Streptococcus pneumoniae is still a leading bacterial pathogen of acute otitis media (AOM), despite the availability of pneumococcal conjugate vaccines (PCVs). We conducted a study on the population structure, antibiotic nonsusceptibility, serotype distribution, and presence of pilus in middle ear fluids ‒ S. pneumoniae isolates recovered from PCV10-vaccinated children with suppurative АОМ in Bulgaria. Non-susceptibility was observed in 68.75% (n = 33) of the isolates, and multidrug resistance (MDR) was detected in 60.4% of the patients. The dual macrolide resistance mechanism was predominant. The most common serotypes were non-PCV10 serotypes 3 (27.1%, n = 13), 19A (25.0%, n = 12), and VT 19F (23.0%, n = 11). Overall, 64.6% were non-PCV10-serotypes. The presence of Pilus type I was observed mostly in the PCV10-serotypes. We found a strong association between clonal complexes (CCs), serotypes, and antimicrobial resistance. Multilocus sequence typing revealed the presence of four CCs: CC320 (39.6%), CC505 (12.5%), CC1377 8.3%), and CC230 (8.3%). The most abundant CC320 comprised MDR 19A and 19F isolates. CC230 clustered MDR isolates from serotypes 19A, 6C, and 14. CC505 and CC1377 were serotype 3 susceptible isolates. The vaccine-induced changes and trends in antimicrobial resistance and clonality must be the focus of systematic investigations.

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