Invasive pneumococcal disease (IPD) due to non-vaccine serotypes after the introduction of pneumococcal conjugate vaccines (PCV) remains a global concern. This study used pathogen genomics to evaluate changes in invasive pneumococcal lineages before, during and after vaccine introduction in South Africa. We included genomes (N = 3104) of IPD isolates from individuals aged <18 years (2005-20), spanning four periods: pre-PCV, PCV7, early-PCV13, and late-PCV13. Significant incidence reductions occurred among vaccine-type lineages in the late-PCV13 period compared to the pre-PCV period. However, some vaccine-type lineages continued to cause invasive disease and showed increasing effective population size trends in the post-PCV era. A significant increase in lineage diversity was observed from the PCV7 period to the early-PCV13 period (Simpson's diversity index: 0.954, 95% confidence interval 0.948-0.961 vs 0.965, 0.962-0.969) supporting intervention-driven population structure perturbation. Increases in the prevalence of penicillin, erythromycin, and multidrug resistance were observed among non-vaccine serotypes in the late-PCV13 period compared to the pre-PCV period. In this work we highlight the importance of continued genomic surveillance to monitor disease-causing lineages post vaccination to support policy-making and future vaccine designs and considerations.

Streptococcus pneumoniae is a leading cause of pneumonia and meningitis worldwide. Many different serotypes co-circulate endemically in any one location(1,2). The extent and mechanisms of spread and vaccine-driven changes in fitness and antimicrobial resistance remain largely unquantified. Here using geolocated genome sequences from South Africa (n = 6,910, collected from 2000 to 2014), we developed models to reconstruct spread, pairing detailed human mobility data and genomic data. Separately, we estimated the population-level changes in fitness of strains that are included (vaccine type (VT)) and not included (non-vaccine type (NVT)) in pneumococcal conjugate vaccines, first implemented in South Africa in 2009. Differences in strain fitness between those that are and are not resistant to penicillin were also evaluated. We found that pneumococci only become homogenously mixed across South Africa after 50 years of transmission, with the slow spread driven by the focal nature of human mobility. Furthermore, in the years following vaccine implementation, the relative fitness of NVT compared with VT strains increased (relative risk of 1.68; 95% confidence interval of 1.59-1.77), with an increasing proportion of these NVT strains becoming resistant to penicillin. Our findings point to highly entrenched, slow transmission and indicate that initial vaccine-linked decreases in antimicrobial resistance may be transient.

Streptococcus dysgalactiae subsp. equisimilis (SDSE) is an emerging cause of human infection with invasive disease incidence and clinical manifestations comparable to the closely related species, Streptococcus pyogenes. Through systematic genomic analyses of 501 disseminated SDSE strains, we demonstrate extensive overlap between the genomes of SDSE and S. pyogenes. More than 75% of core genes are shared between the two species with one third demonstrating evidence of cross-species recombination. Twenty-five percent of mobile genetic element (MGE) clusters and 16 of 55 SDSE MGE insertion regions were shared across species. Assessing potential cross-protection from leading S. pyogenes vaccine candidates on SDSE, 12/34 preclinical vaccine antigen genes were shown to be present in >99% of isolates of both species. Relevant to possible vaccine evasion, six vaccine candidate genes demonstrated evidence of inter-species recombination. These findings demonstrate previously unappreciated levels of genomic overlap between these closely related pathogens with implications for streptococcal pathobiology, disease surveillance and prevention.

Due to the emergence of non-vaccine serotypes in vaccinated populations, Streptococcus pneumoniae remains a major global health challenge despite advances in vaccine development. Serotype 16F is among the predominant non-vaccine serotypes identified among vaccinated infants in South Africa (SA). To characterize lineages and antimicrobial resistance in 16F isolates obtained from South Africa and place the local findings in a global context, we analysed 10 923 S. pneumoniae carriage isolates obtained from infants recruited as part of a broader SA birth cohort. We inferred serotype, resistance profile for penicillin, chloramphenicol, cotrimoxazole, erythromycin and tetracycline, and global pneumococcal sequence clusters (GPSCs) from genomic data. To ensure global representation, we also included S. pneumoniae carriage and disease isolates from the Global Pneumococcal Sequencing (GPS) project database (n=19 607, collected from 49 countries across 5 continents, 1995-2018, accessed 17 March 2022). Nine per cent (934/10923) of isolates obtained from infants in the Drakenstein community in SA and 2 %(419/19607) of genomes in the GPS dataset were serotype 16F. Serotype 16F isolates were from 28 different lineages of S. pneumoniae, with GPSC33 and GPSC46 having the highest proportion of serotype 16F isolates at 26 % (346/1353) and 53 % (716/1353), respectively. Serotype 16F isolates were identified globally, but most isolates were collected from Africa. GPSC33 was associated with carriage [OR (95 % CI) 0.24 (0.09-0.66); P=0.003], while GPSC46 was associated with disease [OR (95 % CI) 19.9 (2.56-906.50); P=0.0004]. Ten per cent (37/346) and 15 % (53/346) of isolates within GPSC33 had genes associated with resistance to penicillin and co-trimoxazole, respectively, and 18 % (128/716) of isolates within GPSC46 had genes associated with resistance to co-trimoxazole. Resistant isolates formed genetic clusters, which may suggest emerging resistant lineages. Serotype 16F lineages were common in southern Africa. Some of these lineages were associated with disease and resistance to penicillin and cotrimoxazole. We recommend continuous genomic surveillance to determine the long-term impact of serotype 16F lineages on vaccine efficacy and antimicrobial therapy globally. Investing in vaccine strategies that offer protection over a wide range of serotypes/lineages remains essential. This paper contains data hosted by Microreact.

