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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.

In food processing, inadequate surface sanitation procedures lead to the formation of biofilms in which bacteria attach and aggregate in a hydrated polymeric matrix of their own synthesis. Formation of these sessile communities and their inherent resistance to existing sanitation procedures and agents are at the root of the risk of bacterial infections for consumers. Due to this existing problem, an effective method for reducing biofilm formation in dairy processing equipment is necessary for dairy products processing. Ultraviolet Pulsed light Technology has shown a positive effect in eliminating microorganism populations on food products. The objective of this work is to evaluate the effect of Pulsed light Technology on a biofilm of different dairy component matrices (e.g. Water (control); whey protein isolates (WPI), lactose, and sweet whey). This evaluation will be performed using the three strains of spore forming Bacillus species most common in commercial milk powder (B. subtilis, B. coagulans, and B. licheniformis). The matrix in which the evaluation was made consisted on allowing the attachment of endospores to on to a square 2.5cm x 2.5cm ASI 304 stainless steel coupon. Four Xenon light treatment levels (no treatment, 5 bursts, 10 seconds, 20 seconds and 30 seconds) were applied to the coupon surfaces using the Xenon model RC847 machine. The attachment of Bacillus to stainless steel in water as matrix was 1000 to 3000/ sq cm as measured in our laboratory. Results showed that there was a significant difference in spore reduction depending on the matrix of the biofilm and with the intensity of the Xenon treatment. Reduction in spores ranged from 1 to 4.7 logarithmic reduction cycles depending on the material of the biofilm, the strain of spores and the intensity of treatment. We conclude that there is significant potential to use this technology in maintaining low spore counts in commercial dairy powders.

Geobacter species are δ-Proteobacteria and are often predominant in the Fe(III) reduction zone of sedimentary environments. Their abilities to remediate contaminated environments and to produce electricity have inspired extensive studies. Cell motility, biofilm formation, and type IV pili, which have been shown to be regulated by chemotaxis genes in other bacteria, all appear important for the growth of Geobacter species in changing environments and for electricity production. The genomes of Geobacter species show the presence of a significant number of chemotaxis gene homologs, suggesting important roles for them in the physiology of Geobacter species, although gene functions are not yet identified. In this study, we focus on identifying chemotaxis components and studying their functions in Geobacter species. We identified a large number of homologs of chemotaxis genes, which are arranged in six or more major clusters in the genomes of Geobacter sulfurreducens, Geobacter metallireducens, and Geobacter uraniireducens. Based on homology to known pathways, functions of some chemotaxis clusters were assigned; others appear to be unique to Geobacter species. We discuss the diversity of chemoreceptors and other signaling proteins as well the regulation of chemotaxis genes in Geobacter species. The functions of chemotaxis genes were studied in G. sulfurreducens, whose genome contains ~ 70 chemotaxis gene homologs, arranged in 6 major clusters. These chemotaxis clusters are also found in other Geobacter species with similar gene order and high level of gene identity, suggesting that our study in G. sulfurreducens could be extrapolated to other Geobacter species. We identified the function of the che5 cluster of G. sulfureducens as regulation of the biosynthesis of extracellular materials. We showed that G. sulfurreducens KN400 is chemotactic, and that this behavior is flagellumdependent. Our preliminary data indicated that G. sulfurreducens may use the che1 cluster, which is found exclusively in Geobacteraceae, to regulate chemotaxis. Our studies demonstrated important roles of chemotaxis genes in Geobacter physiology and their presence in large numbers could be one of the reasons why Geobacter species outcompete other species in bioremediation sites. Further studies are warranted for better understanding of the mechanisms of Che-like pathways and their potential use in optimization of conditions for applications of Geobacter species in bioremediation and electricity generation.

Desulfovibrio alaskensis G20 and other sulfate-reducing bacteria cause significant damage to metal pipelines and other infrastructure through a metabolic pathway that releases toxic hydrogen sulfide into their surroundings. The biocorrosion that results from the release of hydrogen sulfide creates significant economic burden, and can pose health risks for those exposed to this chemical. They are commonly present in the form of biofilms, an extracellular matrix composed of bacterial cells, polysaccharides, proteins, nucleic acids, and other materials. These biofilms are difficult to remove, and they provide protection to the bacteria within from anti-bacterial treatments. Desulfovibrio alaskensis G20 is a strain derived from a wild-type bacterium collected from an oil well corrosion site and is a model organism for understanding biofilm formation of sulfate-reducing bacteria and how these biofilms can be prevented or inhibited by techniques such as cerium oxide nanoparticle coating. To this end, samples of Desulfovibrio alaskensis G20 were grown anaerobically in 24-well and 96-well plates, and the resultant biofilm growth was measured through spectrophotometry. Several different environmental parameters were tested, including temperature, electron donor molecules, basal and enriched growth media, and oxidative stress, revealing several affinities for production of biofilm growth.

E-cigarette (e-cig) use is rising, but much is unknown about the effects of its vapor. This vapor contains chemicals such as propylene glycol, a known antimicrobial, and nicotine, whose derivatives are carcinogenic. Here, we study the effects of vaping on resident bacteria of the oral cavity and on oral cell inflammation. Oral streptococci are major residents in the oral cavity, with S. mutans the primary cause of dental caries. Growth and biofilm formation have been shown to be enhanced upon exposure to traditional cigarette smoke in vitro. In this study, we analyzed the effects of e-cig vapor on growth and biofilm formation in S. mutans, S. sanguinis, and S. gordonii. Organisms and oral epithelial cells were treated using nicotine-free and 3mg nicotine vapor, as well as double-shot menthol freeze flavored 3mg nicotine vapor in a vape chamber designed to phenocopy physiologically relevant exposure. Nicotine-independent inhibition of growth occurred upon exposure in all three bacterial species. Interestingly, biofilm formation was enhanced in the S. mutans while decreased in S. sanguinis and S. gordonii. Epithelial cells showed activation of survival pathways by Western Blot upon exposure to only e-cigarette vapor as well as co-culturing of bacterial and oral epithelial cells at a multiplicity of infection of one The pioneer colonizers S. gordonii and S. sanguinis generally antagonize caries-causing S. mutans, which can become a predominant member of the community under appropriate conditions, leading to dental caries formation. The observed decrease in the biofilm formation of the commensals S. sanguinis and S. gordonii upon e-cig vapor exposure indicates the opportunistic colonization of S. mutans, whose biofilm-forming abilities increased. Following e-cig usage, dental caries and cancer in the oral epithelium may result from this dysbiosis of the microbiome.

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