Disruption of Quorum Sensing in Staphylococcus aureus...
Transcript of Disruption of Quorum Sensing in Staphylococcus aureus...
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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
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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
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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.
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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.
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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.
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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.
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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).
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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)
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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,
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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.
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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
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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.
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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
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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.
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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
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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
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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
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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
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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.
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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.
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Figure 2. Fluorescence excitation and emission spectra of SYTO 9 and propidium iodide (Thermo Fisher Scientific, 2004)
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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