ABSTRACT. Objective. Kingella kingae often colonizes the oropharyngeal and respiratory tracts of children but infrequently causes invasive disease. In mid-October 2003, 2 confirmed and 1 probable case of K kingae osteomyelitis/septic arthritis occurred among children in the same 16-to 24-month-old toddler classroom of a child care center. The objective of this study was to investigate the epidemiology of K kingae colonization and invasive disease among child care attendees.Methods. Staff at the center were interviewed, and a site visit was performed. Oropharyngeal cultures were obtained from the staff and children aged 0 to 5 years to assess the prevalence of Kingella colonization. Bacterial isolates were subtyped by pulsed-field gel electrophoresis (PFGE), and DNA sequencing of the 16S rRNA gene was performed. A telephone survey inquiring about potential risk factors and the general health of each child was also conducted. All children and staff in the affected toddler classroom were given rifampin prophylaxis and recultured 10 to 14 days later. For epidemiologic and microbiologic comparison, oropharyngeal cultures were obtained from a cohort of children at a control child care center with similar demographics and were analyzed using the same laboratory methods. The main outcome measures were prevalence and risk factors for colonization and invasive disease and comparison of bacterial isolates by molecular subtyping and DNA sequencing.Results. The 2 confirmed case patients required hospitalization, surgical debridement, and intravenous antibiotic therapy. The probable case patient was initially misdiagnosed; MRI 16 days later revealed evidence of ankle osteomyelitis. The site visit revealed no obvious outbreak source. Of 122 children in the center, 115 (94%) were cultured. Fifteen (13%) were colonized with K kingae, with the highest prevalence in the affected toddler classroom (9 [45%] of 20 children; all case patients tested negative but had received antibiotics). Six colonized children were distributed among the older classrooms; 2 were siblings of colonized toddlers. No staff (n ؍ 28) or children aged <16 months were colonized. Isolates from the 2 confirmed case patients and from the colonized children had an indistinguishable PFGE pattern. No risk factors for invasive disease or colonization were identified from the telephone survey. Of the 9 colonized toddlers who took rifampin, 3 (33%) remained positive on reculture; an additional toddler, initially negative, was positive on reculture. The children of the control child care center demonstrated a similar degree and distribution of K kingae colonization; of 118 potential subjects, 45 (38%) underwent oropharyngeal culture, and 7 (16%) were colonized with K kingae. The highest prevalence again occurred in the toddler classrooms. All 7 isolates from the control facility had an indistinguishable PFGE pattern; this pattern differed from the PFGE pattern observed from the outbreak center isolates. 16S rRNA gene sequencing demonstrated that the outbreak K kingae...