Columbia agar with 5 sheep blood

Susceptibility to antibiotics was tested using Columbia agar with 5% sheep blood (bioMerieux, Marcy l'Etoile, France) and according to EUCAST 2015 recommendations (https://www.eucast.org) as described by Elsawi et al. [11 (link)]. The following antibiotics were tested: penicillin G, amoxicillin, daptomycin, ciprofloxacin, ceftazidime, ceftriaxone, clindamycin, doxycycline, fosfomycin, imipenem, linezolid, oxacillin, rifampicin, teicoplanin, tobramycin, trimethoprim‐sulfamethoxazole, and vancomycin using ETEST® (bioMerieux, Marcy l'Etoile, France).

The material to microbiological tests included nasal and pharyngeal swabs collected from patients before a planned vaccination against S. pneumoniae (0m-1st test) and 1-2 months after vaccination (Im-2nd test). The swabs were collected using a kit with a transport medium, and next a culture of microorganisms was performed on the following media: Columbia agar with 5% sheep blood, Mannitol salt agar, McConkey, Chocolate agar, and Sabouraud (bioMerieux, France), and the phenotypic identification with kits: the APINH, the APIStrep, the APIE, the APINE APIStaph, and the API AUX (bioMerieux, France). The numeric code of the identified strain was read out with the apiweb™ program (bioMerieux, France). The tests were conducted according to the routinely applied microbiological diagnostics.

The following reagents were used: acetonitrile (Sigma-Aldrich, Saint Louis, MO, USA); BHI or BHI agar (Brain Heart Infusion, OXOID, Basingstoke, UK); Columbia agar with 5% sheep blood (BioMérieux, Warszawa, Poland); Bradford reagent (Protein Assay Dye Reagent Concentrate, Bio-Rad, München, Germany); GelCode blue stain reagent (Thermo Scientific, Rockford, IL, USA); human neutrophil elastase and elastase fluorogenic substrate MeoSuc-Ala-Ala-Pro-Val-AMC (Calbiochem, San Diego, CA, USA); Hank’s Buffer with Ca²⁺, Mg²⁺ (HBSS, Lonza, Walkersville, MD, USA); kanamycin and erythromycin (ROTH, Karlsruhe, Germany); Percoll (GE Healthcare, Uppsala, Sweden); dextran (Sigma, St. Louis, MO, USA); PBS (IITD PAN, Wroclaw, Poland); propidium iodide (Invitrogen by Thermo Fisher Scientific, Eugene, OR, USA); Trypan blue (ICN Biomedicals, Eschwege, Germany); sequencing grade modified porcine trypsin (Promega, Madison, WI, USA); Zwittergent 3-14 (Calbiochem, Darmstadt, Germany).