RESUMEN
Chronic wounds typically comprise of necrotic tissue and dried secretions, often culminating in the formation of a thick and tough layer of dead skin known as eschar. Removal of eschar is imperative to facilitate wound healing. Conventional approach for eschar removal involves surgical excision and grafting, which can be traumatic and frequently leads to viable tissue damage. There has been growing interest in the use of enzymatic agents for a gentler approach to debridement, utilizing proteolytic enzymes. In this study, a purified intracellular recombinant serine protease from Bacillus sp. (SPB) and its cream formulation were employed to evaluate their ability to degrade artificial wound eschar; composed of collagen, fibrin, and elastin. Degradation was assessed based on percentage weight reduction of eschar biomass, analysis via sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and scanning electron microscopy (SEM). Both SPB and its cream formulation were able to degrade up to 50â¯% artificial wound eschar, with the SPB cream maintaining its degradation efficiency for up to 24â¯hours. Additionally, the SPB-based cream demonstrated the ability to hydrolyze proteinaceous components of eschars individually (fibrin and collagen) as determined through qualitative assessment. These findings suggest that SPB holds promise for the debridement of wound eschar.
Asunto(s)
Bacillus , Desbridamiento , Fibrina , Serina Proteasas , Cicatrización de Heridas , Serina Proteasas/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Fibrina/metabolismo , Bacillus/enzimología , Humanos , Colágeno/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Recombinantes/metabolismo , Elastina/metabolismoRESUMEN
In nature, bacteria are ubiquitous and can be categorized as beneficial or harmless to humans, but most bacteria have one thing in common which is their ability to produce biofilm. Biofilm is encased within an extracellular polymeric substance (EPS) which provides resistance against antimicrobial agents. Protease enzymes have the potential to degrade or promote the growth of bacterial biofilms. In this study, the effects of a recombinant intracellular serine protease from Bacillus sp. (SPB) on biofilms from Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa were analyzed. SPB was purified using HisTrap HP column and concentrated using Amicon 30 ultra-centrifugal filter. SPB was added with varying enzyme activity and assay incubation period after biofilms were formed in 96-well plates. SPB was observed to have contrasting effects on different bacterial biofilms, where biofilm degradations were observed for both 7-day-old A. baumannii (37.26%) and S. aureus (71.51%) biofilms. Meanwhile, SPB promoted growth of P. aeruginosa biofilm up to 176.32%. Compatibility between protein components in S. aureus biofilm with SPB as well as a simpler membrane structure morphology led to higher biofilm degradation for S. aureus compared to A. baumannii. However, SPB promoted growth of P. aeruginosa biofilm due likely to its degrading protein factors that are responsible for biofilm detachment and dispersion, thus resulting in more multi-layered biofilm formation. Commercial protease Savinase which was used as a comparison showed degradation for all three bacterial biofilms. The results obtained are unique and will expand our understanding on the effects that bacterial proteases have toward biofilms.