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Background: Ralstonia pickettii is a Gram-negative bacillus commonly found in soil and moist environments; however, R. pickettii is rarely isolated from clinical specimens. In August 2001, a cluster of R. pickettii bacteremia occurred among neonatal intensive care unit (NICU) infants at a California hospital., Methods: A case-control study was conducted to determine risk factors for infection. A case was a NICU patient with R. pickettii bacteremia. Controls were NICU infants with negative blood cultures drawn during the same time period. A detailed environmental investigation was also conducted., Results: We identified 18 patients with 19 distinct episodes of R. pickettii bacteremia from July 30 through August 30, 2001. All cases had intravascular access at the time of bacteremia. Although the case-control study did not implicate any statistically significant risk factors, the most likely source of the outbreak was the heparin flush prepared in the hospital pharmacy. This is supported by the following: (1) the heparin flush was the only substance introduced directly into the bloodstream of all case infants; (2) the heparin flush was used exclusively by the NICU; and (3) no further cases were identified after the heparin flush was discontinued. Cultures of remaining heparin flush and environmental cultures from the NICU were negative for R. pickettii., Conclusions: This unusual outbreak of R. pickettii bacteremia was most likely caused by contaminated heparin flush and ended after the heparin flush was discontinued.