Horizontal gene transfer transforms commensal E. faecalis into multidrug resistance (MDR) opportunistic pathogens causing diseases such as infective endocarditis (IE), septicemia, and urinary tract infections (UTI) (4,1). E. faecalis are among the top three leading causes of hospital-acquired infections and pheromone responsive plasmids (pCF10) are the most extensively characterized conjugative plasmids in E. faecalis infection (2,4). E. faecalis is a potential future public health concern because of the co-occurrence factors of antibiotic resistance and virulence traits (6)Plasmid-free commensal E. faecalis form homogenous biofilms that have a uniform distribution of the bacterial cell and a fluid-like movement (22). The introduction of the pheromone responsive plasmid pCF10 leads to the formation of heterologous rigid structures within the biofilm (22). In the current work, the timeline of biofilm tower formation was characterized. Tower formation was not observed in the commensal strain. The pCF10-containing bacteria formed a rigid base layer on day 1 and small aggregates on day 1. pCF10-containing biofilm forms heterologous towers on days two and three. Interestingly, mixed biofilms with both plasmid-containing and plasmid-free bacteria developed tower-like structures as early as day 1 and had larger resulting structures by day three. In the mixed population, we hypothesize that the induction of aggregation substance and cell clumping during plasmid transfer may further contribute to structure formation (5,10). Plasmid-free mCherry-labeled bacteria could be observed in the viscous biofilms between heterologous rigid structures; however, the rigid structures were predominantly composed of plasmid-containing cells. Occasionally, mCherry cells were observed in the rigid structures, we hypothesize that these cells represent transconjugants, where pCF10 was transferred by conjugation to mCherry-plasmid-free OG1RF. The formation of rigid structures can protect bacteria from antibiotics by reducing the penetration of the antibiotic but binding and sequestration of the antibiotic in the outer layers. Antibiotic resistance increased in the pCF10-containing biofilms as rigid structures were formed. We hypothesize that underflow, like that found in the gastrointestinal tract, the heterologous rigid structures may form protected microenvironments for sensitive regions of the biofilms. In future studies, fluorescently labeled antibiotics will be used to access the formation of protected microenvironments in biofilms underflow. Previous studies in the laboratory demonstrated that the presence of pCF10 protects E. faecalis from hydrogen peroxide oxidative stress. E. faecalis produces hydrogen peroxide. Higher levels of hydrogen peroxide can be detected in rigid structures. The presence of pCF10 is known to increase the size of heart vegetations during endocarditis and hydrogen peroxide is a known activator of platelet activation (12,19). In these studies, the presence of pCF10 increased platelet activation in pCF10 containing biofilms. Software toolboxes are currently being developed to quantitate visual observations. The role of hydrogen peroxide is supported in our preliminary experiment revealing catalase treatment reduced platelet activation. Studies are ongoing to mutate the aroc and menB gene of E. faecalis, which contribute to hydrogen peroxide production (35). We will compare platelet activation in knockout (double or single) E. faecalis and the wild-type strain. For future studies, several of the preliminary data need to be repeated to further the study. We will repeat quantitative hydrogen peroxide production in the catalase experiments. We will also finish knocking out the aroc and men B gene of E. faecalis responsible for hydrogen peroxide and then compare platelet activation to the control strain. / Microbiology and Immunology