No description available.

Polymicrobial communities of bacterial and fungal species are present on the skin and mucosal surfaces of the body. Invasive infections caused by Candida species are commonly seen in immunocompromised individuals (HIV, transplants, cancer) and ranks as the third leading cause of infection in hospitalized patients. C. albicans is a polymorphic opportunistic fungus that infects critically ill patients and has the ability to change its morphology from yeast to hyphal form.The morphogenesis of C. albicans is a major aspect of its virulence and is regulated by quorum sensing (QS) molecules they produce, as well as the presence of neighboring microbes.In this study, we examined two transporter mutants, cdr4 and ssu1, for their ability to form biofilms in the presence of cyclic-di-GMP and 3-oxo-12-homoserine lactone. To quantify biomass, wild type and mutant cells were grown overnight at 30˚C in YPD. The cells were washed, counted and diluted to a desired density of 106 cells/ml in medium 199, pH7.5. Cells were added to 96-well plates pre-incubated with 5% fetal bovine serum at densities of 105, 104, and 103 cells/well and allowed to adhere at 37˚C for one hour. The wells were then covered with fresh M199 media containing the QS molecule and monitored for 48 hours at 37˚C. After this time, the media and planktonic cells were removed. The biofilms were fixed with methanol, dried, then stained with 0.05% crystal violet. Bulk biomass was assessed by spectrophotometry. We did observe a difference in biofilm density when incubated in the presence of cyclic-diGMP. We noted that for the wild type and ssu1 strain their biofilms biomass increased by as much as 10% at 104 and 103 cell densities when compared with the control. While the cdr4 strain had a slight reduction in biofilm density when cyclic-diGMP was present. This result also indicates a potentially positive role in which cyclic-diGMP can help C. albicans develop denser biofilms, potentially in the presence of bacteria like P. aeruginsa, which secrete cyclic di-GMP, but kill hyphal forms of C. albicans. Additionally, it has been shown that C. albicans mutants lacking the hybrid histidine kinase, Chk1p, are refractory to the effects of farnesol, a QS molecule that inhibits morphogenesis.Given that mutations in CDR4 and SSU1 impact the QS response in C. albicans, we investigated whether these genes were regulated through two-component signaling by Chk1. To assess CDR4 and SSU1 expression, wild type and mutant strains were grown overnight in YPD media at 30˚C. Cells were then harvested and RNA was obtained by acid phenol extraction. Using RT-PCR, we determined both CDR4 and SSU1 expression is reduced or highly repressed in the chk1, ypd1, and skn7 null strains. These results suggest the two genes are downstream targets in a pathway regulated by Chk1p. The finding that QS proceeds through a two-component pathway can be exploited in antifungal drug development. Given that two-component signaling is absent in mammalian cells, development of novel compounds that interfere with this pathway may be a useful alternative for treating patients with candidiasis.

An alarming problem has plagued both human and veterinary healthcare for decades: the ever-increasing presence of antibiotic resistant bacteria. Specifically, Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-resistant Staphylococcus pseudintermedius (MRSP) are of great concern. A new compound, aptly named, LavenGel has recently been developed and demonstrates treatment potential to effectively inhibit the growth of at least two Staphylococcus species: S. aureus and S. pseudintermedius. LavenGel has been previously shown in our lab to inhibit a variety of microbes, particularly S. aureus. However, the molecular pathway that LavenGel utilizes to inhibit S. aureus and whether this inhibition could be translated to other Staphylococcus species, particularly in animals, has yet to be investigated. The major aims of this study are to demonstrate and quantify the efficacy of LavenGel in preventing S. aureus and S. pseudintermedius growth and to understand the specific S. aureus cellular mechanisms that LavenGel impacts. In order to quantitatively represent the effectiveness of LavenGel for veterinary purposes, biofilms of S. pseudintermedius were treated at different phases of development. LavenGel inhibited both the attachment of cells to form biofilms, as well as the eradication of pre-existing biofilms. Minimum inhibitory concentrations and minimum bacterial concentrations were determind for S. pseudintermedius. To better understand the impact that LavenGel may have in human healthcare, a panel of genes expressed under LavenGel treatment were examined. LavenGel does not induce the typical SOS response in S. aureus that is seen when using other leading bactericidal treatments for Staphylococcus infections which have also been shown to induce resistance. LavenGel could potentially help solve the bacterial resistance issue by working against the bacterial cell membrane instead of inducing the typical SOS response. The threat of antibiotic resistant bacteria is a constant concern in the scientific and healthcare community. The implications of this study dictate LavenGel is a highly effective, all-natural, unique option for treating common Staphylococcus infections in both veterinary and human healthcare and shows promise as a treatment that, as of yet, does not induce bacterial resistance. LavenGel could prove to be a powerful tool in the future of medical management of bacterial infections.

