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Item Global Distribution of Invasive Serotype 35D Streptococcus pneumoniae Isolates following Introduction of 13-Valent Pneumococcal Conjugate Vaccine.(American Society for Microbiology, 2018-06-25) Lo SW; Gladstone RA; van Tonder AJ; Hawkins PA; Kwambana-Adams B; Cornick JE; Madhi SA; Nzenze SA; du Plessis M; Kandasamy R; Carter PE; Eser ÖK; Ho PL; Elmdaghri N; Shakoor S; Clarke SC; Antonio M; Everett DB; von Gottberg A; Klugman KP; McGee L; Breiman RF; Bentley SDA 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.Item SeroBA: rapid high-throughput serotyping of Streptococcus pneumoniae from whole genome sequence data.(Microbiology Society, 2018-06-15) Epping L; van Tonder AJ; Gladstone RA; The Global Pneumococcal Sequencing Consortium; Bentley SD; Page AJ; Keane JAStreptococcus pneumoniae is responsible for 240 000-460 000 deaths in children under 5 years of age each year. Accurate identification of pneumococcal serotypes is important for tracking the distribution and evolution of serotypes following the introduction of effective vaccines. Recent efforts have been made to infer serotypes directly from genomic data but current software approaches are limited and do not scale well. Here, we introduce a novel method, SeroBA, which uses a k-mer approach. We compare SeroBA against real and simulated data and present results on the concordance and computational performance against a validation dataset, the robustness and scalability when analysing a large dataset, and the impact of varying the depth of coverage on sequence-based serotyping. SeroBA can predict serotypes, by identifying the cps locus, directly from raw whole genome sequencing read data with 98 % concordance using a k-mer-based method, can process 10 000 samples in just over 1 day using a standard server and can call serotypes at a coverage as low as 15-21×. SeroBA is implemented in Python3 and is freely available under an open source GPLv3 licence from: https://github.com/sanger-pathogens/seroba.Item Effect of Vaccination on Pneumococci Isolated from the Nasopharynx of Healthy Children and the Middle Ear of Children with Otitis Media in Iceland.(American Society for Microbiology, 2018-11-27) Quirk SJ; Haraldsson G; Erlendsdóttir H; Hjálmarsdóttir MÁ; van Tonder AJ; Hrafnkelsson B; Sigurdsson S; Bentley SD; Haraldsson Á; Brueggemann AB; Kristinsson KGVaccination with pneumococcal conjugate vaccines (PCVs) disrupts the pneumococcal population. Our aim was to determine the impact of the 10-valent PCV on the serotypes, genetic lineages, and antimicrobial susceptibility of pneumococci isolated from children in Iceland. Pneumococci were collected between 2009 and 2017 from the nasopharynges of healthy children attending 15 day care centers and from the middle ears (MEs) of children with acute otitis media from the greater Reykjavik capital area. Isolates were serotyped and tested for antimicrobial susceptibility. Whole-genome sequencing (WGS) was performed on alternate isolates from 2009 to 2014, and serotypes and multilocus sequence types (STs) were extracted from the WGS data. Two study periods were defined: 2009 to 2011 (PreVac) and 2012 to 2017 (PostVac). The overall nasopharyngeal carriage rate was similar between the two periods (67.3% PreVac and 61.5% PostVac, P = 0.090). Vaccine-type (VT) pneumococci decreased and nonvaccine-type (NVT) pneumococci (serotypes 6C, 15A, 15B/C, 21, 22F, 23A, 23B, 35F, and 35B) significantly increased in different age strata post-PCV introduction. The total number of pneumococci recovered from ME samples significantly decreased as did the proportion that were VTs, although NVT pneumococci (6C, 15B/C, 23A, and 23B) increased significantly. Most serotype 6C pneumococci were multidrug resistant (MDR). Serotype 19F was the predominant serotype associated with MEs, and it significantly decreased post-PCV introduction: these isolates were predominantly MDR and of the Taiwan19F-14 PMEN lineage. Overall, the nasopharyngeal carriage rate remained constant and the number of ME-associated pneumococci decreased significantly post-PCV introduction; however, there was a concomitant and statistically significant shift from VTs to NVTs in both collections of pneumococci.Item Diverse Streptococcus pneumoniae Strains Drive a Mucosal-Associated Invariant T-Cell Response Through Major Histocompatibility Complex class I-Related Molecule-Dependent and Cytokine-Driven Pathways.