Accurate serotype identification of Streptococcus pneumoniae using nanopore Cas9-targeted serotype identification (nCATSerotyping)

Abstract

Streptococcus pneumoniae (pneumococcus) is a leading cause of community-acquired pneumonia and invasive diseases, particularly among children and the elderly. The introduction of pneumococcal conjugate vaccines has significantly reduced invasive pneumococcal disease, but the prevalence of non-vaccine serotypes and newly emerging serotypes is increasing globally. Thus, accurate serotyping is essential for epidemiological surveillance and the development of next-generation multivalent pneumococcal vaccines. Conventional serotyping methods, including multiplex polymerase chain reaction (mPCR), monoclonal antibody (mAb) assays, and Quellung reaction using rabbit antisera, are limited by serotype coverage and cross-reactivity, making the detection of new or emerging serotypes challenging. In this study, we developed a nanopore Cas9-targeted serotyping (nCATSerotyping) platform, which employs Cas9-mediated enrichment of the capsular polysaccharide synthesis locus followed by Oxford Nanopore sequencing. Applying this method to 276 clinical pneumococcal isolates collected in South Korea (2018–2020), we achieved a serotyping success rate of 97.10% (268∕276), significantly outperforming conventional methods such as mAb and mPCR, which identified only 76.45% (211∕276) of isolates. Whole-genome sequencing of the remaining eight non-typeable isolates revealed them to be non-pneumococcal (oral streptococci), confirming 100% accuracy for S. pneumoniae serotyping. Importantly, our method identified emerging and underrepresented serotypes, including serotype 13 and null capsule clade strains. nCATSerotyping offers a rapid, accurate, and comprehensive solution for pneumococcal serotyping, with significant advantages in identifying novel and non-typeable strains. This scalable platform will be a valuable tool for global serotype surveillance and next-generation multivalent pneumococcal vaccine development. Copyright © 2025 Ji et al.