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.

Genomic evolution, transmission and pathogenesis of Streptococcus pneumoniae, an opportunistic human-adapted pathogen, is driven principally by nasopharyngeal carriage. However, little is known about genomic changes during natural colonisation. Here, we use whole-genome sequencing to investigate within-host microevolution of naturally carried pneumococci in ninety-eight infants intensively sampled sequentially from birth until twelve months in a high-carriage African setting. We show that neutral evolution and nucleotide substitution rates up to forty-fold faster than observed over longer timescales in S. pneumoniae and other bacteria drives high within-host pneumococcal genetic diversity. Highly divergent co-existing strain variants emerge during colonisation episodes through real-time intra-host homologous recombination while the rest are co-transmitted or acquired independently during multiple colonisation episodes. Genic and intergenic parallel evolution occur particularly in antibiotic resistance, immune evasion and epithelial adhesion genes. Our findings suggest that within-host microevolution is rapid and adaptive during natural colonisation.

Whole genome sequencing (WGS)-based approaches for pneumococcal capsular typing have become an alternative to serological methods. In silico serotyping from WGS has not yet been applied to long-read sequences produced by third-generation technologies. The objective of the study was to determine the capsular types of pneumococci causing invasive disease in Catalonia (Spain) using serological typing and WGS and to compare the performance of different bioinformatics pipelines using short- and long-read data from WGS. All invasive pneumococcal pediatric isolates collected in Hospital Sant Joan de Déu (Barcelona) from 2013 to 2019 were included. Isolates were assigned a capsular type by serological testing based on anticapsular antisera and by different WGS-based pipelines: Illumina sequencing followed by serotyping with PneumoCaT, SeroBA, and Pathogenwatch vs MinION-ONT sequencing coupled with serotyping by Pathogenwatch from pneumococcal assembled genomes. A total of 119 out of 121 pneumococcal isolates were available for sequencing. Twenty-nine different serotypes were identified by serological typing, with 24F (n = 17; 14.3%), 14 (n = 10; 8.4%), and 15B/C (n = 8; 6.7%) being the most common serotypes. WGS-based pipelines showed initial concordance with serological typing (>91% of accuracy). The main discrepant results were found at the serotype level within a serogroup: 6A/B, 6C/D, 9A/V, 11A/D, and 18B/C. Only one discrepancy at the serogroup level was observed: serotype 29 by serological testing and serotype 35B/D by all WGS-based pipelines. Thus, bioinformatics WGS-based pipelines, including those using third-generation sequencing, are useful for pneumococcal capsular assignment. Possible discrepancies between serological typing and WGS-based approaches should be considered in pneumococcal capsular-type surveillance studies.

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.

A newly recognized pneumococcal serotype, 35D, which differs from the 35B polysaccharide in structure and serology by not binding to factor serum 35a, was recently reported. The genetic basis for this distinctive serology is due to the presence of an inactivating mutation in wciG, which encodes an O-acetyltransferase responsible for O-acetylation of a galactofuranose. Here, we assessed the genomic data of a worldwide pneumococcal collection to identify serotype 35D isolates and understand their geographical distribution, genetic background, and invasiveness potential. Of 21,980 pneumococcal isolates, 444 were originally typed as serotype 35B by PneumoCaT. Analysis of the wciG gene revealed 23 isolates from carriage (n = 4) and disease (n = 19) with partial or complete loss-of-function mutations, including mutations resulting in premature stop codons (n = 22) and an in-frame mutation (n = 1). These were selected for further analysis. The putative 35D isolates were geographically widespread, and 65.2% (15/23) of them was recovered after the introduction of pneumococcal conjugate vaccine 13 (PCV13). Compared with serotype 35B isolates, putative serotype 35D isolates have higher invasive disease potentials based on odds ratios (OR) (11.58; 95% confidence interval[CI], 1.42 to 94.19 versus 0.61; 95% CI, 0.40 to 0.92) and a higher prevalence of macrolide resistance mediated by mefA (26.1% versus 7.6%; P = 0.009). Using the Quellung reaction, 50% (10/20) of viable isolates were identified as serotype 35D, 25% (5/20) as serotype 35B, and 25% (5/20) as a mixture of 35B/35D. The discrepancy between phenotype and genotype requires further investigation. These findings illustrated a global distribution of an invasive serotype, 35D, among young children post-PCV13 introduction and underlined the invasive potential conferred by the loss of O-acetylation in the pneumococcal capsule.

