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Surgical site infection remains a complication of spine surgery despite routine use of prophylactic antibiotics. Retrospective clinical studies of intrawound vancomycin use have documented a decreased prevalence of surgical site infection after spine surgery. The purpose of the present study was to assess the efficacy of intrawound vancomycin powder in terms of eradicating a known bacterial surgical site contamination in a rabbit spine surgery model.Twenty New Zealand White rabbits underwent lumbar partial laminectomy and wire implantation. The surgical sites were inoculated, prior to closure, by injecting 100 μL of cefazolin-sensitive and vancomycin-sensitive Staphylococcus aureus (S. aureus) (1 × 108 colony-forming units [CFU]/mL) into the wound. Preoperative cefazolin was administered to all rabbits, and vancomycin powder (100 mg) was placed into the wound of ten rabbits prior to closure. The rabbits were killed on postoperative day four, and tissue and wire samples were obtained for bacteriologic assessment. An independent samples t test was used to assess mean group differences, and a Fisher exact test was used to assess differences in categorical variables.The vancomycin-treated and the control rabbits were similar in weight (mean [and standard deviation], 4.1 ± 0.5 kg and 4.0 ± 0.4 kg, respectively; p = 0.60) and sex distribution and had similar durations of surgery (21.7 ± 7.7 minutes and 16.9 ± 6.7 minutes; p = 0.15). The bacterial cultures of the surgical site tissues were negative for all ten vancomycin-treated rabbits and positive for all ten control rabbits (p < 0.0001). Bacterial growth occurred in thirty-nine of forty samples from the control group but in zero of forty samples from the vancomycin group (p < 0.0001). All blood and liver samples were sterile. No rabbit had evidence of sepsis or vancomycin toxicity. Gross examination of the surgical sites showed no differences between the groups.In a rabbit spine-infection model, intrawound vancomycin powder in combination with preoperative cefazolin eliminated S. aureus surgical site contamination. All rabbits that were managed with only prophylactic cefazolin had persistent S. aureus contamination.This animal study supports the findings in prior clinical reports that intrawound vancomycin powder helps reduce the risk of surgical site infections.