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.

In vitro Activity of Ceftaroline Against an International Collection of Kingella kingae Isolates Recovered From Carriers and Invasive Infections.

BACKGROUND: Improvements in blood culture techniques and molecular-based diagnostics have led to increased recognition of Kingella kingae as an invasive human pathogen causing bacteremia, septic arthritis, osteomyelitis and endocarditis in young children. Serious disease and potentially life-threatening complications of infection due to K. kingae necessitate timely identification and appropriate antimicrobial therapy. Ceftaroline is a fifth-generation broad spectrum cephalosporin that possesses activity against Gram-negative and Gram-positive pathogens similar to third-generation cephalosporins, but also includes methicillin-resistant Staphylococcus aureus . This study reports the in vitro activity of ceftaroline and comparator agents against an international collection of K. kingae isolates. METHODS: A collection of 308 K. kingae isolates was obtained primarily from children with bacteremia, endocarditis, osteoarticular infections or from asymptomatic pediatric carriers. Isolates were tested for antibiotic susceptibility using Clinical and Laboratory Standard Institute broth microdilution methodology and screened for ß-lactamase production using a nitrocefin chromogenic test. RESULTS: Ceftaroline inhibited all K. kingae isolates at ≤0.06 mg/L (MIC 50/90 , 0.015/0.03 mg/L). Ceftaroline MICs were similar to results with ceftriaxone (MIC 50/90 , 0.015/0.015 mg/L), meropenem (MIC 50/90 , 0.015/0.015 mg/L) and ampicillin-sulbactam (MIC 50/90 , 0.06/0.06 mg/L). Ceftaroline MICs were slightly lower than MICs for cefuroxime and amoxicillin/clavulanate (MIC 50/90 , 0.06/0.12 mg/L). MICs were high for clindamycin (MIC 50/90 , 2/4 mg/L) and oxacillin (MIC 50/90 , 4/8 mg/L). Sixteen isolates (5.2%) yielded a positive nitrocefin test indicating production of ß-lactamase; ceftaroline demonstrated equivalent MICs against ß-lactamase - positive and ß-lactamase - negative strains (MIC 50/90 , 0.015/0.3 mg/L). CONCLUSIONS: The potent activity of ceftaroline against this large international collection of K. kingae isolates supports further clinical evaluation in children.

The Past, Present, and Future of Kingella kingae Detection in Pediatric Osteoarthritis.

As a result of the increasing use of improved detection methods, Kingella kingae, a Gram-negative component of the pediatric oropharyngeal microbiota, is increasingly appreciated as the prime etiology of septic arthritis, osteomyelitis, and spondylodiscitis in children aged 6 to 48 months. The medical literature was reviewed to summarize the laboratory methods required for detecting the organism. Kingella kingae is notoriously fastidious, and seeding skeletal system samples onto solid culture media usually fails to isolate it. Inoculation of synovial fluid aspirates and bone exudates into blood culture vials enhances Kingella kingae recovery by diluting detrimental factors in the specimen. The detection of the species has been further improved by nucleic acid amplification tests, especially by using species-specific primers targeting Kingella kingae's rtxA, groEL, and mdh genes in a real-time PCR platform. Although novel metagenomic next-generation technology performed in the patient's plasma sample (liquid biopsy) has not yet reached its full potential, improvements in the sensitivity and specificity of the method will probably make this approach the primary means of diagnosing Kingella kingae infections in the future.

Kingella kingae Reveals Its Secrets.

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Pharyngeal Colonization by Kingella kingae, Transmission, and Pathogenesis of Invasive Infections: A Narrative Review.

