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Catheter-related bloodstream infections (CRBSI) are a common problem in patients after central venous catheterization. Using DNA analysis we compared bacteria found on the tip of central venous catheters removed because of clinical signs of CRBSI with bacteria found on needle, dilator, and guidewire used for insertion of these catheters. In five of seven central venous catheters removed because of clinical signs of CRBSI, bacteria on the catheter tip were genetically identical to bacteria found on the insertion device, proving that catheter contamination in these cases was caused by contacting bacteria during the initial puncture. These findings may be important for antibiotic prophylaxis or therapy in patients at risk for CRBSI.