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Saureusly, Disrupt Quorum agatsonis/Docs/STEMPoster.pdf · PDF file 2018. 5. 24. · Quorum Sensing Quorum sensing is the method by which bacteria within a biofilm express genes collectively

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  • Saureusly, Disrupt

    Quorum Se Staphyl aureus

  • Stop It! ion of nsing in ococcus Biofilms

  • Introduction

  • Researchable Question

     Will an S. aureus culture exhibit cell death if silver, copper, or zinc

    ions are bound to autoinducing peptide (AIP) and inserted into the

    culture?

  • Hypothesis

     If silver, copper, or zinc ions are bound to AIP and inserted into an S.

    aureus culture, then cell death will occur.

  • Background

  • Quorum Sensing

     Quorum sensing is the method

    by which bacteria within a

    biofilm express genes

    collectively.

     In pathogenic bacteria, such

    as S. aureus, these genes

    regulate the production of

    virulence factors.

     Autoinducing peptide, or AIP-

    1, is the autoinducer of the

    agr (accessory gene regulator)

    quorum sensing system (Kong

    et al., 2006) of S. aureus.

    Figure 1. Structure of AIP-1. The top image shows the

    atomic structure of AIP-1 while the bottom image shows

    the amino acid structure. (Kjaerulff et al., 2013)

  • Materials and Methods

  • Materials

    Table 1. List of materials used in the project and their sources

    Materials

    Salt and

    glucose

    medium

    Incubator UV/VIS

    spectrometer

    Flask

    stoppers

    Micropipette

    tips

    Silver nitrate Biological

    safety hood

    (BSL-2)

    Centrifuge Flasks Staphyloccoc

    us aureus

    Zinc acetate Balance LS 55

    fluorescence

    spectrometer

    Weighing

    boats

    TSB (tryptic

    soy broth)

    Copper

    sulfate

    hexahydrate

    Pipet gun Culture tubes Double-

    ended

    spatula

    SYTO 9 dye

    Deionized

    water

    Refrigerator Pipettes Micropipette PI (propidium

    iodide dye)

  • Metal Ion Solution Creation 96-Well Plate Preparation

  • Figure 2. Flowchart showing how metal ion solutions were prepared

    Figure 3. Flowchart depicting how the metal ion plate was prepared

  • Metal Ion Exposure Tests

  • Figure 4. Flowchart depicting how the timed metal ion exposure experiments were conducted

  • Results

  • Definition of RatioG/R

     The live/dead assay involved two dyes.

     SYTO 9 (abbreviated to G, because of its ability to stain live cells

    green) and propidium iodide (abbreviated R, because of its ability to

    stain dead cells red) were used to determine the overall viability of

    the cells.

     Forming RatioG/R by dividing the intensity of the green fluorescence

    (510 – 540 nm) by the intensity of the red fluorescence (620 – 650 nm)

    allows the viability of the bacterial cells to be assessed.

  • 96-Well Plate Test

    Figure 5. The RatioG/R of the control group consisting of 15 wells of S.

    aureus, no metal ions, and no AIP in the 96-well plate experiment Figure 6. The RatioG/R of the silver group consisting of 15 wells of S.

    aureus, silver ions, and no AIP in the 96-well plate experiment

  • Figure 7. The RatioG/R of the zinc group consisting of 15 wells of S.

    aureus, zinc (II) ions, and no AIP in the 96-well plate experiment

    Figure 8. The RatioG/R of the copper group consisting of 15 wells of

    S. aureus, copper (II) ions, and no AIP in the 96-well plate

    experiment

  • First Metal Ion Exposure Test

    Figure 9. A comparison of the three groups (the control group, the copper group, and the zinc group) in the first fluorescence

    spectroscopy experiment

  • Figure 10. A comparison of the two groups (the control group and the copper group) in the second fluorescence

    spectroscopy experiment

    Second Metal Ion Exposure Test

  • Conclusions

  • 96-Well Plate Data Analysis

     The positive slopes for the silver and zinc groups (0.0287 and 0.0433

    respectively) were unexpected because both silver and zinc have been

    proven to be antimicrobials (see Figures 6 and 7).

     One possible cause of these results could have been that the plate

    was not left to incubate for an extensive period of time.

     The slope of the copper group (see Figure 8), -0.0244, indicates that

    copper ions could react with bacterial cells more quickly than zinc

    ions or silver ions can.

  • Metal Ion Exposure Tests Analysis

     In the first timed exposure experiment, both the zinc group and the

    copper group showed a negative trend in RatioG/R as time elapsed (see

    Figure 9).

     For the copper group, the steepest decrease in RatioG/R occurred from

    30 minutes to 90 minutes, suggesting that most of copper’s

    antimicrobial activity occurs early on when exposed to bacteria.

     The zinc group appeared to have its steepest decrease in RatioG/R occur from 30 to 120 minutes.

     RatioG/R at 90 minutes for zinc showed an unusually sharp increase.

    The conditions under which the zinc group was incubating should not

    have been conducive for bacterial growth.

  •  The copper group in the second timed experiment showed a negative

    trend from 0 minutes to 116 minutes (see Figure 10), while the copper

    group in the first experiment showed a steeper negative trend from 30

    to 90 minutes and a less steep negative trend onwards.

     These results still suggest that copper has an increased period of

    antibacterial activity early on.

     The copper group of the second fluorescence spectroscopy experiment

    showed a period of inactivity from 129 to 160 minutes, which is

    similar to the first spectroscopy experiment in which the copper group

    showed a period of inactivity after 100 minutes.

  • Future Work

  •  Determine a procedure to purify AIP.

     Conduct the same experiments but with added AIP.

     Determine a procedure to conduct the experiments using silver.

     Conduct experimentation using different metal ions.

  • Preparation of the Metal Ion Plate and Modelling of Copper Binding to AIP-1

    Figure 11. Photograph of Achilles Gatsonis

    preparing the 96-well plate for the metal ion test

    (Jared Watson)

    Figure 12. Model of Cu2+ binding to AIP-1 (John Cvitkovic)

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