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Yagupsky, P. Pharyngeal Colonization by Kingella kingae. Encyclopedia. Available online: https://encyclopedia.pub/entry/20814 (accessed on 03 January 2025).
Yagupsky P. Pharyngeal Colonization by Kingella kingae. Encyclopedia. Available at: https://encyclopedia.pub/entry/20814. Accessed January 03, 2025.
Yagupsky, Pablo. "Pharyngeal Colonization by Kingella kingae" Encyclopedia, https://encyclopedia.pub/entry/20814 (accessed January 03, 2025).
Yagupsky, P. (2022, March 21). Pharyngeal Colonization by Kingella kingae. In Encyclopedia. https://encyclopedia.pub/entry/20814
Yagupsky, Pablo. "Pharyngeal Colonization by Kingella kingae." Encyclopedia. Web. 21 March, 2022.
Pharyngeal Colonization by Kingella kingae
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This entry is adapted from the peer-reviewed paper 10.3390/microorganisms10030637

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 pili carriage transmission children invasive disease

1. Introduction

Shortly after birth, the skin and the upper respiratory, gastrointestinal, and female genital tracts of a newborn child become gradually coated with a variety of bacterial species, including many potentially dangerous organisms [1]. Despite their distant taxonomic position and vast biological differences, bacteria such as Haemophilus influenzae type b and Streptococcus pneumoniae colonizing the pharyngeal epithelium display homologous components as pili or antiphagocytic polysaccharide capsules [2]. These striking similarities result from a convergent evolution to adapt to the shared environment, adhere to the mucosal layer, avoid being washed out, and subvert the mucosal immune response.
The composition of the human microbiota is in a continuous dynamic state: organisms are acquired, eradicated, and re-acquired several times in a lifetime, and strains within a given species exhibit a remarkable turnover, indicating that the antigenic variability of virulent factors enables the acquisition of heterologous strains [3][4]. Bacterial colonization is asymptomatic in most cases, and the number of colonized but healthy individuals is enormous compared to those with clinical infections. An invasive disease usually occurs when colonizing bacteria breach the mucosal layer and penetrate the bloodstream. This event may result in the hematogenous dissemination and seeding of the organism to distant sites, causing focal infections. The colonized upper respiratory mucosa is also the source of person-to-person transmission of the bacterium through buccal and respiratory secretions, enabling its spread in the population.

2. Kingella kingae: An Oropharyngeal Resident

Similar to many other members of the Neisseriaceae family of Gram-negative organisms, K. kingae also colonizes the upper respiratory epithelium. In a large prospective study in which pairs of oropharyngeal and nasopharyngeal specimens were obtained from asymptomatic daycare center attendees, K. kingae grew in 109 out of 624 (17.5%) oropharyngeal samples. In contrast, the bacterium was not isolated from the nasopharynx, indicating a restricted ecological niche [5]. This finding was corroborated in a separate study in which 4472 oropharyngeal and nasopharyngeal specimens were sequentially collected from a cohort of 716 young children. Overall, 388 (8.7%) oropharyngeal cultures, but only a single nasopharyngeal culture, recovered K. kingae [6].
The colonization of the oropharynx by K. kingae organisms plays a double role. On the one hand, the exposed oropharyngeal surfaces are the natural reservoir of the bacterium from which it may be transmitted by contaminated buccal and upper respiratory secretions, and thus disseminated. On the other hand, the colonized oropharyngeal epithelium is the stepping-stone from which the bacterium translocates to the bloodstream and disseminates to remote body sites. In-depth study of the colonization phenomenon is, therefore, crucial to understanding the transmission of K. kingae and the pathogenesis of invasive infections.