Streptococcus pneumoniae (pneumococcus) is a leading vaccine-preventable cause of childhood invasive disease. Nigeria has the second highest pneumococcal disease burden globally, with an estimated ~49 000 child deaths caused by pneumococcal infections each year. Ten-valent pneumococcal conjugate vaccine (GSK; PCV10) was introduced in December 2014 in a phased approach. However, few studies have characterized the disease-causing pneumococci from Nigeria. This study assessed the prevalence of serotypes, antibiotic susceptibility and genomic lineages using whole genome sequencing and identified lineages that could potentially escape PCV10 (GSK). We also investigated the potential differences in pneumococcal lineage features between children with and without sickle cell disease. A collection of 192 disease-causing pneumococcal isolates was obtained from Kano (n=189) and Abuja (n=3) states, Nigeria, between 1 January 2014 and 31 May 2018. The majority (99 %, 190/192) of specimens were recovered from children aged 5 years or under. Among them, 37 children had confirmed or traits of sickle cell disease. Our findings identified 25 serotypes expressed by 43 Global Pneumococcal Sequence Clusters (GPSCs) and 85 sequence types (STs). The most common serotypes were 14 (18 %, n=35), 6B (16 %, n=31), 1 (9 %, n=17), 5 (9 %, n=17) and 6A (9 %, n=17); all except serotype 6A are included in PCV10 (GSK). PCV10 (SII; PNEUMOSIL) and PCV13 formulations include serotypes 6A and 19A which would increase the overall coverage from 67 % by PCV10 (GSK) to 78 and 82 %, respectively. The pneumococcal lineages were a mix of globally spreading and unique local lineages. Following the use of PCV10 (GSK), GPSC5 expressing serotype 6A, GPSC10 (19A), GPSC26 (12F and 46) and GPSC627 (9L) are non-vaccine type lineages that could persist and potentially expand under vaccine-selective pressure. Approximately half (52 %, 99/192) of the pneumococcal isolates were resistant to the first-line antibiotic penicillin and 44 % (85/192) were multidrug-resistant. Erythromycin resistance was very low (2 %, 3/192). There was no significant difference in clinical manifestation, serotype prevalence or antibiotic resistance between children with and without traits of or confirmed sickle cell disease. In summary, our findings show that a high percentage of the pneumococcal disease were caused by the serotypes that are covered by currently available vaccines. Given the low prevalence of resistance, macrolide antibiotics, such as erythromycin, should be considered as an option to treat pneumococcal disease in Nigeria. However, appropriate use of macrolide antibiotics should be vigilantly monitored to prevent the potential increase in macrolide resistance.

BACKGROUND: In early 2021, the 10-valent Pneumococcal conjugate vaccine (PCV10) was replaced with 13-valent (PCV13) by the federal directorate of immunization (FDI), Pakistan. We assessed the impact of a higher valent vaccine, PCV13, on the serotype distribution of nasopharyngeal carriage in rural Pakistan. METHODS: Children <2 years were randomly selected from two rural union councils of Matiari, Sindh in Pakistan between September-October,2022. Clinical, sociodemographic and vaccination histories were recorded. Nasopharyngeal swabs were collected and processed at Infectious Disease Research Laboratory, Aga Khan University, Karachi. Whole genome sequencing was performed on the culture positive isolates. RESULTS: Of the 200 children enrolled, pneumococcus was detected in 140(70 %) isolates. Majority of age-eligible children (60.1 %,110/183) received 3 PCV13 doses. PCV10 carriage declined from 13.2 %(78/590) in 2017/18 to 7.2 % (10/140) in 2022, additional PCV13 serotypes (3, 6A/6C and 19A) decreased from 18.5 %(109/590) to 11.4 %(16/140) while non-PCV13 serotypes increased from 68.3 %(403/590) to 81.4 %(114/140). There were 88.5 %(n = 124), 80.7 %(n = 113), 55.0 %(n = 77), and 46.0 %(n = 65) isolates predicted to be resistant to cotrimoxazole, penicillin(meningitis cut-off), tetracycline, and erythromycin respectively. CONCLUSION: Replacing PCV10 with PCV13 rapidly decreased prevalence of PCV13 carriage among vaccinated children in Matiari, Pakistan. Vaccine-driven selection pressure may have been responsible for the increase of non-PCV13 serotypes.

The evolutionary histories of the antibiotic-resistant Streptococcus pneumoniae lineages PMEN3 and PMEN9 were reconstructed using global collections of genomes. In PMEN3, one resistant clade spread worldwide, and underwent 25 serotype switches, enabling evasion of vaccine-induced immunity. In PMEN9, only 9 switches were detected, and multiple resistant lineages emerged independently and circulated locally. In Germany, PMEN9’s expansion correlated significantly with the macrolide:penicillin consumption ratio. These isolates were penicillin sensitive but macrolide resistant, through a homologous recombination that integrated Tn1207.1 into a competence gene, preventing further diversification via transformation. Analysis of a species-wide dataset found 183 acquisitions of macrolide resistance, and multiple gains of the tetracycline-resistant transposon Tn916, through homologous recombination, often originating in other streptococcal species. Consequently, antibiotic selection preserves atypical recom- bination events that cause sequence divergence and structural variation throughout the S. pneumoniae chromosome. These events reveal the genetic exchanges between species normally counter-selected until perturbed by clinical interventions.Competing Interest StatementNJC has consulted for Antigen Discovery Inc. NJC has received an investigator-initiated award from GlaxoSmithKline.

Objective We reported a novel tetracycline-resistant gene in Streptococcus pneumoniae and investigated its temporal spread in relation to nationwide clinical interventions.Methods We whole genome sequenced 12,254 pneumococcal isolates from twenty-nine countries on an Illumina HiSeq Sequencer. Serotypes, sequence types and antibiotic resistance were inferred from genomes. Phylogeny was built based on single-nucleotide variants. Temporal changes of spread were reconstructed using a birth-death model.Results We identified tet(S/M) in 131 pneumococcal isolates, 97 (74%) caused invasive pneumococcal diseases among young children (59% HIV-positive, where HIV status was available) in South Africa. A majority of tet(S/M)-positive isolates (129/131) belong to clonal complex (CC)230. A global phylogeny of CC230 (n=389) revealed that tet(S/M)-positive isolates formed a sub-lineage that exhibited multidrug-resistance. Using the genomic data and a birth-death model, we detected an unrecognised outbreak of this sub-lineage in South Africa between 2000 and 2004 with an expected secondary infections (R) of ~2.5. R declined to ~1.0 in 2005 and &lt;1.0 in 2012. The declining epidemic coincided and could be related to the nationwide implementation of anti-retroviral treatment (ART) for HIV-infected individuals in 2004 and PCVs in late 2000s. Capsular switching from vaccine serotype 14 to non-vaccine serotype 23A was observed within the sub-lineage.Conclusions The prevalence of tet(S/M) in pneumococci was low and its dissemination was due to an unrecognised outbreak of CC230 in South Africa prior to ART and PCVs. However, capsular switching in this multidrug-resistant sub-lineage highlighted its potential to continue to cause disease in the post-PCV13 era.

