Abstract
Chronic wounds are a major global health problem with the presence of biofilm significantly contributing to wound chronicity. Current treatments are ineffective in resolving biofilm and simultaneously killing the bacteria; therefore, effective biofilm-resolving drugs are needed. We have previously shown that, together with α-tocopherol, N-acetyl-cysteine (NAC) significantly improves the healing of biofilm-containing chronic wounds, in a diabetic mouse model we developed, by causing disappearance of the bacteria and breakdown of the extracellular polymeric substance (EPS). We hypothesize that NAC creates a microenvironment that affects bacterial survival and EPS integrity. To test this hypothesis, we developed an in vitro biofilm system using microbiome taken directly from diabetic mouse chronic wounds. For these studies, we chose mice in which chronic wound microbiome was rich in Pseudomonas aeruginosa (97%). We show that NAC at concentrations with pH < pKa causes bacterial cell death and breakdown of EPS. When used before biofilm is formed, NAC leads to bacterial cell death whereas treatment after the biofilm is established NAC causes biofilm dismantling accompanied by bacterial cell death. Mechanistically, we show that NAC can penetrate the bacterial membrane, increase oxidative stress, and halt protein synthesis. We also show that low pH is important for the actions of NAC and that bacterial death occurs independently of the presence of biofilm. In addition, we show that both the acetyl and carboxylic groups play key roles in NAC functions. The results presented here provide insight into the mechanisms by which NAC dismantles biofilm and how it could be used to treat chronic wounds after debridement (NAC applied at the start of culture) or without debridement (NAC applied when biofilm is already formed). This approach can be taken to develop biofilm from microbiome taken directly from human chronic wounds to test molecules that could be effective for the treatment of specific biofilm compositions.
Highlights
Chronic wounds are a major global health problem
We investigated the effects of NAC on biofilm developed from the microbiome taken from chronic wounds
We have previously developed a db/db-/- mouse model for chronic wounds in which the wounds become chronic, presenting all the abnormalities found in human chronic wounds, including the presence of a biofilm with bacteria that are found in chronic wounds in humans [24]
Summary
Chronic wounds are a major global health problem. The care for chronic wounds leads to a large financial and psychological problem to individuals and an economic burden to the society. Defective mesenchymal stem cells [7,8,9], degradation of growth factors [10], diminished capacity of fibroblast proliferation and migration [11], and downregulation of wound-associated keratin and its heteropolymers [12] result in impairment of healing. These conditions promote pathogen colonization and strong biofilm formation in the wound bed, which further delay the healing [13, 14]
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