3. Mechanism of Colonization

To colonize the oropharynx, K. kingae employs type IV pili that anchor the planktonic bacterium to the mucosal surfaces, and thus avoids being removed by saliva and respiratory fluids [7]. Elaboration of these pili is encoded in a chromosomal gene cluster, similar to that found in other Gram-negative pathogens, and in two other genes located in physically separated chromosomal regions, namely pilC1 and pilC2 [7][8]. The chromosomal gene cluster consists of a pilA1 gene that encodes the major pilin subunit and two additional genes, named pilA2 and fimB. The function of fimB is unknown, and it does not appear to be required for pilus expression or attachment [7]. The pilA1 gene sequence shows marked between-strain variation, and the pilA1 subunit exhibits vast differences in antibody reactivity, suggesting that this exposed virulence factor is subjected to selective pressure by the host’s immune system [9]. The pilC1 subunit is needed for twitching motility and adherence, whereas pilC2 has only a minor role in motility and no effect on adherence [8]. The expression of pili in K. kingae is finely regulated by the σ54, pilS, and pilR genes [9], and most oropharyngeal isolates and those derived from bacteremic patients express pili. In contrast, those samples isolated from skeletal system infections or endocarditis are usually non-piliated [10]. This finding suggests that piliation promotes K. kingae colonization and facilitates the initial bloodstream invasion but is disadvantageous for invading deep body structures.
In addition to the pili, K. kingae elaborates a trimeric autotransporter protein named Knh (Kingella NhhA homolog), which is essential for the strong anchoring of the organism to the oropharyngeal epithelium [11]. However, the carbohydrate capsule conceals the Knh element, rendering it inaccessible for attachment to the host’s cells. Porsch et al. have proposed that, following an initial weak adherence of the long pili to the epithelial surface, a strong retraction of these filaments displaces the capsule and exposes the Knh protein, which may then firmly stick to the mucosal surface [11].

4. Immunity to Colonization and Infection

The crucial role played by the immune system in preventing oropharyngeal K. kingae colonization and subsequent invasive disease is supported by the fact that adults with a variety of immunosuppressive conditions are at increased risk of K. kingae disease [12]. In a longitudinal study in which serum antibody levels against K. kingae outer-membrane proteins were measured by an ELISA test, IgG levels were high at 2 months of age and gradually diminished thereafter, reaching a nadir level at 6–7 months, then remaining low until the age of 18 months, followed by an increase in 24-month-old children. IgA levels were lowest at 2 months and slowly increased between 4 and 7 months of age. A further increment of both antibody types was measured in children aged ≥24 months [13].
This pattern is consistent with protection from colonization and invasive disease by vertically transmitted immunity and limited exposure to K. kingae in early infancy. Vanishing maternal antibodies and increasing social contacts result in exposure to the organism in the second life semester, with corresponding increasing IgG and IGA levels. While the colonization and attack rates of disease are high in the second year, antibody levels remain high and stable. Colonization rates, and the incidence of invasive infections and IgG levels decline in older children as they reach immunological maturity. Because asymptomatic K. kingae carriage is common in early childhood, whereas invasive infections are exceptional, it is postulated that pharyngeal colonization is the immunizing event.
Similar to other respiratory pathogens such as pneumococci and H. influenzae type b, K. kingae elaborates a polysaccharide capsule and secretes an exopolysaccharide. Both components inhibit the host’s immune response [14][15][16], enabling colonization of the upper respiratory tract, protecting the organism from phagocytosis by blood leukocytes and tissue macrophages, and facilitating the invasion of deep tissues. The maturation of the T-cell independent arm of the immune system, which is responsible for producing antibodies to polysaccharide antigens, is delayed in humans until the age of 2–4 years [2], explaining the increased susceptibility of young children to both colonization and disease. It should be noted that whereas the prevalence of K. kingae colonization reaches its peak and remains steady during the second year of life, the age-related curve of invasive disease is markedly skewed to the left: >75% of affected children are aged <18 months, and >95% are younger than 48 months, indicating that resistance to invasive infections is acquired before immunity to mucosal colonization [12].