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. Most NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5’ end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (eight forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

Pneumococcal conjugate vaccines (PCV) have reduced pneumococcal diseases globally. Despite this, much remains to be learned about their effect on pathogen population structure. Here we undertook whole genome sequencing of 660 pneumococcal strains from asymptomatic carriers to investigate population restructuring in pneumococcal strains sampled before and after PCV13 introduction in a previously vaccine-naïve setting. We show substantial decreasing frequency of vaccine-type (VT) strains and their strain diversity post-vaccination in the vaccinated but not unvaccinated age groups indicative of direct but limited or delayed indirect effect of vaccination. Clearance of identical VT serotypes associated with multiple lineages occurred regardless of their genetic background. Interestingly, despite the increasing frequency of non-vaccine type (NVT) strains through serotype replacement, the serotype diversity was not fully restored to the levels observed prior to vaccination implying limited serotype replacement. The frequency of antibiotic resistant strains was low and remained largely unchanged post-vaccination but intermediate-penicillin-resistant lineages were reduced in the post vaccine population. Significant perturbations marked by changing frequency of accessory genes associated with diverse functions especially mobile genetic elements and bacteriocin activity were detected. This phylogenomic analysis demonstrates early vaccine-induced pneumococcal population restructuring not only at serotype but also accessory genome level.Author summary Different formulations of PCVs have been effective in reducing the invasive pneumococcal disease burden globally. Clinical trials have started to indicate high impact and effectiveness of PCV13 in Sub Saharan Africa (SSA) but there is limited understanding of how the introduction of PCVs alters the population structure of pneumococcal strains at serotype and genomic level. Here we investigated this using pneumococcal strains sampled pre‐ and post-PCV13 introduction from a previously vaccine naïve setting in Northern Malawi. Our findings reveal decrease in frequency of VT serotypes and their associated lineages in the largely vaccinated under-five population but not older individuals indicating a direct but limited or delayed indirect protection. The diversity of serotypes also decreased post-vaccination in VT strains in the under-fives but there was no change in NVT strains suggesting incomplete serotype replacement. At the genomic level, logistic regression revealed changing frequency of accessory genes largely associated with mobile genetic elements but such changes did not include any antibiotic resistance genes. These findings show significant perturbations at serotype and accessory genome level in carried pneumococcal population after two years from PCV13 introduction but the pneumococcal population was still perturbed and had not returned to a new equilibrium state.

Streptococcus pneumoniae serotype 3 remains a significant cause of morbidity and mortality worldwide, despite inclusion in the 13-valent pneumococcal conjugate vaccine (PCV13). Serotype 3 increased in carriage since the implementation of PCV13 in the United States, while invasive disease rates remain unchanged. We investigated the persistence of serotype 3 in carriage and disease, through genomic analyses of a global sample of 301 serotype 3 isolates of the Netherlands3–31 (PMEN31) clone CC180, combined with associated patient data and PCV utilization among countries of isolate collection. We assessed phenotypic variation between dominant clades in capsule charge (zeta potential), capsular polysaccharide shedding, and susceptibility to opsonophagocytic killing, which have previously been associated with carriage duration, invasiveness, and vaccine escape. We identify a recent shift in the CC180 population attributed to a lineage termed Clade II, which was estimated by Bayesian coalescent analysis to have first appeared in 1968 [95% HPD: 1939–1989] and increased in prevalence and effective population size thereafter. Clade II isolates are divergent from the pre-PCV13 serotype 3 population in non-capsular antigenic composition, competence, and antibiotic susceptibility, the last resulting from the acquisition of a Tn916-like conjugative transposon. Differences in recombination rates among clades correlated with variations in the ATP-binding subunit of Clp protease as well as amino acid substitutions in the comCDE operon. Opsonophagocytic killing assays elucidated the low observed efficacy of PCV13 against serotype 3. Variation in PCV13 use among sampled countries was not independently correlated with the CC180 population shift; therefore, genotypic and phenotypic differences in protein antigens and, in particular, antibiotic resistance may have contributed to the increase of Clade II. Our analysis emphasizes the need for routine, representative sampling of isolates from disperse geographic regions, including historically under-sampled areas. We also highlight the value of genomics in resolving antigenic and epidemiological variations within a serotype, which may have implications for future vaccine development.Author Summary Streptococcus pneumoniae is a leading cause of bacterial pneumoniae, meningitis, and otitis media. Despite inclusion in the most recent pneumococcal conjugate vaccine, PCV13, serotype 3 remains epidemiologically important globally. We investigated the persistence of serotype 3 using whole-genome sequencing data form 301 isolates collected among 24 countries from 1993–2014. Through phylogenetic analysis, we identified three distinct lineages within a single clonal complex, CC180, and found one has recently emerged and grown in prevalence. We then compared genomic difference among lineages as well as variations in pneumococcal vaccine use among sampled countries. We found that the recently emerged lineage, termed Clade II, has a higher prevalence of antibiotic resistance compared to other lineages, diverse surface protein antigens, and a higher rate of recombination, a process by which bacteria can uptake and incorporate genetic material from its surroundings. Differences in vaccine use among sampled countries did not appear to be associated with the emergence of Clade II. We highlight the need to routine, representative sampling of bacterial isolates from diverse geographic areas and show the utility of genomic data in resolving epidemiological differences within a pathogen population.

The polysaccharide (PS) capsule is essential for immune evasion and virulence of Streptococcus pneumoniae. Existing pneumococcal vaccines are designed to elicit anti-capsule antibodies, however, the effectiveness of these vaccines is being challenged by the emergence of new capsule types or variants. Herein, we characterize a newly discovered capsule type, 33E, that appears to have repeatedly emerged from vaccine type 33F via an inactivation mutation in the capsule glycosyltransferase gene, wciE. Structural analysis demonstrated that 33E and 33F share an identical repeat unit backbone [→5)-β-D-Galf2Ac-(1→3)-β-D-Galp-(1→3)-α-D-Galp-(1→3)-β-D-Galf-(1→3)-β-D-Glcp-(1→], except that a galactose (α-D-Galp) branch is present in 33F but not in 33E. Though the two capsule types were indistinguishable using conventional typing methods, the monoclonal antibody Hyp33FM1 selectively bound 33F but not 33E pneumococci. Further, we confirmed that wciE encodes a glycosyltransferase that catalyzes the addition of the branching α-D-Galp and that its inactivation in 33F strains results in the expression of the 33E capsule type. Though 33F and 33E share a structural and antigenic similarity, our pilot study suggested that immunization with a 23-valent pneumococcal PS vaccine containing 33F PS didn't significantly elicit cross-opsonic antibodies to 33E. New conjugate vaccines that target capsule type 33F may not necessarily protect against 33E. Therefore, studies of new conjugate vaccines require knowledge of the newly identified capsule type 33E and reliable pneumococcal typing methods capable of distinguishing it from 33F.

