Transcript Escherichia coli

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.