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

Within-host pneumococcal serotype 3 genetic diversity and evolution during a one-year prolonged carriage episode in a healthy adult | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Within-host pneumococcal serotype 3 genetic diversity and evolution during a one-year prolonged carriage episode in a healthy adult Lusako Sibale, Stephanie Lo, Newton Kalata, Tinashe Nyazika, Ndaona Mitole, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6313580/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Oct, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Streptococcus pneumoniae can rapidly evolve within hosts through genetic mutations, recombination, and mobile genetic elements, enabling adaptation to antibiotics and immune pressures. Here, we detail a longitudinal phenotypic and genomic analysis of a pneumococcal serotype 3 prolonged carriage episode (> 335 days) in a healthy HIV-uninfected adult in Malawi. Whole Genome Sequencing (WGS) of single-colony culture isolates confirmed persistent carriage of a novel multidrug-resistant serotype 3 strain (GPSC10-ST18362), outcompeting other transiently acquired serotypes during the study period. Sequentially sampled isolates showed between 2 to 11 single nucleotide polymorphism (SNP) differences randomly distributed across the genome with no evidence of recombination, but high mutation rates were observed in genes associated with antimicrobial resistance. Further analysis of the sequenced plate sweep samples revealed intrahost single nucleotide variants in several genes associated with survival, including bacterial metabolism, virulence, DNA synthesis and repair, and oxidative stress defence. The study has demonstrated the prolonged carriage of a novel pneumococcal serotype 3 (GPSC10-ST18362) in a healthy adult, revealing its association with multidrug resistance and potential within-host adaptive mechanisms. Health sciences/Medical research/Genetics research Biological sciences/Genetics/Microbial genetics/Bacterial genetics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Clinically relevant bacterial species have small, haploid genomes and exhibit low mutation rates 1 , 2 . However, due to their large population sizes and rapid growth, even these low mutation rates can lead to significant genetic diversity during infections 3 . Additional diversity is generated through mechanisms such as hypermutation, recombination, and integrating mobile genetic elements 4 , 5 . Single-nucleotide variant mutations and small indels are commonly identified as the primary drivers of within-host evolution in bacterial populations, whereas recombination events and mobile genetic element insertions occur less frequently but are still significant 3 . In S. pneumoniae , base substitutions occur approximately once every 15 weeks, while recombination, though less frequent, introduces large-scale genetic changes with an average of 72 SNPs per event 6 . This enhanced genomic plasticity facilitates adaptation to antibiotics and vaccine escape, demonstrating the pathogen’s capacity for rapid evolution in response to selective pressures. In other respiratory pathogens, such as Mycobacterium tuberculosis and SARS-CoV-2, prolonged infection drives within-host evolution resulting in more virulent strains 7 , 8 . Longitudinal carriage studies are important in elucidating potential factors that may explain the dynamics of pneumococcal colonisation, such as the carriage duration, colonisation density, multiple carriage, and clone succession driven by the within-host bacterial evolution. Understandably, most studies on pneumococcal carriage dynamics have primarily focused on children due to the reported high invasive disease burden and carriage prevalence 9 , 10 , 11 , 12 , 13 . However, the old paradigm of short pneumococcal carriage durations in adults 11 , 13 , 14 , 15 , 16 , 17 appears to be shifting, with a recent longitudinal study showing carriage durations of up to 4 months in adults 18 . Such prolonged pneumococcal carriage in adults may constitute reservoirs for continued transmission and circulation of serotypes, especially those less targeted by the PCVs 19 , in high carriage prevalence and disease-burdened settings. Adult pneumococcal carriage has historically been considered transient, though persistent carriage prevalence of vaccine-escape pneumococcal serotype 3 has been reported 19 . Pneumococcal serotype 3 is included in PCV13 but evades host antibody-mediated clearance 20 , 21 . Its capsular polysaccharide's distinctive structure and electrochemical properties significantly impede opsonophagocytosis 22 , an essential anti-bacterial mechanism against pneumococcus. Moreover, pneumococcal serotype 3 is associated with a case fatality rate of 30–47% in critical cases 23 . The persistence and adaptability of serotype 3, even post-vaccine introduction, highlight serious concerns about its clonal evolution and vaccine escape. Therefore, we investigated whether prolonged nasopharyngeal serotype 3 S. pneumoniae carriage would promote genomic diversification or the emergence of antimicrobial-resistance in an adult with long-term carriage. We used samples from an individual recruited under an adult longitudinal pneumococcal carriage study 24 , who had cultured-confirmed pneumococcal serotype 3 isolates in all his longitudinal samples, spanning 335 days. We conducted phenotypic and genomic analysis across all the longitudinal samples to ascertain prolonged carriage and assess within-host genetic diversity and evolution. Results Identification of pneumococcal serotype 3 isolates High pneumococcal carriage prevalence and prolonged episodes have been shown to provide a reservoir for potential genetic exchange and the development of AMR 25 , 26 . Global surveillance has demonstrated persistent pneumococcal carriage duration of an upper bound of ~ 258.3 days in children and adults (Supplementary Table 1). In an adult longitudinal pneumococcal carriage study conducted in Malawi 24 , The median carriage duration of phenotypic serotype 3 was 29 days (95% CI 8–51) among 27 episodes (n = 21) in the study (Fig. 1 A). However, in samples collected from a healthy female PCV-unvaccinated 30-year-old HIV-uninfected adult (participants’ characteristics are detailed in Supplementary Table 2), we persistently identified the presence of pneumococcus serotype 3 for approximately one year (Fig. 1 A). Moreover, this pneumococcal carriage episode duration of approximately a year is longer than any previously reported serotype 3 carriage durations, with an upper bound of ~ 100 days among children and adults (Supplementary Table 3). We further observed fluctuations of pneumococcal density during the presumed prolonged pneumococcal carriage episode, with a median carriage density of 33,500 CFU/ml (range from 1,340 CFU/ml to 36,850,000 CFU/ml) (Fig. 1 B). Together this observation suggest potential adaption and evolution of the bacteria within the adult host environment. Therefore, we performed both whole plate sweep and single colony-derived sequencing to understand the potential within-host adaptation and evolution associated with this presumed prolonged carriage. Fourteen whole plate sweep samples were sequenced and used in the final analysis (Fig. 1 C). In addition, we managed to sequence thirteen single colony-derived isolates. Out of the thirteen samples sequenced, eleven passed single colony quality control and were used in the subsequent analysis detailed below (Fig. 1 C). Genotypic characterisation of persistently carried pneumococcal serotype 3 isolates Genomic data availability has allowed pneumococcus to be clustered based on their genetic background, notably the Global Pneumococcal Sequencing Cluster approach (GPSC) using POPulation Partitioning Using Nucleotide Kmers (PopPUNK) 27 and Public databases for molecular typing and microbial genome diversity (PubMLST) 28 . These clusters are essential in determining pneumococcal virulence, invasiveness, and potential vaccine escape 29 . Using in silico serotyping tools, the persistently carried isolates were confirmed as serotype 3. Furthermore, using the GPSC PopPUNK 27 , it was established that all the isolates belonged to GPSC10. Moreover, in a global phylogeny of serotype 3, the persistently carried isolates were closely related to lineage GPSC10 and multilocus sequence type 700 (ST700) 30 , circulating only in Africa based on the Global Pneumococcal Sequencing database (Fig. 2 A). GPSC10 (CC230) is a recombinogenic lineage that is globally spreading and capable of expressing multiple serotypes (Fig. 2 B) 31 . Furthermore, using PubMLST 28 these isolates were clustered as novel sequence type 18362 (ST18362) with a single locus variant (SLV) to GPSC10-ST700. These data confirm prolonged pneumococcal carriage with a novel serotype 3 sub-lineage that belongs to a highly diverse and recombinant lineage circulating in Africa. Antimicrobial resistance profiling of GPSC10-ST18362 pneumococcal serotype 3 Persistent bacterial carriage plays a crucial role in maintaining pathogen populations, allowing extended exposure to selective pressures that contribute to antimicrobial resistance 32 , promoting their evolutionary success 25 . We, therefore, hypothesised that prolonged serotype 3 carriage creates an evolutionary advantage for the development and retention of AMR, enabling persistence within hosts. We demonstrated that all isolates, even though susceptible to ceftriaxone and erythromycin, were resistant to tetracycline, oxacillin, and cotrimoxazole and had reduced susceptibility to benzylpenicillin, and hence were classified as multi-drug resistant (MDR) (Supplementary Fig. 1A). Moreover, based on the Global Pneumococcal Sequence database among the dominant circulating serotype 3 lineages (GPSC12, GPSC51, GPSC10, GPSC83 and GPSC43), only GPSC10-serotype 3 were shown to be non-susceptible to penicillin and amoxicillin using the CDC pipeline 33 , 34 MIC prediction (Supplementary Fig. 1B). In addition, all GPSC10-serotype 3 from the GPS database were tetracycline and cotrimoxazole resistant, hence classified as MDR. Together, this suggests a potential genomic and phenotypic interplay between prolonged carriage and AMR, which could help promote bacterial persistence and resistance within the host. Within-host genetic evolution of GPSC10-ST18362 pneumococcal serotype 3 during prolonged carriage S. pneumoniae , a pathogen capable of significant genomic changes, can persist within host environments through mutations, recombination, and genome rearrangement 6 , 25 . We therefore hypothesised that prolonged carriage would be associated with within-host genomic changes that support its continued persistence. We observed no evidence of recombination (Fig. 3 A), and we confirmed it by running Genealogies Unbiased By recomBinations In Nucleotide Sequences (Gubbins) 35 . However, we observed a single nucleotide polymorphism (SNP) distance among all the isolates ranging from 2 to 11 SNPs (Fig. 3 A). This nucleotide substitution rate is higher than the previously reported nucleotide substitution per year 6 , 36 . Furthermore, high mutations were observed in genes that encode penicillin-binding protein 2x (PBP2x) and pneumococcal surface protein A ( pspA ) (Fig. 3 B). Pneumococcal surface protein A was associated with a high abundance of non-synonymous amino acid changes (Fig. 3 C). We further assessed potential genome rearrangement using a Bacterial Pan Genome Analysis Pipeline (Panaroo pipeline) 37 . We observed gene deletions over time, with the majority of genes deleted conferring to hypothetical proteins (Fig. 3 E). However, we also observed the deletion of genes that conferred to ATP-binding cassette transporters ( yusV, yfhA, fepD_2, yhfQ ), Antitoxin ( pezA ), and Toxin ( pezT ) (Fig. 3 E). In addition, these genes were present within the Tn 5253 -like ICE (defective chloramphenicol and tetracycline resistance-conferring element 38 ) and between complete copies of the genes coding for a toxin-antitoxin system ( pezAT ) (Fig. 3 D). The pezAT system is known to stabilise the mobile elements within the pneumococcal host 39 , 40 . Deletion of a single pair of pezAT operons has been associated with resilience to lysis, enhanced genetic competence, and resistance to Beta-Lactam (β-lactams) antibiotics 41 . Together, these findings reveal evidence of adaptive evolution through nucleotide substitutions, significant non-synonymous mutations, and targeted gene loss. Presence of capsular locus and virulence genes associated with adaptation in GPSC10-ST18362 pneumococcal serotype 3 The pneumococcal capsule is a major contributor to S. pneumoniae virulence and vaccine escape 42 . Pneumococcal lineage recombination affects carriage duration and pneumococcal capsule size 26 . Recent data from Malawi shows a vaccine escape serotype 3 GPSC10-ST700, with a distinct CPS locus deletion associated with increased antimicrobial resistance and lower susceptibility to opsonophagocytic killing 30 . The GPSC10-ST18362 differs from GPSC10-ST700 by a single variant but shares a similar antimicrobial resistance profile. We, therefore, hypothesised that the persistently carried GPSC10-ST18362 would exhibit similar CPS locus deletions that could support its adaptation