Kingella Species

Microbiology

K. kingae is a facultative anaerobic β-hemolytic gram-negative bacterium that appears as pairs and short chains of 4–8 plump bacilli with tapered ends ( Fig. 176.1 ). Some strains resist decolonization and may be misclassified as gram-positive, but electron microscopy shows the characteristic gram-negative cell wall structure.

K. kingae grows on routine laboratory media such as trypticase soy agar with 5% added hemoglobin (blood-agar), chocolate-agar plates, and GC-based media as small grey colonies producing marked pitting on the agar plate surface. Most K. kingae strains grow well on Mueller-Hinton agar with 5% added defibrinated horse blood and 20 mg/L of β-NAD and on Thayer-Martin agar but fail to develop on MacConkey or Krigler agar. Growth is enhanced in a CO ₂ -enriched atmosphere. The organism is encapsulated, non-motile, exhibits positive-oxidase and catalase-negative reactions, produces acid from glucose and maltose but not from other sugars, exhibits positive alkaline and acid phosphatase activity, and hydrolyzes indoxyl phosphate and L-prolyl-β-naphthylamide. K. kingae should be suspected on the bases of the characteristic Gram stain morphology, β-hemolysis, positive oxidase, and a negative catalase tests, and its identification can be confirmed by commercial systems, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) spectrometry, 16S rDNA gene sequencing, and molecular tests that amplify species-specific targets.

Virulence And Pathogenesis

The pathogenesis of invasive K. kingae disease is believed to begin with the colonization of the oropharyngeal mucosal surface, and organisms recovered from normally sterile sites are genotypically identical to those isolated from the oropharynx. Contrarily to other pathogens of respiratory origin such as Haemophilus , Moraxella , and the pneumococcus, K. kingae is not recovered from nasopharyngeal cultures.

Colonization involves an initial loose attachment to epithelial cells by type IV pili and a trimeric autotransporter protein called Knh. Following this, the pili retract, pulling the organism closer to the host cell, thus displacing the polysaccharide capsule and exposing a trimeric autotransporter Knh adhesin that mediates high-strength and tight adherence to the host mucosal surface . Most pharyngeal isolates and those recovered from bacteremic children express pili, whereas the majority of strains associated with osteoarticular infections or endocarditis are non-piliated, suggesting that piliation is not only important in establishing K. kingae colonization of the upper respiratory epithelium but is also important in the initial stages of bloodstream invasion, but is unfavorable to the organism for invading joint, bone, and cardiac tissues. Remarkably, the bacterial density of K. kingae organisms on the oropharyngeal surface in children with skeletal infections does not differ from that found in healthy carriers, suggesting that the intrinsic virulence of the strain, antecedent or concurrent viral infections, and other factors play an important role in the transition from asymptomatic carriage to disease.

The organism produces a potent pore-forming 100 kD extracellular toxin that belongs to the repeats-in-toxin (RTX) family, identical to that elaborated by K. negevensis and homologous to the RTX toxins of Haemophilus influenzae and Neisseria meningitidis , suggesting that toxin encoding genes have been acquired by horizontal transfer from a donor bacterium. ^{, ,} The RTX toxin disrupts mucosal epithelial cells, enables bacterial survival in the hostile bloodstream environment, and causes damage to skeletal tissues, promoting colonization of bones and joints. The RTX toxin also is present in cytotoxic outer membrane vesicles (OMVs) that pinch out of K. kingae outer membrane during growth. OMVs are internalized by human osteoblasts and synovial cells, and this mechanism of toxin delivery may contribute to the pathogenesis of K. kingae osteomyelitis. Intraperitoneal inoculation of 7-day-old rats with an invasive K. kingae strain derived from a child with arthritis resulted in a rapidly fatal illness consisting of abdominal wall necrosis, peritonitis, bacteremia, and seeding of internal organs, whereas inactivation of the RTX encoding operon renders the organism unable to cause invasive infection in laboratory animals. ^, Remarkably, intraperitoneal or intra-articular inoculation of 21-day-old rats with the wild-type strain did not result in observable disease, bacteremia, or arthritis, indicating that the age-dependent susceptibility to invasive infections of humans is reproduced in the experimental model.

