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Publisher's version (útgefin grein), Understanding the structure of a bacterial population is essential in order to understand bacterial evolution. Estimating the core genome (those genes common to all, or nearly all, strains of a species) is a key component of such analyses. The size and composition of the core genome varies by dataset, but we hypothesized that the variation between different collections of the same bacterial species would be minimal. To investigate this, we analyzed the genome sequences of 3,118 pneumococci recovered from healthy individuals in Reykjavik (Iceland), Southampton (United Kingdom), Boston (United States), and Maela (Thailand). The analyses revealed a "supercore" genome (genes shared by all 3,118 pneumococci) of 558 genes, although an additional 354 core genes were shared by pneumococci from Reykjavik, Southampton, and Boston. Overall, the size and composition of the core and pan-genomes among pneumococci recovered in Reykjavik, Southampton, and Boston were similar. Maela pneumococci were distinctly different in that they had a smaller core genome and larger pan-genome. The pan-genome of Maela pneumococci contained several >25 Kb sequence regions (flanked by pneumococcal genes) that were homologous to genomic regions found in other bacterial species. Overall, our work revealed that some subsets of the global pneumococcal population are highly heterogeneous, and our hypothesis was rejected. This is an important finding in terms of understanding genetic variation among pneumococci and is also an essential point of consideration before generalizing the findings from a single dataset to the wider pneumococcal population., This work was supported by a Wellcome Trust Biomedical Research Fund award (04992/Z/14/Z) to MM, KJ, and AB; a Wellcome Trust Research Fellowship (083511/Z/07/Z) to AB; and a University of Oxford John Fell Fund award (123/734) to AB. Core funding for the Sanger Institute was provided by the Wellcome Trust (098051). Support from the Eimskipa University Fund was received by SQ and KK. The Icelandic vaccine impact study was an investigator-initiated study funded by GlaxoSmithKline Biologicals SA and the Landspítali University Hospital Research Fund to KK, AH, HE, SB, and AB.

The bacterial core genome is of intense interest and the volume of whole genome sequence data in the public domain available to investigate it has increased dramatically. The aim of our study was to develop a model to estimate the bacterial core genome from next-generation whole genome sequencing data and use this model to identify novel genes associated with important biological functions. Five bacterial datasets were analysed, comprising 2096 genomes in total. We developed a Bayesian decision model to estimate the number of core genes, calculated pairwise evolutionary distances (p-distances) based on nucleotide sequence diversity, and plotted the median p-distance for each core gene relative to its genome location. We designed visually-informative genome diagrams to depict areas of interest in genomes. Case studies demonstrated how the model could identify areas for further study, e.g. 25% of the core genes with higher sequence diversity in the Campylobacter jejuni and Neisseria meningitidis genomes encoded hypothetical proteins. The core gene with the highest p-distance value in C. jejuni was annotated in the reference genome as a putative hydrolase, but further work revealed that it shared sequence homology with beta-lactamase/metallo-beta-lactamases (enzymes that provide resistance to a range of broad-spectrum antibiotics) and thioredoxin reductase genes (which reduce oxidative stress and are essential for DNA replication) in other C. jejuni genomes. Our Bayesian model of estimating the core genome is principled, easy to use and can be applied to large genome datasets. This study also highlighted the lack of knowledge currently available for many core genes in bacterial genomes of significant global public health importance., Author Summary Whole genome sequencing has revolutionised the study of pathogenic microorganisms. It has also become so affordable that hundreds of samples can reasonably be sequenced in an individual project, creating a wealth of data. Estimating the bacterial core genome – traditionally defined as those genes present in all genomes – is an important initial step in population genomics analyses. We developed a simple statistical model to estimate the number of core genes in a bacterial genome dataset, calculated pairwise evolutionary distances (p-distances) based on differences among nucleotide sequences, and plotted the median p-distance for each core gene relative to its genome location. Low p-distance values indicate highly-conserved genes; high values suggest genes under selection and/or undergoing recombination. The genome diagrams depict areas of interest in genomes that can be explored in further detail. Using our method, we analysed five bacterial species comprising a total of 2096 genomes. This revealed new information related to antibiotic resistance and virulence for two bacterial species and demonstrated that the function of many core genes in bacteria is still unknown. Our model provides a highly-accessible, publicly-available tool to use on the vast quantities of genome sequence data now available.

Background: One of the most important global pathogens infecting all age groups is Streptococcus pneumoniae (the ‘pneumococcus’). Pneumococci reside in the paediatric nasopharynx, where they compete for space and resources, and one competition strategy is to produce a bacteriocin (antimicrobial peptide or protein) to attack other bacteria and an immunity protein to protect against self-destruction. We analysed a collection of 336 diverse pneumococcal genomes dating from 1916 onwards, identified bacteriocin cassettes, detailed their genetic composition and sequence diversity, and evaluated the data in the context of the pneumococcal population structure. Results: We found that all genomes maintained a blp bacteriocin cassette and we identified several novel blp cassettes and genes. The composition of the ‘bacteriocin/immunity region’ of the blp cassette was highly variable: one cassette possessed six bacteriocin genes and eight putative immunity genes, whereas another cassette had only one of each. Both widely-distributed and highly clonal blp cassettes were identified. Most surprisingly, one-third of pneumococcal genomes also possessed a cassette encoding a novel circular bacteriocin that we called pneumocyclicin, which shared a similar genetic organisation to well-characterised circular bacteriocin cassettes in other bacterial species. Pneumocyclicin cassettes were mainly of one genetic cluster and largely found among seven major pneumococcal clonal complexes. Conclusions: These detailed genomic analyses revealed a novel pneumocyclicin cassette and a wide variety of blp bacteriocin cassettes, suggesting that competition in the nasopharynx is a complex biological phenomenon.