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Introduction (ID 42)
O021 - RAPID, INTERACTIVE AND SECURE ANALYSIS OF PNEUMOCOCCAL SEQUENCING DATA FOR ON-SITE EPIDEMIOLOGY (ID 303)
Abstract
Background
Genomic surveillance of pneumococci promises to revolutionize how we trace the global spread of strains, detect resistance, and make decisions on how to control disease. However, complex and cumbersome methods, barriers to data integration, and reproducibility issues with currently used genotyping schemes have prevented the translation of technological innovation into the clinic.
Methods
Our core method, PopPUNK, uses a machine learning approach to produce a stable, consistent genotyping scheme reflecting pneumococcal population biology. We have extended this software to use customised genome sketching techniques to enhance its flexibility, scalability, and portability. We also provide a machine-learning based approach for determining susceptibility to antimicrobials.
Results
We have defined the structure of the pneumococcal species by applying our method to over 50,000 globally sampled genomes. Users can rapidly integrate their own sequences into the context of this global population, directly from the browser, and visualise the results without the need for bioinformatics expertise. Phylogenetic analysis of sub-populations, for example to determine outbreak characteristics, has also been optimised and automated. Unlike some other platforms, users will not be required to give away any rights to their data, and sequence data never leaves the local machine.
Conclusions
Our software suite empowers local epidemiologists to perform analysis onsite, regardless of prior expertise, available computational resources, or location.
O022 - CHARACTERISATION OF PNEUMOCOCCAL WITHIN-HOST DIVERSITY DURING TREATMENT, TRANSMISSION AND CARRIAGE VIA DEEP POPULATION SEQUENCING (ID 374)
Abstract
Background
Typically studies investigating the genomics of Streptococcus pneumoniae have used whole genome sequencing (WGS) of single colony isolates obtained by sequencing DNA after selecting an individual colony from a culture plate. Such studies provide limited insight into within-host genomic diversity, and can obscure cases of multiple carriage.
Methods
More than 3500 nasopharyngeal swab samples from the longitudinal Maela pneumococcal colonization study were plated onto selective Columbia nalidixic acid agar plates. All colonies were harvested from plates and DNA extracted for deep within-host population sequencing.
Results
We demonstrate that population sequencing can accurately identify instances of multiple colonization and antimicrobial resistance elements. The increased resolution detected a greater prevalence of the invasive serotype 1 during carriage, and an elevated rate of transmission from mothers to their children in the first year of the child’s life. Accurate data on the administration of antimicrobials indicated that infants were at risk of both the acquisition and colonization of a multi drug resistant bacterium following antimicrobial treatment. Rates of co-colonisation imply that in the absence of treatment, susceptible lineages outcompete resistant lineages within the host.
Conclusions
The improved resolution and additional insights into co-colonisation, antimicrobial resistance, within-host selection and transmission indicate that deep within-host population sequencing can provide a general approach to enhance the genomic surveillance of S. pneumoniae and other pathogens.
O023 - GENOME-WIDE ASSOCIATION STUDY OF PNEUMOCOCCAL CARRIAGE AMONG NEPALESE CHILDREN. (ID 435)
Abstract
Background
Determining the host molecular genetic characteristics associated with pneumococcal colonisation may inform the development of new clinical interventions which could interrupt pneumococcal transmission and progression to disease. We performed a genome-wide association study to identify the genetic variants associated with pneumococcal carriage.
Methods
DNA samples collected from healthy Nepalese children were genotyped using Illumina Global Screening Arrays. Array data underwent QC and filtering before undergoing imputation using the HRC R1.1 2016 reference panel. Nasopharyngeal swabs collected from participants were processed for the presence of pneumococci by conventional microbiological techniques. Association analysis was performed using PLINK 2.
Results
Following filtering, 1355 carriers (cases) and 766 non-carriers (controls) were analysed. 10 variants within a single region on chromosome 12, were associated with pneumococcal carriage (p<10-8). The variant that had the strongest association with pneumococcal carriage (MAF carriers = 0.07 vs MAF non-carriers = 0.13, OR 0.52, 95% CI 0.42-0.64, p=2.3x10-8), was located in PPFIA2 (PTPRF Interacting Protein Alpha 2).
Conclusions
We identified host genetic variants in PPFIA2 (which encodes a protein involved in cell adhesion, integrin expression, and cell-matrix interactions) are associated with pneumococcal carriage. Further studies confirming this association and the biological role in pneumococcal carriage are needed.
O024 - PHYLOGENETIC INFERENCE OF THE TRANSMISSION DIRECTION OF PNEUMOCOCCAL INFECTIONS, A VALIDATION STUDY (ID 782)
Abstract
Background
Sustaining herd protection via reduced-dose schedules of pneumococcal vaccine may ameliorate some of the associated costs but necessitates a better understanding of Streptococcus pneumoniae (Sp) transmission. We ran a pilot study to explore a phylogenetic approach for detecting pneumococcal occurrence and direction of transmission.
Methods
Five putative transmission pairs and their same days’ nasopharyngeal swabs were selected from a longitudinal household study of asymptomatic carriage. Deep-sequencing of the infecting Sp populations was performed from culture sweeps and the direction of transmission was inferred using Phyloscanner. Transmission bottlenecks were assessed by comparing the frequency of single nucleotide polymorphism (SNPs) within transmission pairs.
Results
Phylogenetic clustering of all five transmission pairs was concordant with epidemiological information. The concordance of the direction of transmission inferred from phylogenies and from epidemiological criteria depended on the genome fragment size and the minimum number of SNPs included; with seemingly increasing concordance if phylogenetic inference is based on longer fragment sizes. In 4 of 5 instances, the putative source of transmission had higher number of unique SNPs than the recipient.
