1. Background BIOFILMS 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). 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 can form wherever there is a smooth surface and an environment conductive to bacteria growth. Biofilms are the most common cause of infections in hospitals and are often found in contact lens cases (Al- Fattani & Douglas, 2004; Wu et al., 2010). 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). 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. Montana State University Purpose of Research To answer the following question: Does a cleavage fragment of Lacritin inhibit biofilm formation? The aim of the project was to investigate the use of Lacritin to inhibit biofilm formation rather than prevent biofilm formation. Establishing Lacritin as an inhibitor of biofilm formation has implications for cost effective uses in the clinical setting. (McKown et al., 2014) Hypothesis Past research has found that a cleavage fragment of Lacritin is bactericidal. Lacritin targets cell-to-cell communication and the extra- cellular matrix of bacterial cells, qualities that have been proposed as enabling biofilm antibacterial resistance. Thus, it was hypothesized that Lacritin would inhibit Escherichia coli biofilm formation. Procedure A 96-well microtiter biofilm assay was used. The wells were inoculated with E. coli cells and experimental mixture was added The plate was incubated overnight to allow for biofilm growth and inhibition The wells were washed to flush out any planktonic bacteria, leaving only biofilms. The plate was developed and growth was marked with a purple dye. A plate reader was used to read the amount of light the contents of each well absorbed. Experiments OPTIMIZATION It was necessary to determine the dilution of E. coli cells that would yield the best results and most accurate data. 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. CONCENTRATION CURVE Optimization established an initial relationship between N- 80 and biofilm inhibition. Wanted to confirm that N-80 inhibited biofilm growth. Data Statistical Analysis EXPERIMENTAL TABLES OPTIMIZATION An 89% difference was found between biofilm growth with versus without N-80 at an E. coli cell concentration of 1:100. This prompted further investigation at the 1:100 E. coli cell concentration. CONCENTRATION CURVE A multiple regression analysis was conducted to determine the relationship between protein concentration and percent absorbance. A negative relationship was revealed between protein concentration and percent absorbance (R- squared = 0.8, p < 0.001). 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 N- 80 protein. There was some biofilm growth with N-80. It is reasonable to conclude that N-80, a cleavage fragment of Lacritin, is capable of inhibiting Escherichia coli biofilm growth, supporting the hypothesis. 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. CONCENTRATION CURVE OPTIMIZATION References 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, 2437- 2437. 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. 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