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THE DISCOVERY OF QUORUM QUENCHING SOIL MICROBES FOR THE DEVELOPMENT OF ANTIMICROBIAL COMPOUNDS

BACTERIAL SILENCING:. THE DISCOVERY OF QUORUM QUENCHING SOIL MICROBES FOR THE DEVELOPMENT OF ANTIMICROBIAL COMPOUNDS. Sonia Rao. Quorum Quenching. Enzymes interfere with Quorum sensing, inhibiting virulence New class of pharmaceuticals Increase in antibiotic resistant. Lactonases.

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THE DISCOVERY OF QUORUM QUENCHING SOIL MICROBES FOR THE DEVELOPMENT OF ANTIMICROBIAL COMPOUNDS

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  1. BACTERIAL SILENCING: THE DISCOVERY OF QUORUM QUENCHING SOIL MICROBES FOR THE DEVELOPMENT OF ANTIMICROBIAL COMPOUNDS Sonia Rao

  2. Quorum Quenching • Enzymes interfere with Quorum sensing, inhibiting virulence • New class of pharmaceuticals • Increase in antibiotic resistant

  3. Lactonases • Removes lactone ring from the AHL molecule • AiiA gene - Erwinia carotovora , Pseudomonas aeruginosa

  4. Acylases • Detach nitrogen bond to form a fatty acid chain and homoserine lactone • PvdQ and QuiP - P. aeruginosa

  5. Hypothesis • Soil communities would contain quorum quenching isolates • Late successional communities would contain greater number of quorum quenching isolates than early successional communities

  6. Variables • Independent - Successional stage - Competitive nature • Dependent -Quorum quenching isolates -Frequency of natural communities to produce quorum quenchers • Constant: - Incubation time for soil samples and isolates - Amount of x-gal

  7. Method • C.violaceum 12472, 31532, CV026 • A.tumefaciens A136, KYC6 • X-gal, antibiotics

  8. Method • 1 gram of 10 soil sample cultured -R2A broth, vortex • 0.1 ml added to R2A plate

  9. Method • C.violaceum 12472 next to isolate • A.tumefaciens A136 • TraR gene detects AHL C6-C14 • lacZ gene which expresses β-galactosidase

  10. Method • A.tumefaciens A136 bioassay • 4.5 mg/ml of tetracycline and 50 mg/ ml of spectinomycin • 50 microliters x-gal (5-bromo-4-chloro-3-indolyl-galactopyranoside) • 3 replicates • Frozen in glycerin

  11. Method • CV026 and 31532 • A136 and KYC6

  12. Method • Gene sequencing • Cells suspended in 10 µl of sterile water • 10 µl of sterile water, 2 µl of 27 F primer, 2 µl of 1392 primer, 4 µl of a master mix, 10 µl of cells culture PCR program: 95 °C – 5.5 min 58 °C – 30 sec 72 °C – 6.5 min

  13. Method • Gel electrophoresis • 2 µl Ethidium bromide • 2 µl added to eppendorf tubes-2nd PCR

  14. Method • Gene sequencing lab • BLAST ( Basic Local Alignment Search Tool) Database

  15. Summation of Method Culturing of Soil Samples and isolates C.Violaceum 12472 bioassay A.tumefaciens A136 bioassay Gene Sequencing and BLAST

  16. Results • 10 Isolates • Late Successional communities have greater number of quorum quenching isolates than early succession communities (p = 0.03). • Gene sequencing- inconclusive

  17. Results

  18. Results

  19. Conclusions • Soil isolates that produce quorum quenching enzymes was supported • Late succession communities would have a greater number of isolates that quorum quench than early succession communities was supported (p = 0.03).

  20. Applications • Sources for quorum quenching microbes • Increase in antibiotics resistant bacteria

  21. Future Studies • Identifying the quorum quenching enzyme • Inducing E.coli to produce the enzyme

  22. Acknowledgments • Chris Reeves and Kim Griggs • Professor Robert McLean • Professor Dave Westenberg • Dr. Chris Waters • Diane Ayers

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