Fruit Juice Suppression of Staph. Biofilm Formation

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

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

3. 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

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

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

6. 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

7. 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

8. 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

9. Materials • 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 • Staph. E culture • KlettSpectrophotometer (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

10. 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%.

11. Concentration Chart

12. 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.

13. 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.

14. 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

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

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

18. Analysis and Stats • Did either cranberry or cherry juice significantly effect Staph. survivorship? • Single factor ANOVA- Ps of 0.001092 and6.66702E-6, respectively • Significant for both • Rejectthe null, acceptthealternate for both • Dideithercranberry or cherry juice significantlyeffectStaph. biofilms? • Single factor ANOVA- Psof2.00086E-10 and5.47719E-10, respectively • Significant for both • Reject the null, accept the alternate for both

19. 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

20. 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

21. 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

22. 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, ManjeetBainsa, UyenNguyenc, 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. MyJoveCorporation, 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.