K. kingae elaborates a polysaccharide capsule anchored to the cell wall, of which four chemically different types (a–d) have been described to date. ^, While capsules c and d are associated with pharyngeal colonization, 95% of invasive isolates elaborate either capsule types a or b, suggesting a potential target for a potential vaccine. ^, Among 567 K. kingae isolates from invasive and carried sites, only 2 (0.4%) were found to be non-encapsulated, indicating the importance of the polysaccharide capsule both in colonization and disease. ^, K. kingae also produces a galactan exopolysaccharide of two different structures, which acts synergistically with the capsule to protect the organism against complement-mediated lysis and to achieve full virulence in an infant rat model of infection. ^, The capsule prevents neutrophil binding to the bacterium and also interferes with the neutrophil oxidative burst response, while the exopolysaccharide confers resistance to antibacterial peptides and blocks phagocytosis. ^, The exopolysaccharide also inhibits biofilm formation, limiting the growth of competing flora and facilitating mucosal colonization by K. kingae .

K. kingae is naturally competent, and uptake of DNA by horizontal gene transfer represents an important source of genomic diversity, resulting in remarkable genomic heterogeneity of the species. ^, However, not all strains colonizing the pharynx are capable of causing disease in the human host and murine models. ^, A few clones have caused a disproportionate fraction of invasive diseases worldwide for >2 decades, while others are commonly carried by healthy children but rarely are isolated from infected sites and/or have a more restricted geographical or temporal distribution. Clone K isolates are associated with bloodstream infection (BSI), clone N organisms with osteoarticular invasion, and clone P with bacterial endocarditis, indicating tropism for specific host tissues. Each clone is characterized by a distinct allele combination of the RTX, housekeeping genes, and porin- and capsule-encoding genes. This linkage disequilibrium indicates that some K. kingae clones are the subject of positive selection, resisting the dislocating effect of horizontal gene transfer. These successful clones exhibit notable stability over prolonged periods and have undergone clonal expansion and wide geographic dispersion. ^,

Children with K. kingae disease frequently exhibit symptoms of nonspecific viral upper respiratory infection, or buccal ulcers caused by herpetic gingivostomatitis, varicella, and especially hand-foot-and-mouth disease or herpangina, suggesting that a breach of the mucosal lining facilitates invasion.. ^, The ensuing BSI may result in symptoms or more frequently seeds to distant sites such as joints, bones, intervertebral disks, or the endocardium for which K. kingae shows a peculiar affinity. ^, Except for endocardial infections, the bacteremic phase usually is transient and short living and, when a focal skeletal infection is detected, blood cultures usually are negative.

Immunity

In a longitudinal study, titers of immunoglobulin G (IgG) antibodies against K. kingae outer membrane proteins were high at 2 months of age, reached a nadir at the age of 6–7 months, remained low until the age of 18 months, and increased at 24 months, whereas serum IgA levels were lowest at 2 months and also peaked in the second year. The low rate of disease, absence of pharyngeal carriage, and high levels of IgG but no IgA antibodies in early infancy suggest protection conferred by maternal antibodies. The high pharyngeal prevalence of K. kingae and the increased incidence of invasive disease among children 6–24 months old coincide with the age at which antibody levels are lowest. Increasing antibody levels in older children presumably reflect immunologic maturation and cumulative experience with K. kingae through colonization or with cross-reacting antigens, resulting in a reduction of carriage and burden of disease. Exposed epitopes, however, are polymorphic; the immune response appears to be incomplete and is strain-specific.

Epidemiology

Carriage

Pharyngeal colonization usually commences after the age of 6 months, ^, sharply increases between the ages of 12 through 24 months and declines thereafter, paralleling the age-related incidence of invasive disease ( Fig. 176.2 ). The prevalence of carriage among adults is low, and the colonization is short and usually related to intimate contact with young children, and especially with childcare center attendees. ^, Prevalence studies have shown colonization rates of 23% among young children in New Zealand’s South Island, 9%–13% in southern Israel, ^{, ,} 13% in Western Norway, 9% in Geneva (Switzerland), 5.1% in France, and low to nil in Vancouver (Canada) and Sidney (Australia). Although this wide range may reflect true prevalence disparities, differences in detection methods (culture vs. molecular tests) and in the study population features (age, childcare attendance patterns, antibiotic consumption) may account for differences. Colonization by K. kingae is characterized by the frequent turnover of strains after weeks or months of carriage. ^{, ,}

