Arbo , Ma. Adelle Gia T. Arteta , Allen O. Belmonte, Pauline Marie C.

1. Quorum Sensing Inhibitionof Vibrio alginolyticus from Caulerpa lentillifera (Lato) on Pseudomonas aeruginosa Arbo, Ma. AdelleGia T. Arteta, Allen O. Belmonte, Pauline Marie C.

2. What is Quorum Sensing? • accumulation of signaling molecules enabling a single cell to sense the population density (Bhattacharya et al, 2008, pp. 30) • production and release of chemical signal molecules called autoinducers to coordinate gene expression • significant in biofilm formation and virulence of some microorganisms

3. Pseudomonas aeruginosa • an opportunistic bacterium that uses quorum sensing in the formation of biofilms, swarming motility, expolysaccharide production, and cell aggregation. (Bhattacharyya et al, 2008, pp. 30-33) Figure 1. Pseudomonas aeruginosa (http://textbookofbacteriology.net/pseudomonas.html)

4. Related Studies • Givskov, Michael et al (2006) • quorum sensing signaling systems of pathogens were used as central regulators for the expression of virulence factors • Quorum sensing inhibitors - compounds are capable of reducing virulence and be used as treatment

5. Joint, Ian et al (2007) • the green seaweed Ulva has been shown to detect N-acylhomoserine lactones (AHLs)

6. Seaweeds • suitable substrate for epibiotic organisms because it releases a large amount of carbon into the surrounding environment http://davaoorientalinvest.com/main/?page_id=121 Figure 2. Seaweeds

7. Caulerpa lentillifera • abundant in the Philippines and cultivated as food and sold fresh and salted http://www.mesa.edu.au/marine_algae/gallery.asp) Figure 3. Caulerpa lentillifera

8. Vibrio alginolyticus • a gram negative bacterium usually isolated from the surface of marine organisms such as seaweeds and stony corals • Some Vibrio species have quorum sensing inhibiting ability

9. Statement of the Problem Main Problem: Is Vibrio alginolyticus from seaweed, Caulerpa lentillifera capable of inhibiting the quorum sensing of Pseudomonas aeruginosa?

10. Specific Problems: • 1. Are there zones of inhibition exhibited by Vibrio alginolyticuson Chromobacterium violaceum after agar overlay technique? • 2. Are there zones of inhibition exhibited by Vibrio alginolyticus on Pseudomonas aeruginosa after agar overlay technique?

11. Significance of the Study • seaweed farmers • antimicrobial agents • economy • Pharmacology

12. Scope and Limitation Limitation Scope • seaweed Caulerpa lentillifera • use of indicator Chromobacterium violaceum 12472 • use of lb broth • Quorum sensing inhibition assay • antibacterial assay • time • observation time

13. Research Design R X1 O1 R X2 O2 • Where: • O1& O2 = posttest observation • X1= quorum sensing inhibition assay • X2= antibacterial assay

14. Research Paradigm Linking Variables Agar overlay of Pseudomonas aeruginosa and Chromobacterium violaceum Zones of inhibition of Pseudomonas aeruginosa and Chromobacterium violaceum 12472 Quorum sensing systems of Pseudomonas aeruginosa; Chromobacteriumviolaceum 12472, Vibrio alginolyticus Signaling molecules and growth conditions of the bacteria Independent Variables Dependent Variables Extraneous Variables Figure 4. Research Paradigm

15. Treatment/ General Procedure Isolation of Vibrio alginolyticus Figure 5. Taking samples of bacteria from Caulerpa lentillifera Figure 6. Agar plate inoculated with bacteria from Caulerpa lentillifera

16. A. Quorum Sensing Inhibition Assay Preparation of Marine agar plates and lb broth Inoculation of Chromobacterium violaceum 12472 and Vibrio alginolyticus on lb broth and marine agar, respectively Figure 7. Plate inoculated with C. violaceum and P. aeruginosa Figure 8. Inoculating lb broth with C. violaceum 24- Hour incubation Overlay of lb broth on the marine agar plates inoculated with Vibrio alginolyticus 24- Hour incubation Figure 10. Overlay of lb broth on marine agar after 24 hours. Figure 9. Test tube inoculated with C. violaceum Observation and Data Gathering

17. B. Agar Overlay of Pseudomonas aeruginosa Preparation of marine agar plates and trypticase soy broth Inoculation of Vibrio alginolyticus on marine agar and of Pseudomonas aeruginosa on trypticase soy agar Figure 11. TSB inoculated with Pseudomonas aeruginosa Figure 12 Marine agar inoculated with Vibrio alginolyticus 24-hour incubation Overlay of trypticase soy on marine agar plates 24-hour incubation Observation and Data Gathering Figure 13. Agar overlay of Pseudomonas aeruginosa Figure 14. Overlay of TSB on marine agar after 24 hours

18. Results and Discussion There was no noticeable zone of inhibition induced by Vibrio alginolyticus on both Chromobacterium violaceum and Pseudomonas aeruginosa. Figure 15. Marine agar plate of Vibrio alginolyticus after agar overlay of Chromobacterium violaceum Figure 16. Marine agar plate of Vibrio alginolyticus after agar overlay of Chromobacterium violaceum

19. no verification that organisms had quorum sensing at that time • mutations of organisms • population of organisms • conditions of bacterial growth

20. Recommendations • Further understanding of quorum sensing • isolate more organisms • exploit other techniques

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