Streptococcus pneumoniae is a leading cause of pneumonia and meningitis worldwide. Many different serotypes co-circulate endemically in any one location. The extent and mechanisms of spread, and vaccine-driven changes in fitness and antimicrobial resistance (AMR), remain largely unquantified. Using geolocated genome sequences from South Africa (N=6910, 2000-2014) we developed models to reconstruct spread, pairing detailed human mobility data and genomic data. Separately we estimated the population level changes in fitness of strains that are (vaccine type, VT) and are not (non-vaccine type, NVT) included in the vaccine, first implemented in 2009, as well as differences in strain fitness between those that are and are not resistant to penicillin. We estimated that pneumococci only become homogenously mixed across South Africa after about 50 years of transmission, with the slow spread driven by the focal nature of human mobility. Further, in the years following vaccine implementation the relative fitness of NVT compared to VT strains increased (RR: 1.29 [95% CI 1.20-1.37]) - with an increasing proportion of these NVT strains becoming penicillin resistant. Our findings point to highly entrenched, slow transmission and indicate that initial vaccine-linked decreases in AMR may be transient. Copyright The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5' end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

The introduction of pneumococcal conjugate vaccines (PCV7, PCV10, PCV13) around the world has proved successful in preventing invasive pneumococcal disease. However, immunization against Streptococcus pneumoniae has led to serotype replacement by non-vaccine serotypes, including serotype 15A. Clonal complex 63 (CC63) is associated with many serotypes and has been reported in association with 15A after introduction of PCVs. A total of 865 CC63 isolates were included in this study, from the USA (n=391) and a global collection (n=474) from 1998-2019 and 1995-2018, respectively. We analysed the genomic sequences to identify serotypes and penicillin-binding protein (PBP) genes 1A, 2B and 2X, and other resistance determinants, to predict minimum inhibitory concentrations (MICs) against penicillin, erythromycin, clindamycin, co-trimoxazole and tetracycline. We conducted phylogenetic and spatiotemporal analyses to understand the evolutionary history of the 15A-CC63 sub-lineage. Overall, most (89.5 %, n=247) pre-PCV isolates in the CC63 cluster belonged to serotype 14, with 15A representing 6.5 % of isolates. Conversely, serotype 14 isolates represented 28.2 % of post-PCV CC63 isolates (n=618), whilst serotype 15A isolates represented 65.4 %. Dating of the CC63 lineage determined the most recent common ancestor emerged in the 1980s, suggesting the 15A-CC63 sub-lineage emerged from its closest serotype 14 ancestor prior to the development of pneumococcal vaccines. This sub-lineage was predominant in the USA, Israel and China. Multidrug resistance (to three or more drug classes) was widespread among isolates in this sub-lineage. We show that the CC63 lineage is globally distributed and most of the isolates are penicillin non-susceptible, and thus should be monitored.

Streptococcus pneumoniae can produce a wide breadth of antigenically diverse capsule types, a fact that poses a looming threat to the success of vaccines that target pneumococcal polysaccharide (PS) capsule. Yet, many pneumococcal capsule types remain undiscovered and/or uncharacterized. Prior sequence analysis of pneumococcal capsule synthesis (cps) loci suggested the existence of capsule subtypes among isolates identified as "serotype 36" according to conventional capsule typing methods. We discovered these subtypes represent two antigenically similar but distinguishable pneumococcal capsule serotypes, 36A and 36B. Biochemical analysis of their capsule PS structure reveals that both have the shared repeat unit backbone [→5)-α-d-Galf-(1→1)-d-Rib-ol-(5→P→6)-β-d-ManpNAc-(1→4)-β-d-Glcp-(1→] with two branching structures. Both serotypes have a β-d-Galp branch to Ribitol. Serotypes 36A and 36B differ by the presence of a α-d-Glcp-(1→3)-β-d-ManpNAc or α-d-Galp-(1→3)-β-d-ManpNAc branch, respectively. Comparison of the phylogenetically distant serogroup 9 and 36 cps loci, which all encode this distinguishing glycosidic bond, revealed that the incorporation of Glcp (in types 9N and 36A) versus Galp (in types 9A, 9V, 9L, and 36B) is associated with the identity of four amino acids in the cps-encoded glycosyltransferase WcjA. Identifying functional determinants of cps-encoded enzymes and their impact on capsule PS structure is key to improving the resolution and reliability of sequencing-based capsule typing methods and discovering novel capsule variants indistinguishable by conventional serotyping methods.

Invasive pneumococcal disease remains one of the leading causes of morbidity and mortality worldwide. In Russia, 13- valent pneumococcal conjugate vaccine (PCV13) was introduced into the childhood immunization programme nationwide in 2014. As part of the Global Pneumococcal Sequencing Project (GPS), we used genome data to characterize 179 pneumococcal isolates collected from Russia in 2011-2018 to investigate the circulating pneumococcal strains using a standardized genomic definition of pneumococcal lineages (global pneumococcal sequence clusters, GPSCs), prevalent serotypes and antimicrobial resistance profiles.We observed high serotype and lineage diversity among the 179 isolates recovered from cerebrospinal fluid (n=77), nasopharyngeal swabs (n=99) and other non-sterile site swabs (n=3). Overall, 60 GPSCs were identified, including 48 clonal complexes (CCs) and 14 singletons, and expressed 42 serotypes (including non-typable). Among PCV13 serotypes, 19F, 6B and 23F were the top three serotypes while 11A, 15B/C and 8 were the top three among non-PCV13 serotypes in the collection. Two lineages (GPSC6 and GPSC47) expressed both PCV13 and non-PCV13 serotypes that caused invasive disease, and were penicillin- and multidrug-resistant (MDR), highlighting their potential to adapt and continue to cause infections under vaccine and antibiotic selective pressure. PCV13 serotypes comprised 92 % (11/12) of the CSF isolates from the children aged below 5 years; however, the prevalence of PCV13 serotype isolates dropped to 53 % (31/58) among the nasopharyngeal isolates. Our analysis showed that 59 % (105/179) of the isolates were predicted to be non-susceptible to at least one class of antibiotics and 26 % (46/179) were MDR. Four MDR lineages (GPSC1, GPSC6, GPSC10 and GPSC47) accounted for 65 % (30/46) of the MDR isolates and expressed PCV13 serotypes (93 %, 28/30).This study provides evidence of high genetic and serotype diversity contributed by a mix of globally spreading and regionally circulating lineages in Russia. The observations suggest that the PCV13 vaccine could be important in reducing both invasive disease and antimicrobial resistance. We also identify potential lineages (GPSC6 and GPSC47) that may evade the vaccine.

