Population-level comparisons of prokaryotic genomes must take into account the substantial differences in gene content resulting from horizontal gene transfer, gene duplication and gene loss. However, the automated annotation of prokaryotic genomes is imperfect, and errors due to fragmented assemblies, contamination, diverse gene families and mis-assemblies accumulate over the population, leading to profound consequences when analysing the set of all genes found in a species. Here, we introduce Panaroo, a graph-based pangenome clustering tool that is able to account for many of the sources of error introduced during the annotation of prokaryotic genome assemblies. Panaroo is available at https://github.com/gtonkinhill/panaroo.

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.

The effect of second-generation pneumococcal conjugate vaccines on invasive pneumococcal disease (IPD) strain distributions have not yet been well described. We analysed IPD isolates recovered from children aged <5 years through Active Bacterial Core surveillance before (2008–2009; n = 828) and after (2011–2013; n = 600) 13-valent pneumococcal conjugate vaccine (PCV13) implementation. We employed conventional testing, PCR/electrospray ionization mass spectrometry and whole genome sequence (WGS) analysis to identify serotypes, resistance features, genotypes, and pilus types. PCV13, licensed in February 2010, effectively targeted all major 19A and 7F genotypes, and decreased antimicrobial resistance, primarily owing to removal of the 19A/ST320 complex. The strain complex contributing most to the remaining β-lactam resistance during 2011–2013 was 35B/ST558. Significant emergence of non-vaccine clonal complexes was not evident. Because of the removal of vaccine serotype strains, positivity for one or both pilus types (PI-1 and PI-2) decreased in the post-PCV13 years 2011–2013 relative to 2008–2009 (decreases of 32–55% for PI-1, and >95% for PI-2 and combined PI-1 + PI-2). β-Lactam susceptibility phenotypes correlated consistently with transpeptidase region sequence combinations of the three major penicillin-binding proteins (PBPs) determined through WGS analysis. Other major resistance features were predictable by DNA signatures from WGS analysis. Multilocus sequence data combined with PBP combinations identified progeny, serotype donors and recipient strains in serotype switch events. PCV13 decreased the frequency of all PCV13 serotype clones and concurrently decreased the frequency of strain subsets with resistance and/or adherence features conducive to successful carriage. Our results serve as a reference describing key features of current paediatric IPD strains in the USA after PCV13 implementation.