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The Role of Bacterial Biofilms in Chronic Infections


Biofilm is the virulence factor that is responsible for chronic infection in diseases such as Cystic Fibrosis (CF) and chronic wounds. In this thesis, we examine the role of AlgX, a required protein for alginate biosynthesis in P. aeruginosa. We show that the absence of AlgX resulted in the loss of mucoidy and in silico studies demonstrated that AlgX binds alginate. Alanine mutations demonstrated that K396, T398, W400, and R406 are important for alginate binding. Alginate rescue assays confirm the importance of these amino acid residues for alginate biosynthesis and acetylation. This is the first functional demonstration of AlgX role in alginate biofilm biosynthesis and acetylation.

Biofilm-producing bacteria and redox imbalance are the leading factors that turn acute wounds into chronic wounds. We demonstrated that LIGHT-/- mouse wounds contain elevated levels of reactive oxygen species (ROS). To see whether chronic wounds can be generated, we increased the redox imbalance in the LIGHT-/- wounds and infecting the wounds with biofilm-forming bacteria. We demonstrated that by using these conditions, we could induce chronic wounds in the LIGHT-/- mouse model 100% of the time. These wounds do not re-epithelialize, contain high bacterial burden, and sustained multi-species bacterial infections that are biofilm-forming and antibiotic-resistant.

We also demonstrated that increasing the redox imbalance was sufficient to turn db/db wounds into chronic wounds. These wounds sustained spontaneous biofilm- producing bacterial infections. To verify that redox imbalance is critical for chronicity, we treated chronic wounds with antioxidants and found that oxidative stress was highly reduced, biofilm production was decreased, and bacteria became more sensitive to antibiotics. This is the first demonstration that chronic wounds can be generated and reversed by manipulating the wound's redox microenvironment in an animal model.

Norspermidine has been shown to negatively affect the structure of extracellular polymeric substances (EPS). Using the bacteria that colonized db/db chronic wounds, we demonstrated that 2 mM norspermidine reduces the amount of adherent biofilms produced by exudate bacterial communities. Furthermore, application of norspermidine increases the bactericidal effect of gentamicin. We believe that norspermidine could be used in combination with bactericidal antibiotics to control biofilm infection in dermal chronic wounds.

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