5. Colonization and Transmission

A prospective study was conducted in the school year 1993–1994 among two cohorts of young children attending a daycare facility in southern Israel to investigate the dynamics of colonization and transmission of K. kingae in children attending out-of-home care facilities [5]. Oropharyngeal specimens were obtained biweekly over 11 months and seeded on the selective blood-agar-vancomycin (BAV) medium [5]. The recovered K. kingae isolates were originally studied by pulsed-field gel electrophoresis (PFGE) and ribotyping techniques with multiple restriction enzymes, and immunoblotting with rabbit immune serum [17]. A strict criterion consisting of complete DNA band identity by the three typing methods was employed to characterize the isolates and prove the person-to-person transmission of the strains. More recently, the isolates were retested by PFGE with the highly discriminative EagI enzyme, which is the currently recommended tool for PFGE analysis of the species. The results of this latter analysis are depicted in Figure 1.
/media/item_content/202203/623922279e07amicroorganisms-10-00637-g001.png
Figure 1. Dissemination of K. kingae clones among two cohorts of attendees at an Israeli daycare center. Horizontal lanes: individual attendees. Each star represents a positive pharyngeal culture, while the different colors represent distinct PFGE clones.
Thirty-five of 48 (73%) children carried K. kingae organisms at least once, and, on average, 28% of the attendees were colonized at any point in time [5]. Individual attendees exhibited sporadic, intermittent, or continuous carriage, and residing strains were frequently substituted by new strains after weeks or months, showing that similar to other respiratory bacteria, K. kingae’s carriage is a dynamic phenomenon with frequent turnover of colonizing organisms [17]. However, it should be pointed out that in the study, a single colony per positive culture was typed, and therefore simultaneous carriage of multiple strains and/or persistence of a previously carried organism at a low and undetectable level cannot be ruled out.
Overall, five distinct PFGE clones were detected in the daycare center during the study period. Four of these clones, namely H, K, N, and U, appeared sequentially in the facility and gradually colonized multiple attendees, while the prevalence of previously carried strains decreased [17] (Figure 1). These observations suggest that prolonged colonization induces an immune response that is strain-specific and eradicates or diminishes the density of the carried strain but does not prevent colonization by an antigenically different organism. Remarkably, although these four highly invasive clones were responsible for over half of the invasive diseases in Israel between 1991 and 2012 [18], none of the colonized daycare attendees developed a clinical K. kingae infection during the follow-up period.
In a secondary analysis performed on the aforementioned southern Israel cohort study [19], the temporal dynamics of K. kingae carriage were investigated in children among whom the bacterium was isolated on >1 occasion [6]. The proportion of PFGE-similar strains was determined for pairs of positive cultures separated by ≤2 months (short-term intervals) and for those separated by ≥5 months (long-term intervals). The fraction of pairs of similar strains out of the total number of pairs was assessed by PFGE analysis for short-term and long-term intervals, and then compared. Of the short-term interval paired isolates, 17 of 19 (89.5%) yielded genotypically similar clones, while only 20 of 91 (22.0%) long-term interval pairs yielded similar clones (p < 0.001), indicating that, over time, colonization enables the eradication of the carried organism, facilitating the later acquisition of a different strain [6].

