Background/Objectives: Accurately determining the bacterial bioburden that survives preoperative skin preparation (PSP) is critical in understanding PSP efficacy and its limitations. Clinical PSP approval relies on a bacterial sampling method described in the American Society for Testing and Materials (ASTM) standard E1173-15. Though common, this technique may overlook deep-dwelling skin bacteria. The objective of this study was to test the hypothesis that deep-dwelling skin flora would survive PSP, and more growth would be detected using a destructive sampling method compared with ASTM E1173-15. Methods: Twelve female participants with a scheduled deep inferior epigastric perforator (DIEP) artery flap procedure at the Huntsman Cancer Institute in Salt Lake City, UT, were enrolled between January and August 2024. PSP was performed using three 26 mL ChloraPrep applicators (2% CHG), and excess tissue was collected. Bacteria in the skin were quantified using a destructive sampling method and ASTM E1173-15, and bioburden outcomes were compared. Two participants were excluded from the quantitative analysis. Results: Bacteria survived PSP in every participant. A greater diversity and more bacteria were quantified with destructive sampling than ASTM E1173-15 (p < 0.01). Generally, anaerobic bioburden values were higher than aerobic bioburden values. Higher bioburden correlated with processing more skin from a participant. Genotypic identification of select isolates identified Staphylococcus epidermidis and Cutibacterium acnes (formerly known as Propionibacterium acnes) as surviving bacteria, among others. Immunofluorescence revealed bacteria in all skin layers. No participant exhibited clinical signs of infection in the abdominal region. Human data corroborated previous porcine data collected using destructive skin sampling after PSP. Conclusions: Clinical PSP application does not create a sterile field. Destructive skin sampling techniques may be more effective than ASTM E1173-15 at resolving bacterial PSP survivors contributing to SSI risk.

Medical device companies and regulatory bodies rely on a nondestructive bacterial sampling technique specified by the American Society for Testing and Materials (ASTM E1173-15) to test preoperative skin preparations (PSPs). Despite the widespread use of PSPs, opportunistic skin-flora pathogens remain the most significant contributor to surgical site infections, suggesting that the ASTM testing standard may be underreporting true dermal bioburden. We hypothesized that ASTM E1173-15 may fail to capture deep skin-dwelling flora. To test this hypothesis, we applied ASTM E1173-15 and a full-thickness skin sampling technique, which we established previously through application to the backs of seven pigs (Yorkshire/Landrace hybrid) following a clinically used PSP (4% chlorhexidine gluconate). The results showed that samples quantified using the full-thickness skin method consistently cultured more bacteria than the ASTM standard, which principally targeted surface-dwelling bacteria. Following PSP, the ASTM standard yielded 1.05 ± 0.24 log₁₀ CFU/cm², while the full-thickness tissue method resulted in 3.24 ± 0.24 log₁₀ CFU/cm², more than a 2 log₁₀ difference (p < 0.001). Immunofluorescence images corroborated the data, showing that Staphylococcus epidermidis was present in deep skin regions with or without PSP treatment. Outcomes suggested that a full-thickness sampling technique may better evaluate PSP technologies as it resolves bioburdens dwelling in deeper skin regions.