Knowledge of pneumococcal lineages, their geographic distribution and antibiotic resistance patterns, can give insights into global pneumococcal disease. We provide interactive bioinformatic outputs to explore such topics, aiming to increase dissemination of genomic insights to the wider community, without the need for specialist training. We prepared 12 country-specific phylogenetic snapshots, and international phylogenetic snapshots of 73 common Global Pneumococcal Sequence Clusters (GPSCs) previously defined using PopPUNK, and present them in Microreact. Gene presence and absence defined using Roary, and recombination profiles derived from Gubbins are presented in Phandango for each GPSC. Temporal phylogenetic signal was assessed for each GPSC using BactDating. We provide examples of how such resources can be used. In our example use of a country-specific phylogenetic snapshot we determined that serotype 14 was observed in nine unrelated genetic backgrounds in South Africa. The international phylogenetic snapshot of GPSC9, in which most serotype 14 isolates from South Africa were observed, highlights that there were three independent sub-clusters represented by South African serotype 14 isolates. We estimated from the GPSC9-dated tree that the sub-clusters were each established in South Africa during the 1980s. We show how recombination plots allowed the identification of a 20 kb recombination spanning the capsular polysaccharide locus within GPSC97. This was consistent with a switch from serotype 6A to 19A estimated to have occured in the 1990s from the GPSC97-dated tree. Plots of gene presence/absence of resistance genes ( tet , erm , cat ) across the GPSC23 phylogeny were consistent with acquisition of a composite transposon. We estimated from the GPSC23-dated tree that the acquisition occurred between 1953 and 1975. Finally, we demonstrate the assignment of GPSC31 to 17 externally generated pneumococcal serotype 1 assemblies from Utah via Pathogenwatch. Most of the Utah isolates clustered within GPSC31 in a USA-specific clade with the most recent common ancestor estimated between 1958 and 1981. The resources we have provided can be used to explore to data, test hypothesis and generate new hypotheses. The accessible assignment of GPSCs allows others to contextualize their own collections beyond the data presented here.

Background Streptococcus pneumoniae is among the most common causes of invasive bacterial infections in children, including pneumonia, bacteremia, and meningitis. Over 90 different serotypes (ST) of pneumococcus exist, with enrichment of some ST within specific invasive phenotypes. Other than capsular genes, molecular determinants of particular invasive phenotypes remain largely unknown. Although vaccination targeting especially invasive ST capsular antigens has successfully decreased the incidence of invasive pneumococcal disease (IPD), new ST have emerged, suggesting methods to target other aspects of pneumococcal invasiveness are needed. Methods Pneumococcal isolates from IPD were collected from children presenting to Primary Children’s Hospital from 1996–2018. All viable isolates underwent next-generation sequencing (Illumina), quality control filtering for contamination and low coverage, de novo genome assembly with SPADES, and annotation with PROKKA. Clinical phenotypes were manually validated with physician chart review. Isolates were serotyped via Quelling and in silico using SeroBA. ROARY was used for pan-genome assembly, and SCOARY for microbial genome-wide association studies. RAxML was used for phylogenetic analysis. Results A total of 354 viable pneumococcal isolates were available for genomic analysis including a spectrum of invasive phenotypes: pneumonia (n = 138, of which 54 were complicated by empyema), CNS infection (n = 50), SSTI/bone infections (n = 42), and isolated bacteremia (n = 68). Thirteen samples were censored for poor coverage or genetic contamination. Invasive isolates spanned 37 capsular ST. The pneumococcal pan-genome comprised 6462 genes, of which only 23% were shared by at least 99% of samples. Phylogenetic relatedness resulted in clustering of some ST (e.g., ST1, ST3), whereas others (eg ST19A) were more broadly distributed. Empyema and meningitis phenotypes were distributed across the phylogenetic tree, but enriched in distinct clusters that crossed ST clusters. Genes involved in empyemagenic pneumococcal capsule production, and those implicated in sensing of preferred sugars or non-preferred sugar metabolism were statistically correlated with the empyema phenotype. Conclusion There is marked genetic diversity among invasive pneumococcal isolates, potentially contributing to the variability of disease phenotypes observed. Clustering of invasive phenotypes across ST suggests a genetic signature for invasive phenotypes other than capsule genes alone, further supported by enrichment of specific genes associated with alternative sugar metabolism in empyema isolates. Critical determinants of invasive phenotypes will inform future efforts at disease prevention and treatment.