BACKGROUND: Serotype 24F is one of the emerging pneumococcal serotypes after the introduction of pneumococcal conjugate vaccine (PCV). We aimed to identify lineages driving the increase of serotype 24F in France and place these findings into a global context. METHODS: Whole-genome sequencing was performed on a collection of serotype 24F pneumococci from asymptomatic colonisation (n=229) and invasive disease (n=190) isolates among individuals younger than 18 years in France, from 2003 to 2018. To provide a global context, we included an additional collection of 24F isolates in the Global Pneumococcal Sequencing (GPS) project database for analysis. A Global Pneumococcal Sequence Cluster (GPSC) and a clonal complex (CC) were assigned to each genome. Phylogenetic, evolutionary, and spatiotemporal analysis were conducted using the same 24F collection and supplemented with a global collection of genomes belonging to the lineage of interest from the GPS project database (n=25 590). FINDINGS: Serotype 24F was identified in numerous countries mainly due to the clonal spread of three lineages: GPSC10 (CC230), GPSC16 (CC156), and GPSC206 (CC7701). GPSC10 was the only multidrug-resistant lineage. GPSC10 drove the increase in 24F in France and had high invasive disease potential. The international dataset of GPSC10 (n=888) revealed that this lineage expressed 16 other serotypes, with only six included in 13-valent PCV (PCV13). All serotype 24F isolates were clustered in a single clade within the GPSC10 phylogeny and long-range transmissions were detected from Europe to other continents. Spatiotemporal analysis showed GPSC10-24F took 3-5 years to spread across France and a rapid change of serotype composition from PCV13 serotype 19A to 24F during the introduction of PCV13 was observed in neighbouring country Spain. INTERPRETATION: Our work reveals that GPSC10 alone is a challenge for serotype-based vaccine strategy. More systematic investigation to identify lineages like GPSC10 will better inform and improve next-generation preventive strategies against pneumococcal diseases. FUNDING: Bill & Melinda Gates Foundation, Wellcome Sanger Institute, and the US Centers for Disease Control and Prevention.

Streptococcus pneumoniae is a key contributor to childhood morbidity and mortality in Papua New Guinea (PNG). For the first time, whole genome sequencing of 174 isolates has enabled detailed characterisation of diverse S. pneumoniae causing invasive disease in young children in PNG, 1989-2014. This study captures the baseline S. pneumoniae population prior to the introduction of 13-valent pneumococcal conjugate vaccine (PCV13) into the national childhood immunisation programme in 2014. Relationships amongst lineages, serotypes and antimicrobial resistance traits were characterised, and the population was viewed in the context of a global collection of isolates. The analyses highlighted adiverse S. pneumoniae population associated with invasive disease in PNG, with 45 unique Global Pneumococcal Sequence Clusters (GPSCs) observed amongst the 174 isolates reflecting multiple lineages observed in PNG that have not been identified in other geographic locations. The majority of isolates were from children with meningitis, of which 52% (n=72) expressed non-PCV13 serotypes. Over a third of isolates were predicted to be resistant to at least one antimicrobial. PCV13 serotype isolates had 10.1 times the odds of being multidrug-resistant (MDR) compared to non-vaccine serotype isolates, and no isolates with GPSCs unique to PNG were MDR. Serotype 2 was the most commonly identified serotype; we identified a highly clonal cluster of serotype 2 isolates unique to PNG, and a distinct second cluster indicative of long-distance transmission. Ongoing surveillance, including whole-genome sequencing, is needed to ascertain the impact of the national PCV13 programme upon the S. pneumoniae population, including serotype replacement and antimicrobial resistance traits.

BACKGROUND: Pneumococcal disease is a leading cause of bacterial pneumonia and invasive bacterial disease among children globally. The reason some strains of pneumococci are more likely to cause disease, and how interventions such as vaccines and antibiotics affect pneumococcal strains is poorly understood. We aimed to identify genetic regions under selective pressure and those associated with disease through the analysis of pneumococcal whole-genome sequences. METHODS: Whole-genome sequencing was performed on pneumococcal isolates collected between January, 2005, and May, 2018, in Kathmandu, Nepal, which included programmatic ten-valent pneumococcal conjugate vaccine (PCV10) introduction in 2015. Isolates were from three distinct cohorts: nasopharyngeal swabs of healthy community-based children, nasopharyngeal swabs of children admitted to hospital with pneumonia, and sterile-site cultures from children admitted to hospital. Across these cohorts we examined serotype distribution, antibiotic resistance, strain distribution, and regions of recombination to determine genes that were undergoing diversifying selection. Genome-wide association studies comparing pneumonia and sterile-site isolates with healthy carriage were used to determine novel variants associated with disease. FINDINGS: After programmatic introduction of PCV10, there was a decline in vaccine covered serotypes; however, strains that had expressed these serotypes continued to exist in the post-PCV population. We identified GPSC9 to be a strain of concern due to its high prevalence in disease, multidrug resistance, and ability to switch to an unencapsulated phenotype via insertion of virulence factor pspC into the cps locus. Antibiotic resistance loci to co-trimoxazole were found to be prevalent (pre-PCV10 78% vs post-PCV10 81%; p=0·27) and increasingly prevalent to penicillin (pre-PCV10 15% vs post-PCV10 32%; p<0·0001). Regions with multiple recombinations were identified spanning the surface protein virulence factors pspA and pspC and antibiotic targets pbpX, folA, folC, folE, and folP. Furthermore, we identified variants in lacE2 to be strongly associated with isolates from children with pneumonia and PRIP to be strongly associated with isolates from sterile sites. INTERPRETATION: Our work highlights the effect of pneumococcal conjugate vaccines, antibiotics, and host-pathogen interaction in pneumococcal variation, and the pathogen's capability of adapting to these factors at both population-wide and strain-specific levels. Ongoing surveillance of disease-associated strains and further investigation of lacE2 and PRIP as serotype-independent targets for therapeutic interventions is required. FUNDING: Gavi, The Vaccine Alliance; WHO; Bill & Melinda Gates Foundation; Wellcome Sanger Institute; and US Centers for Disease Control and Prevention.

