1

Disruption of Quorum Sensing in Staphylococcus aureus

Biofilms

Achilles Gatsonis

Gatsonis 2

Table of Contents

Abstract ........................................................................................................................................... 4

Literature Review............................................................................................................................ 5

Overview of Biofilms ................................................................................................................. 5

The Cycle of Biofilm Formation................................................................................................. 5

Stage 1: Attachment ................................................................................................................ 5

Stage 2: Growth ...................................................................................................................... 6

Stage 3: Detachment ............................................................................................................... 6

Importance of Biofilms ............................................................................................................... 6

How Biofilms Impact the Environment .................................................................................. 6

How Biofilms Impact Industry ............................................................................................... 7

How Biofilms Impact the Medical Field ................................................................................ 7

Quorum Sensing.......................................................................................................................... 8

LuxS Quorum Sensing ............................................................................................................ 8

Agr Quorum Sensing .............................................................................................................. 8

LuxI/LuxR Quorum Sensing................................................................................................... 9

Staphylococcus aureus ................................................................................................................ 9

Importance of S. aureus ........................................................................................................ 10

Quorum Sensing in S. aureus................................................................................................ 10

Agr Quorum Sensing in S. aureus ........................................................................................ 10

LuxS Quorum Sensing in S. aureus ...................................................................................... 11

Metal Bacterial Toxicity ........................................................................................................... 12

Silver ..................................................................................................................................... 12

Copper ................................................................................................................................... 12

Zinc ....................................................................................................................................... 13

Plan ............................................................................................................................................... 14

Researchable Question .............................................................................................................. 14

Hypothesis................................................................................................................................. 14

Methodology ............................................................................................................................. 14

Materials and Methods .................................................................................................................. 16

Gatsonis 3

Results ........................................................................................................................................... 21

Conclusions ................................................................................................................................... 25

References ..................................................................................................................................... 27

Appendix A ................................................................................................................................... 29

Acknowledgements ................................................................................................................... 29

Appendix B ................................................................................................................................... 30

Limitations and Assumptions ................................................................................................... 30

Appendix C ................................................................................................................................... 31

Project Notes ............................................................................................................................. 31

Knowledge Gaps ................................................................................................................... 31

Journal Articles ..................................................................................................................... 32

Websites ................................................................................................................................ 55

Books .................................................................................................................................... 59

Appendix D ................................................................................................................................... 62

Raw Data Tables ....................................................................................................................... 62

Gatsonis 4

Abstract

Roughly 80% of all infections are caused by biofilms. Biofilm-related infections are

much more difficult to treat than other infections because biofilms are resistant to antibiotics, and

their removal can entail surgery. Staphylococcus aureus, which often causes these infections,

produces autoinducing peptide (AIP), a quorum sensing signal. Quorum sensing is responsible

for the production of virulence factors, which make infected individuals exhibit symptoms. It

was theorized that binding metal ions to AIP would directly expose cells within a biofilm to toxic

metal ions. Three experiments were conducted using a live/dead assay (Thermo Fisher, 2004). In

the first experiment, S. aureus was exposed to copper ions, zinc ions, silver ions, or no metal ions

for 15 minutes. In the second experiment, S. aureus was exposed to copper ions, zinc ions, or no

metal ions for a 210-minute period. A third experiment was conducted in which S. aureus was

exposed to either copper ions or no metal ions for a 160-minute period. This research could be

beneficial in developing a novel method to destroy biofilms.

Gatsonis 5

Literature Review

Overview of Biofilms

A biofilm is an immobile colony of cells within a layer of extracellular polymeric

substance (EPS). A biofilm has a complex structure in which a large community of bacterial cells

can thrive. This structure can take numerous shapes depending on environmental conditions, but

most commonly assumes the shape of a pillar or mushroom (Archer et al., 2011). A network of

channels within this structure allows the deepest cells of a biofilm to have access to essential

nutrients. Because of its intricate structure, a biofilm has a low metabolic rate and its membrane

acts as a diffusion barrier; these two characteristics allow a biofilm to resist antimicrobial activity

much more effectively than planktonic, free-floating bacteria (Archer et al., 2011). A biofilm is a

safe, nutritive environment in which many bacterial cells are sustained.

The Cycle of Biofilm Formation

Biofilms form through a several-step process. The first stage of biofilm formation,

attachment, occurs when planktonic bacteria adhere to a surface. The second stage of the cycle,

growth, happens when these bacteria form a biofilm. The third stage of the cycle, detachment, is

the process by which other biofilms are be generated from the first biofilm (“Biofilm basics,”

2003).

Stage 1: Attachment

During the attachment stage of biofilm formation, a conditioning layer is formed by

planktonic bacteria. This layer gains an electrical charge which attracts other bacterial cells

(“Steps in Biofilm Formation,” 2017). The cells start producing EPS, which firmly attaches the

cells to the surface and to each other within a sticky matrix.

Gatsonis 6

Stage 2: Growth

During the growth stage of biofilm formation, EPS production continues. As more EPS is

produced, the bacterial colony forms a structure with a network of channels capable of

transporting nutrients and removing toxins. This structure allows for the rapid multiplication of

bacteria.

Stage 3: Detachment

Bacterial cells must detach from the biofilm for the production of separate biofilms.

There are multiple ways for bacteria to detach from a biofilm, including mass detachment, in

which a large group of cells detach, and seeding dispersal, in which individual bacterial cells are

released (“Biofilm basics,” 2003).

Importance of Biofilms

Biofilms play an important role in many areas. A few of these areas are the environment,

industry, and medical field.

How Biofilms Impact the Environment

Biofilms have an important role in their environments and can cause both positive and

negative effects. Biofilms can positively contribute to their environments by forming the basis of

many food webs and, by decontaminating soil through their ability to process and degrade waste

(“Biofilm basics,” 2003). However, they can facilitate the movement of toxic substances, deplete

oxygen from bodies of water, and cause dangerous algal blooms, more commonly known as “red

tides” (“Biofilm basics,” 2003). Biofilms have the capability to sustain or destroy their

environments.

Gatsonis 7

How Biofilms Impact Industry

The presence of biofilms is typically disastrous in industrial settings. In the United States,

biofilm formation is responsible for billions of dollars of equipment damage and decreased

productivity (“Biofilm basics,” 2003). While they can form on many kinds of machines, biofilms

are most effective at colonizing machinery used in water-based industries, where they

contaminate the water (“Biofilm basics,” 2003). Biofilms can also damage pipes and factory

equipment by causing blockages and corroding metals. Biofilm formation in industry damages

machinery and hinders productivity.

How Biofilms Impact the Medical Field

Biofilms are particularly dangerous in medical settings. Biofilms effectively colonize the

human body, forming on the arteries, lungs, skin, teeth, nostrils, and many other locations

(“Biofilm basics,” 2003). Biofilms can also form on indwelling medical devices, such as

catheters, and are responsible for over one billion dollars of damage annually in the United States

(Resch, Rosenstein, Nerz, & Gotz, 2005). Furthermore, approximately 80% of all infections are

thought to be caused by biofilms (Feng et al., 2015). Biofilm-related infections are much more

difficult to treat than other infections because biofilms exhibit extreme resistance to antibiotics

and the removal of a biofilm can require surgery (Archer et al., 2011). Potentially lethal illnesses

such as bacterial endocarditis, Legionnaire’s disease, and cystic fibrosis can all result from

biofilm formation in the human body (“Biofilm basics,” 2003). While bacteria naturally inhabit

the human body, the formation of a biofilm can be deadly because of the persistence and lethality

of biofilm-related infections.

Gatsonis 8

Quorum Sensing

Quorum sensing is the mechanism by which the bacterial cells of a biofilm regulate gene

expression cooperatively. The cells in a biofilm carry out this cell-to-cell communication using

signaling molecules called autoinducers, also known as AIs (Kong & Otto, 2006). However, the

biofilm must be at a threshold population in order for quorum sensing to occur; the

transcriptional regulator involved in quorum sensing only activates when AIs accumulate to a

certain concentration within the biofilm (Kong & Otto, 2006). Quorum sensing allows a biofilm

to orchestrate widespread gene expression.

LuxS Quorum Sensing

LuxS quorum sensing occurs in both Gram-positive and Gram-negative bacteria and is

regulated by the LuxS protein, also known as AI-2 synthase (Keersmaecker, Sonck, &

Vanderleyden, 2006). Interestingly, bacteria possessing a LuxS homologue can respond to the

AI-2 produced by other species of bacteria, supporting the idea that the LuxS quorum sensing

system allows for interspecies communication (Bassler, 1999).

Agr Quorum Sensing

Agr quorum sensing occurs only in Gram-positive bacteria and consists of two

transcripts, RNAII and RNAIII. Their transcription is carried out by P2 (promoter 2) and P3

(promoter 3) respectively. RNAIII contains agrA, agrB, agrC, and agrD, which are four

accessory genes. The expression of agrB and agrD produces autoinducing peptide, the signal

which allows for agr quorum sensing to occur (Kong & Otto, 2006).

Gatsonis 9

Autoinducing Peptide 1

Autoinducing peptide 1, or AIP-1, is the

autoinducer of the agr quorum sensing system

(Kong & Otto, 2006). AIP-1 is composed of eight

amino acids which form a macrocycle and an

exocyclic tail (see Figure 1). Although the structure

of AIP-1 remains consistent in each strain of Staphylococcus, the sequence of amino acids varies

(Kong & Otto, 2006). AIP-1 is the quorum-sensing signal responsible for the activation of the

agr system in Staphylococcus.

