Isolation and Serotyping of Streptococcus pneumoniae

The European Intervention Study (EURIS) manual was used for isolation of bacterial strains [20 (link), 21 (link)]. Nasopharyngeal samples were transferred to the laboratory using transport media swabs (Copan 114C, Copan Diagnostics, Inc., Corona, USA), and inoculated within 3 to 6 h after the arrival. Samples were cultured on 5% defibrinated sheep blood agar (Oxoid Ltd., UK) supplemented with gentamicin (5 mg/L) and incubated at 35–37 °C under anaerobic conditions for 18 to 24 h. Samples were also cultured on sheep blood agar (Oxoid Ltd., UK) with optochin disks (AB Biodisk, Sola, Sweden) (5 μg) and incubated in 5% CO₂ at 35–37 °C for 18–24 h. Strains were identified as S. pneumoniae by colony morphology, negative catalase reaction, optochin susceptibility, agglutination in the Pneumo-Kit slidex test (BioMèrieux, Missouri, USA), and by the bile solubility test [22 ]. Isolates were serotyped by the Quellung reaction using serotype-specific antisera (SSI Diagnostica, Denmark).