With the appreciation of Kingella kingae as a prime etiology of osteoarticular infections in young children, there is an increasing interest in the pathogenesis of these diseases. The medical literature on K. kingae's colonization and carriage was thoroughly reviewed. Kingella kingae colonizes the oropharynx after the second life semester, and its prevalence reaches 10% between the ages of 12 and 24 months, declining thereafter as children reach immunological maturity. Kingella kingae colonization is characterized by the periodic substitution of carried organisms by new strains. Whereas some strains frequently colonize asymptomatic children but are rarely isolated from diseased individuals, others are responsible for most invasive infections worldwide, indicating enhanced virulence. The colonized oropharyngeal mucosa is the source of child-to-child transmission, and daycare attendance is associated with a high carriage rate and increased risk of invasive disease. Kingella kingae elaborates a potent repeat-in-toxin (RTXA) that lyses epithelial, phagocytic, and synovial cells. This toxin breaches the epithelial barrier, facilitating bloodstream invasion and survival and the colonization of deep body tissues. Kingella kingae colonization and carriage play a crucial role in the person-to-person transmission of the bacterium, its dissemination in the community, and the pathogenesis of invasive infections.

Changing aetiology of paediatric septic arthritis.

The management of septic arthritis in children requires the prompt administration of antibiotic therapy and the identification of the causative pathogen. In the past, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae and Haemophilus influenzae type b were considered the main causative agents of the disease, but a substantial fraction of presumptive joint infections remained unconfirmed by conventional bacteriologic cultures. In the last two decades, our knowledge of the aetiology of paediatric infectious arthritis has substantially changed as the result of the implementation of vaccination programmes against H. influenzae type b and pneumococci, and by the use of improved detection methods. In 1988, the inoculation of synovial fluid aspirates into blood culture vials revealed that Kingella kingae, a commensal member of the oropharyngeal microbiota, was the prime aetiology of skeletal system infections in children aged 6-48 months. The clinical presentation of K. kingae arthritis is subtle, and the disease is frequently missed by classic clinical and laboratory diagnostic criteria. Many children are afebrile, the acute phase reactants levels and the white blood cell counts in the blood and synovial fluid specimens are frequently normal, requiring a high clinical acumen. Increasing use of sensitive molecular methods in recent years, and particularly nucleic acid amplification tests that target K. kingae-specific genes, has further improved the detection of this elusive pathogen, demonstrated that it is responsible for 30-93% of all cases of septic arthritis below 4 years of age and reduced the fraction of culture-negative infections.

Review highlights the latest research in Kingella kingae and stresses that molecular tests are required for diagnosis.

AIM: The aim of this study was to provide an update on paediatric Kingella kingae infections. METHODS: We used the PubMed database to identify studies published in English, French and Spanish up to 15 November 2020. RESULTS: Kingella kingae colonised the oropharynx after the age of 6 months, and the mucosal surface was the portal of entry of the organism to the bloodstream and the source of child-to-child spread. Attending day care centres was associated with increased carriage rate and transmission and disease outbreaks were detected in day care facilities. Skeletal system infections were usually characterised by mild symptoms and moderately elevated inflammation markers, requiring a high clinical suspicion index. The organism was difficult to recover in cultures and molecular tests significantly improve its detection. Kingella kingae was generally susceptible to beta-lactam antibiotics, and skeletal diseases and bacteraemia responded to antimicrobial, leaving no long-term sequelae. However, patients with endocarditis frequently experienced life-threatening complications and the case fatality rate exceeded 10%. CONCLUSION: Kingella kingae was the prime aetiology of skeletal system infections in children aged 6-48 months. Paediatricians should be aware of the peculiar features of this infection and the need to use molecular tests for diagnosis.

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.

Outbreaks of Kingella kingae Infections in Daycare Centers Suggest Tissue Tropism of the Causative Strains.

