Diabetic neuropathic foot ulcers are a major healthcare burden. These chronic wounds always have a bacterial load, and although normal flora is not harmful, increased tissue burden may impede healing before clinical signs of infection are evident. In this study, chronic noninfected diabetic neuropathic foot ulcers (those with adequate blood supply and pressure offloading) were assessed for bacterial burden immediately before the application of a skin substitute. Eight patients with diabetic neuropathic foot ulcers greater than 1 cm2 and free of necrotic tissue had 3-mm tissue biopsies taken from the ulcer base for quantitative bacteriology. Five of the eight patients (75%) had greater or equal to 10(5) colony forming units/gram organisms present despite the absence of clinical signs of infection. Wound healing rates were linked to bacterial load as determined from quantitative biopsy--no growth was associated with a wound healing rate of 0.2 cm per week, 10(5) to 10(6) colony forming units/gram was associated with a healing rate of 0.15 cm per week, and greater than 10(6) colony forming units/gram was associated with 0.05 cm/per week healing rate. High bacterial burden impeded healing both before and after the application of the skin substitute. The authors will change their clinical practice to assess all diabetic neuropathic foot ulcers using quantitative skin biopsies before applying skin substitutes. All patients will be treated with combination antibiotics and repeat biopsies obtained with decreased bacterial burden (< 10(6) colony forming units/gram) prior to using any bioengineered skin substitute or growth factor treatment.