The diffusion of antibiotics through the biofilm pores produced by Pseudomonas
aeruginosa using a synthetic neutrophil extracellular trap
Team Nanobots
Mark Ly, Fahima Nakitende, and Shannon Wesley
Theme: Nanoscience
Subtheme: Nanoparticle systems for studying biomolecules
Keywords: Pseudomonas aeruginosa biofilm, copper-oxide nanowire bundles, neutrophil
extracellular trap, cystic fibrosis, ciprofloxacin/tobramycin
PROPOSAL SUMMARY
INTRODUCTION AND BACKGROUND
We propose to study the control of Pseudomonas aeruginosa infections by using
nanowires to deliver antibiotics across the Pseudomonas aeruginosa’s biofilm to treat chronic
infections. According to previous research, among 40 patients suffering from cystic fibrosis,
77% were infected by Pseudomonas aeruginosa, even though 71% of these patients exhibited a
normal immune response (Neter 1974; Diaz et al. 1970; Diaz and Neter 1970). Consequently,
conventional antibiotic therapy was ineffective against the Pseudomonas aeruginosa infection.
Thus, we will use copper-oxide nanowire bundles (CuO NWB) to penetrate the biofilm produced
by Pseudomonas aeruginosa to deliver antibiotics and kill the bacteria residing inside the
biofilm.
Cystic fibrosis is an inherited disease that causes a thick, sticky mucus build up in the
lungs, which is an ideal environment for Pseudomonas aeruginosa biofilms to fluorish.
Pseudomonas aeruginosa infections form biofilms to defend themselves against human
immunity and conventional antibiotic therapy (Costerton et al. 1999; Nickel et al. 1985). This is
important because a Pseudomonas aeruginosa’s biofilm forms a 200 µm barrier of sugars and
carbohydrates to prevent antibiotics from reaching the pathogens (Hanlon et al. 2001; Anwar and
Costerton 1992; Costerton et al. 1987; Govan and Deretic 1996).
Earlier studies have used diffusion as a way to enter the pathogen’s biofilm layer to kill
it, which is the method we propose to use in this study. This is because Pseudomonas
aeruginosa’s biofilm consists of numerous pores and channels, which can be used for the
diffusion of nanowires (Costerton et al. 1999; Stewart 1996).
Nanowires have been used in various biological applications due to their size and
selectivity (Li et al. 2010; Bao et al. 2008; Lu et al. 2007). These wires have a diameter of the
nanometer scale, which can have different shapes and properties depending on the material they
are made of. And, we can arrange these nanowires into nanowire bundles to form a synthetic
mesh-like net that will increase the available surface area for antibiotic attachment.
Metal ions have been used by Harrison (2005) to kill the pathogen by diffusing metal
ions through the Pseudomonas aeruginos’s biofilm. In addition, the results showed that a high
concentration and long exposure time of various metal ions were able to completely eliminate the
biofilm. And, the lengthy exposure time was a result of cationic binding of the metal ions to the
biofilm that restricted the rate of penetration. Harrison (2005) also determined that 40 mM of
lead, 120 mM of zinc, 140 mM of cobalt and nickel, and 300 mM of aluminum metal ions were
required to destroy a biofilm, whereas only 30 mM of copper was required to have the same
outcome. Thus, we decided to make our nanowires out of copper because copper was effective at
low concentrations and non toxic to biological systems. Therefore, we will use electrostatics to
compose CuO NWB antibiotic carriers. We predict the antibiotics will occupy the positive
charge on the CuO NWBs and will not adhere to the surface of the biofilm during diffusion.
Previous research performed by Walters et al. (2002) demonstrated tobramycin and
ciprofloxacin eventually penetrated the biofilm, but failed to kill Pseudomonas aeruginosa once
inside the biofilm. As indicated by Figure 1, both ciprofloxacin and tobramycin did not increase
Pseudomonas aeruginosa’s mortality rate over a 100 hour exposure time.
Figure 1. Killing of P. aeruginosa in biofilms in exposure to ciprofloxacin (A) and killing
of P. aeruginosa in biofilms in exposure to tobramycin (B). Filled squares were the treatment
and the unfilled were the controls (Walter et al. 2002)
Thus, the ineffectiveness of these antibiotics was not because of poor penetration, but
oxygen limitation and low metabolic activity inside the biofilm. A lack of oxygen within the
biofilm either prevented Pseudomonas aeruginosa growth or the action of the antibiotics;
whereas, free swimming bacterial cells were susceptible to tobramycin and ciprofloxacin. For
these reasons, we decided to use these two antibiotics in conjunction with our CuO NWBs to kill
the pathogen inside the biofilm. These bundles will not block the Pseudomonas aeruginosa
biofilm pores because of their nanometer size. Therefore, oxygen transport will be restored,
metabolic activity will continue as normal, and we will be able to spread the antibiotics over a
larger area. So, by combining these antibiotics to our CuO NWBs we are creating a synthetic
neutrophil extracellular trap (synNET).
According to Brinkmann (2004), white blood cells in a
Figure 2. Neutrophil extracellular
trap (NET).
normal immune response form a neutrophil extracellular trap (NET)
by secreting proteins and chromatin. This NET traps the pathogen
and prevents damage to the adjacent tissues at the site of infection.