potential. Using a capsular locus phylogeny, we assessed the capsular locus similarity with global pneumococcal serotype 3. We observed that the capsular locus of GPSC10-ST18362 clustered with GPSC10 serotype 3 capsules, and each major lineage had a distinct capsular locus (Fig. 4 A). Moreover, GPSC10-ST18362 exhibited a capsular deletion of the wzg , wzh , wzd and wze genes similar to GPSC10-ST700. In contrast, these deleted genes were present in the global lineage clonal complex 180 (CC180)-GPSC12 or Netherlands 3 -31 or PMEN31 (Fig. 4 B). PMEN31’s cps locus is publicly available as CR931634 43 and contains the majority of serotype 3’s that have been sequenced and analysed to date 44 . Other virulence genes like cbpD , lytA , and lytC have been associated with biofilm formation, fratricide, and adaptation 45 . Notably, using the Mass screening of contigs for antimicrobial resistance or virulence genes (ABRicate) virulence factor database ( VFDB ) 46 , we observed that GPSC10-ST18362 had a combination of cbpD, cbpG, lytC , and lytA virulence genes that could be essential in there adaptation, along with ply, nanB, nanA, pfbA, psaA, pspA, lytB, pce, and pavA virulence genes (Supplementary Fig. 2). Collectively, the findings show that GPSC10-ST18362 had CPS locus deletions associated with adaption similar to the vaccine escape GPSC10-ST700, as well as possessing a combination of adaptation-related virulence genes. Within-host competition and colonisation advantage of GPSC10-ST18362 pneumococcal serotype 3 Within-host competition among pneumococcal lineages is a key driver of genomic evolution, shaping colonisation dynamics and adaptation 47 . During multiple carriage, strains compete for resources and niches, with genetic adaptations and virulence factors influencing colonisation advantage 48 – 50 . External pressures like antibiotics and vaccines further shape these competitive interactions, which can disrupt co-colonisation and favour lineages with adaptive advantages 45 . GPSC10-ST18362 exemplifies this evolutionary process since we have shown that its persistence is linked to lineage-specific genomic traits that support competitive dominance. Genetic factors associated with immune evasion, biofilm formation, and antimicrobial resistance may confer a colonisation advantage, enabling prolonged carriage and increasing opportunities for within-host evolution and horizontal gene transfer 45 , 50 . We therefore used a whole-plate sweep sequencing approach to investigate transient serotype carriage and co-colonisation dynamics during prolonged carriage of GPSC10-ST18362. We observed transient serotype carriage from other lineages during the follow-up period (Fig. 5 ) without impacting the persistence of GPSC10-ST18362. These findings suggest that GPSC10-ST18362 possesses traits that could potentially provide a within-host competitive advantage for colonisation. Genic and intergenic Intrahost single nucleotide variants (iSNVs) of GPSC10-ST18362 pneumococcal serotype 3 We have established that persistent carriage was associated with within-host competition and genetic changes linked to adaptation. Studies on pneumococcal populations have demonstrated that carriage provides an ideal environment for the emergence of minority variants due to selective pressures within the host, such as immune responses, microbial competition, and antibiotic exposure 32 . Therefore, we hypothesised that this persistent carriage of serotype 3 GPSC10-ST18362 could also be associated with an increased prevalence of minority variants, representing genomic plasticity and adaptation during the carriage episode. We utilised whole plate sweeps sequencing-based approach to quantify unique intrahost single nucleotide variants (iSNVs). We restricted our analysis to iSNVs found in samples involving only a single pneumococcal lineage (GPSC10) and serotype (pneumococcal serotype 3). We observed high intragenic and intergenic iSNVs in transposase and hypothetical genes (Fig. 6 and Supplementary Table 4). However, we also observed iSNVs in genes coding for bacterial metabolism, survival and virulence ( adcAII , zinc-binding lipoprotein AdcAII, phoH , phosphate starvation-inducible protein PhoH and phtB , pneumococcal histidine triad protein PhtB), DNA synthesis and repair ( carA , glutamine-hydrolyzing carbamoyl-phosphate synthase small subunit and pcrA , DNA helicase PcrA) and Oxidative Stress Defense ( gor , glutathione-disulfide reductase) (Fig. 6 A, Supplementary Fig. 3A and Supplementary Table 4). In addition, we observed Intergenic (upstream gene variants) iSNVs for genes coding for bacterial metabolism and host defense ( dhaM , PTS-dependent dihydroxyacetone kinase phosphotransferase subunit DhaM, phoH , phosphate starvation-inducible protein PhoH and phtE , pneumococcal histidine triad protein PhtE), antibacterial properties ( blpU , bacteriocin-like peptide BlpU), DNA repair ( sufD , Fe-S cluster assembly protein SufD, radC , DNA repair protein RadC, dnaG , DNA primase and parC , DNA topoisomerase IV subunit A), ions transport ( pstA , phosphate ABC transporter permease PstA), bacterial protein synthesis ( ylqF , ribosome biogenesis GTPase YlqF and metG , methionine–tRNA ligase), and Peptidoglycan Biosynthesis ( glmU , bifunctional UDP-N-acetylglucosamine diphosphorylase/glucosamine-1-phosphate N-acetyltransferase GlmU) (Fig. 6 B, Supplementary Fig. 3B and Supplementary Table 4). Our findings suggest that the adaptational evolution and within-host competition during this episode resulted in a variable accumulation of potentially advantageous substitutions. Discussion We have demonstrated persistent carriage of a GPSC10 pneumococcal serotype 3 novel sequence type 18362, which was associated with multi-drug resistance within a healthy adult in Malawi. These data shed light on the within-host complex interplay of recombinant bacterial adaptation, virulence, and antimicrobial resistance. Pneumococcal serotype 3 is associated with severe clinical outcomes, including adverse cardiovascular events and higher mortality rates in adults 23 , 51 , 52 . The inclusion of pneumococcal serotype 3 in high-valent pneumococcal conjugate vaccines has been less effective in reducing serotype 3 disease burden compared to other vaccine-included serotypes, as evidenced by persistent serotype 3-associated parapneumonic pleural effusion/empyema, ongoing vaccine evasion, and serotype replacement in adult pneumonia cases 53 – 55 and overall circulation in the community 19 . Serotype 3 capsule is not covalently linked to peptidoglycan and, in turn, facilitates the shedding of serotype 3 capsule, which in turn interferes with bacterial clearance 20 . Moreover, serotype 3 has the lowest surface charge (zeta potential), which plays a crucial role in evading the immune system by reducing phagocytosis and complement deposition 22 . In line with these findings, we identified pneumococcal carriage of serotype 3 for approximately one year in a healthy adult, indicative of poor clearance 56 . The spread of the MDR lineage of GPSC10 serotype 24F has been associated with disease in France 31 , highlighting the expansion potential of GPSC10 lineages. In a global context, the persistent carriage isolates ST18362 clustered with GPSC10. Based on the GPS database, GPSC10 serotype 3 was solely from Africa, suggesting a clonal dissemination within the continent. Moreover, the ST18362 isolates were multi-drug resistant, typical of GPSC10 lineage strains 31 . ST18362 had a single locus variant to GPSC10-ST700. Moreover, the identification of new and emerging variations at the single nucleotide level among serotype 3 has been highlighted as essential in surveillance due to clade expansion 54 . Consequently, prolonged carriage durations of lineages of global concern, such as GPSC10, provide ample time for clonal dissemination and seeding future outbreaks. Pneumococci colonisation success is determined by a complex competitive interaction from both other bacterial species 57 and other pneumococcal lineages 58 , 59 . Several factors have been associated with colonisation success or failure, including capsular thickness 48 , 49 , positive carriage status 59 and competent cells fratricide 50 . Competent cell fratricide is mediated by bacteriocins 60 , as well as a combination of a putative murein hydrolase cbpD , autolysin lytA , and cell wall hydrolase lytC 45 , 50 . We have demonstrated that this persistent ST18362 carriage episode was associated with transient pneumococcal lineages, which did not out-compete its dominance. Moreover, a combination of a putative murein hydrolase cbpD , autolysin lytA , and cell wall hydrolase lytC were present in ST18362. Together, these findings suggest that ST18362 has the potential to withstand within-host competition from other pneumococcal strains during the carriage episode. A copy of the pezAT operon is associated with stabilising mobile elements, moreover, they have been discovered within a putative pneumococcal integrative and conjugative element (ICE) Tn5253 39,40 . In concordance with these results, we observed two copies of the pezAT operon within the Tn5253 -like ICE (defective chloramphenicol and tetracycline resistance-conferring element 38 ). However, the disruption of pezAT has been shown to impact virulence 61 . Moreover, the loss of a single copy of pezAT in pneumococci has been associated with increased changes in the cell wall biosynthesis in that the mutants form shorter chains during the exponential phase, leading to increased colony-forming units 62 . Furthermore, the mutants became more resilient to lysis, enhanced their acquisition of transforming DNA, and became more resistant to antibiotics targeting the cell wall, mainly β-lactam antibiotics 62 . We observed the loss of a single pair of pezAT over time during the carriage episode, which could suggest a potential adaptation mechanism of GPSC10-ST18362. Pneumococcal serotype 3 lineages exhibit substantial genetic diversity, with evidence of frequent horizontal gene transfer and genomic variability, as observed in multiple studies 44 , 54 . Prolonged carriage durations have been associated with increased genetic alterations and adaptive changes 25 , 38 , 63 . We observed genome reduction and higher mutation rates than previously reported within a year 6 , 36 . Specifically, we observed high mutation in genes that encode penicillin-binding protein and pneumococcal surface proteins, which have been associated with pneumococcal adaptation 6 . We further observed genic and intergenic minority variants associated with adaptation from the prolonged carriage episode. Collectively, these data demonstrate potential mutations that could have supported the adaptation of GPSC10-ST18362 within the individual. Despite a comprehensive analysis of persistent within-host pneumococcal carriage of a serotype and lineage of global concern, the study has some limitations. Firstly, we performed broad sequencing, not deep sequencing; hence, we might have potentially underestimated within-host minority variants and multiple carriage events. However, Serocall 64 is robust in detecting multiple pneumococcal serotypes, even at low frequencies. Secondly, we could not fully decipher the genomic variants over time in this current analysis due to the lack of a complete publicly available reference genome for GPSC10 serotype 3. However, the utilisation of both a GPSC10 serotype 24 complete genome reference and capsular locus of GPSC-ST700 together with CR931634 43 as references mitigates this challenge. Our study has demonstrated the persistent carriage of pneumococcal serotype 3 (GPSC10-ST18362) with a novel sequence type within a healthy adult, revealing its association with multidrug resistance and within-host adaptive mechanisms. The findings indicate that GPSC10 serotype 3 lineages can be carried for extended periods, which may facilitate their global spread and provide ample opportunities to acquire diverse serotype cps. Methods Study design and recruitment Nasopharyngeal samples were collected from asymptomatic adults in Blantyre, Malawi 24 . In brief, the study participants were recruited 3 days after screening and then followed up on days 7, 14, 21, and 28 for the first month, and then every month for 12 months. All participants were recruited from the ART clinics and Voluntary Counselling and Testing (VCT) centres at Lighthouse-Queen Elizabeth Central Hospital and Gateway Health Centre in Blantyre. Participants were screened for pneumococcal carriage, using WHO-recommended nasopharyngeal sampling and microbiological culture methods for pneumococcal detection 65 . Inclusion criteria included confirmed pneumococcal carriage in adults aged 18 to 45 years, living with a child under 5 years old, and providing written informed consent.Ethical approvals The study was conducted following good clinical practice (GCP) guidelines and the Declaration of Helsinki. Ethical approval was obtained from the College of Medicine Research Ethics Committee (COMREC) (P.11/18/2532) and Liverpool School of Tropical Medicine Research Ethics Committee (LSTMREC) (19–033). Microbiological culture and density quantification Standard microbiological culture was used to determine the presence of S. pneumoniae from the nasopharyngeal swab 65 . We identified