LuxI/LuxR Quorum Sensing

The LuxI/LuxR quorum sensing system is present only in Gram-negative bacteria and is

regulated by the following two proteins: LuxI (acyl-homoserine-lactone synthase) and LuxR

(acyl-homoserine-lactone regulator). LuxI is an autoinducer synthase which produces the

autoinducer HSL, homoserine lactone. LuxR is a protein which promotes the transcription of an

operon, a section of DNA which contains several genes and is under the control of a promoter,

when HSL is bound to it. The transcription of the operon results in the expression of a gene

(Bassler, 1999).

Staphylococcus aureus

Staphylococcus aureus is a Gram-positive coccus which naturally inhabits the human

body and can cause illness. A Gram-positive bacterium has a thick cell wall of peptidoglycan. A

coccus is a bacterium which has a round shape.

Figure 1. Structure of AIP-1 (Kjaerulff et al., 2013)

Gatsonis 10

Importance of S. aureus

S. aureus is a very common cause of nosocomial infection. More than 500,000

nosocomial infections are caused by S. aureus each year (Balaban, 1998). S. aureus can cause

numerous deadly infections, including meningitis, pneumonia, and toxic shock syndrome

(Balaban, 1998). Furthermore, over 20% of the human population’s nostrils are continuously

colonized by S. aureus; this colonization of the nares has been linked to a higher risk of

nosocomial infection (Archer et al., 2011). Because host immune responses are typically

ineffective against S. aureus biofilms, the host often suffers from chronic infection (Archer et al.,

2011). S. aureus is a widespread bacterium naturally present in many people’s bodies that can

cause life-threatening sicknesses.

Quorum Sensing in S. aureus

Quorum sensing has been observed to occur in S. aureus. Both the agr quorum sensing

system and the LuxS quorum sensing system are present in the coccus. These systems appear to

play a vital role throughout an S. aureus biofilm’s existence.

Agr Quorum Sensing in S. aureus

The agr quorum sensing system is important in the detachment phase of the biofilm

formation cycle. The ability of cells to detach from a biofilm is crucial for a successful infection;

detachment allows cells to form biofilms on other sites, which allows a biofilm-associated

infection to spread throughout the host organism. In S. aureus, the agr quorum sensing system

regulates the production of PSMs, or phenol-soluble modulins, which, through their amphipathic

nature, promote the detachment of cells from a biofilm (Kong & Otto, 2006). Interestingly, one

study revealed that a Staphylococcus mutant without a functional agr system produced a thicker

biofilm, most likely because of the inability of cells to detach from the biofilm (Kong & Otto,

Gatsonis 11

2006). The agr quorum sensing system, through the production of PSMs, allows cells to detach

from a biofilm and spread infection.

The agr quorum sensing system has also been shown to bolster the ability of a

staphylococcal biofilm to defend itself from the human immune system. A Staphylococcus

mutant without a functional agr system was found to be severely hindered in its ability to

successfully react to host immune responses. The mutant was less capable of causing chemotaxis

(the expulsion) of human neutrophils and was less resistant to human antimicrobial peptides in

comparison to a Staphylococcus with a functional agr system (Kong & Otto, 2006). Furthermore,

because the agr system controls the general oxidative stress responses of staphylococcal bacteria,

the mutant was believed to be less effective at dealing with the respiratory burst, or the release of

reactive oxygen species, such as hydrogen peroxide, of human neutrophils (Kong & Otto, 2006).

Thus, the agr system is fundamental to a Staphylococcus biofilm’s ability to protect itself from

the human immune system.

LuxS Quorum Sensing in S. aureus

The LuxS quorum sensing system impacts the density of an S. aureus biofilm. The LuxS

protein regulates the production of PIA (polysaccharide intracellular adhesin), a molecule which

allows the cells within a biofilm to attach to each other more firmly (Kong & Otto, 2006). When

a Staphylococcus mutant without a functional LuxS system was observed, it formed a thicker

biofilm than a wild-type Staphylococcus with a functional LuxS system (Kong & Otto, 2006).

Thus, the LuxS quorum sensing system, much like the agr system, promotes the ability of cells to

detach from a biofilm.

Gatsonis 12

Metal Bacterial Toxicity

Metals have been found to be toxic to numerous bacterial species. The antibacterial

nature of metals can be utilized in the development of methods to inhibit biofilm formation, kill

bacterial cells, and inhibit quorum sensing.

Silver

In the past, silver has been used as an antimicrobial and is one of the most commonly

used antimicrobials today. Silver causes a wide variety of damage to bacteria, including

respiratory inhibition, membrane damage, destruction of the proton motive force, and damage to

membrane proteins (Hobman & Crossman, 2014). Silver can also cause the generation of

hydroxyl radicals, which are harmful to bacteria (Hobman & Crossman, 2014). When applied to

S. aureus in a bactericidal concentration, cell death, inhibition of growth, and cell wall

breakdown was observed (Li et al., 2010). In addition to its lethal combination of effects, silver

is a very versatile antimicrobial. Both Gram-positive and Gram-negative bacteria can be killed

with silver (Hobman & Crossman, 2014). Furthermore, silver does not induce microbial

resistance as strongly as other metals (Hobman & Crossman, 2014). Because of its capability to

inhibit growth and break down membrane proteins, silver has the potential to inhibit quorum

sensing.

Copper

Copper, much like silver, was previously used as an antimicrobial, but has recently lost

popularity. Copper has been shown to have a strong affinity for biological molecules, rapidly

kills both Gram-positive and Gram-negative bacteria, and can cause the generation of hydroxyl

radicals (Hobman & Crossman, 2014). However, S. aureus has developed methods against

copper toxicity. S. aureus exhibits oxidative stress resistance, repairs protein damage caused by

Gatsonis 13

copper, and regulates the intake of hydrogen peroxide, a compound which can form hydroxyl

radicals (Hobman & Crossman, 2014). Despite the numerous defensive mechanisms against

copper toxicity, copper is a viable candidate to inhibit quorum sensing because of its affinity for

biological molecules and its ability to rapidly kill Gram-positive bacteria.

Zinc

Like silver and copper, zinc, although much less commonly, was used as an antimicrobial

in the past. However, because of zinc’s previous unpopularity, many different species of bacteria

have not developed a zinc resistance. Furthermore, zinc is highly toxic to prokaryotes, including

S. aureus, and is an effective antimicrobial at low concentrations (Hobman & Crossman, 2014).

Because of zinc’s high toxicity to prokaryotes and deadliness at low concentrations, zinc is likely

to successfully inhibit quorum sensing or kill cells within a biofilm.

Gatsonis 14

Plan

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 biofilm?

Hypothesis

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

then cell death will occur.

Methodology

S. aureus was grown in tryptic soy broth (TSB) liquid media in a culture tube at 37℃.

This culture was prepared through a process involving centrifugation, resuspension, and an

adjustment of its optical density (Thermo Fisher Scientific, 2004). Afterwards, a 96-well plate

was prepared. 15 metal ion solutions which contained either zinc, copper, or silver ions were

created. For each metal ion solution, three wells were filled with 100 µL of the suspension, 100

µL of a dye solution containing propidium iodide and SYTO 9 dye, and 30 µL of the metal ion

solution. 15 other wells were filled with 100 µL of the bacterial suspension, 100 µL of dye

solution, and 30 µL of a salt and glucose solution. A fluorescence spectrometer was used to

measure the fluorescence emission spectrum of each suspension in the well plate.

In a second experiment, S. aureus was grown in TSB at 37℃. After centrifugation,

resuspension, and an adjustment of its optical density, 1 mL of the suspension was added to each

of three cuvettes containing either 1 mL of the 10 mM zinc solution and 1 mL of the salt and

glucose solution, 1 mL of the 10 mM copper solution and 1 mL of the salt and glucose solution,

or 2 mL of the salt and glucose solution. These cuvettes were left in an incubator at 37℃. Every

30 minutes during a 210-minute period, 100 µL from each of the three cuvettes were pipetted

Gatsonis 15

into a 96-well plate. Afterwards, 100 µL dye solution were pipetted into each of the three wells.

A fluorescence spectrometer was used to measure the fluorescence emission spectrum of each

suspension in the plate. A third experiment similar to the second experiment was conducted.

However, in this experiment, only two cuvettes (one containing 1 mL of the 10 mM copper

solution and 1 mL of the salt and glucose solution and the other containing 2 mL of the salt and

glucose solution) were prepared and the fluorescence emission spectra were measured over a

160-minute period. Additionally, the dye solution used in this experiment was more dilute than

the dye solution used in the second experiment.

Gatsonis 16

Materials and Methods

Culture tubes, pipettes of varying sizes (1 mL, 5 mL, 10 mL, 20 mL), glass cuvettes (3

mL), 18 flasks (10 mL), 15 flask stoppers, a double-ended spatula, 3 weighing boats, two 96-well

plates, glass beakers of varying sizes (25 mL, 250 mL), a micropipette, and micropipette tips

were obtained from Professor Lambert. Staphylococcus aureus was purchased from ATCC

(American Type Culture Collection) by Professor Lambert. Tryptic soy broth (TSB), 0.85%

NaCl solution, bleach, SYTO-9, and propidium iodide were provided by Professor Lambert.

Silver nitrate, zinc acetate, copper sulfate hexahydrate, salt and glucose solution (8 g/L NaCl and

1 g/L glucose), glucose solution (1 M), and deionized water were obtained from Worcester

Polytechnic Institute (WPI). An incubator, a biosafety hood (biological safety level 2), a balance,

a pipet gun, and a refrigerator were provided by Professor Lambert. A UV/VIS spectrometer, a

centrifuge, and an LS 55 fluorescence spectrometer from WPI were used.