Streptococcus pneumoniae (the pneumococcus) is a leading cause of childhood mortality globally and in Cambodia. It is commensal in the human nasopharynx, occasionally resulting in invasive disease. Monitoring population genetic shifts, characterized by lineage and serotype expansions, as well as antimicrobial-resistance (AMR) patterns is crucial for assessing and predicting the impact of vaccination campaigns. We sought to elucidate the genetic background (global pneumococcal sequence clusters; GPSCs) of pneumococci carried by Cambodian children following perturbation by pneumococcal conjugate vaccine (PCV) 13. We sequenced pre-PCV13 (01/2013-12/2015, N=258) and post-PCV13 carriage isolates (01/2016-02/2017, N=428) and used PopPUNK and SeroBA to determine lineage prevalence and serotype composition. Following PCV13 implementation in Cambodia, we saw expansions of non-vaccine type (NVT) serotypes 23A (GPSC626), 34 (GPSC45) and 6D (GPSC16). We predicted antimicrobial susceptibility using the CDC-AMR pipeline and determined concordance with phenotypic data. The CDC-AMR pipeline had >90 % concordance with the phenotypic antimicrobial-susceptibility testing. We detected a high prevalence of AMR in both expanding non-vaccine serotypes and residual vaccine serotype 6B. Persistently high levels of AMR, specifically persisting multidrug-resistant lineages, warrant concern. The implementation of PCV13 in Cambodia has resulted in NVT serotype expansion reflected in the carriage population and driven by specific genetic backgrounds. Continued monitoring of these GPSCs during the ongoing collection of additional carriage isolates in this population is necessary.

Streptococcus pneumoniae is a major human pathogen responsible for over 317000 deaths in children <5 years of age with the burden of the disease being highest in low- and middle-income countries including South Africa. Following the introduction of the 7-valent and 13-valent pneumococcal conjugate vaccine (PCV) in South Africa in 2009 and 2011, respectively, a decrease in both invasive pneumococcal infections and asymptomatic carriage of vaccine-type pneumococci were reported. In this study, we described the changing epidemiology of the pneumococcal carriage population in South Africa, by sequencing the genomes of 1825 isolates collected between 2009 and 2013. Using these genomic data, we reported the changes in serotypes, Global Pneumococcal Sequence Clusters (GPSCs), and antibiotic resistance before and after the introduction of PCV13. The pneumococcal carriage population in South Africa has a high level of diversity, comprising of 126 GPSCs and 49 serotypes. Of the ten most prevalent GPSCs detected, six were predominantly found in Africa (GPSC22, GPSC21, GPSC17, GPSC33, GPSC34 and GPSC52). We found a significant decrease in PCV7 serotypes (19F, 6B, 23F and 14) and an increase in non-vaccine serotypes (NVT) (16F, 34, 35B and 11A) among children <2 years of age. The increase in NVTs was driven by pneumococcal lineages GPSC33, GPSC34, GPSC5 and GPSC22. Overall, a decrease in antibiotic resistance for 11 antimicrobials was detected in the PCV13 era. Further, we reported a higher resistance prevalence among vaccine types (VTs), as compared to NVTs; however, an increase in penicillin resistance among NVT was observed between the PCV7 and PCV13 eras. The carriage isolates from South Africa predominantly belonged to pneumococcal lineages, which are endemic to Africa. While the introduction of PCV resulted in an overall reduction of resistance in pneumococcal carriage isolates, an increase in penicillin resistance among NVTs was detected in children aged between 3 and 5 years, driven by the expansion of penicillin-resistant clones associated with NVTs in the PCV13 era.

The isolation of Streptococcus pneumoniae serotypes in systemic tissues of patients with invasive disease versus the nasopharynx of healthy individuals with asymptomatic carriage varies widely. Some serotypes are hyper-invasive, particularly serotype 1, but the underlying genetics remain poorly understood due to the rarity of carriage isolates, reducing the power of comparison with invasive isolates. Here, we use a well-controlled genome-wide association study to search for genetic variation associated with invasiveness of serotype 1 pneumococci from a serotype 1 endemic setting in Africa. We found no consensus evidence that certain genomic variation is overrepresented among isolates from patients with invasive disease than asymptomatic carriage. Overall, the genomic variation explained negligible phenotypic variability, suggesting a minimal effect on the disease status. Furthermore, changes in lineage distribution were seen with lineages replacing each other over time, highlighting the importance of continued pathogen surveillance. Our findings suggest that the hyper-invasiveness is an intrinsic property of the serotype 1 strains, not specific for a "disease-associated" subpopulation disproportionately harbouring unique genomic variation.

Pneumococcal serotype 35B is an important non-conjugate vaccine (non-PCV) serotype. Its continued emergence, post-PCV7 in the USA, was associated with expansion of a pre-existing 35B clone (clonal complex [CC] 558) along with post-PCV13 emergence of a non-35B clone previously associated with PCV serotypes (CC156). This study describes lineages circulating among 35B isolates in South Africa before and after PCV introduction. We also compared 35B isolates belonging to a predominant 35B lineage in South Africa (GPSC5), with isolates belonging to the same lineage in other parts of the world. Serotype 35B isolates that caused invasive pneumococcal disease in South Africa in 2005-2014 were characterized by whole-genome sequencing (WGS). Multi-locus sequence types and global pneumococcal sequence clusters (GPSCs) were derived from WGS data of 63 35B isolates obtained in 2005-2014. A total of 262 isolates that belong to GPSC5 (115 isolates from South Africa and 147 from other countries) that were sequenced as part of the global pneumococcal sequencing (GPS) project were included for comparison. Serotype 35B isolates from South Africa were differentiated into seven GPSCs and GPSC5 was most common (49 %, 31/63). While 35B was the most common serotype among GPSC5/CC172 isolates in South Africa during the PCV13 period (66 %, 29/44), 23F was the most common serotype during both the pre-PCV (80 %, 37/46) and PCV7 period (32 %, 8/25). Serotype 35B represented 15 % (40/262) of GPSC5 isolates within the global GPS database and 75 % (31/40) were from South Africa. The predominance of the GPSC5 lineage within non-vaccine serotype 35B, is possibly unique to South Africa and warrants further molecular surveillance of pneumococci.

