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.

Osteoarticular infections: Blood as a determinant factor in the isolation of Kingella kingae.

We assessed the capacity of Kingella kingae to grow in blood culture bottles (BCB), taking into account the concentrations of the microorganism and blood in the culture medium. An initial suspension (McFarland 0.5) of 32 strains of K. kingae was serially diluted. One mL of the initial suspension and 1 mL of the subsequent dilutions were inoculated in two BCB, together with 1 mL of human blood in the 2nd BCB. Also, 1mL serial dilutions of human blood were added to BCBs previously inoculated with 1 mL of K. kingae dilution 1/104. In non-blood-supplemented BCB, 23 strains grew with the initial suspension and only one with the first processed dilution, as compared to all strains with the initial suspension and the 3 first dilutions, 22 with the 4th dilution, and one with the 5th dilution in blood-supplemented BCB. In BCB inoculated with K. kingae dilution 1/104 and decreasing concentrations of human blood, all strains grew with blood dilutions 1/2 and 1/4, 26 with dilution 1/8, 19 with dilution 1/16, 10 with dilution 1/32, and none with dilution 1/64. Increasing time to positivity was observed with both decreasing bacterial (p = .001) and blood concentrations (r = -0.632, p < .0001). The addition of human blood was essential to boost the growth of K. kingae in BCB. If replicated in vivo, these findings would increase the isolation of fastidious K. kingae organisms from pediatric osteoarticular exudates.

Phasevarion-Regulated Virulence in the Emerging Pediatric Pathogen Kingella kingae.

Kingella kingae is a common etiological agent of pediatric osteoarticular infections. While current research has expanded our understanding of K. kingae pathogenesis, there is a paucity of knowledge about host-pathogen interactions and virulence gene regulation. Many host-adapted bacterial pathogens contain phase variable DNA methyltransferases (mod genes), which can control expression of a regulon of genes (phasevarion) through differential methylation of the genome. Here, we identify a phase variable type III mod gene in K. kingae, suggesting that phasevarions operate in this pathogen. Phylogenetic studies revealed that there are two active modK alleles in K. kingae Proteomic analysis of secreted and surface-associated proteins, quantitative PCR, and a heat shock assay comparing the wild-type modK1 ON (i.e., in frame for expression) strain to a modK1 OFF (i.e., out of frame) strain revealed three virulence-associated genes under ModK1 control. These include the K. kingae toxin rtxA and the heat shock genes groEL and dnaK Cytokine expression analysis showed that the interleukin-8 (IL-8), IL-1ß, and tumor necrosis factor responses of THP-1 macrophages were lower in the modK1 ON strain than in the modK1::kan mutant. This suggests that the ModK1 phasevarion influences the host inflammatory response and provides the first evidence of this phase variable epigenetic mechanism of gene regulation in K. kingae.

Simplifying the treatment of acute bacterial bone and joint infections in children.

The treatment of acute hematogenous bone and joint infections of children - osteomyelitis (OM), septic arthritis (SA) and OM-SA combination (OM+SA) - has simplified over the past years. The old approach included months-long antibiotic treatment, started intravenously for at least a week, followed by oral completion of the course. Recent prospective randomized trials show that most cases heal with a total course of 3 weeks (OM, OM+SA) or 2 weeks (SA) of an appropriate antibiotic, provided the clinical response is good and C-reactive protein level has normalized. If the prevalence of methicillin-resistant Staphylococcus aureus and Kingella kingae is low, clindamycin and a first-generation cephalosporin are safe, inexpensive and effective alternatives. They should be administered in large doses and four times a day. Clindamycin, vancomycin and expensive linezolid are options against methicillin-resistant Staphylococcus aureus. Extensive surgery beyond a diagnostic sample by aspiration is rarely needed in uncomplicated cases.

Expression of Kingella kingae type IV pili is regulated by sigma54, PilS, and PilR.