6. Detection of K. kingae Colonization

Because of the abundance and complexity of the upper respiratory and buccal microbiota, the isolation and identification of K. kingae in oropharyngeal specimens are notoriously tricky. Although the organism frequently colonizes the oropharynx of young children, its presence in Petri dishes is concealed by the rapid overgrowth of other members of the residing bacterial flora [20].
To facilitate the culture recovery and identification of the species, a selective and differential medium consisting of blood agar with 2 mcg/mL of added vancomycin (BAV medium) has been developed [20]. BAV plates, streaked with oropharyngeal secretions, are incubated for 48 h at 35 °C under aerobic conditions in a 5% CO2-enriched atmosphere [20]. The aerobic conditions suppress the development of anaerobic species; the glycopeptide antimicrobial drug inhibits the growth of Gram-positive bacteria; and the added CO2 enhances the growth of capnophilic K. kingae organisms, whereas the blood component facilitates the visualization of hemolytic colonies [20] (Figure 2).
Figure 2. Oropharyngeal specimen seeded onto selective BAV medium exhibiting growth of β-hemolytic K. kingae colonies.
When the capability of the BAV and the traditional blood agar media for the primary isolation of K. kingae from oropharyngeal cultures were compared in a prospective study, the BAV plate identified 43 of 44 (97.7%) carriers [20]. In contrast, the comparator detected only 10 (22.7%) (p < 0.001 by the Chi-square test) [20]. The BAV medium and a Columbia-agar-based variant [21] have been successfully used in the investigation of the acquisition, prevalence, and transmission of K. kingae in the pediatric population and the investigation of outbreaks of invasive disease in daycare facilities [5][6][19][21][22]. It should be pointed out that chocolate agar media are not suitable for K. kingae detection in primary cultures since they do not reveal the presence of β-hemolytic colonies. The use of chocolate agar probably contributed to the failure to identify respiratory K. kingae carriers in a cluster of infections among attendees at a North Carolina daycare center [23].
In recent years, nucleic acid amplification tests (NAATs) targeting the 16S rRNA or species-specific genes have been introduced into clinical practice to detect fastidious bacteria in normally sterile body fluids and tissues. NAATs enable bacterial identification within hours instead of days and in patients receiving antibiotic therapy [24]. This revolutionary approach is gaining increasing popularity as a sensitive and convenient culture-independent tool for diagnosing invasive K. kingae infections [24][25][26][27][28][29][30] and for identifying K. kingae carriers in prevalence studies [22][31]. However, because only single genes are amplified, NAATs do not discriminate between different K. kingae strains and, thus, have a limited value in investigating complex disease outbreaks [22].
Tests that amplify K. kingae-specific genes have a higher sensitivity than those targeting the broad spectrum 16S rRNA gene [24]. The three species-specific genes that are targeted by the current assays are the rtx operon that encodes the RtxA toxin [32], chaperonin 60 (the cpn60 gene, also known as groEL) [24], and the malate dehydrogenase (mdh) gene [28]. The rtx-based tests do not discriminate between K. kingae and the hemolytic and recently described Kingella negevensis species that also colonizes the pediatric oropharynx [28], and those that amplify the cpn60 target show suboptimal sensitivity due to variability in the gene sequence among K. kingae strains [28]. The novel molecular assay that targets the mdh gene exhibits an optimal sensitivity and specificity and will probably replace the older tests [28].