S. pneumoniae by their morphology and optochin sensitivity. The bile solubility test was used on isolates with no or intermediate (zone diameter < 14mm) optochin susceptibility 65 . Plates showing no S. pneumoniae growth were incubated for a further 24 hours before being reported as negative. A single colony of confirmed pneumococcus was selected and grown on a new SBG plate. Growth from this second plate was used for serotyping by latex agglutination (ImmuLexTM 23-valent Pneumotest; Statens Serum Institut, Denmark). Pneumococcal density was quantified using microbiological culture serial dilutions 66 on a gentamicin-sheep blood agar plate (SBG; 5% sheep blood agar, 5µL gentamicin/mL), and results were reported as colony forming units per millilitre (CFU/ml). Antimicrobial resistance profiling Antimicrobial susceptibility of pneumococcal isolates was assessed by the disk diffusion method (Oxoid, USA) for beta-lactams antibiotic (oxacillin 1µg), MLS B antibiotic (erythromycin 15µg), tetracycline antibiotic (tetracycline 30µg) and Trimethoprim/sulfamethoxazole (co-trimoxazole 1.25–23.75µg). Beta-lactam antibiotic susceptibility was confirmed by benzylpenicillin Etest (bioMérieux, Marcy-I’Étoile, France) minimum inhibitory concentrations (MICs). A Ceftriaxone Etest was also performed when oxacillin 1µg zone diameter < 9 mm according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines 67 . Etest strips were applied to the surface of the plates according to the manufacturer’s recommendations. The MIC was defined as the lowest concentration of the drug where the zone edge intersected the Etest strip. Interpretation of results followed EUCAST guidelines for meningitis breakpoints 67 . S. pneumoniae ATCC 49619 was used as a quality control strain. MDR was defined as non-susceptibility to agents in three or more chemical classes of antibiotics 68 , 69 . DNA extraction and quantification For single isolate culture specimens, 10µl of stored pneumococcal isolates were plated onto Columbia CNA agar containing 5% sheep blood (CBA). These were then incubated overnight at 37 ± 2°C with 5% CO 2 65 . All growth was collected from the plate using 10µl sterile plastic loops into an Eppendorf tube ready for DNA extraction. For plate sweep culture specimens, 100µl of stored respiratory samples (nasopharyngeal swab in STGG with known positive for pneumococcus determined by standard microbiological culture 65 ) were plated onto CBA. These were then incubated overnight at 37 ± 2°C with 5% CO 2 . All growth was collected from the plate using 10µl sterile plastic loops into an Eppendorf tube ready for DNA extraction. QIAamp DNA Mini Kit (Qiagen, Germany) was used for DNA extraction following the manufacturer's instructions. DNA quantity and quality were evaluated by Invitrogen Qubit Fluorometer. Samples that had a total DNA yield of > 2.5µg were submitted for sequencing. Whole genome sequencing Whole genome sequencing (WGS) was performed at the Wellcome Sanger Institute on an Illumina NovaSeq 6000 (Illumina-HTP NovaSeq 6000 Paired-end sequencing). Specifically, the sequencing platform was NovaSeq SP, the read length was 150bp, the plex was 384, and the expected reads per sample were 1.8M. Single colony analysis Quality control of single colonies (Appendix - supplementary methods 31 ) QUAST (QUality ASsessment Tool) v5.2.0 was used to extract assembly quality metrics. Overall sequencing depth of > 20X 70 , total number of contigs of 90% of reads that mapped to S. pneumoniae using Kraken metagenomic classification algorithm (v1.0.0) 71 and > 60% mapping coverage of reference genome (PMEN global clone Spain23F-1, accession number FM211187) 31 . Finally, to account for contamination due to multiple carriage of > 1 pneumococcal strain, the number of heterozygous sites of < 220 or percent of heterozygous sites over the total number of single nucleotide polymorphisms (SNPs) of ≤ 15% were included as previously described 31 . SeroCall 64 was performed as a second stage to confirm multiple serotypes. Phylogeny analysis Genome de novo assemblies were done by Spades (v3.15.4) 72 , and Genome alignments were done by Burrows-Wheeler Aligner (bwa) (v 0.7.17) 73 . S. pneumoniae strain 475 chromosomes, complete genome, serogroup 24 [GenBank: CP046355.1 ] as a GPSC10 reference. Gubbins (v3.2.1) 35 was used to detect recombination, and phylogenetic trees were constructed by RAxML (Randomised Axelerated Maximum Likelihood) 74 . We contextualised the episode to global pneumococcal lineages (n = 20,924 genomes) assigned by popPUNK (v2.6.0) 27 and serotype 3 (n = 593 genomes) isolates that passed the above QC from the Global Pneumococcal Sequencing Project database ( https://data.monocle.sanger.ac.uk/ ). We also included serotype 3 from the parent study (n = 30 genomes) that passed the above QC. The phylogenetic trees overlaid with epidemiological data and in silico output were visualised using ggtree (v3.8.0) 75 . Pan-genome analysis Prokka: Rapid prokaryotic genome annotation (v1.14.5) 76 was used for annotation, and gene clustering analysis was done by panaroo (v1.3.3) 37 and visualised by an interactive viewer for populations of bacterial genomes linked by a phylogeny (phandango) 77 . Genome lineage clustering was done by assigning GPSC using PopPUNK (v2.6.0) 27 . Sequence typing was done by MLSTcheck (v2.23.0) and serotyping by a k-mer-based Pipeline to identify the Serotype from Illumina NGS reads (SeroBA) (v1.0.2) 78 . Detection of recombination was done by Gubbins (v3.2.1) 35 , detection of pairwise snp distance was done by a Pairwise SNP distance matrix from a FASTA sequence alignment (snp-dist) (v0.7.0) 79 and summary of SNPs relative to a first sample as reference sequence using snipit : summarise snps relative to your reference sequence (v1.1.2) 80 . Detection and annotation of majority variants were done by a Bayesian haplotype-based genetic polymorphism discovery and genotyping tool ( freebayes ) and genetic variant annotation, and functional effect prediction toolbox (SnpEff) (v5.2) 81 respectively within a rapid haploid variant calling and core genome alignment tool (snippy) (v4.6.0) 82 . Resistance profiling was done by Mass screening of contigs for antimicrobial resistance or virulence genes (ABRicate v1.0.1) 83 , the comprehensive antibiotic resistance (CARD) database 84 and the Pathogen Watch CDC pipeline 33 . Virulence genes profiling was done using the ABRicate virulence factor database ( VFDB ) and blast using diverse pspA sequences as reference 85 . Capsular locus analysis Genome mapping was done by Burrows-Wheeler Aligner (BWA) (v0.7.17-r1188) 73 . Serotype 3 capsular encoding region capsular locus reference used was NCBI accession: CR931634 43 . Tree constructed by RAxML 74 . The phylogenetic trees were visualised using ggtree (v3.8.0) 75 . We extracted and annotated the capsular locus of serotype 3 in Geneious Prime and visualised it by easyfig (v2.2.2) 86 . Mobile genetic element analysis We assessed the presence of MGE in all isolates from this persistent carriage using ICEfinder and confirmed the element by annotating it using Geneious Prime and Artemis . Tn5253 (GenBank: EU351020.1) was used as a reference to annotate the mobile genetic element and the final illustration visualised by easyfig (v2.2.2) 86 . Plate sweep analysis Lineage deconvolution All pneumococcal genomes that passed quality control (20,924 genomes) from the Global Pneumococcal Sequencing Project database ( https://www.pneumogen.net/gps/ ) were included as a reference for lineage deconvolution. PopPUNK (v2.6.0) 27 was used to reassign lineages to these reference genomes. Using a statistical mixture model, the mSWEEP (v2.0.0) 87 algorithm used read pseudo-alignments output from Themisto (v2.1.0) 88 to quickly estimate the abundance of lineages based on the reference within a mixed sample. mGEM (v1.3.3) 89 algorithm used the resulting likelihood estimates output from mSWEEP to deconvolute the mixed reads into groups based on the lineages. Shovill (v1.1.0)was used for the assembly of binned reads. To reduce the possibility of false positives, lineages were only called if they were present at a relative abundance of greater than 1% (0.01), as described previously 89 . The quality control tool demix_check was run on the resulting lineage-level bins, and high confidence scores of 1 and 2 were retained as reliable identification. Serotyping, serotype abundance and antimicrobial-resistant calls To determine serotypes, SeroCall 64 was run on raw reads samples, and SeroBA (v1.0.2) 78 was run on each deconvoluted lineage to confirm untypable serotypes and serotypes at the sub-serogroup level. Resistance calling was done by ABRicate (v1.0.1) 83 , the comprehensive antibiotic resistance (CARD) database 84 and Pathogen Watch CDC pipeline 33 . Minority variants call To investigate the intrahost evolution, we rigorously employed the LoFreq (v2.1.5) variant calling pipeline on all samples, specifically those with a single identified GPSC lineage and serotype, using mSWEEP (v2.0.0) 87 and SeroCall 64 , respectively. Therefore, plate sweep samples with multiple lineages and serotypes were excluded (Fig. 5 ). We included samples with over 89% of reads mapping to S. pneumoniae using Kraken (v1.0.0) 71 to address contamination (Supplementary Fig. 4). Therefore, the final analysis included 64% (9/14) sampling points. S. pneumoniae strain 475 chromosomes, complete genome, serogroup 24 [GenBank: CP046355.1 ] as a GPSC10 reference to call minority variants, annotate the variants and compute genome coverage. Reads were aligned to the reference genomes using BWA (v0.7.17-r1188) 73 . The Picard tools (v2.23.8) ‘CleanSam’ function was then used to align soft clip reads to the end of contigs and to set the alignment qualities of unaligned reads to zero. The LoFreq pipeline was initially run with a coverage of 20 reads to identify a variant. The resulting variant calls were used along with the read alignment as input to the GATK (Genome Analysis Toolkit Variant Discovery in High-Throughput Sequencing Data) BaseRecalibrator tool (v4.1.9), as suggested in the LoFreq manual , to improve the estimated base quality scores. Finally, the LoFreq pipeline was run for a final time with a coverage requirement of 20 reads. The resulting genetic variant calls were only considered if their maximum allele frequency was = 0.15. The minimum allele frequency was based on Adam Lauring’s rule of thumb 90 “that the coverage should be 10 times the reciprocal of a variant's frequency” . The variants were annotated using SnpEff (v5.2) 81 . Each mutation was counted once per gene or locus tag position during the carriage episode using the first sample sequenced as a reference after annotation with S. pneumoniae strain 475 chromosomes, complete genome, serogroup 24 [GenBank: CP046355.1 ]. Therefore, only a unique mutation to the first sample sequenced are indicated in the subsequent follow-up days. Pneumococcal colonisation episode definition and data presentation A pneumococcal colonisation episode was defined by either the first pneumococcal carriage at study screening or the re-acquisition of pneumococci after pneumococcal clearance. Pneumococcal clearance was defined by detecting negative cultures for any serotype at two consecutive sampling points, as previously described 11 , 13 . R statistical package ggplot2 (v3.4.3) 91 , Inkscape (open-source vector graphics) and BioRender (a web-based tool used to create high-quality scientific illustrations) for visualisation. Declarations Data availability Raw sequencing data are stored with the NCBI under project code PRJNA1137437, with individual accessions given in Supplementary Table 5 for single colony-derived sequences and Supplementary Table 6 for plate sweep-derived sequences. Annotated minority variants are available in Supplementary Table 4. Code availability Manuscript code and R scripts that were used to analyse the datasets are available in the GitHub repository https://github.com/Lusako/SPN3_ST18362_persistent_carriage_manuscript Acknowledgements RSH and KJ were supported by National Institute for Health Research (NIHR) Global Health Research Unit on Mucosal Pathogens using UK aid from the UK Government [16/136/46] and the Medical Research Council (MRC, UK) [MR/T008822/1] awarded to KCJ. The views expressed in this publication are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. 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Available from: http://dx.doi.org/10.1093/nar/gkw1004 Hollingshead SK, Becker R, Briles DE (2000) Diversity of PspA: mosaic genes and evidence for past recombination in Streptococcus pneumoniae. Infect Immun [Internet]. ;68(10):5889–900. Available from: https://journals.asm.org/doi/ 10.1128/iai.68.10.5889-5900.2000?url_ver=Z39. 