Kingella kingae is a member of the Neisseriaceae and is being recognized increasingly as an important cause of serious disease in children. Recent work has demonstrated that K. kingae expresses type IV pili that mediate adherence to respiratory epithelial and synovial cells and are selected against during invasive disease. In the current study, we examined the genome of K. kingae strain 269-492 and identified homologs of the rpoN and the pilS and pilR genes that are essential for pilus expression in Pseudomonas aeruginosa but not in the pathogenic Neisseria species. The disruption of either rpoN or pilR in K. kingae resulted in a marked reduction in the level of transcript for the major pilus subunit (pilA1) and eliminated piliation. In contrast, the disruption of pilS resulted in only partial reduction in the level of pilA1 transcript and a partial decrease in piliation. Furthermore, the disruption of pilS in colony variants with high-density piliation resulted in variants with low-density piliation. Mutations in the promoter region of pilA1 and gel shift analysis demonstrated that both sigma(54) and PilR act directly at the pilA1 promoter, with PilR binding to two repetitive elements. These data suggest that the regulation of K. kingae type IV pilus expression is complex and multilayered, influenced by both the genetic state and environmental cues.

Kingella kingae: condiciones determinantes del crecimiento en botella de hemocultivo / Kingella kingae: specific growth conditions in blood culture bottles

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Identification and characterization of an RTX toxin in the emerging pathogen Kingella kingae.

Kingella kingae is an emerging bacterial pathogen that is increasingly recognized as the causative agent of a variety of pediatric diseases, including septic arthritis and osteomyelitis. The pathogenesis of K. kingae disease is believed to begin with colonization of the upper respiratory tract. In the present study, we examined interactions between K. kingae and cultured respiratory epithelial cells and observed potent cytotoxicity, detected by both microscopy and lactic acid dehydrogenase (LDH) release assays. Experiments with synovial and macrophage cell lines revealed cytotoxicity for these cell types as well. Using mariner mutagenesis and a screen for loss of cytotoxicity, a genetic locus encoding an RTX toxin system was identified. Disruption of the K. kingae RTX locus resulted in a loss of cytotoxicity for respiratory epithelial, synovial, and macrophage cell lines. DNA sequence analysis demonstrated that the RTX locus is flanked by insertion elements and has a reduced G+C content compared to that of the whole genome. Two relatively less invasive Kingella species, K. oralis and K. denitrificans, were found to be noncytotoxic and to lack the RTX region, as determined by LDH release assays and Southern blotting. We concluded that K. kingae expresses an RTX toxin that has wide cellular specificity and was likely acquired horizontally. The possible roles for this toxin in the pathogenesis of K. kingae disease include breaching of the epithelial barrier and destruction of target tissues, such as synovium (joint lining).

Isolation of Kingella kingae from synovial fluids using four commercial blood culture bottles.

According to the literature, Kingella kingae may be an underdiagnosed cause of joint and bone infections in children. The use of the Bactec blood culture system for culture of joint fluids has dramatically improved the isolation of this fastidious bacterium. The aim of this study was to test the recovery rate and detection time of four commercial blood culture systems: three different BacT/Alert (Organon Teknika, USA) bottles and one Bactec (Becton Dickinson Microbiology Systems, USA) bottle, all inoculated with Kingella kingae strains mixed with pooled synovial fluids. For each strain the same inoculum and volume of synovial fluid was distributed into each of the four bottles. All 24 strains tested grew in the BacT/Alert Aerobic (100%) and the BacT/Alert Pedi-BacT (100%) bottles. Twenty-one strains grew in the BacT/Alert FAN aerobic (88%) bottle, and 15 strains grew in the Bactec Plus Aerobic F (63%) bottle, in both systems within 12 days (P<0.01). The Kingella kingae strains were first detected in the BacT/Alert Pedi-BacT bottles (P<0.001). The results were reproducible. The BacT/Alert blood culture bottles were superior to previously described blood culture systems in isolating Kingella kingae from synovial fluid, even with small inoculums and small volumes of synovial fluid.

Evaluation of novel vancomycin-containing medium for primary isolation of Kingella kingae from upper respiratory tract specimens.

A new selective medium (BAV), consisting of trypticase agar with 5% sheep hemoglobin and 2 micrograms of vancomycin per ml, was compared with the routine blood-agar medium for the primary isolation of Kingella kingae from upper respiratory specimens from a population of young children. Infection was detected by the BAV medium in 43 of 44 (98%) cultures positive for K. kingae, and detection of the organism was facilitated by inhibition of gram-positive flora. Infection was detected in only 10 of 44 (23%) positive cultures by the blood-agar medium, and plates were usually covered by abundant normal flora, making the recognition of K. kingae much more difficult. Challenge of the medium with different organisms of respiratory origin showed that the BAV medium was inhibitory for gram-positive cocci and Haemophilus influenzae but that it supported growth of eight K. kingae strains isolated from patients with invasive infections. The new medium appears to be a useful epidemiological tool for studying the respiratory carriage of K. kingae.