Infections of fracture fixation implants and synovial structures are a primary cause of complications, increased treatment costs, and mortality in people and horses. Treatment failure is often due to biofilms that are communities of bacteria that are adhered to a surface or to each other and are surrounded in a self-secreted extracellular matrix. The biofilm matrix protects the indwelling bacteria from being killed by antibiotics and the immune system. Biofilms also stimulate chronic inflammation and tissue destruction, including peri-implant osteolysis and subsequent implant failure and chondromalacia with subsequent osteoarthritis. In horses, the resulting lameness, reduced athletic potential, and poor quality of life may necessitate euthanasia. Equine bone marrow-derived mesenchymal stromal cells (MSC) reduce inflammation and promote healing in musculoskeletal injuries and have recently been discovered to have antimicrobial properties. Equine MSC kill planktonic (free-floating) bacteria and prevent biofilm establishment in laboratory models. MSC from mice and people also promote the transition from acute inflammation to tissue regeneration (resolution of inflammation) by secretion of specialized pro-resolving lipid mediators (SPM). Whether equine MSC can disrupt established biofilms of orthopedic pathogens and modulate the inflammatory response to orthopedic biofilms is unknown.
Using a novel biofilm-MSC co-culture model, our objectives were two-fold. We investigated whether MSC alone or with amikacin sulfate, an antibiotic used to treat equine orthopedic infections, could reduce biomass, pellicle size, and live bacteria of biofilms of orthopedic infectious agents S. aureus and E. coli. Next, we investigated whether MSC could modulate immune response to S. aureus biofilms by reducing secretion of pro-inflammatory cytokines by peripheral blood mononuclear cells (PBMC) and by secreting SPM. MSC demonstrated partial ability to reduce biofilms but performed differently on S. aureus versus E. coli biofilms. Co-culture of biofilms with MSC significantly reduced pellicle area of biofilms of both bacteria, reduced biomass of S. aureus biofilms, and killed live S. aureus bacteria. MSC combined with amikacin also significantly reduced S. aureus biomass to a greater extent compared to amikacin alone. The resolution in detecting differences between groups for E. coli was diminished because of high variation between biofilms treated with MSC between different donors and between control biofilms between experiments.
Using the same experimental system, culture of S. aureus biofilms with MSC in the transwell inserts and PBMC in the bottom wells significantly reduced biofilm size compared to untreated biofilms. Co-culture of MSC and PBMC with S. aureus biofilms also significantly increased detection of multiple SPM on lipid chromatography-mass spectrometry compared to MSC or PBMC cultures alone. Using a commercial equine multiplex bead ELISA, multiple inflammatory cytokines and chemokines were increased when S. aureus biofilms were cultured with MSC and PBMC; however, these were not different from untreated biofilms. Our results indicate that the utility of MSC in combating orthopedic biofilm infections lies in their ability to disrupt the biofilm matrix and promote inflammation resolution. These findings support continued investigation into and optimization of the anti-biofilm mechanisms of MSC. / Doctor of Philosophy / Biofilms are coating layers made by bacteria to protect them from being killed by antibiotics or the immune system. Biofilms result in untreatable infection, chronic inflammation and tissue destruction in people and horses with bone and joint infections. The resulting complications, including pain, reduced mobility, and poor quality of life, may result in horses being euthanized. Equine bone marrow-derived mesenchymal stromal cells (MSC) kill free floating bacteria in laboratory models and reduce inflammation in orthopedic injuries. Whether MSC can disrupt formed biofilms and reduce inflammation resulting from biofilm infections is unknown. Using a laboratory model, our objectives were to determine: 1) whether MSC alone or with an antibiotic used to treat orthopedic infections in horses can disrupt biofilms and kill indwelling live bacteria of orthopedic infectious agents S. aureus and E. coli, and 2) whether MSC can modify the immune response to S. aureus biofilms. MSC demonstrated some biofilm reducing ability but performed differently on S. aureus versus E. coli biofilms. Specifically, MSC reduced the size of biofilms of both bacteria, reduced the coating layer of S. aureus biofilms alone and to a greater extent when combined with the antibiotic, and killed live S. aureus bacteria. Using the same system, culture of MSC with S. aureus biofilms and peripheral blood mononuclear cells (PBMC), a type of white blood cell, reduced biofilm size compared to controls. The addition of MSC and PBMC to S. aureus biofilms also increased detection of fatty acid-derived signals that promote resolution of inflammation, compared to controls. Multiple inflammatory cytokines and chemokines were increased with culture of MSC and PBMC with S. aureus biofilms but were not different from untreated biofilms. These results indicate that MSC may be useful to combat biofilm infections by breaking down the coating layer of biofilms and by promoting resolution of inflammation. Taken together, our results support continued investigation into the potential of MSC as a treatment for orthopedic biofilm infections. The potential of MSC to simultaneously break down biofilms and mitigate inflammation in orthopedic infections would improve cure rates and overall outcomes for horses and people afflicted with orthopedic biofilm infections.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/116058 |
| Date | 18 August 2023 |
| Creators | Khatibzadeh, Sarah M. |
| Contributors | Biomedical and Veterinary Sciences, Bogers, Sophie Helen, Dahlgren, Linda A., Ducker, William A., Caswell, Clayton Christopher |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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