88-2003픯_id=ori%3Arid%3Acrossref.org픯_dat=cr_pub++0pubmed Sullivan MJ, Petty NK, Beatson SA (2011) Easyfig: a genome comparison visualizer. Bioinformatics [Internet]. ;27(7):1009–10. Available from: http://dx.doi.org/10.1093/bioinformatics/btr039 Mäklin T, Kallonen T, David S, Boinett CJ, Pascoe B, Méric G et al (2021) High-resolution sweep metagenomics using fast probabilistic inference. Wellcome Open Res [Internet]. Oct 8 [cited 2023 Jun 28];5(14):14. Available from: https://www.duo.uio.no/handle/10852/92867 Alanko JN, Vuohtoniemi J, Mäklin T, Puglisi SJ (2023) Themisto: a scalable colored k-mer index for sensitive pseudoalignment against hundreds of thousands of bacterial genomes. Bioinformatics [Internet]. ;39(39 Suppl 1):i260–9. Available from: http://dx.doi.org/10.1093/bioinformatics/btad233 Mäklin T, Kallonen T, Alanko J, Samuelsen Ø, Hegstad K, Mäkinen V et al (2021) Bacterial genomic epidemiology with mixed samples. Microb Genom [Internet]. ;7(11). Available from: http://dx.doi.org/10.1099/mgen.0.000691 Lauring AS (2020) Within-Host Viral Diversity: A Window into Viral Evolution. Annu Rev Virol [Internet]. ;7(1):63–81. Available from: http://dx.doi.org/10.1146/annurev-virology-010320-061642 Wickham H (2009) ggplot2: Elegant Graphics for Data Analysis [Internet]. Springer Science & Business Media; 213 p. Available from: https://play.google.com/store/books/details?id=bes-AAAAQBAJ Additional Declarations There is NO Competing Interest. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Published Journal Publication published 07 Oct, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6313580","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":437462911,"identity":"462f9643-b022-4256-8687-271a54cf0c47","order_by":0,"name":"Lusako Sibale","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqklEQVRIiWNgGAWjYBACPgbGNgaGAgY5BnZitbCBtRgwGDMwE68FhAwYEhuI18J/uO3BDwOb9LXNPAZMN9uI0SKR2G7YY5CWu+0wjwFzLnFaGNskeAwOA7XwbmDO3UaUww62Sf4x+J9uRrwWhsQ2aR6DAwkkaAH6xVjGINlw22H+D4dz/xGhhZ//+LOHbyrs5M2OtyU+zjlDhBYUcIBUDaNgFIyCUTAKcAAAgnYxGLukvDwAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-5378-3561","institution":"Malawi-Liverpool Wellcome Research Programme","correspondingAuthor":true,"prefix":"","firstName":"Lusako","middleName":"","lastName":"Sibale","suffix":""},{"id":437462912,"identity":"bd7ce38b-cb5f-4484-96ea-7bf334bc7658","order_by":1,"name":"Stephanie Lo","email":"","orcid":"https://orcid.org/0000-0002-2182-0222","institution":"Wellcome Sanger Institute","correspondingAuthor":false,"prefix":"","firstName":"Stephanie","middleName":"","lastName":"Lo","suffix":""},{"id":437462913,"identity":"5198b129-6b93-4bf1-ac77-f8802b124ab8","order_by":2,"name":"Newton Kalata","email":"","orcid":"","institution":"Malawi-Liverpool Wellcome Research Programme","correspondingAuthor":false,"prefix":"","firstName":"Newton","middleName":"","lastName":"Kalata","suffix":""},{"id":437462914,"identity":"0cf99e79-ffc8-4d88-b1f1-d4d8c388e2ea","order_by":3,"name":"Tinashe Nyazika","email":"","orcid":"","institution":"Department of Biomedical Sciences and Physiology, School of Life Sciences, Faculty of Science and Engineering, University of Wolverhampton","correspondingAuthor":false,"prefix":"","firstName":"Tinashe","middleName":"","lastName":"Nyazika","suffix":""},{"id":437462915,"identity":"0c7d22ea-10c8-4205-a88f-9e00faa76677","order_by":4,"name":"Ndaona Mitole","email":"","orcid":"","institution":"Malawi-Liverpool Wellcome Research 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Programme","correspondingAuthor":false,"prefix":"","firstName":"Aku","middleName":"","lastName":"Kalizang'Oma","suffix":""},{"id":437462922,"identity":"4d394606-e68c-423c-a66f-8c6b9718782e","order_by":11,"name":"Todd Swarthout","email":"","orcid":"","institution":"Research Department of Infection, Division of Infection and Immunity, University College London","correspondingAuthor":false,"prefix":"","firstName":"Todd","middleName":"","lastName":"Swarthout","suffix":""},{"id":437462923,"identity":"51b76010-9944-4a28-bae8-f70c7dc4c819","order_by":12,"name":"Ken Malisita","email":"","orcid":"","institution":"Lighthouse-Queens Elizabeth Hospital and Gateway Health Centre","correspondingAuthor":false,"prefix":"","firstName":"Ken","middleName":"","lastName":"Malisita","suffix":""},{"id":437462924,"identity":"a22dd38d-b355-421a-98b7-a544e97df39a","order_by":13,"name":"Arox Kamng'ona","email":"","orcid":"https://orcid.org/0000-0002-0841-7586","institution":"Kamuzu University of Health Sciences","correspondingAuthor":false,"prefix":"","firstName":"Arox","middleName":"","lastName":"Kamng'ona","suffix":""},{"id":437462925,"identity":"0eb002b6-3a1e-4888-8050-01dd9d3a6983","order_by":14,"name":"Robert Heyderman","email":"","orcid":"https://orcid.org/0000-0003-4573-449X","institution":"University College","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"Heyderman","suffix":""},{"id":437462926,"identity":"c9e63938-0ad3-4eff-82ef-4a147481c470","order_by":15,"name":"Stephen Bentley","email":"","orcid":"https://orcid.org/0000-0001-8094-3751","institution":"Wellcome Trust Sanger Institute","correspondingAuthor":false,"prefix":"","firstName":"Stephen","middleName":"","lastName":"Bentley","suffix":""},{"id":437462927,"identity":"dd12285e-3554-4d16-bfe7-715b02227ef3","order_by":16,"name":"Brenda Kwambana-Adams","email":"","orcid":"","institution":"Malawi-Liverpool Wellcome Research Programme","correspondingAuthor":false,"prefix":"","firstName":"Brenda","middleName":"","lastName":"Kwambana-Adams","suffix":""},{"id":437462928,"identity":"eb3c6d32-b298-41f2-ac19-01aef10419da","order_by":17,"name":"Chrispin Chaguza","email":"","orcid":"","institution":"Department of Epidemiology of Microbial Diseases, Yale School of Public Health, Yale University","correspondingAuthor":false,"prefix":"","firstName":"Chrispin","middleName":"","lastName":"Chaguza","suffix":""},{"id":437462929,"identity":"d1ea12cf-4b9b-437a-9d9e-aee7a1fdd3fe","order_by":18,"name":"kondwani Jambo","email":"","orcid":"https://orcid.org/0000-0002-3195-2210","institution":"Liverpool School of Tropical Medicine","correspondingAuthor":false,"prefix":"","firstName":"kondwani","middleName":"","lastName":"Jambo","suffix":""}],"badges":[],"createdAt":"2025-03-26 15:06:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6313580/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6313580/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-63974-2","type":"published","date":"2025-10-07T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79813587,"identity":"d5bffc15-435b-4765-9123-3273d125de6f","added_by":"auto","created_at":"2025-04-03 07:10:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":230213,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePneumococcal carriage duration, density and sampling timeline.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Pneumococcal carriage durations of PCV13 serotypes among all carriage episodes in the study \u003ca href=\"https://paperpile.com/c/6yJ44z/gBBob\"\u003e\u003csup\u003e24\u003c/sup\u003e\u003c/a\u003e. Highlighted in light red is the persistent carriage episode. We only show phenotypic serotypes included in the PCV13 due to the limitation of the phenotypic serotyping kit. B) Pneumococcal carriage density for the persistent carriage episode (highlighted in light in Fig 1A) over time. C) Sequenced samples for the persistent carriage episode (highlighted in light in Fig 1A) that were used in the final analysis stratified by sequencing type (Whole plate sweep and single colony derived) and day of sample collection (Created with \u003cu\u003ewww.\u003c/u\u003e\u003cu\u003e\u003cem\u003eBioRender.com\u003c/em\u003e\u003c/u\u003e).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6313580/v1/e60a556a8b88d489b013abde.png"},{"id":79812644,"identity":"d5c8fe93-f7f0-49bc-a658-02a0fd88c314","added_by":"auto","created_at":"2025-04-03 07:02:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":87992,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlacement of the ST18362 serotype 3 isolates in the global pneumococcal phylogeny.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) 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 clade 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 novel 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’s. The white colour indicates that the serotype was not defined in the GPS database\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6313580/v1/64c717ce39f2d09d004fb2e0.png"},{"id":79812643,"identity":"d462b5f2-63cc-4a64-9f7d-7a84912b3aaf","added_by":"auto","created_at":"2025-04-03 07:02:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":221825,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntra-host evolution and pan-genome analysis of the novel ST18362 serotype 3. \u003c/strong\u003eA) Summary of single nucleotide substitutions on 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\u003cstrong\u003e \u003c/strong\u003estratified by the day of sample collection.\u003cstrong\u003e \u003c/strong\u003eGene absence indicates deletions of genes over time.\u003cstrong\u003e \u003c/strong\u003eE) Representative plot of the enrolment sample and first sample with gene deletions. The gene deletions were observed within the Tn\u003cem\u003e5253\u003c/em\u003e-like Integrative Conjugative Element (ICE) (defective chloramphenicol and tetracycline resistance-conferring element \u003ca href=\"https://paperpile.com/c/6yJ44z/bV9qb\"\u003e\u003csup\u003e38\u003c/sup\u003e\u003c/a\u003e) and between complete copies of the genes coding for a toxin-antitoxin system (\u003cem\u003epezAT\u003c/em\u003e). Tn5253 (GenBank: EU351020.1) was used as a reference to annotate the ICE and visualise it by easyfig.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6313580/v1/8eecc6b77d30781457b9eaa5.png"},{"id":79812357,"identity":"89051731-23e2-44cd-977b-c42f2d158022","added_by":"auto","created_at":"2025-04-03 06:54:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":93646,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerotype 3 capsular locus phylogeny and illustrations of serotype 3 capsular locus in the context of ST18362.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Global phylogeny of pneumococcal serotype 3 cps locus based on GPS publicly available genomes, overlaid by pneumococcal lineage (GPSC). Highlighting in red is the novel 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.\u003cem\u003e \u003c/em\u003eB) Representative plot of three serotype 3 cps locus; a publicly available reference sequence (CR931634), representative sample from the current study (GPSC10-ST18362) and closely related GPSC10-ST700.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6313580/v1/02684f0dfcde0ef56b43493a.png"},{"id":79813588,"identity":"41829ab7-f08e-46bd-927a-47260c2efd94","added_by":"auto","created_at":"2025-04-03 07:10:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":142392,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIllustration of multiple lineage and serotype carriage.\u003c/strong\u003e Representative plot of multiple lineage and serotype carriage during the persistent carriage episode of ST18362 stratified by antimicrobial resistant genes (AMR genes) and \u003cem\u003ein silico\u003c/em\u003e antimicrobial resistant profile of penicillin minimum inhibitory concentration (penMIC) and trimethoprim/sulfamethoxazole (co-trimoxazole). Numbers under the “GPSC \u0026amp; 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 (Created with \u003ca href=\"http://www.biorender.com/\"\u003ewww.\u003c/a\u003e\u003cu\u003e\u003cem\u003eBioRender.com\u003c/em\u003e\u003c/u\u003e\u003cem\u003e).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6313580/v1/47e3a70177bd6fab2ac01027.png"},{"id":79812647,"identity":"b464f12a-6298-4cd4-ac88-7044294c0a74","added_by":"auto","created_at":"2025-04-03 07:02:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":376628,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenic and intergenic Intrahost single nucleotide variants of the novel ST18362 per kilobase. \u003c/strong\u003eA) Genic number of intrahost single nucleotide variants per 1000 nucleotides (kilobase) 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).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6313580/v1/691cec7d6476f1f3f29204c9.png"},{"id":93009911,"identity":"e7bb482d-39d8-43d1-99c0-4e06ceb86b4f","added_by":"auto","created_at":"2025-10-08 07:09:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2013299,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6313580/v1/fd770f54-12b5-470f-b970-099b2ff13890.pdf"},{"id":79812360,"identity":"846ee32b-b988-4032-9c36-cf58149b87d5","added_by":"auto","created_at":"2025-04-03 06:54:36","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":741682,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6313580/v1/7ea99ab3ed7efea6686dd474.