Pan-genome mediated therapeutic target mining in Kingella kingae and inhibition assessment using traditional Chinese medicinal compounds: an informatics approach.

Kingella kingae causes bacteremia, endocarditis, osteomyelitis, septic arthritis, meningitis, spondylodiscitis, and lower respiratory tract infections in pediatric patients. Usually it demonstrates disease after inflammation of mouth, lips or infections of the upper respiratory tract. To date, therapeutic targets in this bacterium remain unexplored. We have utilized a battery of bioinformatics tools to mine these targets in this study. Core genes were initially inferred from 55 genomes of K. kingae and 39 therapeutic targets were mined using an in-house pipeline. We selected aroG product (KDPG aldolase) involved in chorismate pathway, for inhibition analysis of this bacterium using lead-like metabolites from traditional Chinese medicinal plants. Pharmacophore generation was done using control ZINC36444158 (1,16-bis[(dihydroxyphosphinyl)oxy]hexadecane), followed by molecular docking of top hits from a library of 36,000 compounds. Top prioritized compounds were ZINC95914016, ZINC33833283 and ZINC95914219. ADME profiling and simulation of compound dosing (100 mg tablet) was done to infer compartmental pharmacokinetics in a population of 300 individuals in fasting state. PkCSM based toxicity analysis revealed the compounds ZINC95914016 and ZINC95914219 as safe and with almost similar bioavailability. However, ZINC95914016 takes less time to reach maximum concentration in the plasma and shows several optimal parameters compared to other leads. In light of obtained data, we recommend this compound for further testing and induction in experimental drug design pipeline.Communicated by Ramaswamy H. Sarma.

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.

Whole-Genome Sequencing Reveals Differences among Kingella kingae Strains from Carriers and Patients with Invasive Infections.

As a result of the increasing use of sensitive nucleic acid amplification tests, Kingella kingae is being recognized as a common pathogen of early childhood, causing medical conditions ranging from asymptomatic oropharyngeal colonization to bacteremia, osteoarthritis, and life-threatening endocarditis. However, the genomic determinants associated with the different clinical outcomes are unknown. Employing whole-genome sequencing, we studied 125 international K. kingae isolates derived from 23 healthy carriers and 102 patients with invasive infections, including bacteremia (n = 23), osteoarthritis (n = 61), and endocarditis (n = 18). We compared their genomic structures and contents to identify genomic determinants associated with the different clinical conditions. The mean genome size of the strains was 2,024,228 bp, and the pangenome comprised 4,026 predicted genes, of which 1,460 (36.3%) were core genes shared by >99% of the isolates. No single gene discriminated between carried and invasive strains; however, 43 genes were significantly more frequent in invasive isolates, compared to asymptomatically carried organisms, and a few showed a significant differential distribution among isolates from skeletal system infections, bacteremia, and endocarditis. The gene encoding the iron-regulated protein FrpC was uniformly absent in all 18 endocarditis-associated strains but was present in one-third of other invasive isolates. Similar to other members of the Neisseriaceae family, the K. kingae differences in invasiveness and tropism for specific body tissues appear to depend on combinations of multiple virulence-associated determinants that are widely distributed throughout the genome. The potential role of the absence of the FrpC protein in the pathogenesis of endocardial invasion deserves further investigation. IMPORTANCE The wide range of clinical severities exhibited by invasive Kingella kingae infections strongly suggests that isolates differ in their genomic contents, and strains associated with life-threatening endocarditis may harbor distinct genomic determinants that result in cardiac tropism and severe tissue damage. The results of the present study show that no single gene discriminated between asymptomatically carried isolates and invasive strains. However, 43 putative genes were significantly more frequent among invasive isolates than among pharyngeal colonizers. In addition, several genes displayed a significant differential distribution among isolates from bacteremia, skeletal system infections, and endocarditis, suggesting that the virulence and tissue tropism of K. kingae are multifactorial and polygenic, depending on changes in the allele content and genomic organization. Further analysis of these putative genes may identify genomic determinants of the invasiveness of K. kingae and its affinity for specific body tissues and potential targets for a future protective vaccine.

[An infant with septic arthritis due toKingella kingae]. / Zuigeling met septische artritis doorKingella kingae.