Background Streptococcus pneumoniae is the most common cause of pneumonia in children, including empyema, a severe complication with increasing incidence in the post-pneumococcal vaccine era. Only a subset of > 90 serotypes cause empyema. Virulence determinants of empyema remain largely unknown. Methods We performed Illumina sequencing of invasive Pneumococcal isolates from pediatric patients at Primary Children’s Hospital (Salt Lake City, UT) isolated between 1996–2018, de novo genome assembly (SPADES), annotation (PROKKA), serotyping (Quelling and SeroBA), and pan-genome assembly (ROARY). SCOARY and pyseer were used for microbial GWAS. Maximum likelihood phylogeny was calculated using RAxML/Gubbins. Results 366 pneumococcal isolates were analyzed from 39 serotypes and multiple phenotypes including pneumonia (n=76), empyema (n=63), CNS infection (n=54), and isolated bacteremia (n=79). Serotypes and empyema phenotype clustered roughly by phylogeny. Most analyzed empyema isolates after 2010 were serotype 3 (19/25); prior to PCV-13 introduction serotypes 1 (8/38), 7F (7/38), and 19A (11/38) were more highly represented. Genes implicated in capsule synthesis, transposases, and metabolism were statistically correlated with the empyema phenotype. Conclusion Specific capsular or metabolic genes may confer optimal fitness for pleural disease. Further characterization of these genetic associations is needed and will inform future treatment and prevention. Disclosures Carrie L. Byington, MD, BioFire (Other Financial or Material Support, Royalties for Intellectual Property)IDbyDNA (Advisor or Review Panel member) Krow Ampofo, MBChB, Merck (Grant/Research Support)

Background Streptococcus pneumoniae is the leading cause of meningitis in children and is associated with significant morbidity and mortality. Despite conjugate pneumococcal vaccines that have otherwise been successful in reducing invasive disease caused by vaccine serotypes, the incidence of pneumococcal meningitis remains relatively unchanged. Using genome-wide association, we identified sequence elements associated with pneumococcal central nervous system (CNS) infection. Methods From 1996 to 2018, 366 clinical pneumococcal isolates were collected from pediatric patients and paired with clinical metadata. All isolates underwent whole-genome sequencing and in silico serotyping. Fifty-four clinical isolates were from children with CNS infection. Sequence element enrichment analysis was performed using elastic net and linear mixed modeling statistical methods to find genetic variants associated with CNS infection. We identified associated genes by mapping statistically associated variants to a serotype 19F reference S. pneumoniae genome. Results CNS isolates spanned 22 unique serotypes; the most common were 22F (n = 6), 7F (n = 5), 18C (n = 4), and 35B (n = 4). We identified two intergenic sequence variants and one intragenic sequence variant significantly associated with CNS infection (p < 1.48 × 10-7). The top intragenic candidate in our analysis was located within cpsC, a gene involved in regulation of capsule synthesis. The CNS infection-associated cpsC variant was found in serotypes 22F (n = 1), 18C (n = 4), and 14 (n = 2). Conclusion The diversity of pneumococcal isolates associated with CNS infection suggests the importance of genetic determinants beyond capsular serotype. We identified a number of novel microbial genetic variants significantly associated with pneumococcal CNS infection across serotypes, including a coding mutation in cpsC. Functional characterization of cpsC variants is needed to inform mechanisms contributing to pneumococcal CNS virulence. Disclosures Anne J. Blaschke, MD, PhD, BioFire Diagnostics/Biomerieux: Advisor/Consultant|BioFire Diagnostics/Biomerieux: Grant/Research Support|BioFire Diagnostics/Biomerieux: IP licensed to BioFire Diagnostics through the University of Utah and royalties received through the University of Utah related to the FilmArray|Merck: Advisor/Consultant.

Background Pediatric group A streptococcal peritonitis (GASP) is a rare but serious infection, with few cases reported in the literature. Utah has an unusually high incidence of invasive GAS (iGAS) disease, but the frequency and characteristics of pediatric GASP are unknown. Methods We performed a retrospective chart review to identify GASP in Utah children from 2000-2019. GASP was defined as isolation of GAS from peritoneal fluid or blood and clinical signs of peritonitis. Results : Eleven children with GASP were identified, with slight female predominance (n=6). Median age was 6 years; males were significantly younger than females (1.4 versus 7.2 years, p=0.01). GAS was isolated from 4 of 8 blood and 8 of 11 peritoneal cultures obtained. Peritoneal fluid PCR was positive for GAS in one patient. Ten patients underwent laparotomy. Peri-appendiceal inflammation prompted appendectomy in 7 patients; only one had pathologic findings of acute appendicitis. Four patients developed streptococcal toxic shock syndrome and 7 required intensive care. Non-white race (n=4) and lack of appendectomy (n=5) were associated with more severe outcomes. Median antibiotic duration was 27 days. Median hospitalization was 8 days. All patients survived. Figure 1. Schematic representation of GAS peritonitis patient clinical course. Each patient is represented by a single line. Duration of symptoms prior to hospitalization, as well as duration of hospitalization (day 0 representing admission), intensive care, antibiotic administration, and timing of procedural interventions are noted. Duration of antibiotics after discharge for patient 3 was unable to be verified, as indicated by a question mark. Hospitalization, general pediatric hospital care. PICU, pediatric intensive care unit. IR, interventional radiology. Conclusion We present the largest pediatric case series of GASP to date. Diagnostic hallmarks included gastrointestinal symptoms, fever, systemic inflammation, and peritoneal enhancement without an abdominal source. Peri-appendiceal inflammation was common, although acute appendicitis was rare, and appendectomy was associated with a less severe course. GASP should be considered in patients with acute abdominal processes given increasing incidence of iGAS infections. Disclosures All Authors: No reported disclosures