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BackgroundInvasive Staphylococcus aureus infections are a common cause of morbidity and mortality in children. In the early 2000's the proportion of infections due the methicillin-resistant S. aureus (MRSA) increased rapidly. We described the clinical and molecular epidemiology of invasive S. aureus disease in a pediatric population.MethodsWe prospectively identified children in Utah with invasive S. aureus infections. Medical records were reviewed to determine diagnosis and clinical characteristics. Isolates were genotyped using multi-locus sequence typing. The presence of genes encoding the Panton-Valentine leukocidin (PVL) was determined using polymerase chain reaction.ResultsOver a 4-year period between January 2009 and December 2012, we identified 357 children, hospitalized at Primary Children's Hospital, with invasive S. aureus infections and isolates available for the study. Methicillin-susceptible S. aureus (MSSA) caused 79% of disease, while MRSA caused only 21% of disease. Mortality associated with invasive S. aureus infection was 3.6%. The most common diagnoses were osteoarticular infections (38%) followed by central line associated blood stream infections (19%) and pneumonia (12%). We identified 41 multi-locus sequence types. The majority of isolates belonged to 6 predominant clonal complexes (CC5, CC8, CC15, CC30, CC45, CC59). PVL was present in a minority (16%) of isolates, of which most were ST8 MRSA.ConclusionsMSSA was the primary cause of invasive S. aureus infections at our institution throughout the study period. A limited number of predominant strains accounted for the majority of invasive disease. The classic virulence factor PVL was uncommon in MSSA isolates. Further study is needed to improve our understanding of S. aureus virulence and disease pathogenesis.

Background Following widespread use of the Haemophilus influenzae serotype b (Hib) vaccine, H. influenzae serotype a (Hia) has emerged as an important pathogen in children in some regions. We describe the clinical features and molecular epidemiology of invasive Hia disease in children in Utah over an 11-year period. Methods We identified cases of invasive Hia disease, defined as detection of Hia from a normally sterile site, in children aged Results We identified 51 children with invasive Hia. The average annual incidence was 1.7 cases per 100 000 children aged Conclusions Hia is a significant cause of severe invasive bacterial infection in Utah. The majority of infections were caused by ST62 isolates, a phylogenetic division II Hia type that lacks the IS1016-bexA duplication-deletion. Hia ST62 has not been commonly reported elsewhere, suggesting a unique molecular epidemiology in our population.

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 Antibiotics are prescribed in up to 80% of pediatric intensive care unit (PICU) patients, but multicenter studies systematically evaluating antibiotic indications and appropriateness in this high-utilizing population are lacking. Methods A multicenter point prevalence study was conducted at 10 geographically diverse tertiary care US children’s hospitals. All PICU patients < 21 years of age who were receiving systemic antibiotics at 8:00 AM on each study day were included. Study days occurred in February and March 2019. Data were abstracted by critical care and/or infectious diseases trained clinicians using standardized data collection forms and definitions of antibiotic appropriateness. Results 408 of 732 PICU patients (56%) received 618 antibiotics on the two study days. Empiric therapy for suspected bacterial infections without sepsis was the most common indication for antibiotics (22%), followed by treatment of community-acquired pneumonia and empiric therapy for septic shock (12% each, Figure 1). Overall, 194 antibiotic orders (32%) were classified as inappropriate and 158 patients (39%) received at least one inappropriate antibiotic. Vancomycin, cefepime, and ceftriaxone were the antibiotics most often inappropriately prescribed (Figure 2). Antibiotics prescribed inappropriately for the top 5 indications shown in Figure 1 accounted for 77% of all inappropriate antibiotic use. Prolonged ( >4 days) empiric therapy and prolonged ( >24 hours) post-operative prophylaxis were the most common reasons antibiotics prescribed for these indications were classified as inappropriate. Pneumonia and ventilator-associated infections were the most common infections for which antibiotics were prescribed inappropriately (46%). Reasons for inappropriate antibiotic use included lack of evidence supporting a bacterial infection (no radiographic infiltrate or significant increase in respiratory support) and use of unnecessarily broad antibiotics (Table 1). Conclusion Inappropriate antibiotic use is common in the PICU, particularly for pneumonia. Studies focused on defining optimal treatment strategies, as well as improved diagnostic approaches to curtail prolonged courses of empiric therapy, should be prioritized. Disclosures All authors: No reported disclosures.

