Abstract THE EFFECTS OF VARIOUS STILBENOIDS ON MOTILITY, GROWTH, BIOFILM FORMATION AND

QUORUM SENSING OF ESCHERICHIA COLI O157:H7

AcknowledgementsSpecial thanks to Professor Kendal Beazer, Dr. Nicholaou, Professor Kenton Cummins and Kent Kriddle for their generous guidance and help throughout. Thanks to the Mr.& Mrs. DenkersFamily Foundation for funding this research.

Combrestatin

Resveratrol Viniferin

Polydatin

Introduction

RESULTS

EHEC is an aggressive human pathogen found in contaminated food and water that causes outbreaks worldwide of hemorrhagic colitis and hemolytic uremic syndrome. It colonizes the large intestine where it produces potent Shiga toxins that cause anemia, kidney failure and can result in death. According to CDC there are 73000 illnesses and 60 deaths due to this pathogen annually. The most susceptible population are elderly and children under 5 years old. Antibiotics are known to stimulate more Shiga toxin production and promote more resistant strains of EHEC. Stilbenoids are natural defense mechanisms in plants showing antimicrobial, antioxidative and antifungal properties. They have the potential to disrupt quorum sensing and virulence factors. EHEC regulates quorum sensing via the AHL’s produced by surrounding organisms through Sid A. The Sid A/AHL combination affects gene transcription in an unknown fashion (Fig 1).

AHL Produced by other Bacteria

SidA

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E. coli

AHL

LuxR

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Gene Expression

Gram Negative Cell

LuxI

Figure 1a. In most gram-negative cells acyl –Homoserinelactones (AHL) are produced by the LuxIsynthase and then bind to LuxR. LuxR/AHL combination binds to DNA and activates virulence gene expression.

Figure 1b. E. coli lacks the Acyl-homoserinelactone (AHL) synthasegene. AHL produced by other bacteria bind to the LuxR homologue SidA. The SidA/HSL combination then binds to DNA and affects transcription.

Methods Discussion

Limitations

Figure 5a.DMSO decreased growth at higher concentration, AHSL dissolved in DMSO had no effect .

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Figure 5c.Growth inhibition at 200ug/mL.Resver. completely inhibited growth and Vinif. and Combr.decreased growth.

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Figure 5b.Growth inhibition at 100ug/mL.Resver.had the most effect on growth.

Figure 6a. Swarming motility distance at 24 hrs.AHL, Resver., and Vinif. decrease motility.

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Figure 5d.Growth inhibition at 400ug/mL.Resver., Combr., and Vinif. are inhibiting. Polyd. Is enhancing.

Figure 7a. Swarming motility distance at 48 hrs. Polyd. +AHL increase motility.

Growth Biofilm Motility

Quorum sensing is a bacterial communication system that controls the expression of genes in response to population density. This expression can lead to increased pathogenicity in humans or adherence to treatment vats in the pasteurization process, making quorum sensing a target for the development of new antimicrobial compounds. This study’s goal is to determine and compare the ability of stilbenoids to inhibit quorum sensing, growth, biofilm formation and swarming motility in Escherichia coli O157:H7 (EHEC). MIC panels with varying concentrations of the stilbenoidcompounds will be used to derive a sub-inhibitory dose for testing. N-(3-oxo-octanoyl)-L- homoserinelactone (AHL) was used to induce SidA quorum sensing. Swarming motility was determined on motility agar plates in the presence of the various stilbenoids and AHL. Inhibition of growth was determined by comparing optical densities at 600nm. Biofilm formation was measured by staining adhered cells in the presence of the various stilbenoids and AHL with crystal violet. Resveratrol and viniferin were found to inhibit growth and motility whereas Polydatin enhanced these characteristics. The findings of this study could potentially lead to the development of new routes of treatment for EHEC.

Figure 2. Cell suspensions were added to a 96 well plate. Background controls were added for DMSO and AHL. The stilbenoid compounds were added with and without the addition of AHL. The plate was read every 2hrs over an 18hr period at 600nm.

Figure 3. A 96 well plate was setup using the same format as the growth assay and an initial optical density reading was taken at 600nm. The plate was then allowed to incubate without agitation for 24and 48hrs at 37⁰C. Another reading was taken at 570nm after staining with crystal violet

Figure 4. A broth culture was grown overnight in LB broth at 37⁰C. 5uL of broth suspension was added to motility agar plates containing the stilbenoidsat 50ug/mL concentrations and allowed to grow for 48hrs. Measurements of the motility distance were taken at 24 and 48hrs.

• SidA activation through AHL has no effect on growth, decreases motility, and didn’t lead to biofilm formation.

• Resveratrol, Viniferin and Combrestatin decrease growth• Polydatin enhanced or increased growth• Motility decreased with resveratrol and Viniferin• Polydatin increased motility• Combrestatin had no effect or lightly increased motility

•Not enough replicates for growth and motility•Not enough chemical supply for replicates•Not enough Money for more chemicals•E. coli O157:H7 didn’t make biofilm in any condition•SidA in E. coli O157:H7 doesn’t follow prototypical quorum sensing systems and is poorly understood

Maria Svagr, Angie Cefalo, Dimetri StasinosFaculty Mentor Kendal Beazer MHAMLS(ASCP)CM