Pediatric subspecialty workforce: what is needed to secure its vitality and survival?

IMPACT: The pediatric subspecialty workforce is challenged by shortages and geographic maldistribution of subspecialists. We invited leaders in pediatrics to discuss how the field's vitality and survival can be secured. These leaders presented their own opinions and not the opinion of the society or organization that they are presenting. Early exposure of future trainees to pediatrics and advocacy for improved reimbursement structures, loan repayment, and funded programs for physician scientists will enhance the recruitment and retention of pediatric subspecialists to guarantee advancement of knowledge and the appropriate care of children with chronic and complex diseases.

Acquisition, co-option, and duplication of the rtx toxin system and the emergence of virulence in Kingella.

The bacterial genus Kingella includes two pathogenic species, namely Kingella kingae and Kingella negevensis, as well as strictly commensal species. Both K. kingae and K. negevensis secrete a toxin called RtxA that is absent in the commensal species. Here we present a phylogenomic study of the genus Kingella, including new genomic sequences for 88 clinical isolates, genotyping of another 131 global isolates, and analysis of 52 available genomes. The phylogenetic evidence supports that the toxin-encoding operon rtxCA was acquired by a common ancestor of the pathogenic Kingella species, and that a preexisting type-I secretion system was co-opted for toxin export. Subsequent genomic reorganization distributed the toxin machinery across two loci, with 30-35% of K. kingae strains containing two copies of the rtxA toxin gene. The rtxA duplication is largely clonal and is associated with invasive disease. Assays with isogenic strains show that a single copy of rtxA is associated with reduced cytotoxicity in vitro. Thus, our study identifies key steps in the evolutionary transition from commensal to pathogen, including horizontal gene transfer, co-option of an existing secretion system, and gene duplication.

The Kingella kingae PilC1 MIDAS Motif Is Essential for Type IV Pilus Adhesive Activity and Twitching Motility.

Kingella kingae is an emerging pathogen that has recently been identified as a leading cause of osteoarticular infections in young children. Colonization with K. kingae is common, with approximately 10% of young children carrying this organism in the oropharynx at any given time. Adherence to epithelial cells represents the first step in K. kingae colonization of the oropharynx, a prerequisite for invasive disease. Type IV pili and the pilus-associated PilC1 and PilC2 proteins have been shown to mediate K. kingae adherence to epithelial cells, but the molecular mechanism of this adhesion has remained unknown. Metal ion-dependent adhesion site (MIDAS) motifs are commonly found in integrins, where they function to promote an adhesive interaction with a ligand. In this study, we identified a potential MIDAS motif in K. kingae PilC1 which we hypothesized was directly involved in mediating type IV pilus adhesive interactions. We found that the K. kingae PilC1 MIDAS motif was required for bacterial adherence to epithelial cell monolayers and extracellular matrix proteins and for twitching motility. Our results demonstrate that K. kingae has co-opted a eukaryotic adhesive motif for promoting adherence to host structures and facilitating colonization.

Pathogenic determinants of Kingella kingae disease.

Kingella kingae is an emerging pediatric pathogen and is increasingly recognized as a leading etiology of septic arthritis, osteomyelitis, and bacteremia and an occasional cause of endocarditis in young children. The pathogenesis of K. kingae disease begins with colonization of the upper respiratory tract followed by breach of the respiratory epithelial barrier and hematogenous spread to distant sites of infection, primarily the joints, bones, and endocardium. As recognition of K. kingae as a pathogen has increased, interest in defining the molecular determinants of K. kingae pathogenicity has grown. This effort has identified numerous bacterial surface factors that likely play key roles in the pathogenic process of K. kingae disease, including type IV pili and the Knh trimeric autotransporter (adherence to the host), a potent RTX-family toxin (epithelial barrier breach), and multiple surface polysaccharides (complement and neutrophil resistance). Herein, we review the current state of knowledge of each of these factors, providing insights into potential approaches to the prevention and/or treatment of K. kingae disease.

Surface Anchoring of the Kingella kingae Galactan Is Dependent on the Lipopolysaccharide O-Antigen.