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Within-host pneumococcal serotype 3 genetic diversity and evolution during a one-year prolonged carriage episode in a healthy adult","fulltext":[{"header":"Introduction","content":"\u003cp\u003eClinically relevant bacterial species have small, haploid genomes and exhibit low mutation rates \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. However, due to their large population sizes and rapid growth, even these low mutation rates can lead to significant genetic diversity during infections \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Additional diversity is generated through mechanisms such as hypermutation, recombination, and integrating mobile genetic elements \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Single-nucleotide variant mutations and small indels are commonly identified as the primary drivers of within-host evolution in bacterial populations, whereas recombination events and mobile genetic element insertions occur less frequently but are still significant \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eS. pneumoniae\u003c/em\u003e, base substitutions occur approximately once every 15 weeks, while recombination, though less frequent, introduces large-scale genetic changes with an average of 72 SNPs per event \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. This enhanced genomic plasticity facilitates adaptation to antibiotics and vaccine escape, demonstrating the pathogen\u0026rsquo;s capacity for rapid evolution in response to selective pressures. In other respiratory pathogens, such as \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e and SARS-CoV-2, prolonged infection drives within-host evolution resulting in more virulent strains \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLongitudinal carriage studies are important in elucidating potential factors that may explain the dynamics of pneumococcal colonisation, such as the carriage duration, colonisation density, multiple carriage, and clone succession driven by the within-host bacterial evolution. Understandably, most studies on pneumococcal carriage dynamics have primarily focused on children due to the reported high invasive disease burden and carriage prevalence \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. However, the old paradigm of short pneumococcal carriage durations in adults \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e appears to be shifting, with a recent longitudinal study showing carriage durations of up to 4 months in adults \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Such prolonged pneumococcal carriage in adults may constitute reservoirs for continued transmission and circulation of serotypes, especially those less targeted by the PCVs \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, in high carriage prevalence and disease-burdened settings.\u003c/p\u003e \u003cp\u003eAdult pneumococcal carriage has historically been considered transient, though persistent carriage prevalence of vaccine-escape pneumococcal serotype 3 has been reported \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Pneumococcal serotype 3 is included in PCV13 but evades host antibody-mediated clearance \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Its capsular polysaccharide's distinctive structure and electrochemical properties significantly impede opsonophagocytosis \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, an essential anti-bacterial mechanism against pneumococcus. Moreover, pneumococcal serotype 3 is associated with a case fatality rate of 30\u0026ndash;47% in critical cases \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The persistence and adaptability of serotype 3, even post-vaccine introduction, highlight serious concerns about its clonal evolution and vaccine escape.\u003c/p\u003e \u003cp\u003eTherefore, we investigated whether prolonged nasopharyngeal serotype 3 \u003cem\u003eS. pneumoniae\u003c/em\u003e carriage would promote genomic diversification or the emergence of antimicrobial-resistance in an adult with long-term carriage. We used samples from an individual recruited under an adult longitudinal pneumococcal carriage study\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, who had cultured-confirmed pneumococcal serotype 3 isolates in all his longitudinal samples, spanning 335 days. We conducted phenotypic and genomic analysis across all the longitudinal samples to ascertain prolonged carriage and assess within-host genetic diversity and evolution.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIdentification of pneumococcal serotype 3 isolates\u003c/p\u003e \u003cp\u003eHigh pneumococcal carriage prevalence and prolonged episodes have been shown to provide a reservoir for potential genetic exchange and the development of AMR \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Global surveillance has demonstrated persistent pneumococcal carriage duration of an upper bound of ~\u0026thinsp;258.3 days in children and adults (Supplementary Table\u0026nbsp;1). In an adult longitudinal pneumococcal carriage study conducted in Malawi \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, The median carriage duration of phenotypic serotype 3 was 29 days (95% CI 8\u0026ndash;51) among 27 episodes (n\u0026thinsp;=\u0026thinsp;21) in the study (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). However, in samples collected from a healthy female PCV-unvaccinated 30-year-old HIV-uninfected adult (participants\u0026rsquo; characteristics are detailed in Supplementary Table\u0026nbsp;2), we persistently identified the presence of pneumococcus serotype 3 for approximately one year (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Moreover, this pneumococcal carriage episode duration of approximately a year is longer than any previously reported serotype 3 carriage durations, with an upper bound of ~\u0026thinsp;100 days among children and adults (Supplementary Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further observed fluctuations of pneumococcal density during the presumed prolonged pneumococcal carriage episode, with a median carriage density of 33,500 CFU/ml (range from 1,340 CFU/ml to 36,850,000 CFU/ml) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Together this observation suggest potential adaption and evolution of the bacteria within the adult host environment. Therefore, we performed both whole plate sweep and single colony-derived sequencing to understand the potential within-host adaptation and evolution associated with this presumed prolonged carriage. Fourteen whole plate sweep samples were sequenced and used in the final analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In addition, we managed to sequence thirteen single colony-derived isolates. Out of the thirteen samples sequenced, eleven passed single colony quality control and were used in the subsequent analysis detailed below (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eGenotypic characterisation of persistently carried pneumococcal serotype 3 isolates\u003c/p\u003e \u003cp\u003eGenomic data availability has allowed pneumococcus to be clustered based on their genetic background, notably the Global Pneumococcal Sequencing Cluster approach (GPSC) using POPulation Partitioning Using Nucleotide Kmers (PopPUNK) \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e and Public databases for molecular typing and microbial genome diversity (PubMLST) \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. These clusters are essential in determining pneumococcal virulence, invasiveness, and potential vaccine escape \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Using \u003cem\u003ein silico\u003c/em\u003e serotyping tools, the persistently carried isolates were confirmed as serotype 3. Furthermore, using the GPSC PopPUNK \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, it was established that all the isolates belonged to GPSC10. Moreover, in a global phylogeny of serotype 3, the persistently carried isolates were closely related to lineage GPSC10 and multilocus sequence type 700 (ST700)\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, circulating only in Africa based on the Global Pneumococcal Sequencing database (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). GPSC10 (CC230) is a recombinogenic lineage that is globally spreading and capable of expressing multiple serotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Furthermore, using PubMLST \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e these isolates were clustered as novel sequence type 18362 (ST18362) with a single locus variant (SLV) to GPSC10-ST700. These data confirm prolonged pneumococcal carriage with a novel serotype 3 sub-lineage that belongs to a highly diverse and recombinant lineage circulating in Africa.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAntimicrobial resistance profiling of GPSC10-ST18362 pneumococcal serotype 3\u003c/p\u003e \u003cp\u003ePersistent bacterial carriage plays a crucial role in maintaining pathogen populations, allowing extended exposure to selective pressures that contribute to antimicrobial resistance \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, promoting their evolutionary success \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. We, therefore, hypothesised that prolonged serotype 3 carriage creates an evolutionary advantage for the development and retention of AMR, enabling persistence within hosts. We demonstrated that all isolates, even though susceptible to ceftriaxone and erythromycin, were resistant to tetracycline, oxacillin, and cotrimoxazole and had reduced susceptibility to benzylpenicillin, and hence were classified as multi-drug resistant (MDR) (Supplementary Fig.\u0026nbsp;1A). Moreover, based on the Global Pneumococcal Sequence database among the dominant circulating serotype 3 lineages (GPSC12, GPSC51, GPSC10, GPSC83 and GPSC43), only GPSC10-serotype 3 were shown to be non-susceptible to penicillin and amoxicillin using the CDC pipeline \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e MIC prediction (Supplementary Fig.\u0026nbsp;1B). In addition, all GPSC10-serotype 3 from the GPS database were tetracycline and cotrimoxazole resistant, hence classified as MDR. Together, this suggests a potential genomic and phenotypic interplay between prolonged carriage and AMR, which could help promote bacterial persistence and resistance within the host.\u003c/p\u003e \u003cp\u003eWithin-host genetic evolution of GPSC10-ST18362 pneumococcal serotype 3 during prolonged carriage\u003c/p\u003e \u003cp\u003e \u003cem\u003eS. pneumoniae\u003c/em\u003e, a pathogen capable of significant genomic changes, can persist within host environments through mutations, recombination, and genome rearrangement \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. We therefore hypothesised that prolonged carriage would be associated with within-host genomic changes that support its continued persistence. We observed no evidence of recombination (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), and we confirmed it by running Genealogies Unbiased By recomBinations In Nucleotide Sequences (Gubbins) \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. However, we observed a single nucleotide polymorphism (SNP) distance among all the isolates ranging from 2 to 11 SNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). This nucleotide substitution rate is higher than the previously reported nucleotide substitution per year \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Furthermore, high mutations were observed in genes that encode penicillin-binding protein 2x (PBP2x) and pneumococcal surface protein A (\u003cem\u003epspA\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Pneumococcal surface protein A was associated with a high abundance of non-synonymous amino acid changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further assessed potential genome rearrangement using a Bacterial Pan Genome Analysis Pipeline (Panaroo pipeline) \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. We observed gene deletions over time, with the majority of genes deleted conferring to hypothetical proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). However, we also observed the deletion of genes that conferred to ATP-binding cassette transporters (\u003cem\u003eyusV, yfhA, fepD_2, yhfQ\u003c/em\u003e), Antitoxin (\u003cem\u003epezA\u003c/em\u003e), and Toxin (\u003cem\u003epezT\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). In addition, these genes were present within the Tn\u003cem\u003e5253\u003c/em\u003e-like ICE (defective chloramphenicol and tetracycline resistance-conferring element \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e) and between complete copies of the genes coding for a toxin-antitoxin system (\u003cem\u003epezAT\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The \u003cem\u003epezAT\u003c/em\u003e system is known to stabilise the mobile elements within the pneumococcal host \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Deletion of a single pair of \u003cem\u003epezAT\u003c/em\u003e operons has been associated with resilience to lysis, enhanced genetic competence, and resistance to Beta-Lactam (β-lactams) antibiotics \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Together, these findings reveal evidence of adaptive evolution through nucleotide substitutions, significant non-synonymous mutations, and targeted gene loss.