Within-host genetic diversity of pneumococcal serotype 3 during one-year prolonged carriage in a healthy adult

Abstract

Streptococcus pneumoniae adapts within hosts through genetic variation and genome plasticity, facilitating persistence under antibiotic and immune pressures. Here, we investigated a prolonged pneumococcal carriage episode (>337 days) in a healthy, HIV-uninfected adult in Malawi. Whole-genome sequencing of single-colony isolates and plate sweep samples confirmed persistent colonisation by a multidrug-resistant serotype 3 strain with a distinct sequence type (GPSC10-ST18362), which maintained stable predominance despite transient acquisition of other serotypes. The sequentially sampled isolates showed 2 to 11 single-nucleotide polymorphism differences, no evidence of recombination, and modest gene loss involving mobile genetic elements. The total genome size decreased from 2.06 Mb to 2.03 Mb across isolates. Intrahost single-nucleotide variants were identified in genes related to metabolism, stress response, and DNA repair, but showed no consistent signatures of positive selection. Capsular locus analysis revealed deletions consistent with GPSC10-related vaccine-escape profiles. These findings highlight the capacity of GPSC10-ST18362 to persist asymptomatically for months with limited within-host genomic diversity.

Fig. 1. Pneumococcal carriage duration, density and sampling timeline.

A Pneumococcal carriage durations of PCV13 serotypes among all carriage episodes in the main cohort study. Box plots indicate the median carriage duration for each serotype (centre line), interquartile range (box), and minimum–maximum values (whiskers). Each data point corresponds to an independent carriage episode of PCV13 serotype (n = 83 episodes from 56 adults). The persistent carriage episode is highlighted in light red. Only phenotypic serotypes included in PCV13 are shown, reflecting the limitations of the phenotypic serotyping kit. B Pneumococcal carriage density for the persistent carriage episode (highlighted in light red in A) over time, measured phenotypically and expressed as log₁₀ colony-forming units per millilitre (CFU/ml). C Sequenced samples for the persistent carriage episode (highlighted in light in A) that were used in the final analysis stratified by sequencing type (Whole plate sweep and single-colony-derived) and day of sample collection (“Sampling timeline”, created in BioRender. Mumba, D. (2025) https://BioRender.com/hf82emband is licensed under CC BY 4.0). PCV13, 13-valent pneumococcal conjugate vaccine. Source data are provided as a Source Data file.

Fig. 2. Placement of the ST18362 serotype 3 isolates in the global pneumococcal phylogeny.

A Global serotype 3 phylogeny based on the Global Pneumococcal Sequencing Project (GPS) publicly available genomes, overlaid by metadata, namely pneumococcal lineages based on the GPSC nomenclature and continent of origin. We have highlighted clades in the phylogeny defined by the pneumococcal multilocus sequence typing scheme; this indicates four major clades for sequence type (ST)700, ST180, ST458 and ST260. The persistently carried isolates were assigned as ST18362 (highlighted in red) with a single locus variant to ST700. B Global GPSC10 phylogeny based on GPS publicly available genomes, stratified by serotype and a highlighted clade in red is ST18362 among serotype 3. The white colour indicates that the serotype was not defined in the GPS database. Source data are provided as a Source Data file.

Fig. 3. Intra-host evolution and pan-genome analysis of the ST18362 serotype 3.

A Summary of single-nucleotide substitutions on the specific genome position of persistently carried serotype 3 genomes over time relative to the first sample collected. B Persistently carried serotype 3 single-nucleotide substitutions per gene (each mutation counted once per gene position during the carriage episode). C Persistently carried serotype 3 single-nucleotide substitutions per gene (each mutation counted once per gene position during the carriage episode) stratified by the substitution effect on the gene. D Phylogeny of gene presence and absence of persistent carried pneumococcal serotype 3 stratified by the day of sample collection. Gene absence indicates deletions of genes over time. E Representative plot of the enrolment sample and first sample with gene deletions. The gene deletions were observed within the Tn5253-like Integrative Conjugative Element (ICE) (defective chloramphenicol and tetracycline resistance-conferring element) and between complete copies of the genes coding for a toxin-antitoxin system (pezAT). Tn5253 (GenBank: EU351020.1) was used as a reference to annotate the ICE and visualise it by easyfig. Source data are provided as a Source Data file.

Fig. 4. Serotype 3 capsular locus phylogeny and illustrations of serotype 3 capsular locus in the context of ST18362.

A Global phylogeny of pneumococcal serotype 3 cps locus based on GPS publicly available genomes, overlaid by pneumococcal lineage (GPSC). Highlighting in red is the ST18362 (persistently carried isolates), and in green are other STs defined by a multilocus sequence typing scheme. The serotype 3 cps locus is 18–20 kilobases long, and this phylogeny indicates how distinct serotype 3 lineages harbour unique cps loci. B Representative plot of three serotype 3 cps locus; a publicly available reference sequence (GenBank accession number: CR931634), representative sample from the current study (GPSC10-ST18362) and closely related GPSC10-ST700. Source data are provided as a Source Data file.

Fig. 5. Illustration of multiple lineage and serotype carriage.

Representative plot of multiple lineage and serotype carriage during the persistent carriage episode of ST18362 stratified by antimicrobial resistant genes (AMR genes) and in silico antimicrobial resistant profile of penicillin minimum inhibitory concentration (penMIC) and trimethoprim/sulfamethoxazole (co-trimoxazole). Numbers under the “GPSC & serotype” section represent pneumococcal serotypes; the colour is the GPSC, and the circle size represents abundance within that GPSC. The percentage indicates the abundance of serotype 3 lineage during multiple serotype carriage. (“Illustration of multiple lineage and serotype carriage”, created in BioRender. Mumba, D. (2025) https://BioRender.com/gd2y3n4and is licensed under CC BY 4.0). Source data are provided as a Source Data file.

Fig. 6. Genic and intergenic Intrahost single-nucleotide variants of the ST18362 per kilobase.

A Genic number of intrahost single-nucleotide variants per 1000 nucleotides (kilobases) per sampled days (each mutation counted once per gene or locus tag position during the carriage episode). B Intergenic number of intrahost single-nucleotide variants per kilobase per sampled days (each mutation counted once per gene or locus tag position during the carriage episode). C Summary of single-nucleotide variants for a specific gene of the persistently carried serotype 3 genomes over time relative to the first sample collected (each mutation counted once per gene or locus tag position during the carriage episode). D Summary of intergenic single-nucleotide variants of the persistently carried serotype 3 genomes over time relative to the first sample collected (each mutation counted once per gene or locus tag position during the carriage episode). Source data are provided as a Source Data file.