BACKGROUND: Kingellakingae is the most frequently found pathogen in septic arthritis in children ≤ 4 years. In contrast to more well-known pathogens, K. kingae usually causes mild arthritis symptoms without high fever or elevated infection markers. In the current (general practitioners') guidelines on septic arthritis in children too little attention is given to the indolent symptoms caused by K. kingae. This could cause a delay in the diagnosis and treatment of children with K. kingae arthritis. CASE DESCRIPTION: An 11 months old boy presented to the general practitioner with 6 days of general malaise, upper airway symptoms and a painful, swollen left knee without fever or preceding trauma. Ultrasound of the knee was normal. Blood samples showed mildly elevated infection markers. K. kingae DNA was isolated using an oropharyngeal PCR and the diagnosis K. kingae septic arthritis was made. Antimicrobial therapy was initiated and resulted in a full recovery. CONCLUSION: In children ≤ 4 years with joint symptoms a septic arthritis caused by K. kingaeshould be considered, even if in the absence of evident symptoms of infection.

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.

A multicentre evaluation and expert recommendations of use of the newly developed BioFire Joint Infection polymerase chain reaction panel.

Septic arthritis is a serious condition with significant morbidity and mortality, routinely diagnosed using culture. The FDA has recently approved the rapid molecular BioFire® Joint Infection Panel (BJIP) for synovial fluid. We aimed to evaluate the BJIP compared to culture and its potential use in patient management. A multicentre retrospective evaluation of BJIP was conducted in the UK and Ireland. Positive percent agreement (PPA) and negative percent agreement (NPA) were calculated between the BJIP and routine culture. A multidisciplinary team (MDT) discussion addressing the optimal or potential case use of the assay practice was facilitated. Three hundred ninety-nine surplus synovial fluid samples (~ 70% from native joints) from eight centres were processed using BJIP in addition to routine culture. An increased yield of positive results was detected using BJIP compared to routine culture (98 vs 83), giving an overall PPA of 91.6% and overall NPA of 93% for the BJIP compared to culture results. The BJIP detected resistant markers and additional organisms that could influence antibiotic choices including Neisseria gonorrhoeae and Kingella kingae. The MDT agreed that the assay could be used, in addition to standard methods, in adult and children patients with specialist advice use based on local needs. Rapid results from BJIP were assessed as having potential clinical impact on patient management. Organisms not included in the panel may be clinically significant and may limit the value of this test for PJI.

First documented outbreak of arthritis caused by Kingella kingae in a Spanish childcare center.

BACKGROUND: Recently, Kingella kingae (K. kingae) has been described as the most common agent of skeletal system infections in children 6 months-2 years of age. More exceptional is the clinical presentation in clusters of invasive K. kingae infections. We describe the investigation of the first outbreak of 3 cases of arthritis caused by K. kingae documented in Spain detected in a daycare center in Roses, Girona. PATIENTS AND METHODS: In December of 2015 surveillance throat swabs obtained from all attendees from the same class of the index daycare center were assessed to study the prevalence of K. kingae colonization. The sample was composed of 9 toddlers (range: 16-23 months of age). Investigation was performed by culture and K. kingae-specific RT-PCR. Combined amoxicillin-rifampicin prophylaxis was offered to all attendees who were colonized by K. kingae. Following antimicrobial prophylaxis, a new throat swab was taken to confirm bacterial eradication. RESULTS: K. kingae was detected by RT-PCR throat swabs in the 3 index cases and 5 of the 6 daycare attendees. Cultures were negative in all cases. After administration of prophylactic antibiotics, 3 toddlers were still positive for K. kingae-specific RT-PCR. CONCLUSIONS: Clusters of invasive K. kingae infections can occur in daycare facilities and closed communities. Increased awareness and use of sensitive detection methods are needed to identify and adequately investigate outbreaks of K. kingae disease. In our experience, the administration of prophylactic antibiotics could result in partial eradication of colonization. No further cases of disease were detected after prophylaxis.

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.

Kingella kingae Spondylodiscitis: Treatment Failure With Flucloxacillin.

Kingella kingae infections generally respond well to most beta-lactam antibiotics. We investigated an antibiotic treatment failure in a 3-year-old with K. kingae L3-4 spondylodiscitis. Her disease progressed even after 19 days of high-dose intravenous flucloxacillin. The clinical isolate did not produce a beta-lactamase and despite phenotypic testing and whole-genome sequencing, the mechanism of flucloxacillin resistance remains unknown.

The role of Kingella kingae in pre-school aged children with bone and joint infections.