\u003c/p\u003e \u003cp\u003ePresence of capsular locus and virulence genes associated with adaptation in GPSC10-ST18362 pneumococcal serotype 3\u003c/p\u003e \u003cp\u003eThe pneumococcal capsule is a major contributor to \u003cem\u003eS. pneumoniae\u003c/em\u003e virulence and vaccine escape \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Pneumococcal lineage recombination affects carriage duration and pneumococcal capsule size \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Recent data from Malawi shows a vaccine escape serotype 3 GPSC10-ST700, with a distinct CPS locus deletion associated with increased antimicrobial resistance and lower susceptibility to opsonophagocytic killing \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The GPSC10-ST18362 differs from GPSC10-ST700 by a single variant but shares a similar antimicrobial resistance profile. We, therefore, hypothesised that the persistently carried GPSC10-ST18362 would exhibit similar CPS locus deletions that could support its adaptation potential. Using a capsular locus phylogeny, we assessed the capsular locus similarity with global pneumococcal serotype 3. We observed that the capsular locus of GPSC10-ST18362 clustered with GPSC10 serotype 3 capsules, and each major lineage had a distinct capsular locus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Moreover, GPSC10-ST18362 exhibited a capsular deletion of the \u003cem\u003ewzg\u003c/em\u003e, \u003cem\u003ewzh\u003c/em\u003e, \u003cem\u003ewzd\u003c/em\u003e and \u003cem\u003ewze\u003c/em\u003e genes similar to GPSC10-ST700. In contrast, these deleted genes were present in the global lineage clonal complex 180 (CC180)-GPSC12 or Netherlands \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e-31 or PMEN31 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). PMEN31\u0026rsquo;s cps locus is publicly available as CR931634 \u003csup\u003e43\u003c/sup\u003e and contains the majority of serotype 3\u0026rsquo;s that have been sequenced and analysed to date \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOther virulence genes like \u003cem\u003ecbpD\u003c/em\u003e, \u003cem\u003elytA\u003c/em\u003e, and \u003cem\u003elytC\u003c/em\u003e have been associated with biofilm formation, fratricide, and adaptation \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Notably, using the Mass screening of contigs for antimicrobial resistance or virulence genes (ABRicate) virulence factor database (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eVFDB\u003c/span\u003e) \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, we observed that GPSC10-ST18362 had a combination of \u003cem\u003ecbpD, cbpG, lytC\u003c/em\u003e, and \u003cem\u003elytA\u003c/em\u003e virulence genes that could be essential in there adaptation, along with \u003cem\u003eply, nanB, nanA, pfbA, psaA, pspA, lytB, pce, and pavA\u003c/em\u003e virulence genes (Supplementary Fig.\u0026nbsp;2). Collectively, the findings show that GPSC10-ST18362 had CPS locus deletions associated with adaption similar to the vaccine escape GPSC10-ST700, as well as possessing a combination of adaptation-related virulence genes.\u003c/p\u003e \u003cp\u003eWithin-host competition and colonisation advantage of GPSC10-ST18362 pneumococcal serotype 3\u003c/p\u003e \u003cp\u003eWithin-host competition among pneumococcal lineages is a key driver of genomic evolution, shaping colonisation dynamics and adaptation \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. During multiple carriage, strains compete for resources and niches, with genetic adaptations and virulence factors influencing colonisation advantage \u003csup\u003e\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. External pressures like antibiotics and vaccines further shape these competitive interactions, which can disrupt co-colonisation and favour lineages with adaptive advantages \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. GPSC10-ST18362 exemplifies this evolutionary process since we have shown that its persistence is linked to lineage-specific genomic traits that support competitive dominance. Genetic factors associated with immune evasion, biofilm formation, and antimicrobial resistance may confer a colonisation advantage, enabling prolonged carriage and increasing opportunities for within-host evolution and horizontal gene transfer \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. We therefore used a whole-plate sweep sequencing approach to investigate transient serotype carriage and co-colonisation dynamics during prolonged carriage of GPSC10-ST18362. We observed transient serotype carriage from other lineages during the follow-up period (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) without impacting the persistence of GPSC10-ST18362. These findings suggest that GPSC10-ST18362 possesses traits that could potentially provide a within-host competitive advantage for colonisation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGenic and intergenic Intrahost single nucleotide variants (iSNVs) of GPSC10-ST18362 pneumococcal serotype 3\u003c/p\u003e \u003cp\u003eWe have established that persistent carriage was associated with within-host competition and genetic changes linked to adaptation. Studies on pneumococcal populations have demonstrated that carriage provides an ideal environment for the emergence of minority variants due to selective pressures within the host, such as immune responses, microbial competition, and antibiotic exposure \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Therefore, we hypothesised that this persistent carriage of serotype 3 GPSC10-ST18362 could also be associated with an increased prevalence of minority variants, representing genomic plasticity and adaptation during the carriage episode. We utilised whole plate sweeps sequencing-based approach to quantify unique intrahost single nucleotide variants (iSNVs). We restricted our analysis to iSNVs found in samples involving only a single pneumococcal lineage (GPSC10) and serotype (pneumococcal serotype 3). We observed high intragenic and intergenic iSNVs in transposase and hypothetical genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Supplementary Table\u0026nbsp;4). However, we also observed iSNVs in genes coding for bacterial metabolism, survival and virulence (\u003cem\u003eadcAII\u003c/em\u003e, zinc-binding lipoprotein AdcAII, \u003cem\u003ephoH\u003c/em\u003e, phosphate starvation-inducible protein PhoH and \u003cem\u003ephtB\u003c/em\u003e, pneumococcal histidine triad protein PhtB), DNA synthesis and repair (\u003cem\u003ecarA\u003c/em\u003e, glutamine-hydrolyzing carbamoyl-phosphate synthase small subunit and \u003cem\u003epcrA\u003c/em\u003e, DNA helicase PcrA) and Oxidative Stress Defense (\u003cem\u003egor\u003c/em\u003e, glutathione-disulfide reductase) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;3A and Supplementary Table\u0026nbsp;4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, we observed Intergenic (upstream gene variants) iSNVs for genes coding for bacterial metabolism and host defense (\u003cem\u003edhaM\u003c/em\u003e, PTS-dependent dihydroxyacetone kinase phosphotransferase subunit DhaM, \u003cem\u003ephoH\u003c/em\u003e, phosphate starvation-inducible protein PhoH and \u003cem\u003ephtE\u003c/em\u003e, pneumococcal histidine triad protein PhtE), antibacterial properties (\u003cem\u003eblpU\u003c/em\u003e, bacteriocin-like peptide BlpU), DNA repair (\u003cem\u003esufD\u003c/em\u003e, Fe-S cluster assembly protein SufD, \u003cem\u003eradC\u003c/em\u003e, DNA repair protein RadC, \u003cem\u003ednaG\u003c/em\u003e, DNA primase and \u003cem\u003eparC\u003c/em\u003e, DNA topoisomerase IV subunit A), ions transport (\u003cem\u003epstA\u003c/em\u003e, phosphate ABC transporter permease PstA), bacterial protein synthesis (\u003cem\u003eylqF\u003c/em\u003e, ribosome biogenesis GTPase YlqF and \u003cem\u003emetG\u003c/em\u003e, methionine\u0026ndash;tRNA ligase), and Peptidoglycan Biosynthesis (\u003cem\u003eglmU\u003c/em\u003e, bifunctional UDP-N-acetylglucosamine diphosphorylase/glucosamine-1-phosphate N-acetyltransferase GlmU) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, Supplementary Fig.\u0026nbsp;3B and Supplementary Table\u0026nbsp;4). Our findings suggest that the adaptational evolution and within-host competition during this episode resulted in a variable accumulation of potentially advantageous substitutions.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe have demonstrated persistent carriage of a GPSC10 pneumococcal serotype 3 novel sequence type 18362, which was associated with multi-drug resistance within a healthy adult in Malawi. These data shed light on the within-host complex interplay of recombinant bacterial adaptation, virulence, and antimicrobial resistance.\u003c/p\u003e \u003cp\u003ePneumococcal serotype 3 is associated with severe clinical outcomes, including adverse cardiovascular events and higher mortality rates in adults \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. The inclusion of pneumococcal serotype 3 in high-valent pneumococcal conjugate vaccines has been less effective in reducing serotype 3 disease burden compared to other vaccine-included serotypes, as evidenced by persistent serotype 3-associated parapneumonic pleural effusion/empyema, ongoing vaccine evasion, and serotype replacement in adult pneumonia cases \u003csup\u003e\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e–\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e and overall circulation in the community \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Serotype 3 capsule is not covalently linked to peptidoglycan and, in turn, facilitates the shedding of serotype 3 capsule, which in turn interferes with bacterial clearance \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Moreover, serotype 3 has the lowest surface charge (zeta potential), which plays a crucial role in evading the immune system by reducing phagocytosis and complement deposition \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In line with these findings, we identified pneumococcal carriage of serotype 3 for approximately one year in a healthy adult, indicative of poor clearance \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe spread of the MDR lineage of GPSC10 serotype 24F has been associated with disease in France \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, highlighting the expansion potential of GPSC10 lineages. In a global context, the persistent carriage isolates ST18362 clustered with GPSC10. Based on the GPS database, GPSC10 serotype 3 was solely from Africa, suggesting a clonal dissemination within the continent. Moreover, the ST18362 isolates were multi-drug resistant, typical of GPSC10 lineage strains \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. ST18362 had a single locus variant to GPSC10-ST700. Moreover, the identification of new and emerging variations at the single nucleotide level among serotype 3 has been highlighted as essential in surveillance due to clade expansion \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Consequently, prolonged carriage durations of lineages of global concern, such as GPSC10, provide ample time for clonal dissemination and seeding future outbreaks.\u003c/p\u003e \u003cp\u003ePneumococci colonisation success is determined by a complex competitive interaction from both other bacterial species \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e and other pneumococcal lineages \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Several factors have been associated with colonisation success or failure, including capsular thickness \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, positive carriage status \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e and competent cells fratricide \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Competent cell fratricide is mediated by bacteriocins \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, as well as a combination of a putative murein hydrolase \u003cem\u003ecbpD\u003c/em\u003e, autolysin \u003cem\u003elytA\u003c/em\u003e, and cell wall hydrolase \u003cem\u003elytC\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. We have demonstrated that this persistent ST18362 carriage episode was associated with transient pneumococcal lineages, which did not out-compete its dominance. Moreover, a combination of a putative murein hydrolase \u003cem\u003ecbpD\u003c/em\u003e, autolysin \u003cem\u003elytA\u003c/em\u003e, and cell wall hydrolase \u003cem\u003elytC\u003c/em\u003e were present in ST18362. Together, these findings suggest that ST18362 has the potential to withstand within-host competition from other pneumococcal strains during the carriage episode.\u003c/p\u003e \u003cp\u003eA copy of the \u003cem\u003epezAT\u003c/em\u003e operon is associated with stabilising mobile elements, moreover, they have been discovered within a putative pneumococcal integrative and conjugative element (ICE) \u003cem\u003eTn5253\u003c/em\u003e \u003csup\u003e\u003cem\u003e39,40\u003c/em\u003e\u003c/sup\u003e. In concordance with these results, we observed two copies of the \u003cem\u003epezAT\u003c/em\u003e operon within the \u003cem\u003eTn5253\u003c/em\u003e-like ICE (defective chloramphenicol and tetracycline resistance-conferring element \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e). However, the disruption of \u003cem\u003epezAT\u003c/em\u003e has been shown to impact virulence \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Moreover, the loss of a single copy of \u003cem\u003epezAT\u003c/em\u003e in pneumococci has been associated with increased changes in the cell wall biosynthesis in that the mutants form shorter chains during the exponential phase, leading to increased colony-forming units \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Furthermore, the mutants became more resilient to lysis, enhanced their acquisition of transforming DNA, and became more resistant to antibiotics targeting the cell wall, mainly β-lactam antibiotics \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. We observed the loss of a single pair of \u003cem\u003epezAT\u003c/em\u003e over time during the carriage episode, which could suggest a potential adaptation mechanism of GPSC10-ST18362.