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Investigating Bacterial-Fungal Interactions Within Chronic Wound Microbiomes
Alex Cheong, Chad Johnson, PhD, Hanxiao Wan, Jeniel Nett, MD, PhD, Lindsay Kalan, PhD.
University of Wisconsin-Madison, Madison, WI, USA.

BACKGROUND: Chronic wounds are host to a diverse microbiome. However, it is unclear how these microbial communities, consisting of both bacteria and fungi, interact within the wound environment.
METHODS: An in vitro mixed-species biofilm model was used investigate species-interactions of wound isolates during biofilm formation and high-resolution microscopy was applied to evaluate physical interactions in three-dimensional space.
RESULTS: Interaction between the polymorphic fungus Candida albicans (CA) and the Gram-negative bacterium Citrobacter freundii (CF) isolated from a diabetic foot ulcer was previously reported to cooperatively form biofilms in vitro, where the bacteria adhere to and grow along fungal hyphal scaffolds in established biofilms. We found that CF competes with CA for substrate binding resulting in a significant reduction in colony forming units (CFU) of CA (2.8 log CFU, 95% CI [2.6, 3.0]) when seeded onto established CF biofilms (two-sample t(4) = 45.18, p < 1E-5). We determined that CF competes with Staphylococcus aureus (SA), the most common bacterium found in DFUs, for binding to CA, resulting in a reduction in SA growth (1.3 log CFU, 95% CI [0.6, 2.0]; two-sample t(16) = 3.77, p < .01), without affecting CF or CA proliferation. Adhesion of CF to CA was found to be mannose-sensitive, suggesting that binding may be mediated by type 1 fimbriae. We also observed that CF increases CA hyphae formation, thus we evaluated host responses by exposing human neutrophils to the mono- or co-cultures. We observed an increase in propidium iodide staining associated with neutrophil extracellular trap release in response to the co-culture, suggesting that fungal-bacterial interactions may result in pro-inflammatory phenotypes. Finally, using an ex vivo skin wound model, scanning electron microscopy, and confocal microscopy, we determined CA grows along collagen fibrils and hair shafts and CF adheres within the resulting biofilm.
CONCLUSIONS: The role of fungal-bacterial interactions in chronic wounds have not been widely studied but appear to be crucial to understanding pathogenesis.


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