OBJECTIVES: The Pre-school Osteoarticular Infection (POI) study aimed to describe the burden of disease, epidemiology, microbiology and treatment of acute osteoarticular infections (OAI) and the role of Kingella kingae in these infections. METHODS: Information about children 3-60 months of age who were hospitalized with an OAI to 11 different hospitals across Australia and New Zealand between January 2012 and December 2016 was collected retrospectively. RESULTS: A total of 907 cases (73%) were included. Blood cultures grew a likely pathogen in only 18% (140/781). The peak age of presentation was 12 to 24 months (466/907, 51%) and Kingella kingae was the most frequently detected microorganism in this age group (60/466, 13%). In the majority of cases, no microorganism was detected (517/907, 57%). Addition of PCR to culture increased detection rates of K. kingae. However, PCR was performed infrequently (63/907, 7%). CONCLUSIONS: This large multi-national study highlights the need for more widespread use of molecular diagnostic techniques for accurate microbiological diagnosis of OAI in pre-school aged children. The data from this study supports the hypothesis that a substantial proportion of pre-school aged children with OAI and no organism identified may in fact have undiagnosed K. kingae infection. Improved detection of Kingella cases is likely to reduce the average length of antimicrobial treatment.

Kingella kingae Displaced S. aureus as the Most Common Cause of Acute Septic Arthritis in Children of All Ages.

BACKGROUND: Acute septic arthritis (SA) still remains a challenge with significant worldwide morbidity. In recent years, Kingella kingae has emerged and treatment regimens have become shorter. We aim to analyze trends in SA etiology and management and to identify risk factors for complications. METHODS: Longitudinal observational, single center study of children (<18 years old) with SA admitted to a tertiary care pediatric hospital, from 2003 to 2018, in 2 cohorts, before and after implementation of nucleic acid amplification assays (2014). Clinical, treatment and disease progression data were obtained. RESULTS: A total of 247 children were identified, with an average annual incidence of 24.9/100,000, 57.9% males with a median age of 2 (1-6) years. In the last 5 years, a 1.7-fold increase in the annual incidence, a lower median age at diagnosis and an improved microbiologic yield (49%) was noticed. K. kingae became the most frequent bacteria (51.9%) followed by MSSA (19.2%) and S. pyogenes (9.6%). Children were more often treated for fewer intravenous days (10.7 vs. 13.2 days, P = 0.01) but had more complications (20.6% vs. 11.4%, P = 0.049) with a similar sequelae rate (3.7%). Risk factors for complications were C-reactive protein ≥80 mg/L and Staphylococcus aureus infection, and for sequelae at 6 months, age ≥4 years and CRP ≥ 80 mg/L. CONCLUSIONS: The present study confirms that K. kingae was the most common causative organism of acute SA. There was a trend, although small, for decreasing antibiotic duration. Older children with high inflammatory parameters might be at higher risk of sequelae.

Primary Septic Arthritis Among Children 6 to 48 Months of Age: Implications for PCR Acquisition and Empiric Antimicrobial Selection.

INTRODUCTION: Primary septic arthritis requires unique evaluation and treatment considerations for children in the 6- to 48-month age range because of the spectrum of identified pathogens and high rate of negative cultures. The purpose of this study is to evaluate primary septic arthritis in this age group in order to differentiate children with infection caused by Kingella kingae from those with other confirmed pathogens and those with no identified pathogen. METHODS: Preschool children who underwent multidisciplinary evaluation and treatment for septic arthritis between 2009 and 2019 were retrospectively studied. Three cohorts were established for comparison of clinical and laboratory features of primary septic arthritis: (1) confirmed K. kingae, (2) confirmed other pathogen, and (3) presumed (without identified pathogen). RESULTS: Among 139 children with septic arthritis, 40 (29%) were confirmed K. kingae, 29 (21%) other pathogen, and 70 (50%) presumed. Children with Kingella and those with presumed septic arthritis had significantly lower initial C-reactive protein (4.8 and 4.5 vs. 9.3 mg/dL) and fewer febrile hospital days (0.2 and 0.4 vs. 1.3 d) than children with other confirmed pathogens. Children with other pathogens had higher rates of bacteremia (38% vs. 0%) and positive joint fluid cultures (86% vs. 15%) than that of children with Kingella. The rate of polymerase chain reaction (PCR) acquisition was 38 of 40 (95.0%) Kingella cases, 18 of 29 (62.1%) other pathogen cases, and 33 of 70 (47.1%) presumed cases. CONCLUSIONS: K. kingae was the most commonly identified pathogen among 6-month to 4-year-old children. The Kingella and other identified pathogens in this study serve to guide empiric antimicrobial recommendations for this age range. Because of similarities between children with septic arthritis because of K. kingae and those with no identified pathogen, it is likely that an unrecognized burden of Kingella resides in culture negative cases, particularly if no PCR is sent. Systematic evaluation, including PCR acquisition, and a high index of suspicion for K. kingae are recommended to thoroughly evaluate septic arthritis in preschool children. LEVEL OF EVIDENCE: Level III-Retrospective cohort comparison.