Metal ion solutions were made. The mass of three weighing boats was measured using a

balance. A double-ended spatula, which was washed with deionized water after each use, was

used to collect 0.0509 g silver nitrate, 0.0749 g copper sulfate pentahydrate, and 0.1835 g zinc

acetate separately in the weighing boats. The mass of each compound was measured by using the

balance and subtracting the mass of the weighing boat. One flask was filled with 10 mL

deionized water. Two other flasks were filled with 10 mL salt and glucose solution. The silver

nitrate was poured into the flask containing 10 mL deionized water using a weighing boat. The

copper sulfate pentahydrate and zinc acetate were poured into the remaining two flasks

containing 10 mL salt and glucose solution using two weighing boats.

Five dilutions were conducted. First, 1 mL of each initial solution was pipetted into a

flask containing 9 mL of the metal’s respective solvent (either deionized water or salt and

Gatsonis 17

glucose solution). Then, 0.333 mL of each initial solution was pipetted into a flask containing

9.667 mL of the metal’s respective solvent. Afterwards, 1 mL of each of the first dilutions was

pipetted into a flask containing 9 mL of the metal’s respective solvent. To create a fourth

dilution, 0.333 mL of each of the first dilutions were pipetted into a flask containing 9.667 mL of

the metal’s respective solvent. For the fifth dilution, 1 mL of each of the third dilutions was

pipetted into a flask containing 9 mL of the metal’s respective solvent. Using a 5 mL pipette and

the pipet gun, 30 µL of 1 M glucose solution were added to each silver ion solution. Finally, each

of the 15 flasks was sealed with a flask stopper (Chudobova et al., 2015).

Afterwards, a culture for the cell death assay was prepared while working under the

biosafety hood. A 20 mL pipette and a pipet gun were used to insert 40 mL TSB into a culture

tube. Then, 1 mL S. aureus from a frozen stock solution was inserted into the same culture tube

using a 1 mL pipette and the pipet gun. The culture tube, after being removed from the biosafety

hood, was placed in an incubator for 6 hours at 37℃. Then, the culture was centrifuged at 7,000

x g for 20 minutes. The supernatant was poured into a 25 mL beaker which contained about 5

mL bleach, and the pellet was resuspended in 20 mL salt and glucose solution. Using a 10 mL

pipette and the pipet gun, 3 mL of the suspension were inserted into a glass cuvette. Using a

UV/VIS spectrometer, the optical density at a wavelength of 670 nm of the suspension was

measured. The suspension was then diluted by pouring it off into the beaker of bleach and adding

salt and glucose solution until the suspension had an optical density of 0.15 absorption units at a

wavelength of 670 nm (Thermo Fisher Scientific, 2004).

Then, a combined reagent mixture of SYTO-9 and PI was made (Thermo Fisher Scientific,

2004). Using the micropipette and a new micropipette tip, 6 µL Component A (SYTO 9 dye,

1.67 mM / PI, 1.67 mM) were inserted into a 25 mL tube. Another micropipette tip was attached

Gatsonis 18

to the micropipette and 6 µL Component B (SYTO 9 dye, 1.67 mM / PI, 18.3 mM) were inserted

into the same tube. Finally, 20 mL deionized water were added to the dye solution using two 10

mL pipettes and the pipet gun. The tube of dye solution was wrapped with aluminum foil and

was then stored in the fridge.

A 96-well plate was prepared. After a micropipette tip was attached, a micropipette was used

to insert 100 µL of the cell suspension into 60 of the 96 wells. The outermost wells were not

used. Then, 30 µL of each of the 15 metal ion diluted solutions were pipetted into separate wells

containing the cell suspension. A new micropipette tip was used for each well. This process was

repeated two more times. Using a new micropipette tip each time, 30 µL of the salt and glucose

solution were pipetted into the 15 remaining wells containing the cell suspension. Finally, 100

µL of the dye solution were pipetted into all 60 wells, using a new micropipette tip for each well.

The well plate was left to incubate at room temperature in the dark for 15 minutes (Thermo

Fisher Scientific, 2004). An LS 55 fluorescence spectrometer was used to measure the

fluorescence emission spectrum of each cell suspension in the 60 wells. The fluorescence

spectrometer was set up two have two wavelength (WL) programs, named Green and Red. The

Green WL program had an excitation wavelength of 440 nm, an excitation slit of 2.5 nm, an

emission wavelength of 530 nm, an emission slit of 2.5 nm, and an open emission filter. The Red

WL program had an excitation wavelength of 485, an excitation slit of 2.5 nm, an emission

wavelength of 670 nm, an emission slit of 5.0 nm, and an open emission filter.

A second experiment was conducted. A culture of S. aureus was prepared under a biosafety

hood. A 20 mL pipette and a pipet gun were used to insert 20 mL TSB into a culture tube. Then,

1 mL S. aureus from a frozen stock solution was inserted into the same culture tube using a 1 mL

pipette and a pipet gun. After being removed from the biosafety hood, the culture tube was

Gatsonis 19

placed in an incubator for 3 hours at 37℃. Subsequently, the culture was centrifuged at 7000 x g

for 20 minutes and the pellet was resuspended in 20 mL salt and glucose solution. The

suspension’s optical density at 670 nm was measured using a UV/VIS spectrometer. The optical

density of the suspension was adjusted to 0.45 absorption units through a process of dilution.

Three cuvettes were prepared. 1 mL salt and glucose solution was inserted into each of these

cuvettes using a single 1 mL pipette and a pipette gun. Using the same pipette, 1 more mL salt

and glucose solution was inserted into one cuvette. A new pipette was used to insert 1 mL 10

mM zinc solution into a second cuvette and a third pipette was used to insert 1 mL 10 mM

copper solution into the final cuvette. Then, 1 mL of the suspension was inserted into each

cuvette using a single pipette and the pipet gun. The three cuvettes were left in the incubator at

37℃.

After a micropipette tip was attached, a micropipette was used to insert 100 µL from each

cuvette into individual wells. The micropipette tip was replaced after each use. A fourth

micropipette tip was attached, and the micropipette was used to insert 100 µL of the previously

made dye solution into each filled well. Every half hour, for three and a half hours, wells were

prepared on a 96-well plate following this procedure. The LS 55 fluorescence spectrometer was

used to measure the fluorescence emission spectrum of each well. The start wavelength was

specified as 470 nm, the end wavelength was specified as 700 nm, the excitation wavelength was

specified as 470 nm, the excitation slit was set to 3.5 nm, the emission slit was set to 3.5 nm, and

the scanning speed was set to 500 nm/min.

A third experiment was conducted. A suspension of S. aureus and two cuvettes (one

containing 2 mL salt and glucose solution and the other containing 1 mL salt and glucose

solution and 1 mL 10 mM copper solution) were prepared as previously described in the second

Gatsonis 20

experiment. The dye solution was made exclusively for this experiment (Thermo Fisher

Scientific, 2004). Using the micropipette and a new micropipette tip, 6 µL Component A (SYTO

9 dye, 1.67 mM / PI, 1.67 mM) were inserted into a 50 mL tube. Another micropipette tip was

attached to the micropipette and 6 µL Component B (SYTO 9 dye, 1.67 mM / PI, 18.3 mM)

were inserted into the same tube. Finally, 40 mL deionized water were added to the dye solution

using one 20 mL pipette and the pipet gun. The tube of dye solution was wrapped with aluminum

foil and was stored in the fridge. Using this new dye solution, a 96-well plate was prepared as

described in the second experiment and the LS 55 fluorescence spectrometer was used to

measure the fluorescence emission spectrum of each well over a 160-minute period. The setup

parameters of the fluorescence spectrometer during the third experiment were the same as in the

second experiment.

Gatsonis 21

Results

With a live/dead assay that uses the fluorescence of two dyes, propidium iodide

(abbreviated to R, because of its ability to stain dead cells red) and SYTO 9 (abbreviated to G,

because of its ability to stain living cells green), the overall viability of the grown bacterial cells

was assessed by determining the ratio of green (510 – 540 nm) to red (620 – 650 nm)

fluorescence (SYTO 9 to propidium iodide). The fluorescence excitation and emission spectra of

the two dyes are shown in Figure 2.

In the first experiment, a 96-well plate was prepared to examine the toxicities of three

metal ions, silver, copper (II), and zinc (II), at various concentrations in a S. aureus environment

where no AIP was present. Lines of best fit were generated for each of the three metal ion groups

and for a control group which had no added metal ion. For the control group, RatioG/R has a slope

of -0.0037 (see Figure 3a). For the silver and zinc groups, RatioG/R has a slope of 0.0287 and

0.0433 respectively (see Figures 3b and 3c). However, for the copper group, RatioG/R has a slope

of -0.0244 (see Figure 3d).

In the second experiment, the toxicities of two metal ions, copper (II) and zinc (II), were

examined in an environment where no AIP was present. RatioG/R for the two metal ion groups

and for a control group (in which no metal ions were present) is shown in Figure 4.

In the third experiment, the toxicity of copper (II) was examined in an environment

where no AIP was present. RatioG/R for this group and for a control group (in which no metal

ions were present) is shown in Figure 5.

Gatsonis 22

Figure 2. Fluorescence excitation and emission spectra of SYTO 9 and propidium iodide (Thermo Fisher Scientific, 2004)

Gatsonis 23

Figure 3. a) The RatioG/R of the control group consisting of 15 wells of S. aureus, no metal ions, and no AIP in the first

experiment b) The RatioG/R of the silver group consisting of 15 wells of S. aureus, silver ions, and no AIP in the first experiment c)

The RatioG/R of the zinc group consisting of 15 wells of S. aureus, zinc (II) ions, and no AIP in the first experiment d) The RatioG/R

of the copper group consisting of 15 wells of S. aureus, copper (II) ions, and no AIP in the first experiment

RatioG/R = -0.0037x + 0.9128

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

0 5 10 15

Rati

oG

/R

Sample Number

a) RatioG/R vs. Sample

Number

RatioG/R = 0.0287x + 0.78680.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

0.000 1.000 2.000 3.000

Rati

oG

/R

Concentration of Silver (mM)

b) RatioG/R vs.