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.

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.

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.

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 (the pneumococcus) is a human pathogen of global importance, classified into serotypes based on the type of capsular polysaccharide produced. Serotyping of pneumococci is essential for disease surveillance and vaccine impact measurement. However, the accuracy of serotyping methods can be affected by previously undiscovered variants. Previous studies have identified variants of serotype 14, a highly invasive serotype included in all licensed vaccine formulations. However, the potential of these variants to influence serotyping accuracy and evade vaccine-induced protection has not been investigated. In this study, we screened 1,386 nasopharyngeal swabs from children hospitalized with acute respiratory infection in Papua New Guinea for pneumococci. Swabs containing pneumococci (n = 1,226) were serotyped by microarray to identify pneumococci with a divergent serotype 14 capsule locus. Three serotype 14 variants ('14-like') were isolated and characterized further. The serotyping results of these isolates using molecular methods varied depending on the method, with 3/3 typing as nontypeable (PneumoCaT), 3/3 typing as serotype 14 (seroBA), and 2/3 typing as serotype 14 (SeroCall and quantitative PCR). All three isolates were nontypeable by phenotypic methods (Quellung and latex agglutination), indicating the absence of capsule. Illumina and nanopore sequencing were employed to examine their capsule loci and revealed unique mutations. Lastly, when incubated with sera from vaccinated individuals, the 14-like isolates evaded serotype-specific opsonophagocytic killing. Our study highlights the need for phenotypic testing to validate serotyping data derived from molecular methods. The convergent evolution of capsule loss underscores the importance of studying pneumococcal population biology to monitor the emergence of pneumococci capable of vaccine escape, globally. IMPORTANCE Pneumococcus is a pathogen of major public health importance. Current vaccines have limited valency, targeting a subset (up to 20) of the more than 100 capsule types (serotypes). Precise serotyping methods are therefore essential to avoid mistyping, which can reduce the accuracy of data used to inform decisions around vaccine introduction and/or maintenance of national vaccination programs. In this study, we examine a variant of serotype 14 (14-like), a virulent serotype present in all currently licensed vaccine formulations. Although these 14-like pneumococci no longer produce a serotype 14 capsule, widely used molecular methods can mistype them as serotype 14. Importantly, we show that 14-like pneumococci can evade opsonophagocytic killing mediated by vaccination. Despite the high accuracy of molecular methods for serotyping, our study reemphasizes their limitations. This is particularly relevant in situations where nonvaccine type pneumococci (e.g., the 14-likes in this study) could potentially be misidentified as a vaccine type (e.g., serotype 14).

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.

Streptococcus pneumoniae (pneumococcus) is a major human pathogen producing structurally diverse capsular polysaccharides. Widespread use of highly successful pneumococcal conjugate vaccines (PCVs) targeting pneumococcal capsules has greatly reduced infections by the vaccine types but increased infections by nonvaccine serotypes. Herein, we report a new and the 100th capsule type, named serotype 10D, by determining its unique chemical structure and biosynthetic roles of all capsule synthesis locus (cps) genes. The name 10D reflects its serologic cross-reaction with serotype 10A and appearance of cross-opsonic antibodies in response to immunization with 10A polysaccharide in a 23-valent pneumococcal vaccine. Genetic analysis showed that 10D cps has three large regions syntenic to and highly homologous with cps loci from serotype 6C, serotype 39, and an oral streptococcus strain (S. mitis SK145). The 10D cps region syntenic to SK145 is about 6 kb and has a short gene fragment of wciNα at the 5' end. The presence of this nonfunctional wciNα fragment provides compelling evidence for a recent interspecies genetic transfer from oral streptococcus to pneumococcus. Since oral streptococci have a large repertoire of cps loci, widespread PCV usage could facilitate the appearance of novel serotypes through interspecies recombination.IMPORTANCE The polysaccharide capsule is essential for the pathogenicity of pneumococcus, which is responsible for millions of deaths worldwide each year. Currently available pneumococcal vaccines are designed to elicit antibodies to the capsule polysaccharides of the pneumococcal isolates commonly causing diseases, and the antibodies provide protection only against the pneumococcus expressing the vaccine-targeted capsules. Since pneumococci can produce different capsule polysaccharides and therefore reduce vaccine effectiveness, it is important to track the appearance of novel pneumococcal capsule types and how these new capsules are created. Herein, we describe a new and the 100th pneumococcal capsule type with unique chemical and serological properties. The capsule type was named 10D for its serologic similarity to 10A. Genetic studies provide strong evidence that pneumococcus created 10D capsule polysaccharide by capturing a large genetic fragment from an oral streptococcus. Such interspecies genetic exchanges could greatly increase diversity of pneumococcal capsules and complicate serotype shifts.