The transmission dynamics of Streptococcus pneumoniae in sub-Saharan Africa are poorly understood due to a lack of adequate epidemiological and genomic data. Here we leverage a longitudinal cohort from 21 neighbouring villages in rural Africa to study how closely related strains of S. pneumoniae are shared among infants. We analysed 1074 pneumococcal genomes isolated from 102 infants from 21 villages. Strains were designated for unique serotype and sequence-type combinations, and we arbitrarily defined strain sharing where the pairwise genetic distance between strains could be accounted for by the mean within host intra-strain diversity. We used non-parametric statistical tests to assess the role of spatial distance and prolonged carriage on strain sharing using a logistic regression model. We recorded 458 carriage episodes including 318 (69.4 %) where the carried strain was shared with at least one other infant. The odds of strain sharing varied significantly across villages (χ(2)=47.5, df=21, P-value <0.001). Infants in close proximity to each other were more likely to be involved in strain sharing, but we also show a considerable amount of strain sharing across longer distances. Close geographic proximity (<5 km) between shared strains was associated with a significantly lower pairwise SNP distance compared to strains shared over longer distances (P-value <0.005). Sustained carriage of a shared strain among the infants was significantly more likely to occur if they resided in villages within a 5 km radius of each other (P-value <0.005, OR 3.7). Conversely, where both infants were transiently colonized by the shared strain, they were more likely to reside in villages separated by over 15 km (P-value <0.05, OR 1.5). PCV7 serotypes were rare (13.5 %) and were significantly less likely to be shared (P-value <0.001, OR -1.07). Strain sharing was more likely to occur over short geographical distances, especially where accompanied by sustained colonization. Our results show that strain sharing is a useful proxy for studying transmission dynamics in an under-sampled population with limited genomic data. This article contains data hosted by Microreact.

BACKGROUND: Pneumococcal disease outbreaks of vaccine preventable serotype 4 sequence type (ST)801 in shipyards have been reported in several countries. We aimed to use genomics to establish any international links between them. METHODS: Sequence data from ST801-related outbreak isolates from Norway (n = 17), Finland (n = 11) and Northern Ireland (n = 2) were combined with invasive pneumococcal disease surveillance from the respective countries, and ST801-related genomes from an international collection (n = 41 of > 40,000), totalling 106 genomes. Raw data were mapped and recombination excluded before phylogenetic dating. RESULTS: Outbreak isolates were relatively diverse, with up to 100 SNPs (single nucleotide polymorphisms) and a common ancestor estimated around the year 2000. However, 19 Norwegian and Finnish isolates were nearly indistinguishable (0-2 SNPs) with the common ancestor dated around 2017. CONCLUSION: The total diversity of ST801 within the outbreaks could not be explained by recent transmission alone, suggesting that harsh environmental and associated living conditions reported in the shipyards may facilitate invasion of colonising pneumococci. However, near identical strains in the Norwegian and Finnish outbreaks does suggest that transmission between international shipyards also contributed to those outbreaks. This indicates the need for improved preventative measures in this working population including pneumococcal vaccination.

In 2010, Brazil introduced the 10-valent pneumococcal conjugate vaccine (PCV10) into the national children's immunization programme. This study describes the genetic characteristics of invasive Streptococcus pneumoniae isolates before and after PCV10 introduction. A subset of 466 [pre-PCV10 (2008-2009): n=232, post-PCV10 (2012-2013): n=234;<5 years old: n=310, ≥5 years old: n=156] pneumococcal isolates, collected through national laboratory surveillance, were whole-genome sequenced (WGS) to determine serotype, pilus locus, antimicrobial resistance and genetic lineages. Following PCV10 introduction, in the <5 years age group, non-vaccine serotypes (NVT) serotype 3 and serotype 19A were the most frequent, and serotypes 12F, 8 and 9 N in the ≥5 years old group. The study identified 65 Global Pneumococcal Sequence Clusters (GPSCs): 49 (88 %) were GPSCs previously described and 16 (12 %) were Brazilian clusters. In total, 36 GPSCs (55 %) were NVT lineages, 18 (28 %) vaccine serotypes (VT) and 11 (17 %) were both VT and NVT lineages. In both sampling periods, the most frequent lineage was GPSC6 (CC156, serotypes 14/9V). In the <5 years old group, a decrease in penicillin (P=0.0123) and cotrimoxazole (P<0.0001) resistance and an increase in tetracycline (P=0.019) were observed. Penicillin nonsusceptibility was predicted in 40 % of the isolates; 127 PBP combinations were identified (51 predicted MIC≥0.125 mg l(-1)); cotrimoxazole (folA and/or folP alterations), macrolide (mef and/or ermB) and tetracycline (tetM, tetO or tetS/M) resistance were predicted in 63, 13 and 21.6 % of pneumococci studied, respectively. The main lineages associated with multidrug resistance in the post-PCV10 period were composed of NVT, GPSC1 (CC320, serotype 19A), and GPSC47 (ST386, serotype 6C). The study provides a baseline for future comparisons and identified important NVT lineages in the post-PCV10 period in Brazil.

