Background Pneumococcal conjugate vaccines have reduced the incidence of invasive pneumococcal disease, caused by vaccine serotypes, but non-vaccine-serotypes remain a concern. We used whole genome sequencing to study pneumococcal serotype, antibiotic resistance and invasiveness, in the context of genetic background. Methods Our dataset of 13,454 genomes, combined with four published genomic datasets, represented Africa (40%), Asia (25%), Europe (19%), North America (12%), and South America (5%). These 20,027 pneumococcal genomes were clustered into lineages using PopPUNK, and named Global Pneumococcal Sequence Clusters (GPSCs). From our dataset, we additionally derived serotype and sequence type, and predicted antibiotic sensitivity. We then measured invasiveness using odds ratios that relating prevalence in invasive pneumococcal disease to carriage. Findings The combined collections ( n = 20,027) were clustered into 621 GPSCs. Thirty-five GPSCs observed in our dataset were represented by >100 isolates, and subsequently classed as dominant-GPSCs. In 22/35 (63%) of dominant-GPSCs both non-vaccine serotypes and vaccine serotypes were observed in the years up until, and including, the first year of pneumococcal conjugate vaccine introduction. Penicillin and multidrug resistance were higher ( p < .05) in a subset dominant-GPSCs (14/35, 9/35 respectively), and resistance to an increasing number of antibiotic classes was associated with increased recombination (R 2 = 0.27 p < .0001). In 28/35 dominant-GPSCs, the country of isolation was a significant predictor ( p < .05) of its antibiogram (mean misclassification error 0.28, SD ± 0.13). We detected increased invasiveness of six genetic backgrounds, when compared to other genetic backgrounds expressing the same serotype. Up to 1.6-fold changes in invasiveness odds ratio were observed. Interpretation We define GPSCs that can be assigned to any pneumococcal genomic dataset, to aid international comparisons. Existing non-vaccine-serotypes in most GPSCs preclude the removal of these lineages by pneumococcal conjugate vaccines; leaving potential for serotype replacement. A subset of GPSCs have increased resistance, and/or serotype-independent invasiveness.

Background Invasive pneumococcal disease remains an important health priority owing to increasing disease incidence caused by pneumococci expressing non-vaccine serotypes. We previously defined 621 Global Pneumococcal Sequence Clusters (GPSCs) by analysing 20 027 pneumococcal isolates collected worldwide and from previously published genomic data. In this study, we aimed to investigate the pneumococcal lineages behind the predominant serotypes, the mechanism of serotype replacement in disease, as well as the major pneumococcal lineages contributing to invasive pneumococcal disease in the post-vaccine era and their antibiotic resistant traits. Methods We whole-genome sequenced 3233 invasive pneumococcal disease isolates from laboratory-based surveillance programmes in Hong Kong (n=78), Israel (n=701), Malawi (n=226), South Africa (n=1351), The Gambia (n=203), and the USA (n=674). The genomes represented pneumococci from before and after pneumococcal conjugate vaccine (PCV) introductions and were from children younger than 3 years. We identified predominant serotypes by prevalence and their major contributing lineages in each country, and assessed any serotype replacement by comparing the incidence rate between the pre-PCV and PCV periods for Israel, South Africa, and the USA. We defined the status of a lineage as vaccine-type GPSC (≥50% 13-valent PCV [PCV13] serotypes) or non-vaccine-type GPSC (>50% non-PCV13 serotypes) on the basis of its initial serotype composition detected in the earliest vaccine period to measure their individual contribution toward serotype replacement in each country. Major pneumococcal lineages in the PCV period were identified by pooled incidence rate using a random effects model. Findings The five most prevalent serotypes in the PCV13 period varied between countries, with only serotypes 5, 12F, 15B/C, 19A, 33F, and 35B/D common to two or more countries. The five most prevalent serotypes in the PCV13 period varied between countries, with only serotypes 5, 12F, 15B/C, 19A, 33F, and 35B/D common to two or more countries. These serotypes were associated with more than one lineage, except for serotype 5 (GPSC8). Serotype replacement was mainly mediated by expansion of non-vaccine serotypes within vaccine-type GPSCs and, to a lesser extent, by increases in non-vaccine-type GPSCs. A globally spreading lineage, GPSC3, expressing invasive serotypes 8 in South Africa and 33F in the USA and Israel, was the most common lineage causing non-vaccine serotype invasive pneumococcal disease in the PCV13 period. We observed that same prevalent non-vaccine serotypes could be associated with distinctive lineages in different countries, which exhibited dissimilar antibiotic resistance profiles. In non-vaccine serotype isolates, we detected significant increases in the prevalence of resistance to penicillin (52 [21%] of 249 vs 169 [29%] of 575, p=0•0016) and erythromycin (three [1%] of 249 vs 65 [11%] of 575, p=0•0031) in the PCV13 period compared with the pre-PCV period. Interpretation Globally spreading line...