We reported tet(S/M) in Streptococcus pneumoniae and investigated its temporal spread in relation to nationwide clinical interventions.

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 harboring unique genomic variation.

Multidrug-resistant Streptococcus pneumoniae emerge through the modification of core genome loci by interspecies homologous recombinations, and acquisition of gene cassettes. Both occurred in the otherwise contrasting histories of the antibiotic-resistant S. pneumoniae lineages PMEN3 and PMEN9. A single PMEN3 clade spread globally, evading vaccine-induced immunity through frequent serotype switching, whereas locally circulating PMEN9 clades independently gained resistance. Both lineages repeatedly integrated Tn916-type and Tn1207.1-type elements, conferring tetracycline and macrolide resistance, respectively, through homologous recombination importing sequences originating in other species. A species-wide dataset found over 100 instances of such interspecific acquisitions of resistance cassettes and flanking homologous arms. Phylodynamic analysis of the most commonly sampled Tn1207.1-type insertion in PMEN9, originating from a commensal and disrupting a competence gene, suggested its expansion across Germany was driven by a high ratio of macrolide-to-β-lactam consumption. Hence, selection from antibiotic consumption was sufficient for these atypically large recombinations to overcome species boundaries across the pneumococcal chromosome.

Hyper-virulent Streptococcus pneumoniae serotype 1 strains are endemic in Sub-Saharan Africa and frequently cause lethal meningitis outbreaks. It remains unknown whether genetic variation in serotype 1 strains modulates tropism into cerebrospinal fluid to cause central nervous system (CNS) infections, particularly meningitis. Here, we address this question through a large-scale linear mixed model genome-wide association study of 909 African pneumococcal serotype 1 isolates collected from CNS and non-CNS human samples. By controlling for host age, geography, and strain population structure, we identify genome-wide statistically significant genotype-phenotype associations in surface-exposed choline-binding (P = 5.00 × 10-08) and helicase proteins (P = 1.32 × 10-06) important for invasion, immune evasion and pneumococcal tropism to CNS. The small effect sizes and negligible heritability indicated that causation of CNS infection requires multiple genetic and other factors reflecting a complex and polygenic aetiology. Our findings suggest that certain pathogen genetic variation modulate pneumococcal survival and tropism to CNS tissue, and therefore, virulence for meningitis.

Pakistan is amongst the four countries with the highest number of pneumococcal deaths. While the PCV10 vaccine was introduced in Pakistan in October 2012, data regarding the impact of the vaccine on the population dynamics of Streptococcus pneumoniae in Pakistan remain obscure. Using whole genome sequencing of 190 isolates (nasopharyngeal carriage=75, disease=113, unknown sites=2) collected between 2002 and 2020, this study presents characteristics of pneumococcal strains in Pakistan in the pre- and post-vaccine era. The isolates were characterized on the basis of serotype distribution, genetic lineages (or Global Pneumococcal Sequence Cluster, GPSC) and antibiotic resistance. A high level of diversity in serotype and genetic lineages of pneumococci was observed in Pakistan. Among 190 isolates, we identified 54 serotypes, 67 GPSCs and 116 sequence types (STs) including 23 new STs. The most prevalent GPSCs and their associated serotypes in nasopharyngeal carriage were GPSC54 (expressing serotype 9V), GPSC5 (15A and 7B, and serogroup 24), GPSC25 (15B/15C), GPSC67 (18C) and GPSC376 (6A and 6D). Similarly, among 113 disease-causing isolates, the most prevalent GPSC/serotype combinations were GPSC2 (serotype 1), GPSC10 (serotypes 14, 10A, 19A and 19F), GPSC43 (serotypes 13, 11A, 23B, 35A and 9V), GPSC67 (serotypes 18A and 18C) and GPSC642 (serotype 11A). Of the 190 isolates, the highest levels of resistance were observed against penicillin (58.9 %, n=122), erythromycin (29.5 %, n=56), clindamycin (13.2 %, n=25), co-trimoxazole (94.2 %, n=179) and tetracycline/doxycycline (53.2 %, n=101). A higher proportion of disease-causing isolates were multidrug resistant as compared to carriage isolates (54 % vs 25 %). Our data suggest limited coverage of PCV10 in nasopharyngeal (21.6 %, 16/74) as well as disease-causing (38.1 %, 16/42) isolates among children ≤5 years old; however, higher valent vaccine PCV13 would increase the coverage rates to 33.8 % in nasopharyngeal and 54.8 % in disease-causing isolates, whereas PCV24/25 would offer the highest coverage rates. Owing to the diversity of serotypes observed during the post-vaccine period, the suggested inclusion of serotype in future vaccine formulations will require investigations with larger data sets with an extended temporal window. This article contains data hosted by Microreact.