Background Haemophilus influenzae causes serious invasive disease across all ages, but has not been widely described in neonatal early-onset sepsis (EOS). EOS, likely caused by organisms acquired from the mother, can lead to significant morbidity and mortality, particularly for preterm infants. There are reports of increasing ampicillin resistance in H. influenzae. We describe a case series of EOS due to H. influenzae at our institution. Methods Neonatal H. influenzae EOS was identified based on positive sterile site cultures at Results Twelve neonates with H. influenzae EOS were identified over 11 years. Nine were preterm (24 hours). All infants had signs and symptoms of sepsis within 24 hours of birth. The majority (10/12) had a blood culture positive for H. influenzae from the time of delivery. Two infants had negative delivery blood cultures but a H. influenzae-positive placental culture. No infant had > 1 day of bacteremia. One H. influenzae isolate was serotype b, one serotype c and one non-typeable, but most isolates (9/12) were not serotyped. Only one isolate produced a β-lactamase. All infants were empirically started on ampicillin and gentamicin at delivery. Nine infants underwent lumbar puncture, two were suggestive of meningitis but cultures were negative. Five infants developed interventricular hemorrhage and six required vasoactive medications. No infant died. Conclusion H. influenzae is an infrequent but important cause of neonatal EOS. H. influenzae EOS occurred in infants with known risk factors, including prolonged rupture of membranes, prematurity, and low birth weight. Recognition of H. influenzae as a potential pathogen in EOS has implications for the use of empiric antibiotic therapy particularly ampicillin, in septic neonates. Disclosures All authors: No reported disclosures.

Background Following widespread use of the Haemophilus influenzaeserotype b (Hib) vaccine,H. influenzae serotype a (Hia) has emerged as an important pathogen in children. Rates of Hia disease are particularly high in Utah. We describe the clinical features and molecular epidemiology of invasive Hia disease in children in Utah over 11 years. Methods Cases of invasive Hia disease in children less than 18 years were identified through electronic clinical and microbiology records. Demographic data and clinical outcomes were abstracted from the medical record for all cases. Available Hia isolates were collected for molecular analysis. Isolates were genotyped by multi-locus sequence typing (MLST) and clonal division was determined using sodC PCR. Presence or absence of the putative virulence-associated IS1016-bexA duplication-deletion was evaluated. Results We identified 51 children with invasive Hia between 2007 and 2017. Median age was 11.3 months. The average annual incidence was 1.7 cases per 100,000 children aged Conclusion Hia is an important cause of severe invasive bacterial infection in Utah. Molecular analyses revealed that a majority of infections were caused by ST62 isolates, a clonal division II Hia type lacking the IS1016-bexA duplication-deletion. Hia ST62 has not been commonly reported in other settings, suggesting a unique molecular epidemiology in our population. Disclosures All authors: No reported disclosures.

The murine model of Lyme disease provides a unique opportunity to study the localized host response to similar stimulus, Borrelia burgdorferi, in the joints of mice destined to develop severe arthritis (C3H) or mild disease (C57BL/6). Pathways associated with the response to infection and the development of Lyme arthritis were identified by global gene expression patterns using oligonucleotide microarrays. A robust induction of IFN-responsive genes was observed in severely arthritic C3H mice at 1 wk of infection, which was absent from mildly arthritic C57BL/6 mice. In contrast, infected C57BL/6 mice displayed a novel expression profile characterized by genes involved in epidermal differentiation and wound repair, which were decreased in the joints of C3H mice. These expression patterns were associated with disease state rather than inherent differences between C3H and C57BL/6 mice, because C57BL/6-IL-10−/− mice infected with B. burgdorferi develop more severe arthritis than C57BL/6 mice and displayed an early gene expression profile similar to C3H mice. Gene expression profiles at 2 and 4 wk postinfection revealed a common response of all strains that was likely to be important for the host defense to B. burgdorferi and mediated by NF-κB-dependent signaling. The gene expression profiles identified in this study add to the current understanding of the host response to B. burgdorferi and identify two novel pathways that may be involved in regulating the severity of Lyme arthritis.