Conclusions
This study is one of the first to attempt inference of transmission directionality from bacteria using whole-genome NGS data. While our sample size was small, we show promise for detecting linked pneumococcal infection and their direction of transmission, particularly if read length is high.
O025 - MECHANISMS OF VIRULENCE REGULATION AND GLOBAL DISTRIBUTION OF VACCINE CANDIDATE ANTIGENS IN THE HIGH VIRULENT STREPTOCOCCUS PNEUMONIAE STRAINS (ID 285)
Abstract
Background
Metabolic flexibility is a prerequisite for successful transition of Streptococcus pneumoniae (Spn) from colonizing to invasive state. The aim of this study was to investigate the common metabolic features of pneumococcus invasive strains and propose vaccine candidate antigens.
Methods
The dataset (1058 PubMLST Spn strains) was stratified by genotype and included all known epidemically significant invasive and non-invasive clones. Genomes (495) were analyzed with GenomeComparator. Core gene variant matrix was used to run machine learning algorithms (RandomForest, XGBoost) to find gene variants associations with genotypes and invasiveness. Topology and composition of phylogenetic trees clusters comparison was performed using Visual TreeCmp.
Results
Phylogenetic analysis (based on MLST concatenates) revealed clustering of global pneumococcal population into the three groups (A, B1, B2), which may be associated with virulence. 181 genes were significant for the formation of A/B1/B2 groups: the components of various metabolic pathways (fatty acid biosynthesis, phenylalanine, biotin, arginine, galactose, fructose, mannose, purine bases, pyruvate, cysteine and methionine, alanine, aspartate and glutamate) and different virulence factors. Invasiveness is regulated by network of interacting genes variants: strH (exo-β-D-N-acetylglucosaminidase), carbohydrate metabolism components (gnd, dexB), aromatic amino acid synthesis pathways (aroE), relA regulatory gene (GTP pyrophosphokinase), peptidoglycan synthesis and cell division genes (murD, pbp1A), regulatory genes of polysaccharide capsule expression (wzg-wzh-wze).
Conclusions
Carbohydrates and aromatic amino acids metabolism pathways are involved in the regulation of cellular processes and phenotypic features of pneumococcus, including virulence. We achieved the list of vaccine candidates, which must be confirmed by reverse vaccinology.
O026 - WEIGHT SYSTEM ADJUSTS INVASIVE PNEUMOCOCCAL DISEASE SURVEILLANCE DATA TO REPRESENT THE GENOMIC COMPONENTS OF CARRIAGE POPULATION (ID 785)
Abstract
Background
Invasive pneumococcal disease datasets can reflect the carriage population, but with invasive serotypes overrepresented. To enrich the genomic data-usage of population-based invasive disease data for studying evolutionary dynamics, we aimed to develop a weight system to adjust the invasive disease data to represent carriage population.
Methods
We calculated the weight for each serotype as the ratio of percentage of the serotype in the carriage data to its percentage in all invasive cases, during the pre-vaccine era 1998-2000, in the US. We applied the weights to the accessory gene absence/presence matrix for each sequence in the invasive dataset (n = 11,784), after we conducted a bioinformatic pipeline to generate the set of accessory genes existing in 5% to 95% of the bacteria population.
Results
The weight system significantly improved the correlation of accessory gene frequency between invasive data and carriage population, for pre-vaccine, post-PCV7, and post-PCV13. The correlations of gene frequency between different vaccine periods in invasive data became more similar to those in carriage population. As the negative frequency dependent selection is one of the evolutionary mechanisms for Streptococcus Pneumonia, the accessory gene frequency “rebounce” after vaccine perturbance found in previous studies was observed in the invasive data during post-PCV13, after the weights were applied.
Conclusions
The proper weight system can adjust invasive disease datasets to represent the genomic components of the carriage population of Streptococcus Pneumoniae. Our methods enrich the value of genomic sequences from invasive disease surveillance, as it is common, easy to collect, and of direct interest of public health.
O027 - DIFFERENTIAL EXPRESSION OF COMPETENCE GENES ASSOCIATED WITH VARIATION IN TRANSFORMATION FREQUENCIES AMONG SEROTYPE 3 STREPTOCOCCUS PNEUMONIAE (ID 176)
Abstract
Background
Recombination has shaped the evolutionary history of Streptococcus pneumoniae, allowing rapid adaptation to interventions. Pneumococci can undergo recombination through natural competence – a state in which bacteria can uptake DNA from the environment. Recombination frequencies vary considerably across pneumococcal populations; yet the underlying mechanisms for these variations are not well understood.
Methods
We sought to elucidate differences in transformation frequency and the underlying genetic mechanisms among strains from two distinct phylogenetic clades of Serotype 3 belonging clonal complex (CC) 180 and a well-characterized non-CC180 comparison strain D39. To this end, we performed in-vitro competence assays and measured differential expression of early and late competence genes using RNA-sequencing.
Results
We found consistent differences in transformation frequencies among CC180 clades and D39 (Figure 1), which correlated with variation in differentially expressed genes during competence. While all strains exhibited a similar response to competence stimulating peptide for early competence genes, expression of late competence genes encoding the DNA uptake apparatus, DNA repair and recombination proteins needed for recombination were attenuated among strains demonstrating lower transformation frequencies. We also observed differences in expression of genes linked to bacteriocin production, which may partially explain observed population genomic-level differences. Further genomic analysis suggests variation in promoter sequences governing late competence genes may be slowing transition from early to late components of the competence pathway.
Conclusions
We show that there is considerable variation in competence even among closely related strains and that this variation may be the result of subtle genomic differences.