\u003c/p\u003e \u003cp\u003ePneumococcal serotype 3 lineages exhibit substantial genetic diversity, with evidence of frequent horizontal gene transfer and genomic variability, as observed in multiple studies\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Prolonged carriage durations have been associated with increased genetic alterations and adaptive changes\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. We observed genome reduction and higher mutation rates than previously reported within a year \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Specifically, we observed high mutation in genes that encode penicillin-binding protein and pneumococcal surface proteins, which have been associated with pneumococcal adaptation\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. We further observed genic and intergenic minority variants associated with adaptation from the prolonged carriage episode. Collectively, these data demonstrate potential mutations that could have supported the adaptation of GPSC10-ST18362 within the individual.\u003c/p\u003e \u003cp\u003eDespite a comprehensive analysis of persistent within-host pneumococcal carriage of a serotype and lineage of global concern, the study has some limitations. Firstly, we performed broad sequencing, not deep sequencing; hence, we might have potentially underestimated within-host minority variants and multiple carriage events. However, Serocall \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e is robust in detecting multiple pneumococcal serotypes, even at low frequencies. Secondly, we could not fully decipher the genomic variants over time in this current analysis due to the lack of a complete publicly available reference genome for GPSC10 serotype 3. However, the utilisation of both a GPSC10 serotype 24 complete genome reference and capsular locus of GPSC-ST700 together with CR931634 \u003csup\u003e43\u003c/sup\u003e as references mitigates this challenge.\u003c/p\u003e \u003cp\u003eOur study has demonstrated the persistent carriage of pneumococcal serotype 3 (GPSC10-ST18362) with a novel sequence type within a healthy adult, revealing its association with multidrug resistance and within-host adaptive mechanisms. The findings indicate that GPSC10 serotype 3 lineages can be carried for extended periods, which may facilitate their global spread and provide ample opportunities to acquire diverse serotype cps.\u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003eStudy design and recruitment\u003c/p\u003e\u003cp\u003eNasopharyngeal samples were collected from asymptomatic adults in Blantyre, Malawi \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In brief, the study participants were recruited 3 days after screening and then followed up on days 7, 14, 21, and 28 for the first month, and then every month for 12 months. All participants were recruited from the ART clinics and Voluntary Counselling and Testing (VCT) centres at Lighthouse-Queen Elizabeth Central Hospital and Gateway Health Centre in Blantyre. Participants were screened for pneumococcal carriage, using WHO-recommended nasopharyngeal sampling and microbiological culture methods for pneumococcal detection\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Inclusion criteria included confirmed pneumococcal carriage in adults aged 18 to 45 years, living with a child under 5 years old, and providing written informed consent.Ethical approvals\u003c/p\u003e\u003cp\u003e The study was conducted following good clinical practice (GCP) guidelines and the Declaration of Helsinki. Ethical approval was obtained from the College of Medicine Research Ethics Committee (COMREC) (P.11/18/2532) and Liverpool School of Tropical Medicine Research Ethics Committee (LSTMREC) (19–033).\u003c/p\u003e\u003cp\u003eMicrobiological culture and density quantification\u003c/p\u003e\u003cp\u003eStandard microbiological culture was used to determine the presence of \u003cem\u003eS. pneumoniae\u003c/em\u003e from the nasopharyngeal swab \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. We identified \u003cem\u003eS. pneumoniae\u003c/em\u003e by their morphology and optochin sensitivity. The bile solubility test was used on isolates with no or intermediate (zone diameter \u0026lt; 14mm) optochin susceptibility \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Plates showing no \u003cem\u003eS. pneumoniae\u003c/em\u003e growth were incubated for a further 24 hours before being reported as negative. A single colony of confirmed pneumococcus was selected and grown on a new SBG plate. Growth from this second plate was used for serotyping by latex agglutination (ImmuLexTM 23-valent Pneumotest; Statens Serum Institut, Denmark). Pneumococcal density was quantified using microbiological culture serial dilutions \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e on a gentamicin-sheep blood agar plate (SBG; 5% sheep blood agar, 5µL gentamicin/mL), and results were reported as colony forming units per millilitre (CFU/ml).\u003c/p\u003e\u003cp\u003eAntimicrobial resistance profiling\u003c/p\u003e\u003cp\u003eAntimicrobial susceptibility of pneumococcal isolates was assessed by the disk diffusion method (Oxoid, USA) for beta-lactams antibiotic (oxacillin 1µg), MLS\u003csub\u003eB\u003c/sub\u003e antibiotic (erythromycin 15µg), tetracycline antibiotic (tetracycline 30µg) and Trimethoprim/sulfamethoxazole (co-trimoxazole 1.25–23.75µg).\u003c/p\u003e\u003cp\u003eBeta-lactam antibiotic susceptibility was confirmed by benzylpenicillin Etest (bioMérieux, Marcy-I’Étoile, France) minimum inhibitory concentrations (MICs). A Ceftriaxone Etest was also performed when oxacillin 1µg zone diameter \u0026lt; 9 mm according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. Etest strips were applied to the surface of the plates according to the manufacturer’s recommendations. The MIC was defined as the lowest concentration of the drug where the zone edge intersected the Etest strip.\u003c/p\u003e\u003cp\u003eInterpretation of results followed EUCAST guidelines for meningitis breakpoints \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eS. pneumoniae\u003c/em\u003e ATCC 49619 was used as a quality control strain. MDR was defined as non-susceptibility to agents in three or more chemical classes of antibiotics \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e,\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDNA extraction and quantification\u003c/p\u003e\u003cp\u003eFor single isolate culture specimens, 10µl of stored pneumococcal isolates were plated onto Columbia CNA agar containing 5% sheep blood (CBA). These were then incubated overnight at 37 ± 2°C with 5% CO\u003csub\u003e2\u003c/sub\u003e \u003csup\u003e65\u003c/sup\u003e. All growth was collected from the plate using 10µl sterile plastic loops into an Eppendorf tube ready for DNA extraction. For plate sweep culture specimens, 100µl of stored respiratory samples (nasopharyngeal swab in STGG with known positive for pneumococcus determined by standard microbiological culture \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e) were plated onto CBA. These were then incubated overnight at 37 ± 2°C with 5% CO\u003csub\u003e2\u003c/sub\u003e. All growth was collected from the plate using 10µl sterile plastic loops into an Eppendorf tube ready for DNA extraction. QIAamp DNA Mini Kit (Qiagen, Germany) was used for DNA extraction following the manufacturer's instructions. DNA quantity and quality were evaluated by Invitrogen Qubit Fluorometer. Samples that had a total DNA yield of \u0026gt; 2.5µg were submitted for sequencing.\u003c/p\u003e\u003cp\u003eWhole genome sequencing\u003c/p\u003e\u003cp\u003eWhole genome sequencing (WGS) was performed at the Wellcome Sanger Institute on an Illumina NovaSeq 6000 (Illumina-HTP NovaSeq 6000 Paired-end sequencing). Specifically, the sequencing platform was NovaSeq SP, the read length was 150bp, the plex was 384, and the expected reads per sample were 1.8M.\u003c/p\u003e\u003cp\u003eSingle colony analysis\u003c/p\u003e\u003cp\u003eQuality control of single colonies (Appendix - supplementary methods\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e)\u003c/p\u003e\u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eQUAST\u003c/span\u003e (QUality ASsessment Tool) v5.2.0 was used to extract assembly quality metrics. Overall sequencing depth of \u0026gt; 20X \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e, total number of contigs of \u0026lt; 500, and total length of the assembled genome size between 1.9–2.35 Mb were retained. To account for contamination \u0026gt; 90% of reads that mapped to \u003cem\u003eS. pneumoniae\u003c/em\u003e using Kraken metagenomic classification algorithm (v1.0.0) \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e and \u0026gt; 60% mapping coverage of reference genome (PMEN global clone Spain23F-1, accession number FM211187) \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Finally, to account for contamination due to multiple carriage of \u0026gt; 1 pneumococcal strain, the number of heterozygous sites of \u0026lt; 220 or percent of heterozygous sites over the total number of single nucleotide polymorphisms (SNPs) of ≤ 15% were included as previously described\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. SeroCall\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e was performed as a second stage to confirm multiple serotypes.\u003c/p\u003e\u003cp\u003ePhylogeny analysis\u003c/p\u003e\u003cp\u003eGenome de novo assemblies were done by Spades (v3.15.4) \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e, and Genome alignments were done by Burrows-Wheeler Aligner (bwa) (v 0.7.17) \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eS. pneumoniae\u003c/em\u003e strain 475 chromosomes, complete genome, serogroup 24 [GenBank: \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCP046355.1\u003c/span\u003e] as a GPSC10 reference. Gubbins (v3.2.1) \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e was used to detect recombination, and phylogenetic trees were constructed by RAxML (Randomised Axelerated Maximum Likelihood) \u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. We contextualised the episode to global pneumococcal lineages (n = 20,924 genomes) assigned by popPUNK (v2.6.0) \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e and serotype 3 (n = 593 genomes) isolates that passed the above QC from the Global Pneumococcal Sequencing Project database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://data.monocle.sanger.ac.uk/\u003c/span\u003e\u003cspan address=\"https://data.monocle.sanger.ac.uk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e We also included serotype 3 from the parent study (n = 30 genomes) that passed the above QC. The phylogenetic trees overlaid with epidemiological data and \u003cem\u003ein silico\u003c/em\u003e output were visualised using ggtree (v3.8.0) \u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePan-genome analysis\u003c/p\u003e\u003cp\u003eProkka: Rapid prokaryotic genome annotation (v1.14.5) \u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e was used for annotation, and gene clustering analysis was done by panaroo (v1.3.3) \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e and visualised by an interactive viewer for populations of bacterial genomes linked by a phylogeny (phandango) \u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. Genome lineage clustering was done by assigning GPSC using PopPUNK (v2.6.0) \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Sequence typing was done by MLSTcheck (v2.23.0) and serotyping by a k-mer-based Pipeline to identify the Serotype from Illumina NGS reads (SeroBA) (v1.0.2) \u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. Detection of recombination was done by Gubbins (v3.2.1) \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, detection of pairwise snp distance was done by a Pairwise SNP distance matrix from a FASTA sequence alignment (snp-dist) (v0.7.0) \u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e and summary of SNPs relative to a first sample as reference sequence using \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003esnipit\u003c/span\u003e: summarise snps relative to your reference sequence (v1.1.2) \u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e. Detection and annotation of majority variants were done by a Bayesian haplotype-based genetic polymorphism discovery and genotyping tool (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003efreebayes\u003c/span\u003e) and genetic variant annotation, and functional effect prediction toolbox (SnpEff) (v5.2) \u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e respectively within a rapid haploid variant calling and core genome alignment tool (snippy) (v4.6.0) \u003csup\u003e\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e. Resistance profiling was done by Mass screening of contigs for antimicrobial resistance or virulence genes (ABRicate