Comparative genotyping of clinical isolates of Streptococcus pneumoniae for understanding their evolutionary lineages

Streptococcus pneumoniae (pneumococcus) is a Gram-positive coccus that primarily colonises the nasopharyngeal region of healthy individuals. Its distinct polysaccharide capsule enhances virulence, allowing it to evade the immune system. This can lead to severe conditions such as septicaemia and meningitis in immunocompromised or older individuals, as well as in children under five years of age, who are particularly vulnerable. Pneumococcal conjugate vaccines (PCV) have been introduced to target the most common disease-associated serotypes. However, vaccine selection pressure has led to the replacement of previously dominant vaccine serotypes (VTs) by non-vaccine types (NVTs). Therefore, the replacement of VTs to NVTs in the bacterial population is of great concern because the currently available pneumococcal vaccines offer protection only against the 24 pneumococcal serotypes, while there are 101 serotypes that have been known. As a result, serotyping and genotyping are essential for epidemiological surveillance and vaccine assessment. This study aimed to determine the genetic variation of serotypes between and within serotypes or serogroups among a collection of clinical S. pneumoniae isolates. In this study, 101 clinical isolates of S. pneumoniae were examined. Sequetyping and sequential conventional multiplex PCR (cmPCR) were used for serotyping, while multilocus sequence typing (MLST) and multiple locus variable number tandem repeat analysis (MLVA) were used for genotyping. Sequetyping identified serotypes for 99 clinical isolates (98%), while sequential cmPCR identified 91 clinical isolates (90.1%). The cpsB gene sequetyping method produced results largely comparable to the sequential cmPCR, with further discrimination of sub-serogroups among the clinical isolates. In the MLST analysis, the major clonal complex, CC320 (25.3%) showed a significant association with the Taiwan19F-14 clone. The second-largest group, CC9 (13.2%) exhibited a notable association with the England14-9. The integration of MLVA revealed a subset of clinical isolates characterised by shared genotypic features but variations in capsular variants, suggesting the possibility of capsule-switching phenomena within MLST-based lineages. Specifically, MLST CC320, MLST CC9, and MLST CC66 showed evidence of capsular switching. The MLVA typing model established in this study serves as a reference dataset for the local population of clinical isolates, complementing MLST for pneumococcal surveillance in facilities lacking whole genome sequencing (WGS) expertise. Furthermore, the MLVA analysis demonstrates its ability to differentiate significant subgroups within strains that share the same sequence type (ST) and can potentially deduce the ST based on the MLVA type (MT). In conclusion, this research provides valuable insights into pneumococcal epidemiology, improving the understanding of pneumococcal transmission patterns and persistence. It emphasises the importance of continuous surveillance in the face of evolving pneumococcal dynamics.

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