pH in the bacteria-contaminated wound and its impact on clostridium histolyticum collagenase activity: implications for the use of collagenase wound debridement agents

Abstract

Purpose: We sought to understand the influence of pH on Clostridium collagenase activity, using both in vitro and in vivo, and to understand the influence of bacterial contamination on pH in vivo.

Design: Artificial wound eschar was used for the assessment of debridement efficacy in vitro and the wound fluid of a contaminated pig wound model was used for examining pH during healing using in vivo techniques.

Methods: We used a collagen-based artificial wound eschar to test collagenase activity in the collagenase product under various pH conditions. We evaluated bacterial contaminated wounds, using a pig wound model with a bacterial load including Pseudomonas aeruginosa, coagulase-negative staphylococci, and Fusobacterium sp to track the pH of wounds in relation to bacterial load.

Results: The pH levels in the wound fluid were all above neutral. They varied from 9.2 on day 1 to 8.3 on day 10. Collagenase achieved its highest activity around a pH of 8.5 when tested in Tris-buffered saline. Using artificial wound eschar, the optimal pH range for C collagenase was determined to be more than a pH of 6. The total initial microbial load (day 0) was higher than levels at any other time during the study. The levels of P aeruginosa began to decrease on day 1, but by day 4 the total pseudomonas population had rebounded to near day 0 levels. This was followed by a distinct decrease and by day 21 levels were lower than those on day 0. The coagulase negative staphylococci (CNS) population behaved differently than the pseudomonas population, decreasing and remaining decreased relative to day 0 through day 14. However, by day 21 the CNS population had increased to near day 0 levels. This greatly influenced the increase in total bioburden by day 21, indicating that it was primarily due to the increase in CNS.

Conclusion: The data demonstrate that wound pH in a model of contaminated pig wounds is alkaline during the first 10 days of healing. As healing progresses, pH decreases with no significant change in the level of bacterial bioburden. C collagenase exhibited robust activity in the pH range found in this contaminated pig wound model, suggesting it can effectively debride necrotic tissue in the environment found in most chronic wounds in humans.