v1.0.1) \u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e, the comprehensive antibiotic resistance (CARD) database \u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e and the \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePathogen Watch\u003c/span\u003e CDC pipeline \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Virulence genes profiling was done using the ABRicate virulence factor database (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eVFDB\u003c/span\u003e) and blast using diverse \u003cem\u003epspA\u003c/em\u003e sequences as reference\u003csup\u003e\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eCapsular locus analysis\u003c/p\u003e\u003cp\u003eGenome mapping was done by Burrows-Wheeler Aligner (BWA) (v0.7.17-r1188) \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. Serotype 3 capsular encoding region capsular locus reference used was NCBI accession: CR931634 \u003csup\u003e43\u003c/sup\u003e. Tree constructed by RAxML \u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. The phylogenetic trees were visualised using ggtree (v3.8.0) \u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. We extracted and annotated the capsular locus of serotype 3 in \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGeneious Prime\u003c/span\u003e and visualised it by easyfig (v2.2.2) \u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMobile genetic element analysis\u003c/p\u003e\u003cp\u003eWe assessed the presence of MGE in all isolates from this persistent carriage using \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eICEfinder\u003c/span\u003e and confirmed the element by annotating it using \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGeneious Prime\u003c/span\u003e and \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eArtemis\u003c/span\u003e. Tn5253 (GenBank: EU351020.1) was used as a reference to annotate the mobile genetic element and the final illustration visualised by easyfig (v2.2.2) \u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePlate sweep analysis\u003c/p\u003e\u003cp\u003eLineage deconvolution\u003c/p\u003e\u003cp\u003eAll pneumococcal genomes that passed quality control (20,924 genomes) from the Global Pneumococcal Sequencing Project database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.pneumogen.net/gps/\u003c/span\u003e\u003cspan address=\"https://www.pneumogen.net/gps/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e were included as a reference for lineage deconvolution. PopPUNK (v2.6.0) \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e was used to reassign lineages to these reference genomes. Using a statistical mixture model, the mSWEEP (v2.0.0) \u003csup\u003e\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e algorithm used read pseudo-alignments output from Themisto (v2.1.0) \u003csup\u003e\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e to quickly estimate the abundance of lineages based on the reference within a mixed sample. mGEM (v1.3.3) \u003csup\u003e\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u003c/sup\u003e algorithm used the resulting likelihood estimates output from mSWEEP to deconvolute the mixed reads into groups based on the lineages. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eShovill\u003c/span\u003e (v1.1.0)was used for the assembly of binned reads.\u003c/p\u003e\u003cp\u003eTo reduce the possibility of false positives, lineages were only called if they were present at a relative abundance of greater than 1% (0.01), as described previously \u003csup\u003e\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u003c/sup\u003e. The quality control tool \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003edemix_check\u003c/span\u003e was run on the resulting lineage-level bins, and high confidence scores of 1 and 2 were retained as reliable identification.\u003c/p\u003e\u003cp\u003eSerotyping, serotype abundance and antimicrobial-resistant calls\u003c/p\u003e\u003cp\u003eTo determine serotypes, SeroCall \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e was run on raw reads samples, and SeroBA (v1.0.2) \u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e was run on each deconvoluted lineage to confirm untypable serotypes and serotypes at the sub-serogroup level. Resistance calling was done by ABRicate (v1.0.1) \u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e, the comprehensive antibiotic resistance (CARD) database \u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e and \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePathogen Watch\u003c/span\u003e CDC pipeline \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMinority variants call\u003c/p\u003e\u003cp\u003eTo investigate the intrahost evolution, we rigorously employed the LoFreq (v2.1.5) variant calling pipeline on all samples, specifically those with a single identified GPSC lineage and serotype, using mSWEEP (v2.0.0) \u003csup\u003e\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e and SeroCall \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e, respectively. Therefore, plate sweep samples with multiple lineages and serotypes were excluded (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). We included samples with over 89% of reads mapping to \u003cem\u003eS. pneumoniae\u003c/em\u003e using Kraken (v1.0.0) \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e to address contamination (Supplementary Fig.\u0026nbsp;4). Therefore, the final analysis included 64% (9/14) sampling points. \u003cem\u003eS. pneumoniae\u003c/em\u003e strain 475 chromosomes, complete genome, serogroup 24 [GenBank: \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCP046355.1\u003c/span\u003e] as a GPSC10 reference to call minority variants, annotate the variants and compute genome coverage.\u003c/p\u003e\u003cp\u003eReads were aligned to the reference genomes using BWA (v0.7.17-r1188) \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. The Picard tools (v2.23.8) ‘CleanSam’ function was then used to align soft clip reads to the end of contigs and to set the alignment qualities of unaligned reads to zero. The LoFreq pipeline was initially run with a coverage of 20 reads to identify a variant. The resulting variant calls were used along with the read alignment as input to the GATK (Genome Analysis Toolkit Variant Discovery in High-Throughput Sequencing Data) BaseRecalibrator tool (v4.1.9), as suggested in the \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eLoFreq manual\u003c/span\u003e, to improve the estimated base quality scores. Finally, the LoFreq pipeline was run for a final time with a coverage requirement of 20 reads. The resulting genetic variant calls were only considered if their maximum allele frequency was \u0026lt; = 0.5 and minimum was \u0026gt; = 0.15. The minimum allele frequency was based on Adam Lauring’s rule of thumb \u003csup\u003e\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e\u003c/sup\u003e \u003cem\u003e“that the coverage should be 10 times the reciprocal of a variant's frequency”\u003c/em\u003e. The variants were annotated using SnpEff (v5.2) \u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eEach mutation was counted once per gene or locus tag position during the carriage episode using the first sample sequenced as a reference after annotation with \u003cem\u003eS. pneumoniae\u003c/em\u003e strain 475 chromosomes, complete genome, serogroup 24 [GenBank: \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCP046355.1\u003c/span\u003e]. Therefore, only a unique mutation to the first sample sequenced are indicated in the subsequent follow-up days.\u003c/p\u003e\u003cp\u003ePneumococcal colonisation episode definition and data presentation\u003c/p\u003e\u003cp\u003eA pneumococcal colonisation episode was defined by either the first pneumococcal carriage at study screening or the re-acquisition of pneumococci after pneumococcal clearance. Pneumococcal clearance was defined by detecting negative cultures for any serotype at two consecutive sampling points, as previously described \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. R statistical package ggplot2 (v3.4.3) \u003csup\u003e\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e\u003c/sup\u003e, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eInkscape\u003c/span\u003e (open-source vector graphics) and \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eBioRender\u003c/span\u003e (a web-based tool used to create high-quality scientific illustrations) for visualisation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eRaw sequencing data are stored with the NCBI under project code PRJNA1137437, with individual accessions given in Supplementary Table 5 for single colony-derived sequences and Supplementary Table 6 for plate sweep-derived sequences. Annotated minority variants are available in Supplementary Table 4.\u003c/p\u003e\n\u003ch2\u003eCode availability\u003c/h2\u003e\n\u003cp\u003eManuscript code and R scripts that were used to analyse the datasets are available in the GitHub repository https://github.com/Lusako/SPN3_ST18362_persistent_carriage_manuscript\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eRSH and KJ were supported by National Institute for Health Research (NIHR) Global Health Research Unit on Mucosal Pathogens using UK aid from the UK Government [16/136/46] and the Medical Research Council (MRC, UK) [MR/T008822/1] awarded to KCJ. The views expressed in this publication are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. We are grateful for the support of the Sanger Institute core informatics and sequencing teams.\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eConceptualisation: LLS and KCJ; Data curation: LLS; Data analysis: LLS; Funding acquisition: KCJ and RSH; Investigation: NM, AK, MK, GS; Methodology: LLS, CC, AK, KCJ; Project administration: NK, NM; Resources: SWL, SDB, CC; Supervision: KCJ, CC, SWL, SDB, BAK; Validation: CC, SWL, AK; Visualisation: LLS; Writing\u0026mdash;original draft: LLS, CC, KCJ; Writing\u0026mdash;review \u0026amp; editing; LLS, SWL, NK, TKN, NM, VD, AK, MK, GS, JP, AK, TDS, NF, KM, AWK, RSH, BAK, SDB, CC, KCJ.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDettman JR, Sztepanacz JL, Kassen R (2016) The properties of spontaneous mutations in the opportunistic pathogen Pseudomonas aeruginosa. 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Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://play.google.com/store/books/details?id=bes-AAAAQBAJ\u003c/span\u003e\u003cspan address=\"https://play.google.com/store/books/details?id=bes-AAAAQBAJ\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6313580/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6313580/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e can rapidly evolve within hosts through genetic mutations, recombination, and mobile genetic elements, enabling adaptation to antibiotics and immune pressures. Here, we detail a longitudinal phenotypic and genomic analysis of a pneumococcal serotype 3 prolonged carriage episode (\u0026gt;\u0026thinsp;335 days) in a healthy HIV-uninfected adult in Malawi. Whole Genome Sequencing (WGS) of single-colony culture isolates confirmed persistent carriage of a novel multidrug-resistant serotype 3 strain (GPSC10-ST18362), outcompeting other transiently acquired serotypes during the study period. Sequentially sampled isolates showed between 2 to 11 single nucleotide polymorphism (SNP) differences randomly distributed across the genome with no evidence of recombination, but high mutation rates were observed in genes associated with antimicrobial resistance. Further analysis of the sequenced plate sweep samples revealed intrahost single nucleotide variants in several genes associated with survival, including bacterial metabolism, virulence, DNA synthesis and repair, and oxidative stress defence. The study has demonstrated the prolonged carriage of a novel pneumococcal serotype 3 (GPSC10-ST18362) in a healthy adult, revealing its association with multidrug resistance and potential within-host adaptive mechanisms.\u003c/p\u003e","manuscriptTitle":"Within-host pneumococcal serotype 3 genetic diversity and evolution during a one-year prolonged carriage episode in a healthy adult","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-03 06:54:31","doi":"10.21203/rs.3.rs-6313580/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ec6c3d1b-55ab-47b0-9492-d41a3c84e2a3","owner":[],"postedDate":"April 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46581214,"name":"Health sciences/Medical research/Genetics research"},{"id":46581215,"name":"Biological sciences/Genetics/Microbial genetics/Bacterial genetics"}],"tags":[],"updatedAt":"2025-10-08T07:08:48+00:00","versionOfRecord":{"articleIdentity":"rs-6313580","link":"https://doi.org/10.1038/s41467-025-63974-2","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-10-07 04:00:00","publishedOnDateReadable":"October 7th, 2025"},"versionCreatedAt":"2025-04-03 06:54:31","video":"","vorDoi":"10.1038/s41467-025-63974-2","vorDoiUrl":"https://doi.org/10.1038/s41467-025-63974-2","workflowStages":[]},"version":"v1","identity":"rs-6313580","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6313580","identity":"rs-6313580","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}