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Funder: Bill and Melinda Gates Foundation (Bill & Melinda Gates Foundation), 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.

Characteristics and assembly statistics for the ST217 study isolates. (DOCX 166Â kb)

Characteristics and assembly statistics for the ST217 study isolates. (DOCX 166Â kb)

Distribution of genes associated with virulence and colonisation in the ST217 in serotype 1 isolates. Presence of the genes was screened in all the isolates using BLAST as described in the methods section. (PDF 1702Â kb)

Deletion of the immunoglobulin A (iga) protease gene in the ST217 isolates. Genomic comparison of one of the serotype 1 isolates (ERS194295) that lacked the iga gene against the TIGR4 reference S. pneumoniae genome with an intact iga gene in its chromosome to determine the location and structure of the genomic deletion in the ST217 isolates. Sequence comparison was performed by BLASTN and visualised with Artemis Comparison Tool (ACT). (PDF 528Â kb)

BACKGROUND: Pneumococcus kills over one million children annually and over 90 % of these deaths occur in low-income countries especially in Sub-Saharan Africa (SSA) where HIV exacerbates the disease burden. In SSA, serotype 1 pneumococci particularly the endemic ST217 clone, causes majority of the pneumococcal disease burden. To understand the evolution of the virulent ST217 clone, we analysed ST217 whole genomes from isolates sampled from African and Asian countries. METHODS: We analysed 226 whole genome sequences from the ST217 lineage sampled from 9 African and 4 Asian countries. We constructed a whole genome alignment and used it for phylogenetic and coalescent analyses. We also screened the genomes to determine presence of antibiotic resistance conferring genes. RESULTS: Population structure analysis grouped the ST217 isolates into five sequence clusters (SCs), which were highly associated with different geographical regions and showed limited intracontinental and intercontinental spread. The SCs showed lower than expected genomic sequence, which suggested strong purifying selection and small population sizes caused by bottlenecks. Recombination rates varied between the SCs but were lower than in other successful clones such as PMEN1. African isolates showed higher prevalence of antibiotic resistance genes than Asian isolates. Interestingly, certain West African isolates harbored a defective chloramphenicol and tetracycline resistance-conferring element (Tn5253) with a deletion in the loci encoding the chloramphenicol resistance gene (cat pC194), which caused lower chloramphenicol than tetracycline resistance. Furthermore, certain genes that promote colonisation were absent in the isolates, which may contribute to serotype 1's rarity in carriage and consequently its lower recombination rates. CONCLUSIONS: The high phylogeographic diversity of the ST217 clone shows that this clone has been in circulation globally for a long time, which allowed its diversification and adaptation in different geographical regions. Such geographic adaptation reflects local variations in selection pressures in different locales. Further studies will be required to fully understand the biological mechanisms which makes the ST217 clone highly invasive but unable to successfully colonise the human nasopharynx for long durations which results in lower recombination rates.

Intragenic number of observed and expected numbers of SNPs in the ST217 isolates. All the genes in the ST217 isolates, sizes, observed (OSNP) and expected (ESNP) number of SNPs, ratio OSNP to ESNP and products of each gene are summarised. (DOCX 315Â kb)

Deletion of the immunoglobulin A (iga) protease gene in the ST217 isolates. Genomic comparison of one of the serotype 1 isolates (ERS194295) that lacked the iga gene against the TIGR4 reference S. pneumoniae genome with an intact iga gene in its chromosome to determine the location and structure of the genomic deletion in the ST217 isolates. Sequence comparison was performed by BLASTN and visualised with Artemis Comparison Tool (ACT). (PDF 528Â kb)

Intragenic number of observed and expected numbers of SNPs in the ST217 isolates. All the genes in the ST217 isolates, sizes, observed (OSNP) and expected (ESNP) number of SNPs, ratio OSNP to ESNP and products of each gene are summarised. (DOCX 315Â kb)