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Background Statistical Analysis OPTIMIZATION An 89% difference was found between biofilm growth with versus without N-80 at an E. coli cell concentration of 1:100. Purpose of Research BIOFILMS Montana State University Biofilms are colonies of bacteria that are highly resistant to antibiotics (Sayen, 2014). Biofilms are formed when planktonic, free floating, bacteria attach to a smooth surface and form a colony (O'Toole, 2011). Hypothesis To answer the following question: Does a cleavage fragment of Lacritin inhibit biofilm formation? Past research has found that a cleavage fragment of Lacritin is bactericidal. A multiple regression analysis was conducted to determine the relationship between protein concentration and percent absorbance. Establishing Lacritin as an inhibitor of biofilm formation has Thus, it was hypothesized that Lacritin would inhibit implications for cost effective uses in the clinical setting. Escherichia coli biofilm formation. A negative relationship was revealed between protein concentration and percent absorbance (Rsquared = 0.8, p < 0.001). A 96-well microtiter biofilm assay was used. Biofilms can form wherever there is a smooth surface and an environment conductive to bacteria growth. • The wells were inoculated with E. coli cells and experimental mixture was added It was necessary to determine the dilution of E. coli cells that would yield the best results and most accurate data. • The plate was incubated overnight to allow for biofilm growth and inhibition • Too few cells would be overpowered by the N-80 protein and would not allow inhibition. • Too many cells would overwhelm the N-80 protein and would yield inconclusive data. Biofilms pose a significant health hazard (e.g., eye infections) (Wilcox, 2013). LACRITIN Lacritin is a naturally occurring protein. Lacritin is produced in the Lacrimal gland and released to the surface of the eye in the form of tears (Peisong, 2007). CONCENTRATION CURVE The aim of the project was to investigate the use of Lacritin Lacritin targets cell-to-cell communication and the extrato inhibit biofilm formation rather than prevent biofilm cellular matrix of bacterial cells, qualities that have been formation. proposed as enabling biofilm antibacterial resistance. These colonies are capable of cell-to-cell communication and form a thick matrix around the colony that assist in antibiotic resistance (Patel, 2005; (Ren et al., 2005). Biofilms are the most common cause of infections in hospitals and are often found in contact lens cases (AlFattani & Douglas, 2004; Wu et al., 2010). This prompted further investigation at the 1:100 E. coli cell concentration. Procedure Experiments OPTIMIZATION • The wells were washed to flush out any planktonic bacteria, leaving only biofilms. CONCENTRATION CURVE • The plate was developed and growth was marked with a purple dye. Optimization established an initial relationship between N80 and biofilm inhibition. • A plate reader was used to read the amount of light the contents of each well absorbed. Wanted to confirm that N-80 inhibited biofilm growth. OPTIMIZATION Data Thus, as the concentration of N-80 protein increases, E. coli biofilm growth decreases. Discussion CONCLUSION There is a statistically significant difference between the absorption with vs. without the addition of the N80 protein. There was some biofilm growth with N-80. CONCENTRATION CURVE It is reasonable to conclude that N-80, a cleavage fragment of Lacritin, is capable of inhibiting Escherichia coli biofilm growth, supporting the hypothesis. Past research (McKown et al., 2014) found that a cleavage fragment of Lacritin, N-80, was antibactericidal. • Escherichia coli cells were found to grow at higher rates when N-80 was removed vs. when it was present. • Lacritin was found to have capabilities to penetrate extracellular matrices and inhibit cell-to-cell communication. APPLICATION The results support the use of Lacritin in a medical setting to treat pre-diagnosed biofilm infections. The use of Lacritin has potential as a cost effective treatment for biofilm infections. References EXPERIMENTAL TABLES (McKown et al., 2014) Charts and Graphs were created by researcher unless otherwise noted Al-Fattani, M., & Douglas, L. (2004). Penetration of Candida Biofilms by Antifungal Agents. Antimicrobial Agents And Chemotherapy, 48(9), 3291-3297. McKown, R., et al. (2014). A Cleavage-potentiated Fragment of Tear Lacritin Is Bactericidal. Journal of Biological Chemistry, 289(32), 22172-82. O'toole, G. (2011). Microtiter Dish Biofilm Formation Assay. Journal of Visualized Experiments, 47, 24372437. Patel, R. (2005). Biofilms and Antimicrobial Resistance. Clinical Orthopaedics and Related Research, 437, 41-47. doi: 10.1097/01.blo.0000175714.68624.74 Peisong Ma, Ningning Wang, Robert L. McKown, Ronald W. Raab, and Gordon W. Laurie (2007). Focus on Molecules: Lacritin. Experimental Eye Research, 86(3), 457-458 Ren, D., Zuo, R., Barrios, A., Bedzyk, L., Eldridge, G., Pasmore, M., & Wood, T. (2005). Differential Gene Expression for Investigation of Escherichia coli Biofilm Inhibition by Plant Extract Ursolic Acid. Applied and Environmental Microbiology, 71(7), 4022-4034. Sayen, S. (2014). Biofilm Control and Antimicrobial Agents. Hoboken: Apple Academic Press. Willcox, M. (2013). Microbial Adhesion to Silicone Hydrogel Lenses. Eye & Contact Lens: Science & Clinical Practice, 39(1), 60-65. Wu, Y., Zhu, H., Willcox, M., & Stapleton, F. (2010). Removal of Biofilm from Contact Lens Storage Cases. Investigative Ophthalmology & Visual Science, 51(12), 6329-6333.