BACKGROUND: Although Kingella kingae is recognized as an important pediatric pathogen, our knowledge of the virulence factors involved in the invasion of specific host's tissues is limited. Outbreaks of K kingae infections in daycare centers represent natural experiments in which a single virulent strain, introduced into a cohort of susceptible young children, causes multiple infections. If K kingae strains exhibit tissue tropism, the syndromes observed in a given cluster of cases would be relatively homogeneous. METHODS: Clinical data of all the K kingae outbreaks known to date were gathered and analyzed. The clinical syndromes diagnosed in the affected attendees were classified as septic arthritis, osteomyelitis, tenosynovitis, soft tissue infection, bacteremia with no focal disease, endocarditis, and meningitis, and computed separately. To assess the similarity of the clinical syndromes detected within outbreaks, we used the Cramer V statistic, which is a measure of the association between 2 nominal variables and, for the purposes of the study, between the detected clinical syndromes and the outbreaks. RESULTS: A total of 23 outbreaks involving 61 attendees were identified. The mean±SD attack rate in the affected classrooms was 15.8% ± 4.8%, and the K kingae colonization rate among the attendees was 54.8% ± 25.3%. Seventy-two separate foci of infection were diagnosed. Osteomyelitis and septic arthritis were the most common clinical syndromes and were diagnosed in 26 children each, followed by tenosynovitis in 4 children. The clinical syndromes diagnosed among attendees to the same classroom showed a statistically significant tendency to be similar (P = .015). CONCLUSIONS: The distribution of clinical syndromes in clusters of K kingae infections differs from that of sporadic cases. The causative strains combine enhanced virulence and high transmissibility, and show tropism toward bones, joints, and tendon sheaths. This information can be used to identify virulence factors associated with invasion of these specific host tissues.

Laboratory Diagnosis of Human Brucellosis.

The clinical presentation of brucellosis in humans is variable and unspecific, and thus, laboratory corroboration of the diagnosis is essential for the patient's proper treatment. The diagnosis of brucellar infections can be made by culture, serological tests, and nucleic acid amplification assays. Modern automated blood culture systems enable detection of acute cases of brucellosis within the routine 5- to 7-day incubation protocol employed in clinical microbiology laboratories, although a longer incubation and performance of blind subcultures may be needed for protracted cases. Serological tests, though they lack specificity and provide results that may be difficult to interpret in individuals repeatedly exposed to Brucella organisms, nevertheless remain a diagnostic cornerstone in resource-poor countries. Nucleic acid amplification assays combine exquisite sensitivity, specificity, and safety and enable rapid diagnosis of the disease. However, long-term persistence of positive molecular test results in patients that have apparently fully recovered is common and has unclear clinical significance and therapeutic implications. Therefore, as long as there are no sufficiently validated commercial tests or studies that demonstrate an adequate interlaboratory reproducibility of the different homemade PCR assays, cultures and serological methods will remain the primary tools for the diagnosis and posttherapeutic follow-up of human brucellosis.

Microbiological Diagnosis of Skeletal System Infections in Children.

BACKGROUND: If not timely diagnosed and adequately treated, skeletal system infections in children may result in severe and permanent disability. Prompt identification of the etiology of the disease and determination of its antibiotic susceptibility are crucial for the successful management of septic arthritis, osteomyelitis, and spondylodiscitis. However, the bacteriological diagnosis of these infections has been traditionally limited by the low yield of conventional cultures and, on average, one-third of cases of pediatric joint and bone infections remained unconfirmed. OBJECTIVE: To review the medical literature to summarize the current approach diagnosing the pediatric skeletal system infections. METHODS: The relevant publications for the last three decades were reviewed. RESULTS: In recent years, the detection of skeletal system pathogens has been revolutionized by the use of improved laboratory methods, including seeding of synovial fluid and bone exudates into blood culture vials, and the development and implementation of sensitive nucleic acid amplification assays. These advances have resulted in the recognition of Kingella kingae as the predominant etiology of hematogenous infections of bones, joints, intervertebral discs and tendon sheaths in children aged 6-48 months, and reduced the fraction of culture-negative osteoarthritis. CONCLUSION: As the exudate and tissue samples obtained from young children with skeletal system infections are frequently insufficient for a comprehensive laboratory workup, physicians should take in consideration the patient's age, predisposing medical conditions and possible exposure to zoonotic organisms, and use a judicious combination of Gram's stain, culture on blood culture vials, and molecular tests to maximize the microbiological diagnosis of these diseases.