Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (Bruker Daltonics, Bremen, Germany) (MALDI-TOF MS) with MALDI-Biotyper 3.0 software (Bruker Daltonics, Bremen, Germany) according to the procedure described earlier [30 (link)]. The correctness and reliability of the abovementioned identifications were expressed in the form of a point indicator, as presented previously [31 ]. Only H. parainfluenzae isolates with identification scores >1.999 on the basis of protein profile were taken for further analysis.