NETs bind to bacterial cells, prevent the spread of infection, and kill
the pathogen using antimicrobial agents (toxins, chemicals, or
antibodies) (Weinrauch et al. 2002). As a result of these above
advantageous properties, we will use a bundle of nanowires to
imitate a NET and trap the pathogen.
According to Tirouvanziam (2007), NETs are rapidly killed by toxins released by the
bacterial cells making them ineffective. This is why patients with cystic fibrosis are
continuously suffering from recurring episodes of Pseudomonas aeruginosa infection. So, could
CuO NWBs carrying antibiotics diffuse through the porous structure of Pseudomonas
aeruginosa’s biofilm and act as a synNET?
We hypothesize that CuO NWBs will imitate a NET and carry antibiotics through the
biofilm of Pseudomonas aeruginosa to kill the pathogen from the inside. If this synNET carrying
antibiotics diffuses through the biofilm successfully and if the antibiotics detach from the
synNET inside the biofilm, we predict no growth on the antibiotic/CuO NWB agar plates.
PROPOSED RESEARCH
The aim of our study is to determine the effectiveness of ciprofloxacin and tobramycin
coupled CuO NWBs against Pseudomonas aeruginosa biofilm growth.
We will grow Pseudomonas aeruginosa biofilms every three days to avoid biofilm
degradation which usually occurs at about 48 hours and to ensure a constant 24 hour aged
biofilm for testing (D. Storey, pers. Commun.). Also, we will synthesize one set of CuO NWBs
for each antibiotic.
Pseudomonas aeruginosa Biofilm Synthesis
We will follow Harrison’s (2006) procedure shown in Figure 1 to grow Pseudomonas
aeruginosa biofilms using the Calgary Biofilm Device (CBD) with the exception of using the
scanning electron microscopy method.
Figure 1. The experimental design to synthesize Pseudomonas aeruginosa biofilms, using LuriaBurtani Broth (LB) and the Calgary Biofilm Device (CBD) system; and examination of the
biofilm’s structure using confocal laser scanning microscopy (CLSM) equipment (Harrison,
2006).
A confocal laser scanning microscope (CLSM) and its 3D imaging properties will be
used to examine the biofilm’s structure and confirm the presence of pores. Only biofilm
structures containing pores will be used in this research study to make sure diffusion transport is
accessible.
Ciprofloxacin and Tobramycin
We will purchase 100 mg of deprotonated (negatively charged) ciprofloxacin and
tobramycin from Sigma-Aldrich.
Walter’s et al. (2002) procedure will be used to test ciprofloxacin and tobramycin against
Pseudomonas aeruginosa biofilms as our control. This will ensure that these two antibiotics are
ineffective against Pseudomonas aeruginosa biofilms individually.
Synthesis of CuO NWBs
We will follow the methods outlined by Li et al. (2010) to synthesize the CuO NWBs.
We will create a one dimensional nanowire and combine it with an aqueous solution of CuCl2.
This composite solution will be vacuum filtered and dried in an oven to produce our nanowires
composition. The initial template will be removed with a solution of NaOH and the copper-oxide
nanowire bundles will dry a final time. Creating our CuO NWBs will take 24 hours to complete.
We will determine the structure and correct chemical composition of our CuO NWBs by using
transmission electron microscopy (TEM) and X-ray diffraction (XRD). This will guarantee our
CuO NWBs are self assembled correctly.
Synthesis of synNETs
To attach our antibiotics, we will use electrostatic interactions (CuO NWBs positive
charge with the antibiotics negative charge) to couple with our CuO NWBs. A solution of
ciprofloxacin/tobramycin will be cast onto the CuO NWBs separately and then slowly
evaporated to produce our synNETs. These synNETs will be stored in a refrigerator (4℃).
We will follow Li’s et al. (2010) characterization method to characterize our synNET.
Fourier transform-IR (FT-IR) spectra will be performed using a Nicolet Avatar mass
spectrometer from the University of Calgary’s Chemical Instrumentation Facility to compare
three different peaks: the CuO NWBs individually, the antibiotic individually, and the antibiotic
coupled with our CuO NWBs. If our antibiotics coupled with our CuO NWBs successfully, we
expect to see a spectra that combines both individual peaks: CuO NWBs and antibiotic.
One factor that could affect our results is the strength of the electrostatic interaction
between the antibiotics and the CuO NWBs. If the interaction is too weak then the CuO NWB
may attach to the biofilm layer instead of the antibiotic, and the CuO NWB will not deliver the
antibiotic across the biofilm layer.
Controls and Test Samples
Pseudomonas aeruginosa biofilms in 96 welled MBEC plates will be randomly exposed to each
parameter listed in Table 1 below:
Table 1. Experimental group parameters tested with Pseudomonas aeruginosa biofilms.