The colonized pharyngeal mucosa is the gateway for the spread of K. kingae organisms to spread from child to child by droplet transmission. Close contact between siblings and playmates, upper respiratory infections, and childcare center attendance increase the risk for acquisition of new strains. ^{, ,} Childcare attendance is strongly and independently associated with K. kingae colonization, ^, and an average carriage rate of 28% was found in a longitudinal study conducted among attendees to an Israeli childcare facility. In recent years, clusters of invasive infections have been associated with childcare centers in the US, ^, Western Europe, ^, and Israel. These outbreaks are characterized by the simultaneous or consecutive occurrence of multiple cases of invasive K. kingae disease within a period of up to 4 weeks with an average attack rate among attendees of 16%, and frequently are precipitated by seasonal viral respiratory illnesses or hand-foot-and-mouth disease. When oropharyngeal specimens obtained from asymptomatic children attending classrooms where K. kingae outbreaks occurred were tested using sensitive molecular assays, up to 93% were found to be colonized by organisms genomically identical to those recovered from index cases, indicating the outbreak strain’s colonization fitness, high transmissibility and enhanced virulence. ^,

Invasive Infections

Because of the fastidious nature of K. kingae , the vast majority of invasive infections caused by the organism have been reported from North America, Western Europe, and Israel, where advanced detection methods, including automated blood culture systems and nucleic acid amplification assays, are widely available. However, the recovery of K. kingae from Latin American countries, sub-Saharan Africa, India, and Iran in recent years indicates that the organism has a worldwide distribution.

In a study performed among the pediatric population of the Geneva area in Switzerland, the risk of a child colonized by K. kingae to develop an invasive infection was estimated to be <1% per year. ⁴⁸

K. kingae infections are almost limited to young children. Of a total of 151 culture-proven patients identified over 28 years in southern Israel, only one was an adult; 149 of the 150 children were <4 years, yielding an annual incidence of invasive disease of 9.4/100,000 in this age group. Because of the suboptimal detection of the organism in culture, this figure should be considered only a minimal estimate, the true incidence probably 4- to 5-fold higher. Infection is rare in the first 6 months of age, reaches a peak at the end of the first year of life, and sharply decreases with increasing age ( Fig. 176.3 ).

Most infected children <4 years are otherwise healthy, whereas older children and adults often have immunosuppressing conditions, liver cirrhosis, diabetes, sickle cell anemia, malignancies, or antecedent cardiac valve pathology. ^{, ,} In a multicenter Israeli study, none of 291 previously healthy children with a culture-proven K. kingae infection were >48 months of age, whereas 9 of 22 (41%) children with underlying diseases were >48 months of age. The disease is more common among males, with a male-to-female ratio of 1.3.

Invasive K. kingae infections occur throughout the year and are most common during November and December and least common between February and April. ^, Since the carriage rate of the organism remains stable throughout the year, it has been suggested that the observed seasonality of invasive disease is related to the breach of pharyngeal epithelium associated with viral respiratory infections during the cold months. Recent or concomitant rhinorrhea, pharyngitis, stomatitis, or diarrhea is recorded in the majority of patients. ^{, ,} It has also been proposed that the beginning of the school year in the fall in Northern Hemisphere countries exposes immunologically naïve childcare center attendees to transmissible K. kingae organisms.

Clinical Manifestations

Children with K. kingae infections other than endocarditis usually are considered to be only mildly or moderately ill. ^{, ,} Most children have a low-grade fever (mean 38.2°C), but 23% are afebrile, and acute phase reactants frequently are normal or only slightly elevated. ^{, ,} In a series of children with culture-proven K. kingae disease, 172 of 301 (57%) had a white blood cell (WBC) count <15,000/μL and 18 of 82 (22%) had normal C-reactive protein (CRP) values. Lack of fever, a normal WBC count, and CRP level do not rule out the diagnosis of K. kingae infection in a young child with skeletal system complaints.

In the 321 children with invasive K. kingae infection enrolled in a nationwide collaborative study in Israel, osteoarticular was observed in 169 (53%), bacteremia with no focus in 140 (44%), endocarditis in 8 (2%), and pneumonia in 1 (1%). In a comprehensive review of 520 children with K. kingae osteoarticular infection, isolated septic arthritis was the most common presentation (65% of patients), followed by concomitant bone and joint involvement in 18%, isolated osteomyelitis in 12%, and spondylodiskitis in 4%.

