CC180 clade dynamics do not universally explain Streptococcus pneumoniae serotype 3 persistence post-vaccine: a global comparative population genomics study

Abstract

Background: Clonal complex 180 (CC180) is currently the major clone of serotype 3 Streptococcus pneumoniae (Spn) causing disease among children and adults worldwide. The 13-valent pneumococcal conjugate vaccine (PCV13) does not have significant efficacy against serotype 3 despite polysaccharide inclusion in the vaccine. It was hypothesized that PCV13 may effectively control Clade I of CC180 but that Clades III and IV are resistant, provoking a population shift that enables serotype 3 persistence. This has been observed in the United States, England, and Wales but not Spain. We tested this hypothesis further utilizing a dataset from Portugal to conduct our population genomics and molecular epidemiology comparative study.

Methods: We performed whole-genome sequencing (WGS) of 501 serotype 3 strains from Portugal isolated from patients with pneumococcal infections between 1999 and 2020. The draft genomes underwent phylogenetic analyses, pangenome profiling, and a genome-wide association study (GWAS). We also completed antibiotic susceptibility testing and compiled over 2600 serotype 3 multilocus sequence type 180 (MLST180) WGSs to perform global comparative genomics.

Findings: Relative to strains from all other lineages, CC180 Clades I, II, III, IV, and VI strains trend towards a decreased association with invasive disease cases compared to non-invasive pneumonia cases (binomial logistic regression, odds ratio or OR = 0.59, 95% confidence interval or CI = [0.34, 0.98], P = 0.046) and adult patients compared to paediatric patients (binomial logistic regression, OR = 0.34, 95% CI = [0.098, 0.92], P = 0.054). The serotype 3 CCs shifted post-PCV13 such that Clades I-VI comprise the majority of post-PCV13 lineages (binomial logistic regression, OR = 7.33, 95% CI = 4.36, 12.80, P < 0.0001), with Clade I representing 54% (220/404) of all post-PCV13 strains. As observed elsewhere, Clade I strains from Portugal are largely antibiotic-sensitive and carry the ΦOXC141 prophage. However, strains from Portugal and Spain, where Clade I remains dominant post-PCV13, have larger pangenomes and are associated with the presence of two genes encoding hypothetical proteins.

Interpretation: Clade I became dominant in Portugal post-PCV13, despite the burden of the prophage and antibiotic sensitivity. The additional accessory genome content may mitigate these fitness costs. Regional differences in Clade I prevalence and pangenome heterogeneity suggest that clade dynamics is not a generalizable approach to understanding serotype 3 vaccine escape.

Funding: National Institute of Child Health and Human Development, Pfizer, and Merck Sharp & Dohme.

Keywords: Clade I; Invasive pneumococcal disease; Molecular epidemiology; PCV13; Serotype 3; Streptococcus pneumonia.

Fig. 1

Clade dynamics of 501 serotype 3 Streptococcus pneumoniae (Spn) strains from Portugal. a. Maximum-likelihood phylogenetic tree of the 501 serotype 3 isolates generated from core genome single nucleotide polymorphisms. The tree scale represents substitutions per site. The legends indicate strain clonal complex (CC) in shades of red, Azarian et al. clade definitions in shades of grey, Kwun et al. clade definitions in shades of blue, and Global Pneumococcal Sequence Clusters (GPSCs) in orange. Subsequent references to clades refer to the Kwun et al. definition. Undefined CCs denote either a double-locus variant or a single-locus variants of two different CCs. b. Pie charts indicating the distribution of clades by disease manifestation and age in 2016–2019. c. Bar charts demonstrating differences in CC and clade proportions before and after the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13).

Fig. 2

Antibiotic resistance and phage diversity in serotype 3 strains from Portugal. The maximum-likelihood phylogenetic tree from Fig. 1a labelled to indicate the presence of various antibiotic resistance genetic determinants, penicillin-binding protein gene alleles (pbp), altered antibiotic susceptibility phenotypes, Tn916-like transposons, and the ΦOXC141 prophage. The asterisk denotes a strain carrying a partial Tn1207.1 sequence. The tree scale represents substitutions per site. 501 serotype 3 strains shown. Classifications were determined according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines for all antibiotics except for chloramphenicol, which were made according to Clinical and Laboratory Standards Institute (CLSI) guidelines.

Fig. 3

Comparative genomics of a global dataset of serotype 3 multilocus sequence type 180 (MLST180) strains. a. Maximum-likelihood phylogenetic tree of 2663 serotype 3 MLST180 strains based on core genome single nucleotide polymorphisms. The tree scale represents substitutions per site. The heritability estimate is indicated by h², the phenotype of interest is strain country of origin. b. A t-distributed stochastic neighbour embedding (tsne) plot of the 2663 serotype 3 MLST180 strains using the pangenome content of each strain. c. Pangenome size according to the number of genes for Clade I serotype 3 MLST180 strains. Each dot denotes the mean, and the error bars indicate the standard deviation. The P-value reflects the results of a Kruskal–Wallis test with Dunn's multiple comparison correction relative to Portugal. d. Results of a genome-wide association study (GWAS) to identify genes linked to strains from Spain and Portugal amongst the 2663 serotype 3 MLST180 strains. The top two most statistically significant hits from Clade I are shown.