Optochin sensitivity test

S. pneumoniae isolates were identified by colony morphology, the alpha hemolysis test, the optochin sensitivity test (Oxoid, Basingstoke, UK), bile solubility, the catalase test, and the Omni antiserum assay (Statens Serum Institute, Copenhagen, Denmark). Conventional microbiological methods were used in combination with the VITEK® 2 microbial analysis system according to the manufacturer’s instructions (bioMérieux, La Balme-les-Grottes, France).

All clinical samples were collected by routine methods and later inoculated on Columbia agar plates (Oxoid, UK) supplemented with 5 % sheep blood. Plates were incubated at 37 °C in 5 % CO₂ for 18–24 h. Pneumococcal isolates were identified by the optochin sensitivity test (Oxoid, UK), the bile solubility test and real-time PCR specific for lytA [26 ]. We serotyped the isolates by latex agglutination and quellung reaction with the sets of pooled and individual antisera (SSI, Denmark) [27, 28 (link)]. Subsequently, all the isolates were stored at −80 °C in 10 % skim milk and 15 % glycerol solution.
Antibiotic susceptibility testing against penicillin (PEN), cefotaxime (CTX), erythromycin (ERY), sulfamethoxazole/trimethoprim (SXT), chloramphenicol (CAT) and tetracycline (TET) was conducted by disc diffusion method, E-tests, broth microdilutions or agar dilutions using Mueller–Hinton agar (Oxoid, UK) supplemented with 5 % sheep blood. S. pneumoniae ATCC 49619 was used as the control reference strain. The results were interpreted using Clinical and Laboratory Standards Institute (CLSI) guidelines (M100-ED28 : 2018) [29 ]. Penicillin resistance was defined as an MIC of ≥0.12 µg ml⁻¹, according to meningitis breakpoints.