Concentration of Silver

RatioG/R = 0.0433x +

0.86020.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

0.000 5.000 10.000

Rati

oG

/R

Concentration of Zinc (mM)

c) RatioG/R vs.

Concentration of Zinc

RatioG/R = -0.0244x + 0.9783

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

0.000 1.000 2.000 3.000

Rati

oG

/R

Concentration of Copper (mM)

d) RatioG/R vs.

Concentration of Copper

Gatsonis 24

Figure 4. A comparison of the three groups (the control group, the copper group, and the zinc group) in the second experiment

Figure 5. A comparison of the two groups (the control group and the copper group) in the third experiment

Gatsonis 25

Conclusions

In the first experiment, the silver group (see Figure 3b) and the zinc group (see Figure 3c)

both have positive slopes, 0.0287 and 0.0433 respectively, for RatioG/R. These results are

unexpected because both silver and zinc have been proven to be antimicrobials, which suggests

that RatioG/R for the silver and zinc groups should have decreased as the concentration of the

metal ions increased. However, one possible cause of these results could have been that the plate

was not left to incubate for enough time. Because the plate only incubated for 15 minutes, the

metal ions may not have had the time necessary to cause adequate cell death. For the silver

group, it is also possible that the silver ions could have precipitated with the chlorine from the

salt and glucose solution, which could have caused interference. The negative slope of RatioG/R

of the copper group (see Figure 3d), -0.0244, indicates that copper ions can react with bacterial

cells more quickly than zinc ions or silver ions can.

In the second experiment, both the zinc group and the copper group show an overall

negative trend in RatioG/R as time elapsed (see Figure 4). For the copper group, the steepest

decrease in RatioG/R occurs from 0 minutes to 60 minutes. From 60 minutes to 180 minutes,

however, RatioG/R does not exhibit much variation. These results suggest that copper’s

antibacterial activity occurs early on when exposed to bacteria. The zinc group appears to have

its steepest decrease in RatioG/R occur from 0 to 90 minutes. The zinc group has a longer period

of antimicrobial activity than the copper group, which suggests that copper can react with

bacterial cells more quickly than zinc can. This conclusion is also supported by the data of the

first experiment (see Figures 3c and 3d) because, during the 15-minute incubation period, copper

was able to kill S. aureus, while zinc was unable to. Interestingly, RatioG/R at 60 minutes for the

zinc group shows a sharp increase. This data point is unusual and most likely inaccurate. The

Gatsonis 26

conditions under which the zinc group was incubating should not have been conducive for

bacterial growth, and even if bacterial growth did occur, it would not have happened so rapidly.

Additionally, there is a very sharp decrease in RatioG/R for the zinc group from 60 minutes to 90

minutes, which further suggests that the data point for zinc at 60 minutes is flawed.

In the third experiment, the copper group shows an overall negative trend for RatioG/R

(see Figure 5). From 0 minutes to 129 minutes, RatioG/R of the copper group decreases. After 129

minutes, RatioG/R remains relatively constant, suggesting that the copper group underwent a

period of inactivity after the 129-minute mark. These results are similar to the results of the

second experiment because both copper groups exhibited a period of early antibacterial activity

followed by a period of inactivity.

In all three experiments, RatioG/R of the control groups do not show much variation (see

Figures 3a, 4, and 5). In the first experiment, the control group has a slope of -0.0037, indicating

that each well had a very similar composition. While RatioG/R did increase slightly in the second

experiment and decrease slightly in the third experiment, these changes are negligible in

comparison to the changes in RatioG/R seen in the copper and zinc groups in the second

experiment and the copper group in the third experiment.

Although experimentation involving AIP has not been conducted, it will be carried out in

the future. It is difficult to purify AIP from S. aureus because doing so often involves

lyophilization and the use of filters or cutoff membranes. Furthermore, there are several different

kinds of AIP which are present in S. aureus. Each of these signals has a different molecular

weight, which adds an increased complexity to the purification process. While conclusions

regarding AIP have not been reached with the current data collected, they will be made after

future experimentation.

Gatsonis 27

References

Archer, N. K., Mazaitis, M. J., Costerton, J. W., Leid, J. G., Powers, M. E., & Shirtliff M. E.

(2011). Staphylococcus aureus biofilms: Properties, regulation, and role in human

disease. Virulence, 2(5). doi:10.4161/viru.2.5.17724

Balaban, N. (1998). Autoinducer of Virulence As a Target for Vaccine and Therapy Against

Staphylococcus aureus. Science, 280(5362), 438-440. doi:10.1126/science.280.5362.438

Bassler, B. L. (1999). How bacteria talk to each other: regulation of gene expression by quorum

sensing. Current Opinion in Microbiology, 2(6), 582-587. doi:10.1016/s1369-

5274(99)00025-9

Biofilm Basics. (2003). Retrieved October 19, 2017, from

http://www.biofilm.montana.edu/biofilm-basics/

Chudobova, D., Dostalova, S., Ruttkay-Nedecky, B., Guran, R., Rodrigo, M. A., Tmejova, K.,

Krizkova, S., Zitka, O., Adam, V., Kizek, R. (2015). The effect of metal ions on

Staphylococcus aureus revealed by biochemical and mass spectrometric analyses.

Microbiological Research, 170, 147-156. doi:10.1016/j.micres.2014.08.003

Feng, G., Cheng, Y., Wang, S., Borca-Tasciuc, D. A., Worobo, R. W., & Moraru, C. I. (2015).

Bacterial attachment and biofilm formation on surfaces are reduced by small-diameter

nanoscale pores: how small is small enough? Npj Biofilms and Microbiomes, 1(1).

doi:10.1038/npjbiofilms.2015.22

Hobman, J. L., & Crossman, L. C. (2014). Bacterial antimicrobial metal ion resistance. Journal

of Medicinal Microbiology, 64, 471-497. doi:10.1099/jmm.0.023036-0

Gatsonis 28

HyCa Technologies Pvt. Ltd. (2017). Retrieved October 19, 2017, from

http://www.hycator.com/domain/functional-sectors/biofouling/steps-in-biofilm-

formation.html

Keersmaecker, S. C., Sonck, K., & Vanderleyden, J. (2006). Let LuxS speak up in AI-2

signaling. Trends in Microbiology, 14(3), 114-119. doi:10.1016/j.tim.2006.01.003

Kjaerulff, L., Nielsen, A., Mansson, M., Gram, L., Larsen, T., Ingmer, H., & Gotfredsen, C.

(2013). Identification of Four New agr Quorum Sensing-Interfering Cyclodepsipeptides

from a Marine Photobacterium. Marine Drugs, 11(12), 5051-5062.

doi:10.3390/md11125051

Kong, K., Vuong, C., & Otto, M. (2006). Staphylococcus quorum sensing in biofilm formation

and infection. International Journal of Medical Microbiology, 296(2-3), 133-139.

doi:10.1016/j.ijmm.2006.01.042

Li, W., Xie, X., Shi, Q., Duan, S., Ouyang, Y., & Chen, Y. (2010). Antibacterial effect of silver

nanoparticles on Staphylococcus aureus. BioMetals, 24(1), 135-141. doi:10.1007/s10534-

010-9381-6

Resch, A., Rosenstein, R., Nerz, C., & Gotz, F. (2005). Differential Gene Expression Profiling of

Staphylococcus aureus Cultivated under Biofilm and Planktonic Conditions. Applied and

Environmental Microbiology, 71(5), 2663-2676. doi:10.1128/aem.71.5.2663-2676.2005

Thermo Fisher Scientific. (2004). LIVE/DEAD™ BacLight™ Bacterial Viability Kit, for

microscopy. Retrieved January 10, 2018, from

https://www.thermofisher.com/order/catalog/product/L7007

Gatsonis 29

Appendix A

Acknowledgements

I would like to thank Professor Lambert and Jared Watson for their involvement in this

project. Professor Lambert graciously gave his time, input, lab, and materials, without which this

project would not have been possible. Jared Watson spent many hours guiding and helping me

carry out experimentation. Without either of their support, this work could not have been as

thorough as it currently is. I would also like to thank Mr. Cvitkovic for his model of copper

binding to AIP-1.

Gatsonis 30

Appendix B

Limitations and Assumptions

1. The lab was not always available.

2. Certain materials could not be purchased because of high costs.

3. The temperature and carbon dioxide level displayed on the incubator were accurate.

4. The UV/VIS spectrometer and the LS 55 fluorescence spectrometer gave accurate

readings.

5. The ionic compounds and purchased bacteria were accurately labelled by the suppliers

and were not of incorrect species.

6. Collected data is predictive of future trends.

7. The sample of Staphylococcus aureus tested on is representative of the species.

Gatsonis 31

Appendix C

Project Notes

Knowledge Gaps

Knowledge gap Resolved by Information is located

What metals should be

considered

Reading a journal article Page 5

How to purify AIP Reading a journal article Page 18

Gatsonis 32

Journal Articles

STAPHYLOCOCCUS AUREUS BIOFILMS: PROPERTIES, REGULATION, AND ROLE IN

HUMAN DISEASE

Source citation Archer, N. K., Mazaitis, M. J., Costerton, J. W., Leid, J. G., Powers, M.

