assessing-biofilm-dispersal-in-murine-wounds

Assessing Biofilm Dispersal in Murine Wounds

Biofilm-related infections are implicated in a wide array of chronic conditions such as non-healing diabetic foot ulcers, chronic sinusitis, reoccurring ...

Texas Tech University Health Sciences Center

Chronic wounds will impact 2% of the United States population at some point in their life. These wounds are often associated with a reoccurring, chronic infection caused by a community of microorganisms encased in an extracellular polymeric substance (EPS), or a biofilm. Biofilm-associated microbes can exhibit tolerance to antibiotics, which has prompted researchers to investigate therapeutics that improve antibiotic efficacy. Glycoside hydrolases (GHs), enzymes that target the polysaccharide linkages within the EPS, are one potential adjunctive therapy. In order to develop GH-based therapeutics, it is imperative that we understand whether the composition of biofilm EPS changes based on the environment and/or presence of other microbes. Here, we utilized α-amylase and cellulase to target the polysaccharides within the EPS of mono- and dual-species  and  biofilms in three different models that vary in clinical relevancy. We show that biofilms established in an  well-plate model are not strongly adhered to the polystyrene surface and do not accurately reflect the GH efficacy seen with biofilms grown . However, dispersal efficacy in an  wound microcosm model was more reflective of that seen in a murine wound model. We also saw a striking loss of efficacy for cellulase to disperse  in both mono- and dual species biofilms grown in the wound models, suggesting that EPS constituents may be altered depending on the environment.

Differential Efficacy of Glycoside Hydrolases to Disperse Biofilms.

The complexity of microbial biofilms offers several challenges to the use of traditional means of microbial research. In particular, it can be difficult to calculate accurate numbers of biofilm bacteria, because even after thorough homogenization or sonication, small pieces of the biofilm remain, which contain numerous bacterial cells and result in inaccurately low colony forming units (CFU). In addition, imaging of infected tissue  often results in a disparity between the CFU and the number of bacterial cells observed under the microscope. We hypothesized that this phenomenon is due to the biofilm extracellular polymeric substance decreasing the accessibility of stains and antibodies to the embedded bacterial cells. In this study, we describe incorporating EPS-degrading glycoside hydrolases for CFU determination to obtain a more accurate estimation of the viable cells and for immunohistochemistry to disrupt the biofilm matrix and increase primary antibody binding to the bacterial cells.

Garrett S. Welch

Department of Surgery,
TTUHSC Surgery Burn Center of Research Excellence

Texas Tech University Health Sciences Center

Assessing Biofilm Dispersal in Murine Wounds

Garrett S. Welch

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Texas Tech University Health Sciences Center

Assessing Biofilm Dispersal in Murine Wounds

Department of Surgery,
TTUHSC Surgery Burn Center of Research Excellence