Kingella kingae is a leading cause of bone and joint infections and other invasive diseases in young children. A key K. kingae virulence determinant is a secreted exopolysaccharide that mediates resistance to serum complement and neutrophils and is required for full pathogenicity. The K. kingae exopolysaccharide is a galactofuranose homopolymer called galactan and is encoded by the pamABC genes in the pamABCDE locus. In this study, we sought to define the mechanism by which galactan is tethered on the bacterial surface, a prerequisite for mediating evasion of host immune mechanisms. We found that the pamD and pamE genes encode glycosyltransferases and are required for synthesis of an atypical lipopolysaccharide (LPS) O-antigen. The LPS O-antigen in turn is required for anchoring of galactan, a novel mechanism for association of an exopolysaccharide with the bacterial surface. IMPORTANCE Kingella kingae is an emerging pediatric pathogen and produces invasive disease by colonizing the oropharynx, invading the bloodstream, and disseminating to distant sites. This organism produces a uniquely multifunctional exopolysaccharide called galactan that is critical for virulence and promotes intravascular survival by mediating resistance to serum and neutrophils. In this study, we established that at least some galactan is anchored to the bacterial surface via a novel structural interaction with an atypical lipopolysaccharide O-antigen. Additionally, we demonstrated that the atypical O-antigen is synthesized by the products of the pamD and pamE genes, located downstream of the gene cluster responsible for galactan biosynthesis. This work addresses how the K. kingae exopolysaccharide can mediate innate immune resistance and advances understanding of bacterial exopolysaccharides and lipopolysaccharides.

Kingella kingae PilC1 and PilC2 are adhesive multifunctional proteins that promote bacterial adherence, twitching motility, DNA transformation, and pilus biogenesis.

The gram-negative bacterium Kingella kingae is a leading cause of osteoarticular infections in young children and initiates infection by colonizing the oropharynx. Adherence to respiratory epithelial cells represents an initial step in the process of K. kingae colonization and is mediated in part by type IV pili. In previous work, we observed that elimination of the K. kingae PilC1 and PilC2 pilus-associated proteins resulted in non-piliated organisms that were non-adherent, suggesting that PilC1 and PilC2 have a role in pilus biogenesis. To further define the functions of PilC1 and PilC2, in this study we eliminated the PilT retraction ATPase in the ΔpilC1ΔpilC2 mutant, thereby blocking pilus retraction and restoring piliation. The resulting strain was non-adherent in assays with cultured epithelial cells, supporting the possibility that PilC1 and PilC2 have adhesive activity. Consistent with this conclusion, purified PilC1 and PilC2 were capable of saturable binding to epithelial cells. Additional analysis revealed that PilC1 but not PilC2 also mediated adherence to selected extracellular matrix proteins, underscoring the differential binding specificity of these adhesins. Examination of deletion constructs and purified PilC1 and PilC2 fragments localized adhesive activity to the N-terminal region of both PilC1 and PilC2. The deletion constructs also localized the twitching motility property to the N-terminal region of these proteins. In contrast, the deletion constructs established that the pilus biogenesis function of PilC1 and PilC2 resides in the C-terminal region of these proteins. Taken together, these results provide definitive evidence that PilC1 and PilC2 are adhesins and localize adhesive activity and twitching motility to the N-terminal domain and biogenesis to the C-terminal domain.

Immunization with HMW1 and HMW2 adhesins protects against colonization by heterologous strains of nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) is a common cause of localized respiratory tract disease and results in significant morbidity. The pathogenesis of NTHi disease begins with nasopharyngeal colonization, and therefore, the prevention of colonization represents a strategy to prevent disease. The NTHi HMW1 and HMW2 proteins are a family of conserved adhesins that are present in 75 to 80% of strains and have been demonstrated to play a critical role in colonization of the upper respiratory tract in rhesus macaques. In this study, we examined the vaccine potential of HMW1 and HMW2 using a mouse model of nasopharyngeal colonization. Immunization with HMW1 and HMW2 by either the subcutaneous or the intranasal route resulted in a strain-specific antibody response associated with agglutination of bacteria and restriction of bacterial adherence. Despite the specificity of the antibody response, immunization resulted in protection against colonization by both the parent NTHi strain and heterologous strains expressing distinct HMW1 and HMW2 proteins. Pretreatment with antibody against IL-17A eliminated protection against heterologous strains, indicating that heterologous protection is IL-17A dependent. This work demonstrates the vaccine potential of the HMW1 and HMW2 proteins and highlights the importance of IL-17A in protection against diverse NTHi strains.