E., & Shirtliff M. E. (2011). Staphylococcus aureus biofilms: Properties,

regulation, and role in human disease. Virulence, 2(5).

doi:10.4161/viru.2.5.17724

Source found by Searching “staphylococcus aureus biofilm” on Summon

Source Type Journal article

Keywords Staphylococcus aureus; Biofilm

Reason for interest Wanted to get background knowledge on S. aureus biofilm

Summary This journal article gave helpful background information about S. aureus

biofilms.

Notes - S. aureus is a Gram-positive coccus

- Host immune responses are typically ineffective against S. aureus biofilm

infections and chronic disease often occurs

- A biofilm is a sessile community in which cells are “embedded in a matrix

of extracellular polymeric substance”

- Can form mushroom/pillar shapes

- Channel networks flow through them and allow the deepest members of a

biofilm to have access to nutrients

- Destroying biofilms is difficult and often requires surgery

Gatsonis 33

- Gradients (in oxygen, nutrients, and electron acceptors) can result in

varied gene expression throughout the biofilm

- Extracellular matrix is capable of isolating essential nutrients and biofilms

are capable of evading the antibacterial mechanisms of their host

- Low metabolic rates and the ability of a biofilm to act as a diffusion

barrier to slow down antimicrobial penetration account for a biofilm’s

antimicrobial resistance

- Biofilm can spread by seeding dispersal/cellular detachment

- Micro-colonies can detach by a genetically programmed response which

causes the seeding dispersal process or by fluid forces

- 20-25% of the human population’s nares are persistently colonized by S.

aureus, which is shown to have a causal relationship with a higher risk of

nosocomial infection

Questions N/A

Gatsonis 34

BACTERIAL ANTIMICROBIAL METAL ION RESISTANCE

Source citation Hobman, J. L., & Crossman, L. C. (2014). Bacterial antimicrobial metal

ion resistance. Journal of Medicinal Microbiology, 64, 471-497.

doi:10.1099/jmm.0.023036-0

Source found by Searching “bacterial antimicrobial metal” on Summon

Source Type Journal article

Keywords Bacterial; Antimicrobial; Metal; Ion; Resistance

Reason for interest Wanted to know which metal ions could be potential candidates

Summary This article discussed how metals interact with bacteria.

Notes - Metals such as mercury, arsenic, copper, and silver have been historically

used as antimicrobials

- Some metalloids are still being used as antimicrobials/chemotherapeutics,

and some metals (copper and silver) are being used in agriculture and

medicine and are being promoted as antimicrobials

- Metals and metalloids give off toxic effects by binding to/blocking

functional groups in molecules, being involved in harmful reactions, and

by displacing essential metals in enzymes

- Divalent copper is shown to have a strong affinity for biological

molecules on the Irving-Williams series of ligand affinities

MERCURY

- Inactivates certain enzymes and interferes with protein functions by

binding to thiol/imino nitrogen groups

Gatsonis 35

- Also binds to nucleotides and lipids, which interferes with DNA function

and contributes to lipid peroxidation

- Most toxic metal to E. coli

COPPER

- Acts as an electron donor/acceptor in enzymes, but can also lead to the

generation of harmful hydroxyl radicals

- Rapid killing of bacteria

- Toxic to prokaryotes

- Staphylococcus aureus shows oxidative stress resistance, protein

misfolding repair of transcriptional responses, and hydrogen peroxide

scavenging defense

SILVER

- Ions cause the inhibition of respiration, membrane damage, and

destruction of the proton motive force

- Interact with thiol groups in membrane proteins/enzymes harmfully

- Cause membrane damage in S. aureus, possibly generate hydroxyl radical

ions by releasing iron from proteins by binding thiol groups in S.

epidermidis

- Possibly generate hydroxyl radical ions in S. aureus and E. coli

ZINC

- Less heavily used as an antimicrobial, so not much bacterial resistance to

it

- Can be an effective antimicrobial at a low concentration

Gatsonis 36

- Toxic to prokaryotes

ARSENIC

- Inorganic arsenic has been connected with ROS generation and disruption

of signal transduction pathways

-Bacteria have evolved ways to deal with metal toxicity;

extracellular/intracellular sequestering of the metal, permeability

reduction, altering target sites, resistance mechanisms similar to antibiotic

resistance mechanisms, and (these are the primary ways) efflux of metal

ions and enzymic detoxification

Questions N/A

Gatsonis 37

STAPHYLOCOCCUS QUORUM SENSING IN BIOFILM FORMATION AND INFECTION

Source citation Kong, K., Vuong, C., & Otto, M. (2006). Staphylococcus quorum

sensing in biofilm formation and infection. International Journal of

Medical Microbiology, 296(2-3), 133-139.

doi:10.1016/j.ijmm.2006.01.042

Source found by Searching “Staphylococcus quorum sensing biofilm” on Summon

Source Type Journal article

Keywords agr; luxS; Staphylococcus; Quorum sensing; Biofilm; Infection

Reason for interest Wanted to know more about quorum sensing molecules and which one to

pick

Summary This article was an excellent source of deeper background knowledge on

quorum sensing within an S. aureus biofilm.

Notes - Quorum sensing in staphylococci typically occurs when there is a large

amount of cells in the biofilm

- AIs (autoinducing peptides) are the signals of quorum sensing systems.

They are usually 8 amino acids in length and, while their sequence of

amino acids varies from strain to strain, they retain their cyclical

structure.

- The agr (accessory gene regulation) quorum sensing system consists of

RNAII and RNAIII, which are transcribed (their transcription is under the

auspices of the P2 and P3 promoters, respectively)

- RNAIII contains four genes, agrA, agrB, agrC, and agrD

- The agrB and agrD gene products carry out the production of AIs

Gatsonis 38

- When AIs accumulate to a certain point, a transcriptional regulator is

activated and regulates the expression of various genes

- Quorum sensing seems to take part in many stages of biofilm formation

(these stages are initial attachment, cell-to-cell adhesion and proliferation,

maturation, and detachment)

- agr quorum sensing, however, does not seem to regulate cell-to-cell

adhesion

- This is because the production of PIA (polysaccharide intercellular

adhesin) was seen to be downregulated by the luxS quorum sensing

system

- agr quorum sensing is shown to play a major role in detachment

(detachment allows bacteria to move to other sites, which is very

important in the spread of a biofilm-associated infection)

- An agr mutant was shown to form a thicker biofilm than the natural type-

> this thickness was due to the inability of cells to detach

- This inability likely resulted because production of a group of small

peptides (these were amphipathic peptides, known as phenol-soluble

modulins or PSMs) whose production depended on agr. These peptides

most likely facilitated the detachment of cells due to their amphipathic

nature.

- A time lapse-confocal microscopy study showed that detachment of cells

coincides with agr expression

Gatsonis 39

- A study using an indwelling medical device model showed that the agr

wild-type strain was more effective in infiltrating surrounding tissue than

an agr-negative counterpart

- According to other studies, the inactivation of agr quorum sensing makes

device-related infection more effective (likely because decreased agr

quorum sensing results in thicker biofilm, which increases survivability)

- RNA profiling data showed that RNAII and RNAIII expression was

lower in vivo, which suggests that the agr quorum sensing system may be

unnecessary for staphylococcal infection

- A study to monitor agr quorum sensing activity in abscess formation in a

mouse by using a luciferase-based biosensor conducted by Wright et. al.

showed that two periods of rapid activation of the agr system (3 hours

after infection and 48-72 hours after infection) occurred (the first period

was followed by a rapid decline in activity roughly 7 hours after

infection). The authors interpreted this to mean that the interval between

these two periods was a neutrophil-dependent eclipse stage, which would

eliminate the agr mutant while the wild-type bacteria would cause a

subcutaneous lesion. The authors suggest that an early burst of agr

activity, which would result in excess production of secreted virulence

factors, is essential for longtime survival of bacteria.

- An isogenic agr mutant strain of S. epidermidis exhibited several negative

traits: it couldn’t produce cytokine tumor necrosis factor as well, had a

severely hindered ability to cause chemotaxis of human neutrophils, had

Gatsonis 40

much less resistance to human antimicrobial peptides, and, because agr

controls the general and oxidative stress response, may have been less

effective at fighting the respiratory burst of human neutrophils

THE LUXS QS SYSTEM IN STAPHYLOCOCCUS

- The LuxS protein takes part in the production of AI-2

- A LuxS mutant strain, similarly to an agr mutant strain, exhibits a thicker

biofilm formation and forms a compacter biofilm. However, it was more

effective in its colonization of a central venous catheter infection of an

animal model.

- The LuxS protein seems to transcriptionally regulate the ica gene locus

(which is responsible for intercellular adhesion) and therefore alters PIA

production (PIA is polysaccharide intercellular adhesin)

- There is a common theme of QS-dependent regulation of biofilm

formation and biofilm-associated infection in staphylococci.

- A high-density bacterial population may not reach the threshold necessary

for quorum sensing activation in an open system because AI is constantly

diluted.

- Limited diffusion rather than bacterial population seems to represent the

basis for quorum sensing activation in a closed system.

- This phenomenon can be seen when S. aureus is internalized in epithelial

cells prior to the bacteria’s release from the endosome (a closed system).

In this scenario, the agr system is induced.

CONCLUSIONS

Gatsonis 41

- Quorum sensing systems in Staphylococcus have an enormous impact on

pathogen success during infection by controlling bacterial physiology and

virulence mechanisms.

- Large bacterial population does not always mean there is active quorum

sensing

- Active quorum sensing does not always mean there is increased virulence

- For biofilm-related staphylococci infections to develop, the inactivation

of quorum sensing must occur.

Questions Does AI enter the bacterium after binding?

Gatsonis 42

ANTIBACTERIAL EFFECT OF SILVER NANOPARTICLES ON STAPHYLOCOCCUS AUREUS

Source citation Li, W., Xie, X., Shi, Q., Duan, S., Ouyang, Y., & Chen, Y. (2010).