Knowledge of the in vivo physiology and metabolism of Streptococcus pneumoniae is limited, even though pneumococci rely on efficient acquisition and metabolism of the host nutrients for growth and survival. Because the nutrient-limited, hypoxic host tissues favor mixed-acid fermentation, we studied the role of the pneumococcal pyruvate formate lyase (PFL), a key enzyme in mixed-acid fermentation, which is activated posttranslationally by PFL-activating enzyme (PFL-AE). Mutations were introduced to two putative pfl genes, SPD0235 and SPD0420, and two putative pflA genes, SPD0229 and SPD1774. End-product analysis showed that there was no formate, the main end product of the reaction catalyzed by PFL, produced by mutants defective in SPD0420 and SPD1774, indicating that SPD0420 codes for PFL and SPD1774 for putative PFL-AE. Expression of SPD0420 was elevated in galactose-containing medium in anaerobiosis compared to growth in glucose, and the mutation of SPD0420 resulted in the upregulation of fba and pyk, encoding, respectively, fructose 1,6-bisphosphate aldolase and pyruvate kinase, under the same conditions. In addition, an altered fatty acid composition was detected in SPD0420 and SPD1774 mutants. Mice infected intranasally with the SPD0420 and SPD1774 mutants survived significantly longer than the wild type-infected cohort, and bacteremia developed later in the mutant cohort than in the wild type-infected group. Furthermore, the numbers of CFU of the SPD0420 mutant were lower in the nasopharynx and the lungs after intranasal infection, and fewer numbers of mutant CFU than of wild-type CFU were recovered from blood specimens after intravenous infection. The results demonstrate that there is a direct link between pneumococcal fermentative metabolism and virulence.Streptococcus pneumoniae is the leading cause of pneumonia in children and adults, and it is a major cause of otitis media, meningitis, and septicemia (22). Despite considerable progress in pneumococcal vaccine development, the serotype specificity and the genomic plasticity of the pneumococcus will hamper its success (36). In addition, the increasing frequency of antibiotic resistance (14) makes it important to understand the mechanisms of pathogenesis of pneumococcal disease.Probably one of the most understudied fields of pneumococcal biology is that of how the pathogen generates its metabolic energy. The pneumococcus is strictly fermentative, and sugars are the major sources of energy for biosynthesis and growth (19,52). Therefore, in vivo fitness is determined to a large extent by the processes governed by sugar metabolism. Accumulating evidence suggests that there is a direct link between pneumococcal virulence and sugar metabolism (6,16,20,21,50). This is not limited to proteins involved in polysaccharide degradation, transport, and regulation (16, 31) but also includes those involved in redox metabolism, such as NADH oxidase (Nox) (6), and in pyruvate metabolism (42,50). For example, mutation of nox caused diminished virulence in a systemic infec...

is formed, confirming the thiol peroxidase activity. Levels of TpxD expression and synthesis in vitro were significantly increased in cells grown under aerobic versus anaerobic conditions. The challenge with D39 and TIGR4 with H 2 O 2 resulted in tpxD upregulation, while psaBCA expression was oppositely affected. However, the challenge of ⌬tpxD mutants with H 2 O 2 did not affect psaBCA, implying that TpxD is involved in the regulation of the psa operon, in addition to its scavenging activity. Virulence studies demonstrated a notable difference in the survival time of mice infected intranasally with D39 compared to that of mice infected intranasally with D39⌬tpxD. However, when bacteria were administered directly into the blood, this difference disappeared. The findings of this study suggest that TpxD constitutes a component of the organism's fundamental strategy to fine-tune cellular processes in response to H 2 O 2 .

Knowledge of pneumococcal lineages, their geographic distribution and antibiotic resistance patterns, can give insights into global pneumococcal disease. We provide interactive bioinformatic outputs to explore such topics, aiming to increase dissemination of genomic insights to the wider community, without the need for specialist training. We prepared 12 country-specific phylogenetic snapshots, and international phylogenetic snapshots of 73 common Global Pneumococcal Sequence Clusters (GPSCs) previously defined using PopPUNK, and present them in Microreact. Gene presence and absence defined using Roary, and recombination profiles derived from Gubbins are presented in Phandango for each GPSC. Temporal phylogenetic signal was assessed for each GPSC using BactDating. We provide examples of how such resources can be used. In our example use of a country-specific phylogenetic snapshot we determined that serotype 14 was observed in nine unrelated genetic backgrounds in South Africa. The international phylogenetic snapshot of GPSC9, in which most serotype 14 isolates from South Africa were observed, highlights that there were three independent sub-clusters represented by South African serotype 14 isolates. We estimated from the GPSC9-dated tree that the sub-clusters were each established in South Africa during the 1980s. We show how recombination plots allowed the identification of a 20 kb recombination spanning the capsular polysaccharide locus within GPSC97. This was consistent with a switch from serotype 6A to 19A estimated to have occured in the 1990s from the GPSC97-dated tree. Plots of gene presence/absence of resistance genes ( tet , erm , cat ) across the GPSC23 phylogeny were consistent with acquisition of a composite transposon. We estimated from the GPSC23-dated tree that the acquisition occurred between 1953 and 1975. Finally, we demonstrate the assignment of GPSC31 to 17 externally generated pneumococcal serotype 1 assemblies from Utah via Pathogenwatch. Most of the Utah isolates clustered within GPSC31 in a USA-specific clade with the most recent common ancestor estimated between 1958 and 1981. The resources we have provided can be used to explore to data, test hypothesis and generate new hypotheses. The accessible assignment of GPSCs allows others to contextualize their own collections beyond the data presented here.