Kingella negevensis shares multiple putative virulence factors with Kingella kingae.

Kingella negevensis is a newly described gram-negative bacterium in the Neisseriaceae family and is closely related to Kingella kingae, an important cause of pediatric osteoarticular infections and other invasive diseases. Like K. kingae, K. negevensis can be isolated from the oropharynx of young children, although at a much lower rate. Due to the potential for misidentification as K. kingae, the burden of disease due to K. negevensis is currently unknown. Similarly, there is little known about virulence factors present in K. negevensis and how they compare to virulence factors in K. kingae. Using a variety of approaches, we show that K. negevensis produces many of the same putative virulence factors that are present in K. kingae, including a polysaccharide capsule, a secreted exopolysaccharide, a Knh-like trimeric autotransporter, and type IV pili, suggesting that K. negevensis may have significant pathogenic potential.

Virulence determinants of the emerging pathogen Kingella kingae.

Kingella kingae is a gram-negative coccobacillus that is a fastidious commensal organism in the oropharynx and is being recognized increasingly as a common cause of osteoarticular infections and other invasive diseases in young children. The pathogenesis of K. kingae disease begins with bacterial adherence to respiratory epithelium, followed by translocation across the epithelial barrier, survival in the bloodstream, and dissemination to distant sites, including bones, joints, and the endocardium, among others. Characterization of the determinants of K. kingae pathogenicity has revealed a novel model of adherence that involves the interplay of type IV pili, a non-pilus adhesin, and a polysaccharide capsule and a novel model of resistance to serum killing and neutrophil killing that involves complementary functions of a polysaccharide capsule and an exopolysaccharide. These models likely apply to other bacterial pathogens as well.

Kingella kingae Surface Polysaccharides Promote Resistance to Neutrophil Phagocytosis and Killing.

Bacterial pathogens have evolved strategies that enable them to evade neutrophil-mediated killing. The Gram-negative coccobacillus Kingella kingae is an emerging pediatric pathogen and is increasingly recognized as a common etiological agent of osteoarticular infections and bacteremia in young children. K. kingae produces a polysaccharide capsule and an exopolysaccharide, both of which are important for protection against complement-mediated lysis and are required for full virulence in an infant rat model of infection. In this study, we examined the role of the K. kingae polysaccharide capsule and exopolysaccharide in protection against neutrophil killing. In experiments with primary human neutrophils, we found that the capsule interfered with the neutrophil oxidative burst response and prevented neutrophil binding of K. kingae but had no effect on neutrophil internalization of K. kingae In contrast, the exopolysaccharide resisted the bactericidal effects of antimicrobial peptides and efficiently blocked neutrophil phagocytosis of K. kingae This work demonstrates that the K. kingae polysaccharide capsule and exopolysaccharide promote evasion of neutrophil-mediated killing through distinct yet complementary mechanisms, providing additional support for the K. kingae surface polysaccharides as potential vaccine antigens. In addition, these studies highlight a novel interplay between a bacterial capsule and a bacterial exopolysaccharide and reveal new properties for a bacterial exopolysaccharide, with potential applicability to other bacterial pathogens.IMPORTANCEKingella kingae is a Gram-negative commensal in the oropharynx and represents a leading cause of joint and bone infections in young children. The mechanisms by which K. kingae evades host innate immunity during pathogenesis of disease remain poorly understood. In this study, we established that the K. kingae polysaccharide capsule and exopolysaccharide function independently to protect K. kingae against reactive oxygen species (ROS) production, neutrophil phagocytosis, and antimicrobial peptides. These results demonstrate the intricacies of K. kingae innate immune evasion and provide valuable information that may facilitate development of a polysaccharide-based vaccine against K. kingae.