References

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  11. Porsch, E.A.; Kehl-Fie, T.E.; St. Geme, J.W., 3rd. Modulation of Kingella kingae Adherence to human epithelial cells by type IV Pili, capsule, and a novel trimeric autotransporter. mBio 2012, 3, e00372-12.
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  13. Slonim, A.; Steiner, M.; Yagupsky, P. Immune response to invasive Kingella kingae infections, age-related incidence of disease, and levels of antibody to outer-membrane proteins. Clin. Infect. Dis. 2003, 37, 521–527.
  14. Starr, K.F.; Porsch, E.A.; Heiss, C.; Black, I.; Azadi, P.; St. Geme, J.W., 3rd. Characterization of the Kingella kingae polysaccharide capsule and exopolysaccharide. PLoS ONE 2013, 8, e75409.
  15. Muñoz, V.L.; Porsch, E.A.; St. Geme, J.W., 3rd. Kingella kingae surface polysaccharides promote resistance to neutrophil phagocytosis and killing. mBio 2019, 10, e00631-19.
  16. Muñoz, V.L.; Porsch, E.A.; St. Geme, J.W., 3rd. Kingella kingae surface polysaccharides promote resistance to human serum and virulence in a juvenile rat model. Infect. Immun. 2018, 86, e00100-18.
  17. Slonim, A.; Walker, E.S.; Mishori, E.; Porat, N.; Dagan, R.; Yagupsky, P. Person-to-person transmission of Kingella kingae among day care center attendees. J. Infect Dis. 1998, 78, 1843–1846.
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  19. Yagupsky, P.; Weiss-Salz, I.; Fluss, R.; Freedman, L.; Peled, N.; Trefler, R.; Porat, N.; Dagan, R. Dissemination of Kingella kingae in the community and long-term persistence of invasive clones. Pediatr. Infect. Dis. J. 2009, 28, 707–710.
  20. Yagupsky, P.; Merires, M.; Bahar, J.; Dagan, R. Evaluation of a novel vancomycin-containing medium for primary isolation of Kingella kingae from upper respiratory tract specimens. J. Clin. Microbiol. 1995, 33, 426–427.
  21. Basmaci, R.; Ilharreborde, B.; Bidet, P.; Doit, C.; Lorrot, M.; Mazda, K.; Bingen, E.; Bonacorsi, S. Isolation of Kingella kingae in the oropharynx during K. kingae arthritis on children. Clin. Microbiol. Infect. 2012, 18, e134–e136.
  22. Yagupsky, P.; El Houmami, N.; Fournier, P.-E. Outbreaks of invasive Kingella kingae infections in daycare facilities: Approach to investigation and management. J. Pediatr. 2017, 182, 14–20.
  23. Seña, A.C.; Seed, P.; Nicholson, B.; Joyce, M.; Cunningham, C.K. Kingella kingae endocarditis and a cluster investigation among daycare attendeES. Pediatr. Infect. Dis. J. 2010, 29, 86–88.
  24. Chometon, S.; Benito, Y.; Chaker, M.; Boisset, S.S.; Ploton, C.; Bérard, J.; Vandenesch, F.; Freydiere, A.M. Specific real-time polymerase chain reaction places Kingella kingae as the most common cause of osteoarticular infections in young children. Pediatr. Infect. Dis. J. 2007, 26, 377–381.
  25. Ceroni, D.; Anderson della Llana, R.; Kherad, O.; Lascombes, P.; Renzi, G.; Manzano, S.; Cherkaoui, A.; Schrenzel, J. Comparing the oropharyngeal colonizationisation density of Kingella kingae between asymptomatic carriers and children with invasive osteoarticular infections. Pediatr. Infect. Dis. J. 2013, 32, 412–414.
  26. Rosey, A.L.; Abachin, E.; Quesnes, G.; Cadilhac, C.; Pejin, Z.; Glorion, C.; Berche, P.; Ferroni, A. Development of a braod-range 16S rDNA real-time PCR for the diagnosis of septic arthritis in children. J. Microbiol. Methods. 2007, 68, 88–93.
  27. Moumile, K.; Merckx, J.; Glorion, C.; Berche, P.; Ferroni, A. Osteoarticular infections caused by Kingella kingae in children: Contribution of polymerase chain reaction to the microbiologic diagnosis. Pediatr. Infect. Dis. J. 2003, 22, 837–839.
  28. El Houmami, N.; Durand, G.A.; Bzdrenga, J.; Darmon, A.; Minodier, P.; Seligmann, H.; Raoult, D.; Fournier, P.-E. A new highly sensitive and specific real-time pcr assay targeting the malate dehydrogenase gene of Kingella kingae and application to 201 pediatric clinical specimens. J. Clin. Microbiol. 2018, 56, e00505-18.
  29. Ceroni, D.; Cherkaoui, A.; Kaelin, A.; Schrenzel, J. Kingella kingae spondylodiscitis in young children: Toward a new approach for bacteriological investigations? A preliminary report. J. Child. Orthop. 2010, 4, 173–175.
  30. Juchler, C.; Spyropoulou, V.; Wagner, N.; Merlini, L.; Dhouib, A.; Manzano, S.; Tabard-Fougère, A.; Samara, E.; Ceroni, D. The contemporary bacteriologic epidemiology of osteoarticular infections in children in Switzerland. J. Pediatr. 2018, 194, 190–196.e1.
  31. Masud, S.; Greenman, J.; Mulpuri, K.; Hasan, M.R.; David, M.; Goldfarb, D.M.; Tilley, P.; Gadkar, V.J.; Al-Rawahi, G.N. Asymptomatic pharyngeal carriage of Kingella kingae among young children in Vancouver, British Columbia, Canada. Pediatr. Infect. Dis. J. 2019, 38, 990–993.
  32. Lehours, P.; Freydière, A.M.; Richer, O.; Burucoa, C.; Boisset, S.; Lanotte, P.; Prère, M.F.; Ferroni, A.; Lafuente, C.; Vandenesch, F.; et al. The rtxA toxin gene of Kingella kingae: A pertinent target for molecular diagnosis of osteo- articular infections. J. Clin. Microbiol. 2011, 49, 1245–1250.
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