Streptococcus pneumoniae (the pneumococcus) carriage precedes invasive disease and influences population-wide strain dynamics, but limited data exist on temporal carriage patterns of serotypes due to the prohibitive costs of longitudinal studies. Here, we report carriage prevalence, clearance and acquisition rates of pneumococcal serotypes sampled from newborn infants bi-weekly from weeks 1 to 27, and then bi-monthly from weeks 35 to 52 in the Gambia. We used sweep latex agglutination and whole genome sequencing to serotype the isolates. We show rapid pneumococcal acquisition with nearly 31% of the infants colonized by the end of first week after birth and quickly exceeding 95% after 2 months. Co-colonization with multiple serotypes was consistently observed in over 40% of the infants at each sampling point during the first year of life. Overall, the mean acquisition time and carriage duration regardless of serotype was 38 and 24 days, respectively, but varied considerably between serotypes comparable to observations from other regions. Our data will inform disease prevention and control measures including providing baseline data for parameterising infectious disease mathematical models including those assessing the impact of clinical interventions such as pneumococcal conjugate vaccines.

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 (χ2=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.

Despite contributing to the large disease burden in West Africa, little is known about the genomic epidemiology of Streptococcus pneumoniae which cause meningitis among children under 5 years old in the region. We analysed whole-genome sequencing data from 185 S. pneumoniae isolates recovered from suspected paediatric meningitis cases as part of the World Health Organization (WHO) invasive bacterial diseases surveillance from 2010 to 2016. The phylogeny was reconstructed, accessory genome similarity was computed and antimicrobial-resistance patterns were inferred from the genome data and compared to phenotypic resistance from disc diffusion. We studied the changes in the distribution of serotypes pre- and post-pneumococcal conjugate vaccine (PCV) introduction in the Central and Western sub-regions separately. The overall distribution of non-vaccine, PCV7 (4, 6B, 9V, 14, 18C, 19F and 23F) and additional PCV13 serotypes (1, 3, 5, 6A, 19A and 7F) did not change significantly before and after PCV introduction in the Central region (Fisher's test P value 0.27) despite an increase in the proportion of non-vaccine serotypes to 40 % (n=6) in the post-PCV introduction period compared to 21.9 % (n=14). In the Western sub-region, PCV13 serotypes were more dominant among isolates from The Gambia following the introduction of PCV7, 81 % (n=17), compared to the pre-PCV period in neighbouring Senegal, 51 % (n=27). The phylogeny illustrated the diversity of strains associated with paediatric meningitis in West Africa and highlighted the existence of phylogeographical clustering, with isolates from the same sub-region clustering and sharing similar accessory genome content. Antibiotic-resistance genotypes known to confer resistance to penicillin, chloramphenicol, co-trimoxazole and tetracycline were detected across all sub-regions. However, there was no discernible trend linking the presence of resistance genotypes with the vaccine introduction period or whether the strain was a vaccine or non-vaccine serotype. Resistance genotypes appeared to be conserved within selected sub-clades of the phylogenetic tree, suggesting clonal inheritance. Our data underscore the need for continued surveillance on the emergence of non-vaccine serotypes as well as chloramphenicol and penicillin resistance, as these antibiotics are likely still being used for empirical treatment in low-resource settings. This article contains data hosted by Microreact.