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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)

SyfteAtt undersöka hur tillväxt och succession sker i ett bakteriellt konsortium bestående avvanliga orala bakteriearter vid lågt pH och vid tillgång av glukos i närmiljön.Material & metodKonsortiumet bestod av Streptococcus mutans, Streptococcus intermedius, Streptococcusoralis, Streptococcus gordonii, Veillonella parvula/dispar, Lactobacillus paracasei,Capnocytophaga gingivalis, Fusobacterium nucleatum, och Actinomyces odontolyticus.Denna bakteriekultur sattes till glukosfattiga lösningar vid pH 5.5 och 7.2, samt till englukosrik lösning vid pH 7.2 och inkuberades i 37°C. Efter 0, 24 och 48 timmar analyseradesbakteriekompositionen samt pH-värdet i bakteriekulturen.ResultatDen procentuella pH-sänkningen var störst i bakteriekulturen med initialt pH 7.2 med tillsatsav glukos, och minst i bakteriekulturen med initialt pH 7.2 utan tillsats av glukos. Allabakteriearter förekom i varierande mängd vid odling, men i takt med pH-sänkning sågs enmer homogen mikroflora som nästan uteslutande dominerades av L. paracasei.SlutsatsL. paracasei hade stor förmåga att överleva i detta konsortium oavsett miljöförändring, ochvisade även på högst syratolerans. Det förekom en samexistens mellan L. paracasei och S.gordonii eller S. oralis oavsett bakteriekultur. Övriga bakterier klarade sig inte lika bra somL. paracasei, däribland S. mutans som inte överlevde i bakteriekulturerna i denutsträckningen som förväntades. / IntroductionThe aim of this study was to investigate the outcome of interspecies interactions in vitro in aconsortium made of nine commonly isolated oral bacterial strains under differentenvironmental conditions, such as glucose excess.Material and methodsThe bacterial consortia was comprised of Streptococcus mutans, Streptococcus intermedius,Streptococcus oralis, Streptococcus gordonii, Veillonella parvula/dispar, Lactobacillusparacasei, Capnocytophaga gingivalis, Fusobacterium nucleatum, and Actinomycesodontolyticus. This complex culture was incubated in glucose-limited and glucose-rich mediawith different pH at 37°C for 48 hours during which the bacterial composition and pH wasanalysed at 0, 24, and 48 hours.ResultsThe greatest pH-reduction was seen in the bacterial culture with an initial pH of 7.2 withglucose-excess, and lowest in the culture with an initial pH of 7.2 with glucose-limitation. Allspecies were identified at least once in the experiments, but the diversity was diminishedfollowing pH reduction.ConclusionL. paracasei showed great ability to survive in the bacterial cultures, with the highest acidtolerance, regardless of environmental change. Coexistence occurred between L. paracaseiand S. gordonii or S. oralis regardless of bacterial culture. Other bacteria did not cope asgood as L. paracasei, including S. mutans that did not survive in the bacterial cultures to theextent expected.

Hospital Acquired Infections and equipment contamination are some of the biggest issues faced by the healthcare industry worldwide. These infections generally range from mild to life threatening human infections which lead to increased costs and prolonged hospitalization time. The primary factor which caused these issues were biofilm forming bacteria which are able to withstand medications and defend themselves from various cleaning procedures. Another aspect which make these bacteria troublesome is that they are able to hide inside the biofilm, thus evading a lot of diagnostic tests. The methods used to detect these biofilms are unfortunately toxic to cells and cannot be used in vivo. Though there is enough data on the formation of biofilm on abiotic surfaces, the data present on the biophysical properties, structural organizations within the biofilm or their viscoelastic properties is very limited. In this master’s degree project, a dynamic monitoring platform is made for 2 different strains of the Salmonella Enteritidis bacteria where their structural and biophysical properties was investigated. Each strain lacks either curli or cellulose which are major components responsible for proper biofilm formation so performing these experiments on them gave us important information on how their properties get affected over time. Bacterial growth monitoring for all the strains were performed by measuring the absorbance every hour over a period of 5h and it was observed that all the strains had a very similar growth pattern until the end of the 4th hour after which they showed very mild differences. The next set of experiments involved using an eQCM to monitor the formation of biofilm on the surface of a quartz chip over 48h. There were differences observed in the biofilm formation pattern and mass deposition which provided clues as to how the biofilm formation and their viscoelastic properties are affected due to the mutations. This led to finding further clues regarding which aspect of biofilm formation is targeted by a specific mutant.