Clinical Features and Outcomes of Children with Culture-Negative Septic Arthritis.

BACKGROUND: Septic arthritis is a serious infection, but the results of blood and joint fluid cultures are often negative in children. We describe here the clinical features and management of culture-negative septic arthritis in children at our hospital and their outcomes. METHODS: We performed a retrospective review of a cohort of children with septic arthritis who were hospitalized at Children's Hospital of Philadelphia between January 2002 and December 2014. Culture-negative septic arthritis was defined as a joint white blood cell count of >50000/µL with associated symptoms, a clinical diagnosis of septic arthritis, and a negative culture result. Children with pretreatment, an intensive case unit admission, Lyme arthritis, immunodeficiency, or surgical hardware were excluded. Treatment failure included a change in antibiotics, surgery, and/or reevaluation because of a lack of improvement/worsening. RESULTS: We identified 157 children with septic arthritis. The patients with concurrent osteomyelitis (n = 28) had higher inflammatory marker levels at presentation, had a longer duration of symptoms (median, 4.5 vs 3 days, respectively; P < .001), and more often had bacteremia (46.4% vs 6.2%, respectively; P < .001). Among children with septic arthritis without associated osteomyelitis, 69% (89 of 129) had negative culture results. These children had lower C-reactive protein levels (median, 4.0 vs 7.3 mg/dL, respectively; P = .001) and erythrocyte sedimentation rates (median, 39 vs 51 mm/hour, respectively; P = .01) at admission and less often had foot/ankle involvement (P = .02). Among the children with culture-negative septic arthritis, the inpatient treatment failure rate was 9.1%, and treatment failure was more common in boys than in girls (17.1% vs 3.8%, respectively; P = .03). We found no association between treatment failure and empiric antibiotics or patient age. No outpatient treatment failures occurred during the 6-month follow-up period, although 17% of the children discharged with a peripherally inserted central catheter line experienced complications, including 3 with bacteremia. CONCLUSIONS: The majority of septic arthritis infections at our institution were culture negative. Among patients with culture-negative infection, empiric antibiotics failed for 9% and necessitated a change in therapy. More sensitive diagnostic testing should be implemented to elucidate the causes of culture-negative septic arthritis in children.

Differential recognition of Haemophilus influenzae whole bacterial cells and isolated lipooligosaccharides by galactose-specific lectins.

Bacterial surfaces are decorated with carbohydrate structures that may serve as ligands for host receptors. Based on their ability to recognize specific sugar epitopes, plant lectins are extensively used for bacteria typing. We previously observed that the galactose-specific agglutinins from Ricinus communis (RCA) and Viscum album (VAA) exhibited differential binding to nontypeable Haemophilus influenzae (NTHi) clinical isolates, their binding being distinctly affected by truncation of the lipooligosaccharide (LOS). Here, we examined their binding to the structurally similar LOS molecules isolated from strains NTHi375 and RdKW20, using microarray binding assays, saturation transfer difference NMR, and molecular dynamics simulations. RCA bound the LOSRdKW20 glycoform displaying terminal Galß(1,4)Glcß, whereas VAA recognized the Galα(1,4)Galß(1,4)Glcß epitope in LOSNTHi375 but not in LOSRdKW20, unveiling a different presentation. Binding assays to whole bacterial cells were consistent with LOSNTHi375 serving as ligand for VAA, and also suggested recognition of the glycoprotein HMW1. Regarding RCA, comparable binding to NTHi375 and RdKW20 cells was observed. Interestingly, an increase in LOSNTHi375 abundance or expression of HMW1 in RdKW20 impaired RCA binding. Overall, the results revealed that, besides the LOS, other carbohydrate structures on the bacterial surface serve as lectin ligands, and highlighted the impact of the specific display of cell surface components on lectin binding.