Antibacterial effect of silver nanoparticles on Staphylococcus aureus.

BioMetals, 24(1), 135-141. doi:10.1007/s10534-010-9381-6

Source found by Searching “staphylococcus aureus silver” on Summon

Source Type Journal article

Keywords Staphylococcus aureus; Silver nanoparticles (Ag-NPs); Antibacterial

effect

Reason for interest Wanted to have a better grasp on how silver interacts with S. aureus

Summary This article discussed the effects of the introduction silver nanoparticles

in an S. aureus biofilm.

Notes - Minimum bactericidal concentration of silver nanoparticles was 20 µg/ml

for 12 hours

- Silver nanoparticles kill Gram-positive and Gram-negative bacteria

- Silver, unlike many metals, is not very toxic to mammalian cells

- Silver ions do not induce microbial resistance as strongly as other metals

- When S. aureus cells were treated with silver nanoparticles for 12 hours,

their cell wall broke down and underwent lysis

- According to growth curves observed during the experiment, silver

nanoparticles can inhibit S. aureus growth and kill cells

- ABC transporters in bacteria are responsible for the intake of essential

nutrients and the removal of toxic substances

Questions How was the silver inserted into the biofilm?

Gatsonis 43

HOW BACTERIA TALK TO EACH OTHER: REGULATION OF GENE EXPRESSION BY

QUORUM SENSING

Source citation Bassler, B. L. (1999). How bacteria talk to each other: regulation of gene

expression by quorum sensing. Current Opinion in Microbiology, 2(6),

582-587. doi:10.1016/s1369-5274(99)00025-9

Source found by Searching

Source Type Journal article

Keywords Gene expression; Quorum sensing

Reason for interest Wanted more general knowledge on quorum sensing

Summary This article provided information about a wide variety of quorum sensing

methods which are not exclusive to S. aureus.

Notes - Quorum sensing is responsible for intra- and inter- species cell-to-cell

communication in a biofilm and allows bacteria to form complex

structures

- Gram-negative bacterial communication is regulated by two regulatory

proteins – LuxI and LuxR

- LuxI is the autoinducer synthase which produces acyl-HSL autoinducer

- LuxR is a transcriptional activator protein that promotes transcription of a

structural operon when an autoinducer is bound to it

- Gram-positive bacteria do not use HSLs as signals and do not use

LuxI/LuxR

Gatsonis 44

- They secrete peptide signals through an ABC (ATP-binding cassette)

exporter protein, which are then recognized by sensor kinase proteins

- Afterwards, the sensor kinase proteins interact with cytoplasmic response

regulator proteins

- In staphylococci, the synthesis of RNA III, an untranslated RNA molecule,

is regulated by peptide quorum sensing

- RNA III is the effector of the system and is responsible for positive and

negative regulation of multiple downstream targets

- V. harveyi responded to the AI-2 produced by other bacteria possessing a

luxS homologue, which supports the idea that AI-2 signal response

systems could be a universal way for bacteria of different species to

interact (S. aureus produces luxS homologues)

- Most of the bacterial species containing a luxS gene are capable of

producing AI-2 activity, which was proven by the elimination of AI-2

production when the luxS gene was mutated

- Quorum sensing is responsible for the regulation of virulence factors in

pathogens

- Quorum sensing bacteria typically delay the production of virulence

factors until the biofilm has reached a high enough population so the

infection would be successful

- In S. aureus, the agr quorum sensing system regulates the production of a

multitude of virulence factors necessary for successful infection

Questions N/A

Gatsonis 45

AUTOINDUCER OF VIRULENCE AS A TARGET FOR VACCINE AND THERAPY AGAINST

STAPHYLOCOCCUS AUREUS

Source citation Balaban, N. (1998). Autoinducer of Virulence As a Target for Vaccine

and Therapy Against Staphylococcus aureus. Science, 280(5362), 438-

440. doi:10.1126/science.280.5362.438

Source found by Searching “autoinducer staphylococcus aureus” on Summon

Source Type Journal article

Keywords Autoinducer; Virulence; Staphylococcus aureus

Reason for interest Wanted to see how others dealt with the inhibition of S. aureus

autoinducer

Summary This article discussed RIP being used to inhibit quorum sensing.

Notes - Pathogenic effects of S. aureus are mainly controlled by the production of

bacterial toxin, which is regulated by RNAIII.

- RNAIII is activated by RAP (RNAII activating protein) and a peptide

called RIP (RNAIII inhibiting peptide) inhibits RNAIII.

- Over 500K nosocomial infections per year are caused by S. aureus – these

range from minor skin infections to pneumonia, endocarditis, meningitis,

postoperative wound infections, septicemia, and toxic shock syndrome

- S. aureus mainly causes disease by producing virulence factors such as

hemolysins, enterotoxins, and toxic shock syndrome toxin, which is

controlled by RNAIII (3-5), which is encoded by the agr locus.

Gatsonis 46

- The rnaiii gene is transcribed in culture during the mid-exponential phase

of growth, when it is autoinduced by RAP (RAP continues to be excreted

by the bacteria at a certain concentration threshold)

- Because RIP competes with RAP for activation of RNAIII, it is suggested

that interference with activating the agr system with RIP may inhibit the

expression of virulence factors regulated by rnaiii

Questions N/A

Gatsonis 47

BACTERIAL ATTACHMENT AND BIOFILM FORMATION ON SURFACES ARE REDUCED

BY SMALL-DIAMETER NANOSCALE PORES: HOW SMALL IS SMALL ENOUGH?

Source citation Feng, G., Cheng, Y., Wang, S., Borca-Tasciuc, D. A., Worobo, R. W., &

Moraru, C. I. (2015). Bacterial attachment and biofilm formation on

surfaces are reduced by small-diameter nanoscale pores: how small is

small enough? Npj Biofilms and Microbiomes, 1(1).

doi:10.1038/npjbiofilms.2015.22

Source found by Searching “how do biofilms attach” on Google

Source Type Journal article

Keywords Nanoscale pores; Biofilm formation; Bacterial attachment

Reason for interest Was looking for an explanation on how biofilms initially attach, but

could not find a detailed description in this article

Summary This article discussed the use of nanoscale pores to prevent the growth of

a biofilm on a surface.

Notes - Approximately 80% of all medical infections are due to biofilm formation

Questions N/A

Gatsonis 48

PURIFICATION AND FUNCTIONAL STUDIES OF A POTENT MODIFIED QUORUM-

SENSING PEPTIDE AND A TWO-PEPTIDE BACTERIOCIN IN STREPTOCOCCUS MUTANS

Source citation Petersen, F. C., Fimland, G., & Scheie, A. A. (2006). Purification and

functional studies of a potent modified quorum-sensing peptide and a

two-peptide bacteriocin in Streptococcus mutans. Molecular

Microbiology, 61(5), 1322-1334. doi:10.1111/j.1365-2958.2006.05312.x

Source found by Searching “purification of peptides in quorum sensing” on Summon

Source Type Journal article

Keywords Purification; Quorum-sensing peptide; Two-peptide bacteriocin;

Streptococcus mutans

Reason for interest Wanted to know how to purify peptides

Summary The article discussed the antimicrobial effects of a modified quorum-

sensing peptide and a bacteriocin.

Notes - METHOD TO ISOLATE PEPTIDES FROM SUPERNATANT OF

S.MUTANS GS5 and UA159 (the S. mutans genome sequence reference

strain)

- Cells from the 200ml bacterial culture were pelleted by centrifugation and

ammonium sulfate was added to the supernatant to a concentration of 40%

(w/v)

- After cooling to 4℃, the precipitate was pelleted by centrifugation at

10000g for 20 min

Gatsonis 49

- The ammonium sulfate precipitate was then dissolved in 50ml of 20mM

phosphate buffer (pH 6) and was supplemented with ammonium sulfate to

a concentration of 10% (w/v)

- The precipitate was applied to a 4 ml Phenyl Sepharose 6 Fast Flow

hydrophobic interaction column pre-equilibriated with 10% ammonium

sulfate (w/v) in 20mM phosphate buffer (pH 6)

- The column was then washed with 10ml 20mM phosphate buffer and the

bound material was eluted with 10ml of 70% (v/v) ethanol in water

- Afterwards, the eluate was diluted 5 times in water supplemented with .1%

(v/v) tri-fluoro acetic acid

- The eluate was applied on a 3ml Resource RPC reverse phase column

using the FPLC chromatography system

- Water and 2-propanol supplemented with .1% (v/v) tri-fluoro acetic acid

were used as mobile phases and a linear gradient from 10% to 50% 2-

propanol was used

- The fractions were collected and were then analyzed by mass spectrometry

- Afterwards, they were tested for antimicrobial activity, which was done in

the presence of the complementary peptide of the two-peptide bacteriocin

- If they displayed antimicrobial activity, they were purified further to

homogeneity using a µRPC C2/C18 SC2.1/10 column, water, and 2-

propanol supplemented with .1% (v/v) tri-fluoro acetic acid as mobile

phases, and the SMART-system with UV-detection at 214nm

Gatsonis 50

Questions What is a reverse phase/ µRPC C2/C18 SC2.1/10 column? What is a mobile

phase? What is the FPLC chromatography system? How were the fractions

analyzed by mass spectrometry?

LABORATORY MAINTENANCE OF METHICILLIN-RESISTANT STAPHYLOCOCCUS

AUREUS (MRSA)

Source citation Vitko, N. P. & Richardson, A. R. (2013). Laboratory Maintenance of

Methicillin-Resistant Staphylococcus aureus (MRSA). PubMed, 9. doi:

10.1002/9780471729259.mc09c02s28

Source found by Searching “how to culture s aureus” on Google

Source Type Journal article

Keywords • Staphylococcus aureus; HA-MRSA; CA-MRSA; Growth; Strain

selection; CDM; Freezer stock

Reason for interest Wanted to know basic procedures for culturing S. aureus

Summary This article provides steps on how to culture S. aureus.