Invasive disease caused by Streptococcus pneumoniae (IPD) is one of the leading causes of morbidity and mortality in young children worldwide. In Argentina, PCV13 was introduced into the childhood immunization programme nationwide in 2012 and PCV7 was available from 2000, but only in the private market. Since 1993 the National IPD Surveillance Programme, consisting of 150 hospitals, has conducted nationwide pneumococcal surveillance in Argentina in children under 6 years of age, as part of the SIREVA II-OPS network. A total of 1713 pneumococcal isolates characterized by serotype (Quellung) and antimicrobial resistance (agar dilution) to ten antibiotics, belonging to three study periods: pre-PCV7 era 1998-1999 (pre-PCV), before the introduction of PCV13 2010-2011 (PCV7) and after the introduction of PCV13 2012-2013 (PCV13), were available for inclusion. Fifty-four serotypes were identified in the entire collection and serotypes 14, 5 and 1 represented 50 % of the isolates. Resistance to penicillin was 34.9 %, cefotaxime 10.6 %, meropenem 4.9 %, cotrimoxazole 45 %, erythromycin 21.5 %, tetracycline 15.4 % and chloramphenicol 0.4 %. All the isolates were susceptible to levofloxacin, rifampin and vancomycin. Of 1713 isolates, 1061 (61.9 %) were non-susceptible to at least one antibiotic and 235(13.7 %) were multidrug resistant. A subset of 413 isolates was randomly selected and whole-genome sequenced as part of Global Pneumococcal Sequencing Project (GPS). The genome data was used to investigate the population structure of S. pneumoniae defining pneumococcal lineages using Global Pneumococcal Sequence Clusters (GPSCs), sequence types (STs) and clonal complexes (CCs), prevalent serotypes and their associated pneumococcal lineages and genomic inference of antimicrobial resistance. The collection showed a great diversity of strains. Among the 413 isolates, 73 known and 36 new STs were identified belonging to 38 CCs and 25 singletons, grouped into 52 GPSCs. Important changes were observed among vaccine types when pre-PCV and PCV13 periods were compared; a significant decrease in serotypes 14, 6B and 19F and a significant increase in 7F and 3. Among non-PCV13 types, serogroup 24 increased from 0 % in pre-PCV to 3.2 % in the PCV13 period. Our analysis showed that 66.1 % (273/413) of the isolates were predicted to be non-susceptible to at least one antibiotic and 11.9 % (49/413) were multidrug resistant. We found an agreement of 100 % when comparing the serotype determined by Quellung and WGS-based serotyping and 98.4 % of agreement in antimicrobial resistance. Continued surveillance of the pneumococcal population is needed to reveal the dynamics of pneumococcal isolates in Argentina in post-PCV13. This article contains data hosted by Microreact.

Streptococcus pneumoniae is an important global pathogen that causes bacterial pneumonia, sepsis and meningitis. Beta-lactam antibiotics are the first-line treatment for pneumococcal disease, however, their effectiveness is hampered by beta-lactam resistance facilitated by horizontal genetic transfer (HGT) with closely related species. Although interspecies HGT is known to occur among the species of the genus Streptococcus, the rates and effects of HGT between Streptococcus pneumoniae and its close relatives involving the penicillin binding protein (pbp) genes remain poorly understood. Here we applied the fastGEAR tool to investigate interspecies HGT in pbp genes using a global collection of whole-genome sequences of Streptococcus mitis, Streptococcus oralis and S. pneumoniae. With these data, we established that pneumococcal serotypes 6A, 13, 14, 16F, 19A, 19F, 23F and 35B were the highest-ranking serotypes with acquired pbp fragments. S. mitis was a more frequent pneumococcal donor of pbp fragments and a source of higher pbp nucleotide diversity when compared with S. oralis. Pneumococci that acquired pbp fragments were associated with a higher minimum inhibitory concentration (MIC) for penicillin compared with pneumococci without acquired fragments. Together these data indicate that S. mitis contributes to reduced β-lactam susceptibility among commonly carried pneumococcal serotypes that are associated with long carriage duration and high recombination frequencies. As pneumococcal vaccine programmes mature, placing increasing pressure on the pneumococcal population structure, it will be important to monitor the influence of antimicrobial resistance HGT from commensal streptococci such as S. mitis.

Globally, India has a high burden of pneumococcal disease, and pneumococcal conjugate vaccine (PCV) has been rolled out in different phases across the country since May 2017 in the national infant immunization programme (NIP). To provide a baseline for assessing the impact of the vaccine on circulating pneumococci in India, genetic characterization of pneumococcal isolates detected prior to introduction of PCV would be helpful. Here we present a population genomic study of 480 Streptococcus pneumoniae isolates collected across India and from all age groups before vaccine introduction (2009-2017), including 294 isolates from pneumococcal disease and 186 collected through nasopharyngeal surveys. Population genetic structure, serotype and antimicrobial susceptibility profile were characterized and predicted from whole-genome sequencing data. Our findings revealed high levels of genetic diversity represented by 110 Global Pneumococcal Sequence Clusters (GPSCs) and 54 serotypes. Serotype 19F and GPSC1 (CC320) was the most common serotype and pneumococcal lineage, respectively. Coverage of PCV13 (Pfizer) and 10-valent Pneumosil (Serum Institute of India) serotypes in age groups of ≤2 and 3-5 years were 63-75 % and 60-69 %, respectively. Coverage of PPV23 (Merck) serotypes in age groups of ≥50 years was 62 % (98/158). Among the top five lineages causing disease, GPSC10 (CC230), which ranked second, is the only lineage that expressed both PCV13 (serotypes 3, 6A, 14, 19A and 19F) and non-PCV13 (7B, 13, 10A, 11A, 13, 15B/C, 22F, 24F) serotypes. It exhibited multidrug resistance and was the largest contributor (17 %, 18/103) of NVTs in the disease-causing population. Overall, 42 % (202/480) of isolates were penicillin-resistant (minimum inhibitory concentration ≥0.12 µg ml(-1)) and 45 % (217/480) were multidrug-resistant. Nine GPSCs (GPSC1, 6, 9, 10, 13, 16, 43, 91, 376) were penicillin-resistant and among them six were multidrug-resistant. Pneumococci expressing PCV13 serotypes had a higher prevalence of antibiotic resistance. Sequencing of pneumococcal genomes has significantly improved our understanding of the biology of these bacteria. This study, describing the pneumococcal disease and carriage epidemiology pre-PCV introduction, demonstrates that 60-75 % of pneumococcal serotypes in children ≤5 years are covered by PCV13 and Pneumosil. Vaccination against pneumococci is very likely to reduce antibiotic resistance. A multidrug-resistant pneumococcal lineage, GPSC10 (CC230), is a high-risk clone that could mediate serotype replacement.