Saureusly, Disrupt

Quorum SeStaphylaureus

Stop It!ion of nsing in ococcusBiofilms

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 experimentFigure 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)