Skeletal System Infections

Pyogenic Arthritis

Pyogenic arthritis is the single most common presentation of K. kingae infections and is the most common etiology of joint infections in children aged 6–48 months in countries where universal conjugate H. influenzae type b programs have been implemented. The large weight-bearing knee, ankle, or hip joints are most commonly affected. ^, Less frequently, K. kingae invades the wrist, shoulder, elbow, sacroiliac joint, and small articulations such as the metacarpophalangeal, sternoclavicular, sternocostal, tarsal, and vertebral facet joints, which are rarely infected by other bacteria. ^{, , ,} Involvement of >1 joint is observed in approximately 5% of the cases. Although the onset of the disease is acute and children with arthritis generally are brought to medical attention within 1–3 days, markers of systemic and local inflammation frequently are minor. Lack of leukocytosis has been observed in 76 of 149 (51%) children with K. kingae arthritis, and 18 of 78 (23%) had <50,000 WBC/μL in synovial fluid. In a retrospective study, 30 Swiss children with K. kingae joint infections and 30 children with arthritis caused by traditional pyogenic bacteria were compared using a clinical and laboratory score comprised of temperature >38°C at presentation, serum CRP concentration >5.5 mg/dL, WBC count >14.000 cells/μL, and >150 band forms/μL. Whereas 97% of patients with K. kingae infections had <2 positive predictors, 90% of those with arthritis caused by other bacteria had 3 or 4 positive predictors. However, in a similar study in which 64 French children with K. kingae arthritis diagnosed by a molecular test were compared with 26 children with Staphylococcus aureus infections, those with K. kingae arthritis had a younger age (median 1.4 years vs . 7.9 years; P < .001), shorter hospitalization, and fewer complications; however, the two groups did not differ significantly in the duration of symptoms, body temperature, WBC count, or level of acute-phase reactants. ^,

The synovial fluid Gram-stain examination usually is negative, probably due to the low bacterial concentration in the exudate and the difficulty to visualize the gram-negative organism against the pink-stained fibrin background. ^{, , ,}

Because of the mild symptomatology and benign laboratory findings, K. kingae arthritis affecting the hip joint can be misdiagnosed as transient synovitis. Since toxic synovitis is uncommon below the age of 4 years, young children presenting with an “irritable hip” should be carefully evaluated to exclude K. kingae arthritis, including the performance of blood cultures and a joint tap. The aspirated synovial fluid sample should be inoculated into blood culture vials and submitted, together with an oropharyngeal specimen, for nucleic acid amplification testing with a K. kingae -specific assay.

Osteomyelitis

Similar to bone infections caused by traditional pathogens, K. kingae osteomyelitis affects the long bones of the extremities in the majority of cases, although involvement of the calcaneus, talus, patella, clavicle, sternum, or pelvis also can occur. ^{, ,} Primary involvement of the epiphysis or apophysis of the long bones, which is only exceptionally observed in osteomyelitis caused by other bacteria, is frequently documented in K. kingae infections and is the most common etiology of infection at these sites. ^, Spread to contiguous joints is frequent, especially with infections involving the shoulder or hip ( Fig. 176.4 ). Magnetic resonance imaging (MRI) may show distinctive small abscesses of the not-yet-ossified cartilage accompanied by a minor soft tissue reaction. ^, Patients with K. kingae osteomyelitis have a significantly more prolonged duration of symptoms before admission compared with children with other bacterial osteoarticular infections (9.2 ± 9.4 days vs. 3.2 ± 3.0 days, respectively), and have a lower body temperature (37.7 ± 1.0°C vs . 38.4 ± 0.9°C, respectively). An indolent and subacute clinical course (with a mild increase in laboratory markers of inflammation) leading to epiphyseal or metaphyseal Brodie abscess that fistulizes to the adjacent joint space is not uncommon. In two young children, the osteolytic lesion showed a pathognomonic pattern suggesting Langerhans cell histiocytosis, implying that an active search for the K. kingae by culture and sensitive molecular methods should be performed when considering this rare pathologic diagnosis in a young child. Despite the frequent diagnostic delay of K. kingae osteomyelitis, an evolution towards chronicity and residual orthopedic sequelae are the exception.

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