The spirochete Borrelia burgdorferi, the etiologic agent of Lyme disease, causes severe subacute arthritis in susceptible inbred mouse strains, such as C3H/HeN, but only mild arthritis in resistant strains such as C57BL/6. The degree of Lyme arthritis severity is controlled in part by host genetics and several quantitative trait loci have been identified which contribute to this regulation. In addition, the anti-inflammatory cytokine IL-10 assumes an important role in the control of arthritis in C57BL/6 mice. However, the identification of genes and signaling pathways that dictate arthritis severity has remained elusive. In an attempt to elucidate such genes and pathways, the power of microarray analysis was combined with information gleaned from gene manipulation models. As a result of this approach, two novel gene profiles were identified: an IFN-inducible profile in arthritis-susceptible C3H and IL-10(-/-) mice, and an epidermal/differentiation profile in C57BL/6 mice. Application of this information to TLR2(-/-) mice, which also develop severe arthritis, indicated that they also upregulated IFN-responsive genes. These results provided new insight into the regulation of Lyme arthritis development and illustrated the utility of combining gene expression analyses with genetically manipulated mouse models in unraveling mechanisms underlying specific disease processes.

C57BL/6 mice deficient in TLR2 develop more severe arthritis following infection with Borrelia burgdorferi than do wild-type C57BL/6 mice, and this increase is suppressed by the simultaneous presence of the scid mutation. This suggested a requirement for lymphocytes in the development of subacute Lyme arthritis in TLR2(-/-) mice, a feature not commonly associated with this arthritis. The increased pathology of B. burgdorferi-infected TLR2(-/-) mice was also accompanied by an increase in mononuclear cell infiltration. In this study, T cells were found to be responsible for the increase in mononuclear cells in infected TLR2(-/-) C3H mice. Accordingly, transcripts for the IFN-inducible T cell chemokines, CXCL9 and CXCL10, were greatly enhanced in joint tissue from TLR2(-/-) mice, as were transcripts for a prototypical IFN-inducible gene IFN-gamma-induced GTPase (igtp). Treatment of murine synovial cells with sonicated B. burgdorferi resulted in induction of transcripts for chemokines and other IFN-inducible genes, irrespective of the presence of TLR2. The presence of T lymphocytes greatly enhanced the transcriptional response of synovial cells. These results suggest that the increased inflammatory cell infiltration in TLR2(-/-) C3H mice is the result of localized overproduction of T cell attracting chemokines.

Several quantitative trait loci regulating murine Lyme arthritis severity have been mapped, including a highly significant linkage found on chromosome 5, termed Bb2Bb3. Within this region, the Ncf1 gene of the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase has recently been identified as a major regulator of arthritis severity in rodent models of rheumatoid arthritis, an effect attributed to protective properties of reactive oxygen species. To assess the role of Ncf1 in Lyme arthritis, we introgressed Bb2Bb3 from severely arthritic C3H/He mice onto mildly arthritic C57BL/6 mice. This increased Lyme arthritis severity, whereas the reciprocal transfer conferred protection from disease. A single nucleotide polymorphism was identified in the Ncf1 gene that did not influence the protein sequence or expression of Ncf1. Although polymorphonuclear leukocytes from C57BL/6 mice generated a greater oxidative burst than polymorphonuclear leukocytes from C3H/He mice, studies with the Bb2Bb3 congenic mice demonstrated this difference was not linked to Ncf1 alleles. Furthermore, Lyme arthritis severity was not altered in mice lacking either the Ncf1 or Gp91phox subunits of the NADPH oxidase complex. Together, these results argue that Ncf1 is not a candidate gene for regulation of Lyme arthritis and reveal Lyme arthritis to be independent of NADPH oxidase activity, distinguishing it from other models of rheumatoid arthritis.