Notes - S. aureus is ~0.6µm in diameter

- PREPARING A STREAK PLATE

- Using a sterile wooden applicator, streak out a small amount of S. aureus

from the frozen stock onto a quarter of a small BHI plate.

- This step should be done using aseptic technique.

Gatsonis 51

- Using a new sterile wooden applicator, streak out another quadrant of S.

aureus on another BHI plate by passing the applicator through the first

quadrant multiple times.

- Repeat this process 2 more times using the newly streaked quadrants.

- After this is complete, the plates should be incubated for 16-24 hours at

37℃.

- GROWTH IN LIQUID MEDIA

- Several of these media are Brain Heart Infusion, Tryptic Soy Broth, Tod

Hewitt Broth, and Luria-Bertani Broth.

- The bacteria grows quickly at 37 degrees Celsius with aeration.

- Using aseptic technique, a colony of S. aureus should be transferred from a

streak plate into the broth.

- This transfer is done by tilting a culture tube and rubbing an inoculating

loop along the side of the tube at the liquid-air interface.

- The culture should then be grown overnight for roughly 16-18 hours at

37℃ with shaking at 250rpm (shaking is not necessary because S. aureus

can carry out anaerobic respiration, but the growth rate and maximum

bacterial density may be impacted).

To maximize aeration, the culture tube must be placed on the shaker at an angle.

Questions N/A

Gatsonis 52

REGULATION OF STAPHYLOCOCCUS AUREUS PATHOGENESIS VIA TARGET OF RNAIII-

ACTIVATING PROTEIN (TRAP)

Source citation Balaban, N., Goldkorn, T., Gov, Y., Hirshberg, M., Koyfman, N.,

Matthews, H. R., . . . Uziel, O. (2000). Regulation of Staphylococcus

aureus Pathogenesis via Target of RNAIII-activating Protein

(TRAP). Journal of Biological Chemistry, 276(4), 2658-2667.

doi:10.1074/jbc.m005446200

Source found by Searching “how to purify autoinducer staphylococcus aureus” on Google

Source Type Journal article

Keywords Regulation; Staphylococcus aureus; Pathogenesis; Target of RNAIII-

activating protein

Reason for interest Wanted to know the standard procedure to purify autoinducing peptide in

Staphylococcus aureus

Summary This article discussed the regulation of S. aureus pathogenesis through

the use of a protein called TRAP, which is thought to prevent the

activation of RNAIII by targeting RNAIII-activating protein.

Notes -Method to partially purify AIP from S. aureus

Step 1 – Grow the cells to the post-exponential growth phase

Step 2 – Centrifuge the growth culture at 6000 xg for 10 minutes at 4℃

Step 3 – Collect the supernatant and filter it through a 0.22 µm filter to remove

residual cells

Step 4 – Lyophilize the supernatant using FlexiDry MP lyophilizer and resuspend

the supernatant in water to one-tenth of the original volume

Gatsonis 53

Step 5 – Apply 15 ml of 10x concentrated supernatant to a 3-kDa cutoff

membrane (Centriprep 10 (Amicon))

Step 6 – Collect material smaller than 3 kDa

Questions N/A

Gatsonis 54

ASSESSMENT AND INTERPRETATION OF BACTERIAL VIABILITY BY USING THE

LIVE/DEAD BACLIGHT IN COMBINATION WITH FLOW CYTOMETRY

Source citation Berney, M., Hammes, F., Bosshard, F., Weilenmann, H., & Egli, T.

(2007). Assessment and Interpretation of Bacterial Viability by Using the

LIVE/DEAD BacLight Kit in Combination with Flow Cytometry.

Applied and Environmental Microbiology, 73(10), 3283-3290.

doi:10.1128/aem.02750-06

Source found by Searching “how to use live dead baclight” on Google

Source Type Journal article

Keywords Bacterial viability; Staphylococcus aureus; Baclight; Flow cytometry

Reason for interest Wanted to know the standard procedure to using a live/dead baclight

Summary This article discussed how to use a live/dead backlight.

Notes -STYO9 is a (green fluorescing) nucleic acid stain that enters all cells, allowing

one to get a total count for a biofilm

-Propidium iodide (PI) is a (red fluorescing) nucleic acid stain that enters cells

with damaged cytoplasmic membranes (assumed to be dead)

-Use stock solutions of the stains as proposed by the manufacturer

Questions N/A

Gatsonis 55

Websites

BIOFILM BASICS

Source citation Biofilm Basics. (2003). Retrieved October 19, 2017, from

http://www.biofilm.montana.edu/biofilm-basics/

Source found by Professor Lambert suggested reading about biofilms on Montana State

University’s website

Source Type Website

Keywords N/A (website)

Reason for interest I wanted to get general background information on biofilms and their role

in the world

Summary This website gave a broad overview on all types of biofilms and their role

in the world.

Notes - Biofilms go through three developmental stages: attachment, growth, and

detachment

- ATTACHMENT

- Planktonic bacteria attach to a surface and produce EPS (extracellular

polymeric substances) to colonize the surface

- GROWTH

- The produced EPS allow the colony to form a complex structure

- DETACHMENT

- Bacteria must detach from the biofilm for the production of a separate

biofilm.

Gatsonis 56

- There are multiple ways for bacteria to detach from a biofilm, including

mass detachment, in which large groups of cells detach, and seeding

dispersal, in which individual cells are released.

- ENVIRONMENT

- Biofilms can form the basis of a food web in an environment, as they are

often the food source of larger animals, which in turn are the food source

of even larger animals.

- Biofilms are important in the decontamination of soil, as they process and

degrade the contaminants.

- Biofilms are often found on the roots of plants, where they boost the

plant’s ability to capture nutrients from the soil.

- Biofilms can deplete bodies of water of oxygen.

- Biofilms can facilitate the movement of toxic elements, including mercury

and arsenic.

- Biofilms can cause dangerous algal blooms, more commonly known as

“red tides.”

- MEDICAL

- A biofilm on teeth, known as dental plaque, can lead to tooth decay and

cavities.

- Biofilms can form within heart valves, arteries, the lungs, the skin, and

various other areas of the human body.

- Several diseases biofilms can cause are bacterial endocarditis,

Legionnaire’s disease, and cystic fibrosis.

Gatsonis 57

- While bacterial illness can often be treated with antibiotics, biofilms have

exhibited extreme resistance to antibiotics.

- INDUSTRY

- Biofilms have been used in the treatment of wastewater.

- Biofilms can form on many industrial machines and are especially

effective at colonizing machinery used for water-based processes.

- In water-based industries, biofilms contaminate and foul the water.

- Biofilms can corrode certain metals used in pipes and in factory

machinery.

- Biofilms can cause blockage in pipes.

- Biofilm formation in machinery is responsible for billions of dollars in lost

industrial productivity and damage of equipment.

Questions N/A

Gatsonis 58

STEPS IN BIOFILM FORMATION

Source citation HyCa Technologies Pvt. Ltd. (2017). Retrieved October 19, 2017, from

http://www.hycator.com/domain/functional-sectors/biofouling/steps-in-

biofilm-formation.html

Source found by Searching “biofilm formation steps” on Google

Source Type Website

Keywords N/A (website)

Reason for interest I wanted to know how biofilms are initially formed

Summary This website discussed how biofilms initially attach to a surface and

grow.

Notes - A conditioning layer begins to form on the surface within seconds of the

introduction of the bacteria.

- As the layer forms, an electrical charge develops and attracts bacteria with

opposite charge. The bacterial colony is easily destroyed at this point.

- Within 8-24 hours, the bacteria attach to the surface and to each other with

tendrils or filaments. Extracellular polymeric substances enclose the cells

within a sticky matrix. The nutritive environment allows for the rapid

growth of the biofilm. Channels deliver essential elements to the cells and

carry away toxins. The biofilm is very difficult to destroy at this point.

Questions N/A

Gatsonis 59

Books

BIOCHEMISTRY LABORATORY: MODERN THEORY AND TECHNIQUES

Source citation Boyer, R. F. (2012). Biochemistry laboratory: modern theory and

techniques. Boston, Mass: Prentice Hall.

Source found by Borrowed from Ms. Curran

Source Type Book

Keywords N/A (book)

Reason for interest Had information on plasmids (decided against the use of plasmids, as it

could provide unrealistic results due to overcrowding of AIP).

Summary This book discussed how to use plasmids in an experiment.

Notes - Recombinant DNA is the covalent insertion of DNA fragments from a

different cell/organism into the replicating DNA of another cell

- Plasmids are self-replicating, bacterial, extrachromosal DNA

- Restriction endonucleases are enzymes that catalyze the hydrolysis of

phosphodiester bonds at specific sites in DNA. The result is a cleavage of

both DNA strands in or near the base sequence region.