Nous avons développé une biopuce de microfluidique qui est capable de surveiller continuellement la population de cellules dans les biofilms en conditions d'écoulement laminaire bien contrôlées. Ce dispositif microfluidique est capable de modeler la formation des biofilms linéaires en utilisant une approche de flux basé sur un modèle. Les considérations de conception et méthodologie de fabrication d'un micro-bioréacteur à deux niveaux, inclus le flux basé sur un modèle (FT-μBR) qui génère un flux de croissance du biofilm entouré par les 3 côtés par un flux de confinement et inhibiteur de croissance. Grâce à une combinaison d'expériences et de simulations, nous avons évalué et exploité exhaustivement les paramètres de contrôle pour manipuler les dimensions du modèle de flux de croissance du biofilm. Ce dispositif est ensuite utilisé pour développer des modèles linéaires du biofilm avec des dimensions contrôlables. Une étude de validation de principe utilisant le dispositif démontre son utilité dans la réalisation des mesures de taux de croissance du biofilms dans différents environnements de force de cisaillement. Cela ouvre la voie à des études quantitatives sur les effets de l'environnement des cisaillements locaux sur les propriétés des biofilms et pour la synthèse d'une nouvelle génération de biomatériaux fonctionnels ayant des propriétés contrôlables. / We have developed a microfluidic biochip that is capable of continuously monitoring cell population in biofilms under well-controlled laminar flow conditions. This microfluidic device capable of patterning linear biofilm formations using a flow-templating approach. The design considerations and fabrication methodology of a two level flow-templating micro-bioreactor (FT-μBR) generates a biofilm growth stream surrounded on 3 sides by a growth inhibiting confinement stream. Through a combination of experiments and simulations we comprehensively evaluate and exploit control parameters to manipulate the biofilm growth template stream dimensions. The FT-μBR is then used to grow biofilm patterns with controllable dimensions. A proof-of-principle study using the device demonstrates its utility in conducting biofilm growth rate measurements under different shear stress environments. This opens the way for quantitative studies into the effects of the local shear environment on biofilm properties and for the synthesis of a new generation of functional biomaterials with controllable properties.

The feasibility of simulating solute transport in a channel impregnated witha biofilm is investigated. Biofilm factors relevant for numerical simulation arestudied to determine a suitable range of simulation. Inside the feasable rangeseveral cases are simulated to gain insight of how different factors influence thesolute transport in and around biofilms.

No description available.

No description available.

No description available.

No description available.

Background: The composition of the vaginal microbiota (VMB) in the female genital tract (FGT) can impact the vaginal epithelium and protect against or increase risk of sexually transmitted viral infections. The VMB grows as a biofilm, a complex structure formed by bacteria for increased survival. When the VMB consists of a diverse bacterial community it correlates with pathogenic effects that lead to adverse health conditions and an increased risk of HIV infection. When the VMB contains Lactobacillus species, beneficial health effects and decreased susceptibility to infection are observed. The aim of this project is to optimize an in vitro model of biofilm formation for different bacteria associated with the VMB, identify the effects that biofilm has on vaginal epithelial cells and test biofilm treatment strategies. We hypothesize that a Lactobacillus biofilm will enhance barrier function and decrease cytotoxicity of vaginal epithelial cells whereas dysbiotic biofilm will decrease barrier function and induce cytotoxicity. We also hypothesize that various conditions, such as presence of estradiol and eubiotic short-chain fatty acids, will stimulate Lactobacillus biofilm growth and suppress dysbiotic biofilm growth in a vaginal epithelial cell model. Methods: For optimization of the biofilm model, VK2/E6E7 cells were grown in air-liquid interface (ALI) or liquid-liquid interface (LLI) cultures in presence or absence of L. crispatus, L. iners, G. vaginalis or P. bivia bacteria. Biofilm formation was assessed using FilmTracerTM SYPRO® Ruby biofilm matrix protein stain. Hormone effects were tested by adding estradiol (10-9 M) and progesterone (10-7 M) to culture media. Short-chain fatty acid (SCFA) effects were tested by adding lactic acid, acetic acid, succinic acid and butyric acid in varying concentrations to culture media. Enzyme effects were tested by adding sialidase to Vk2 cells before bacteria inoculation. Results: A novel in vitro model of biofilm formation on vaginal epithelial cells was created. Vk2 cells in ALI and LLI cultures remained viable in anaerobic conditions and showed mucin-1 production in aerobic and anaerobic conditions. Matrix protein staining provided a means to accurately visualize and quantify biofilm formation in this model. L. crispatus and L. iners biofilm growth maintained vaginal epithelial barrier integrity without cytotoxicity. G. vaginalis and P. bivia biofilm growth significantly reduced barrier integrity (p=0.0166, p=0.0115) and increased cytotoxicity (p=0.0024, p<0.0001). Estradiol significantly increased the growth of L. crispatus biofilm in the co-culture system (p<0.0001). Progesterone significantly increased G. vaginalis biofilm growth in the Vk2 cell co-culture (p=0.006). L. crispatus biofilm formation in the estradiol condition, G. vaginalis biofilm formation in the progesterone condition and P. bivia biofilm growth in the normal media condition were significantly decreased in the presence of sialidase (p<0.0001, p=0.0001, p=0.0380). Conclusion: A novel in vitro model of biofilm formation on a vaginal epithelial cell line that can be used to visualize and quantify biofilm growth was generated. This model was used to test various strategies for biofilm enhancement or dissociation. Estradiol enhanced beneficial Lactobacillus biofilm growth, while progesterone enhanced dysbiotic biofilm growth. Mucin- digesting enzyme sialidase was effective at dissociating all biofilms. This model can be used in the future to test different strategies of dysbiotic biofilm dissociation and enhancement of Lactobacillus biofilm in order to investigate treatments for Bacterial Vaginosis (BV) and reduce susceptibility to HIV transmission in women. / Thesis / Master of Science (MSc)