Endovascular Infection with Kingella kingae Complicated by Septic Arthritis in Immunocompromised Adult Patient.

We report a case of Kingella kingae endovascular infection in an immunocompromised elderly patient in Israel who had culture-negative septic arthritis. This case highlights potential sources of metastatic infection other than infective endocarditis, and emphasizes the need for molecular diagnostic methods in detection of pathogens in culture-negative septic arthritis in immunocompromised patients.

Osteoarticular Infections of the Chest Wall Due to Kingella Kingae: A Series of 8 Cases.

Osteoarticular infections of the chest wall are relatively uncommon in pediatric patients and affect primarily infants and toddlers. Clinical presentation is often vague and nonspecific. Laboratory findings may be unremarkable in osteoarticular chest wall infections and not suggestive of an osteoarticular infection. Causative microbes are frequently identified if specific nucleic acid amplification assays are carried out. In the young pediatric population, there is evidence that Kingella kingae is 1 of the main the main causative pathogens of osteoarticular infections of the chest wall.

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.

The first report on epidemiology of oropharyngeal Kingella kingae carriage in Scandinavian children: K. kingae carriage is very common in children attending day care facilities in Western Norway.

Kingella kingae colonizes the upper airways in children and has been recognized as the most common causative agent of osteoarticular infections (OAI) in children below 4 years of age. This is the first Scandinavian study to investigate oropharyngeal K. kingae carriage in healthy children. From June 2015 to August 2016, we recruited 198 healthy children aged 11-14 months from routine consultations at health promotion centers in Hordaland County, Norway for a cross-sectional study. After their parents had provided informed consent; demographic data were registered, and an oropharyngeal swab was collected. The oropharyngeal swab was analyzed with a real-time PCR assay specific to K. kingae targeting the RTX toxin locus. Results showed an asymptomatic carriage rate of 12.6%. A striking and highly significant difference was observed between the children that had started attending day care facilities as compared with children still being at home (33.33% vs 8.5%; p < 0.001). K. kingae is prevalent in young children in Norway. This study emphasize that K. kingae should be considered an important etiological agent in OAI. Transmission seems to be facilitated in day care facilities. The correlation between oropharyngeal carriage and OAI needs to be further explored.

Kingella kingae and Osteoarticular Infections.

OBJECTIVES: In this study, we aimed to contrast the bacteriologic epidemiology of osteoarticular infections (OAIs) between 2 patient groups in successive 10-year periods, before and after the extensive use of nucleic acid amplification assays in the diagnostic process. METHODS: Epidemiologic data and bacteriologic etiologies of all children presenting with OAIs on admission to our institution over 20 years (1997-2016) were assessed retrospectively. The population was divided into 2 cohorts, using the standardized use of polymerase chain reaction as the cutoff point (2007). The conventional cohort included children with OAIs mainly investigated by using classic cultures, whereas the molecular cohort referred to patients also investigated by using molecular assays. RESULTS: Kingella kingae was the most frequently isolated pathogen, responsible for 51% of OAIs, whereas other classic pathogens were responsible for 39.7% of cases in the molecular cohort. A statistically significant increase in the mean incidence of OAIs was observed, as was a decrease in the mean age at diagnosis after 2007. After 2007, the pathogen remained unidentified in 21.6% of OAIs in our pediatric population. CONCLUSIONS: Extensive use of nucleic acid amplification assays improved the detection of fastidious pathogens and has increased the observed incidence of OAI, especially in children aged between 6 and 48 months. We propose the incorporation of polymerase chain reaction assays into modern diagnostic algorithms for OAIs to better identify the bacteriologic etiology of OAIs.