Fruit Juice Suppression of Staph. Biofilm Formation Project By: Warren Sipe Central catholic high School Grade 11

Cherry and Cranberry Juices Pure cherry juice Cranberry Cranberry juice (primary ingredient) Grape juice Apple juice Pear juice Pectin Vitamin C Commercially available

Biofilms Coherent and generally adherent cells Extracellular Polymeric Substance (EPS) 80% of human diseases Phenotypic shift in gene regulation More resistant- 1000x Lateral gene transfer Quorum sensing

Biofilm Inhibition Adherence- conditioning films, polysaccharides Anti-biofilm agents- chemical inhibitors to adherence Other major methods: Surface modification (anti-microbial coatings) Hydrophobicity

Model Bacteria (Staph. E) Staphylococcus epidermidis Extremely common bacteria Frequently used for scientific research Skin flora Gram positive Can be pathogenic

Importance Biofilms- 80% of human infectious disease Past studies on anti-microbial effects of juice Poor understanding of effect on biofilms Important for hospitals Catheters Need for non-toxic, biofilm targeted disinfection

Purpose To determine the effect of cranberry and cherry juices on Staph. E biofilms To determine the effect of cranberry and cherry juices on Staph. E survivorship To relate the biofilm effects to the microbiocidal effects

Hypothesis Null hypothesis- Neither juice will significantly effect Staph. E survivorship or biofilm formation Alternate hypothesis- Both juices will significantly effect Staph. E survivorship and biofilm formation

Materials Staph. E culture Klett Spectrophotometer (reading in KU) Cherry juice Cranberry juice LB agar plates LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride) Sterile dilution fluid (100 mM KH2PO4, 100 mM K2HPO4, 10 mM MgSO4, 1mM NaCl) Sterile pipette tips Micropipettes Vortex Incubator (37 degrees C) Sidearm flask Sterile spreader bars Ethanol 96 well tissue culture treated microtiter dish Crystal violet Acetic acid Microtiter plate absorbance reader

Survivorship Procedure 1. Staph. E was grown overnight in sterile LB Media. 2. The culture was added to fresh media in a sterile sidearm flask. 3. The cultures were placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10⁸ cells/mL. 4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10⁵ cells/mL. 5. The juices were sterilized by means of a 0.2 micron syringe filter 6. The experimental variables were mixed with the appropriate amounts of SDF to create concentrations of 0%, 1%, 10%, and 50%.

Concentration Chart 0% 1% 10% 50% Sterile Dilution Fluid 9.9 ml 9.8 ml Staph. E 0.1 ml Juice 0 ml 1.0 ml 5.0 ml Total volume 10.0 ml

Survivorship Procedure (cont.) 6. The solutions were vortexed and allowed to sit at room temperature for 10 minutes. 7. 100 µL aliquots were removed from the tubes and spread on LB-agar plates. 8. The plates were incubated at 37°C for 48 hours. 9. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

Biofilm Procedure Growing a Biofilm 1. Tubes were prepared according to the dilution chart above. 2. 200 μL from the tubes was added per well in a 96 well dish. 8 replicates were performed from each tube. 3. The microtiter plate was incubated for 48 hours at 37°C. Staining the Biofilm 1. After incubation, the cells were gently removed out by turning the plate and allowing to drip dry. 2. The plate was gently submerged in a small tub of water. The plate was allowed to drip dry. 3. 200 μL of a 0.1% solution of crystal violet in water was added to each well of the microtiter plate.

Biofilm Procedure (cont.) 4. The microtiter plate was incubated at room temperature for 10 minutes. 5. The plate was rinsed by submerging in a tub of water as outlined above. 6. The microtiter plate was turned upside down and dried overnight. Quantifying the Biofilm 1. 200 μL of 30% acetic acid in water was added to each well of the microtiter plate to solubilize the CV. 2. The microtiter plate was incubated at room temperature for 10 minutes. 3. The absorbance of the microtiter plates was quantified in a microtiter plate reader at 550 nm using 30% acetic acid in water as the blank. *credit George O’Toole

P-Value= 0.0010926 P-Value=6.66702E-6

P-Value= 2.00086E-10 P-Value= 5.47719E-10

Analysis and Stats Did either cranberry or cherry juice significantly effect Staph. survivorship? Single factor ANOVA- Ps of 0.001092 and 6.66702E-6, respectively Significant for both Reject the null, accept the alternate for both Did either cranberry or cherry juice significantly effect Staph. biofilms? Single factor ANOVA- Ps of 2.00086E-10 and 5.47719E-10, respectively

Conclusions The null hypothesis was rejected The alternate hypothesis was supported Both juices showed anti-microbial and biofilm inhibitive effects Biofilm effect was greater than survivorship effect for both juices

Possible Limitations Spread plating was not perfectly synchronized Only survivorship, and not growth, was measured Only Staph. E was tested Only cranberry and cherry juices were tested Only one growth time was used for the biofilms

Extensions Efforts will be made to achieve more synchronous spread plating Survivorship and growth will be measured More species will be tested Tests will be done at more stages of growth Other juices will be tested

Resources Special thanks to Dr. Carrie Doonan of CMU for the use of her lab and equipment Bukhari, Mohammad. "Staphylococcus Epidermidis." Staphylococcus Epidermidis. University of Conneticut, 2004. Web. 24 Jan. 2017. Domenico, Phil. "Natural Anti-Biofilm Agents." The Science of Nutrition. N.p., 22 Sept. 2016. Web. 31 Dec. 2016. Fuente-Núñeza, César De La, Victoria Korolikb, Manjeet Bainsa, Uyen Nguyenc, Elena B. M. Breidensteina, Shawn Horsmand, Shawn Lewenzad, and Lori Burrowsc And. "Inhibition of Bacterial Biofilm Formation and Swarming Motility by a Small Synthetic Cationic Peptide." Antimicrobial Agents and Chemotherapy, 01 May 2012. Web. 24 Jan. 2017. O'Toole, George A. "Microtiter Dish Biofilm Formation Assay." Journal of Visualized Experiments : JoVE. MyJove Corporation, 2011. Web. 31 Dec. 2016. Romling, U., and C. Balsabore. "Biofilm Infections, Their Resilience to Therapy and Innovative Treatment Strategies." Journal of Internal Medicine (2012): 541-63. Web. 31 Dec. 2016.

Cranberry Survivorship 1 10 50 Cranberry Biofilm 190 163 143 157 3.022 1.989 2.155 0.834 2 197 172 170 158 3.184 2.914 2.042 0.828 3 185 193 173 175 3.822 2.724 2.697 1.103 4 182 164 178 166 3.41 1.887 2.365 1.214 5 192 147 3.827 2.343 2.202 1.12 6 188 152 168 3.503 2.66 2.647 0.931 Total 1129 1072 963 981 20.768 14.517 14.108 6.03 Average 188.1666667 178.6666667 160.5 163.5 3.461333333 2.4195 2.351333333 1.005 Cherry Survivorship Cherry Biofilm 211 169 150 2.832 2.313 1.159 183 140 151 3.231 3.101 1.537 189 154 142 3.558 2.682 1.404 199 165 161 153 3.199 2.123 1.346 200 184 179 156 2.984 2.046 1.327 177 171 3.043 1.994 1.165 1170 1024 943 916 18.847 14.259 7.938 195 170.6666667 157.1666667 152.6666667 3.141166667 2.3765 1.323