(Oxford University Press, 2018-03-15) Kurioka A; van Wilgenburg B; Javan RR; Hoyle R; van Tonder AJ; Harrold CL; Leng T; Howson LJ; Shepherd D; Cerundolo V; Brueggemann AB; Klenerman PMucosal-associated invariant T (MAIT) cells represent an innate T-cell population that can recognize ligands generated by the microbial riboflavin synthesis pathway, presented via the major histocompatibility complex class I-related molecule (MR1). Streptococcus pneumoniae is a major human pathogen that is also associated with commensal carriage; thus, host control at the mucosal interface is critical. The recognition of pneumococci by MAIT cells has not been defined nor have the genomics and transcriptomics of the riboflavin operon. We observed robust recognition of pneumococci by MAIT cells, using both MR1-dependent and MR1-independent pathways. The pathway used was dependent on the antigen-presenting cell. The riboflavin operon was highly conserved across a range of 571 pneumococci from 39 countries, dating back to 1916, and different versions of the riboflavin operon were also identified in related Streptococcus species. These data indicate an important functional relationship between MAIT cells and pneumococci.Item Polyhydroxyalkanoate beads as a particulate vaccine against Streptococcus pneumoniae and Neisseria meningitidis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Manawatu, New Zealand(Massey University, 2017) González Miró, MajelaStreptococcus pneumoniae and Neisseria meningitidis are the major causes of pneumonia and meningitis, respectively, worldwide. Capsular polysaccharide-protein vaccines (conjugate vaccines) provide protection against these diseases but not protection against infections caused by serotypes and serogroups not included in these vaccines. Proteins have been increasingly considered as antigens for vaccine development due to their more structurally conserved composition when compared to capsular polysaccharides. Proteins subunit vaccines are safe and protective; however, they have limitations such as serotype-dependent immunity, and low immunogenicity of the proteins, requiring adjuvant to be included in these formulations or delivery systems that enhance the desired immune response. In addition, complex production procedures are required, increasing production costs and therefore market prices making these vaccines inaccessible for many people affected by these diseases. Recently, bacterial storage polymer inclusions have been developed as protein antigen carriers. Polyhydroxyalkanoate, in particular 3-polyhydroxybutyrate (PHB) inclusions have been successfully bioengineered to display antigens from pathogens like Mycobacterium tuberculosis and Hepatitis C virus. These particulate vaccine candidates elicited both a Th1 and Th2 immunity patterns combined with a protective immune response against Mycobacterium bovis in mice. This thesis focuses on the study of polyhydroxybutyrate (PHB) beads properties as a carrier/delivery system engineered to display antigens from extracellular bacteria. The antigens Pneumococcal adhesin A, Pneumolysin (proteins) and 19F capsular polysaccharide (CPS) from Streptococcus pneumoniae, and Neisserial adhesin A, factor H binding protein (proteins) and serogroup C CPS from Neisseria meningitidis were displayed on the PHB bead surface. These antigenic proteins were produced as fusion proteins on the PHB bead surface, while the CPS was covalently attached by chemical conjugation. Mice vaccinated with these PHB beads produced strong and antigen-specific antibody levels. In addition, splenocytes from the same mice generated both IL-17A and IFN-ɣ production. The antibodies elicited against antigenic pneumococcal proteins were able to recognise the same protein in the context of an Streptococcus pneumoniae whole cell lysate from more than six different strains, while antibodies produced after vaccination with 19F CPS conjugate to PHB showed high opsonophagocytic titers against the homologous strain. In the case of Neisseria meningitidis, bactericidal antibodies were elicited in mice vaccinated with PHB beads displaying proteinaceous and CPS antigens. Overall, this thesis shows that PHB as particulate vaccine candidate holds the promise of a broadly protective vaccine that can be produced cost-effectively for widespread application to prevent diseases caused by Neisseria meningitidis and Streptococcus pneumoniae.