Molecular and biological characterization of <i>Streptococcus pneumoniae</i> isolates from patients with pneumococcal meningitis

The aim of the study is to provide key characteristics of Streptococcus pneumoniae isolates circulating in Russia in 2015–2020 and isolated from pneumococcal meningitis patients based on high-throughput sequencing data, including global pneumococcal sequence clusters, serotypes, virulence factors and genetic determinants of resistance, in comparison with clinical data on antimicrobial susceptibility. Materials and methods. We studied 68 invasive S. pneumoniae isolates from blood and cerebrospinal fluid of patients with bacterial meningitis in different regions of Russia in 2015–2020. Species identification was performed taking into account the morphology of colonies on blood agar, the presence of α-hemolysis, negative catalase reaction, sensitivity to optoquine, and positive latex-agglutination results. The sensitivity of isolates to antimicrobials was determined by microdilution in broth, and sensitivity categories were determined based on borderline values of minimum inhibitory concentrations (MICs). Whole genome sequencing of S. pneumoniae isolates, analysis of isolates for penicillin-binding protein signature, determination of global pneumococcal sequence clusters, MLST alleles, serotypes, sequence types and acquired resistance genes (mefA, ermB, tetM, folA/P, cat), identification of virulence genes were carried out. Results. Twenty-eight GPSCs, 45 sequence types and 27 serotypes were identified. The coverage rates of PPV-23 and PCV-13 were 78% and 59%, respectively. Serotypes 3 (18%), 19F (9%), 23F (7%) and 15B (6%) were predominant. The GPSC12 lineage (serotype 3) was predominant (43%). Lineages expressing vaccine serotypes GPSC1(19F), GPSC6(14), GPSC13(6A), GPSC904(14) and GPSC10(19F) exhibited multiple antimicrobial resistance, including penicillin resistance. The resistant lineages expressing non-vaccine serotypes were GPSC230 (13) and GPSC177 (35F). In most cases, genotypic and phenotypic resistance to penicillin (increased MICs of β-lactams correlated with types of penicillin-binding proteins), erythromycin (ermB, mefA, ermB/mefA), clindamycin (ermB) and tetracycline (tetM), and trimethoprim-sulfamethoxazole (folA, folP) was found to be consistent. The virulence genes cbpG, lytA, pce/cbpE, pavA, pfbA, ply, hysA, nanA and cps4A were detected in all isolates. Zinc metalloproteinase C was detected in 13% of isolates. Conclusion. A high diversity of serotypes and lineages among pneumococcal isolates from meningitis patients was revealed. Out of the 68 S. pneumoniae isolates from patients with bacterial meningitis, more than 17% belonged to non-vaccine serotypes. The results of phenotypic and genotypic antimicrobial resistance comparison were characterized by good concordance, which indicates the necessity for further study of the possibility of using whole-genome sequencing as a diagnostic tool to identify resistance mechanisms in clinical isolates of pneumococci.