- The cleavage results in one of 2 types of ends in DNA: 1) cohesive

(sticky) ends: a few bases can remain weakly associated by hydrogen

bonding 2) blunt ends: ends that do not overlap

- DNA ligase, an enzyme capable of catalyzing the ATP-dependent

formation of phosphodiester linkages at the insertion sites, can be used to

close the cleavage

- To prepare recombinant DNA:

Gatsonis 60

- 1) Choose and prepare the DNA fragment that will be inserted into the cell

(it can be prepared through chemical synthesis, such as the PCR and

sequence specific primers, action of restriction endonucleases, or

transcription of mRNA catalyzed by reverse transcriptase)

- 2) Choose a proper vector to carry the DNA fragment (the vector can be

plasmid DNA, DNA from a phage, or DNA from yeast artificial

chromosomes. Circular vectors must be linearized in order to accept the

DNA fragment, and, ideally, the same restriction enzyme should be used

to prepare the DNA fragment and vehicle so that there will possibly be

overlapping cohesive ends)

- 3) Insert the DNA fragment into the vector by overlapping cohesive ends

or by modifying blunt ends using homopolymer tails (the final covalent

bonds are formed by DNA ligase, which catalyzes the ATP-dependent

formation of phosphodiester bonds, resulting in a product of recombinant

DNA

- 4) Introduce the hybrid DNA into a host organism (typically a bacterial

cell) where it can be replicated (this process is transformation)

- 5) Develop a method to identify and screen the host cells that accepted and

are replicating the recombinant DNA (usually by testing antibiotic

resistance)

- Plasmids and bacteriophage DNA are common types of cloning vectors in

prokaryotes, while yeast artificial chromosomes and baculovirus are

common vectors for eukaryotic cells

Gatsonis 61

- Plasmids have a closed, circular shape, are double-stranded, and are much

smaller than chromosomal DNA, with molecular weights ranging from 2 x

106 to 20 x 106 (3K to 30K base pairs)

- Bacterial plasmids convey the information to express a phenotype

- Plasmids can be replicated in the cell either through stringent replication,

in which there are only a few copies of the plasmid, or relaxed replication,

in which there are many copies of the plasmid (up to 200). In relaxed

replication, up to 2K to 3K copies may be produced and may compose 30-

40% of the total cellular DNA

- Plasmid cloning vectors should have these properties:

- 1) It should replicated in a relaxed fashion to produce many copies

- 2) It should be small so that it is easier to separate from the chromosomal

DNA, easier to handle w/o physical damage, and so that it probably has

very few sites for attack by restriction endonucleases

- 3) The plasmid should contain markers so that it can be identified in future

generations (try to have at least 2 markers – one to confirm its presence,

and another to confirm the insertion of foreign DNA)

- 4) The plasmid should have only 1 cleavage site for a specific restriction

endonuclease so that there are only 2 ends to which foreign DNA can be

attached (the single restriction site should ideally be within a gene so that

inserting the foreign DNA will inactivate the gene ((insertional marker

inactivation))

Questions N/A

Gatsonis 62

Appendix D

Raw Data Tables

Table 1. Raw data table for the control group in the first experiment. RFU (relative fluorescence units) is an arbitrary unit

determined by the settings on the fluorescence spectrometer.

Table 2. Raw data table for the silver group in the first experiment. RFU (relative fluorescence units) is an arbitrary unit

determined by the settings on the fluorescence spectrometer.

Sample Number Green Fluorescence (RFU) Red Fluorescence (RFU) RatioG/R

1 335.80 377.76 0.89

2 343.09 390.10 0.88

3 324.63 372.92 0.87

4 483.50 417.43 1.16

5 446.45 425.58 1.05

6 421.24 387.32 1.09

7 300.68 422.23 0.71

8 348.58 469.30 0.74

9 340.31 457.74 0.74

10 244.76 381.18 0.64

11 202.33 379.78 0.53

12 447.41 495.23 0.90

13 430.94 429.27 1.00

14 456.27 460.16 0.99

15 426.12 410.61 1.04

Silver Concentration (mM) Green Fluorescence (RFU) Red Fluorescence (RFU) RatioG/R

3.000 419.37 460.18 0.91

3.000 388.68 467.25 0.83

3.000 390.38 470.76 0.83

1.000 401.35 439.91 0.91

1.000 402.35 456.91 0.88

1.000 385.28 464.19 0.83

0.300 336.86 478.50 0.70

0.300 334.51 435.05 0.77

0.300 359.49 450.14 0.80

0.100 320.17 427.80 0.75

0.100 334.75 447.19 0.75

0.100 307.10 359.71 0.85

0.030 335.64 447.86 0.75

0.030 361.42 453.40 0.80

0.030 353.49 430.93 0.82

Gatsonis 63

Table 3. Raw data table for the zinc group in the first experiment. RFU (relative fluorescence units) is an arbitrary unit

determined by the settings on the fluorescence spectrometer.

Table 4. Raw data table for the copper group in the first experiment. RFU (relative fluorescence units) is an arbitrary unit

determined by the settings on the fluorescence spectrometer.

Zinc Concentration (mM) Green Fluorescence (RFU) Red Fluorescence (RFU) RatioG/R

10.000 654.08 532.10 1.23

10.000 672.22 551.87 1.22

10.000 673.11 527.11 1.28

3.000 563.23 500.06 1.13

3.000 566.02 518.43 1.09

3.000 563.35 510.01 1.10

1.000 536.62 482.39 1.11

1.000 562.73 493.77 1.14

1.000 559.59 501.49 1.12

0.300 383.15 450.22 0.85

0.300 325.55 437.62 0.74

0.300 330.86 476.00 0.70

0.100 305.05 387.13 0.79

0.100 277.99 443.67 0.63

0.100 280.67 428.23 0.66

Copper Concentration (mM) Green Fluorescence (RFU) Red Fluorescence (RFU) RatioG/R

3.000 378.57 442.07 0.86

3.000 390.52 429.93 0.91

3.000 385.27 421.28 0.91

1.000 462.88 444.82 1.04

1.000 457.08 427.78 1.07

1.000 458.00 455.36 1.01

0.300 347.06 415.69 0.83

0.300 314.11 397.55 0.79

0.300 328.13 465.59 0.70

0.100 466.19 410.10 1.14

0.100 505.30 465.91 1.08

0.100 491.80 492.43 1.00

0.003 419.17 415.22 1.01

0.003 442.61 439.17 1.01

0.003 455.74 460.02 0.99

Gatsonis 64

Table 5. Raw data table for the control group in the second experiment. RFU (relative fluorescence units) is an arbitrary unit

determined by the settings on the fluorescence spectrometer.

Table 6. Raw data table for the zinc group in the second experiment. RFU (relative fluorescence units) is an arbitrary unit

determined by the settings on the fluorescence spectrometer.

Table 7. Raw data table for the copper group in the second experiment. RFU (relative fluorescence units) is an arbitrary unit

determined by the settings on the fluorescence spectrometer.

Time Elapsed (minutes) Green Fluorescence (RFU) Red Fluorescence (RFU) RatioG/R

0 40667.63 11009.95 3.69

30 59175.53 13520.90 4.38

60 60981.81 13164.70 4.63

90 50231.62 11794.49 4.26

120 58123.04 12679.87 4.58

150 52654.55 11444.82 4.60

180 52914.17 11703.46 4.52

210 45062.66 10544.21 4.27

Time Elapsed (minutes) Green Fluorescence (RFU) Red Fluorescence (RFU) RatioG/R

0 46594.50 11212.55 4.16

30 24066.09 10867.25 2.21

60 47879.58 11465.19 4.18

90 18045.37 9740.23 1.85

120 17136.46 9582.31 1.79

150 15940.01 9177.25 1.74

180 15585.33 8830.40 1.76

210 14011.06 9121.47 1.54

Time Elapsed (minutes) Green Fluorescence (RFU) Red Fluorescence (RFU) RatioG/R

0 18918.67 10776.32 1.76

30 13755.97 9516.31 1.45

60 10401.14 8855.12 1.17

90 9732.11 8523.37 1.14

120 9324.42 8398.16 1.11

150 9100.82 8407.77 1.08

180 8112.27 7815.01 1.04

Gatsonis 65

Table 8. Raw data table for the control group in the third experiment. RFU (relative fluorescence units) is an arbitrary unit

determined by the settings on the fluorescence spectrometer.

Time Elapsed (minutes) Green Fluorescence (RFU) Red Fluorescence (RFU) RatioG/R

0 29654.15 6824.88 4.35

3 29749.75 6859.91 4.34

5 29283.54 6763.32 4.33

9 28710.43 6722.34 4.27

11 28349.12 6608.88 4.29

18 28695.70 6837.25 4.20

23 29071.93 6928.69 4.20

28 28578.27 6801.62 4.20

33 28559.15 6732.78 4.24

42 27213.16 6624.45 4.11

51 26762.51 6704.73 3.99

59 27896.14 6699.68 4.16

68 27546.96 6732.64 4.09

76 27667.62 6753.40 4.10

88 26206.71 6666.58 3.93

86 26313.58 6533.18 4.03

99 25053.87 6444.61 3.89

116 22914.08 6399.38 3.58

131 23667.37 6582.84 3.60

147 24675.57 6368.38 3.87

Gatsonis 66

Table 9. Raw data table for the copper group in the third experiment. RFU (relative fluorescence units) is an arbitrary unit

determined by the settings on the fluorescence spectrometer.

Time Elapsed (minutes) Green Fluorescence (RFU) Red Fluorescence (RFU) RatioG/R

0 28881.69 6828.00 4.23

4 27749.65 6801.07 4.08

8 27544.94 6624.46 4.16

10 27807.42 6585.57 4.22

14 27008.16 6498.41 4.16

19 27060.44 6674.37 4.05

26 25612.76 6652.75 3.85

29 25200.38 6565.60 3.84

36 23564.41 6768.33 3.48

43 22078.78 6299.38 3.50

54 19439.06 6277.98 3.10

62 19499.13 6263.65 3.11

69 19282.71 6208.71 3.11

79 17550.68 6180.74 2.84

89 16751.77 6113.01 2.74

99 15491.60 6047.68 2.56

110 14471.78 5952.03 2.43

129 12748.76 6000.15 2.12

142 12910.08 6049.59 2.13

160 12681.79 6002.92 2.11