TREATMENT
A
Control
B
Antibiotic only
C
Nanowire only
D
Nanowire + Antibiotic
EXPERIMENTAL TREATMENT
PARAMETERS
No antibiotic
No copper-oxide nanowire
Antibiotic
No copper-oxide nanowire
No antibiotic
Copper-oxide nanowire
Antibiotic fused
Copper-oxide nanowire
EXPECTED
RESULT
Growth
Growth
Limited growth
(copper-oxide toxicity)
No growth
(antibiotic penetration)
To prepare each of our test group parameters in Table 1 above, we will follow the
procedures outlined in Table 2 below:
Table 2. Experimental treatment group preparations.
TREATMENT
PREPARATION PROCEDURE
A
Control
Add 200 µL of 0.9% NaCl to 24 wells of a 96-well
MBEC plate of a randomly selected region (A,B,C,
and D) (Fig. 2)
B
Antibiotic only
Add 200 µL of antibiotic to 24 wells of a 96-well
MBEC plate of a randomly selected region (A,B,C,
and D) (Fig. 2)
C
Nanowire only
Add CuO NWBs to 24 wells of a 96-well MBEC
plate containing 200 µL of 0.9% NaCl of a randomly
selected region (A,B,C, and D) (Fig. 2)
D
Nanowire + Antibiotic
Add 200 µL of diluted antibiotic fused copper-oxide
nanowires to 24 wells of a 96-well microtiter plate of
randomly selected regions (A,B,C, and D) (Fig. 2)
Figure 2. A MBEC plate illustrating the regions of the treatment parameters: control, antibiotic
only, nanowire only, and antibiotic coupled CuO NWBs. Each treatment will consist of 24 out
of the 96 wells and will be rotated between regions A, B, C, and D (Ceri et al. 1999).
Experimental Procedure
The first day we will grow Pseudomonas aeruginosa biofilms and synthesize CuO NWBs.
Then, we will routinely repeat the following 3 day outline for the duration of our 8 month
experimentation with 2 days of intermittent data collection.
Day 1
-
Confirm Pseudomonas aeruginosa biofilm growth using CLSM and
collect A treatment results
Confirm the presence of copper on the CuO NWBs using TEM & XRD
Synthesize ciprofloxacin and tobramycin coupled CuO NWBs
Confirm proper coupling using FT-IR
Start B treatment and incubate 24 hours (35℃)
Day 2
-
Sonicate biofilm pegs, start C treatment, and incubate 24 hours (35℃)
Sonicate biofilm pegs, start D treatment, and incubate 24 hours (35℃)
Collect B treatment results
Day 3
-
Collect C treatment results
Collect D treatment results
Grow Pseudomonas aeruginosa biofilms
-
Sample Size
We expect limited growth once we penetrate the biofilm layer of Pseudomonas
aeruginosa with our synNET. To determine the sample size (N) we will use a chi-squared
logistical model to determine the noncentrality parameter (λ) using the formula N = λ / w2. The
effect size (w) is the effect we are interested in detecting. With our growth and no growth model
we expect to see a medium effect size of 0.3. With 14 degrees of freedom in our experimental
design, we obtained a noncentrality parameter of 27.2. The sample size that is required for our
experiment is 303. Thus, we will need at least 13 MBEC plates to get the required sample size.
Analysis and Interpretation
Harrison’s (2006) viable cell counting procedure will be used to determine the mean
amount of Pseudomonas aeruginosa biofilm growth after being exposed to three different
treatments (Fig. X).
Figure 3. Diagram of experimental procedure used for group tests B, C, and D (Table 1). Step D
above will incubate with antibiotics for group tests B and D and with copper oxide nanowires for
group tests C and D (Herrmann, 2010). Cross out G
From our collected data, a one-way fixed effect ANOVA statistical test will be performed
to compare the mean colony forming units (CFUs) for our three different treatments. The
ANOVA statistical test will determine any growth differences between our antibiotic only, CuO
NWBs only, and synNETs.
Our expected results from the ANOVA statistical test should reveal 100% growth in the
control and antibiotic only treatments, less than 100% growth in the CuO NWBs only treatment,
and 0% growth in the antibiotic coupled CuO NWBs treatment. If our experimental results
correlate with our expected results presented above, then our synNETs can be used as an
alternative therapy for Pseudomonas aeruginosa biofilm infections. In addition, future studies
could be conducted to determine the optimum concentration of CuO NWBs (without using
antibiotic coupling) needed to effectively kill Pseudomonas aeruginosa biofilm growth.
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BUDGET
Summer students: We plan on having 2 summer students for the first year at a rate of $5K/student each
year with a total of $10K. We then plan on hiring 2 graduate students of $13k/student for the second year
for a total of $26K. Over the 2 year period the gross total cost for personal is expect to be $36K without
considering USRA grants and TA positions.
Publication costs: We plan on publishing a paper in the American Society for Microbiology. The cost
associated with publishing this will be $3549.
Equipment costs: We will need to purchase materials to create our own Pseudomonas Aeruginosa
biofilms. The lab of Dr. Douglas Storey at the University of Calgary will provide the bacterial strain. 630
LB Agar plates ($2393.20), 100 mg of Tobramycin and 100 mg of ciprofloxacin (Total $198.90), will be
purchased from Sigma-Aldrich. There will be a rental fee associated with the use of the X-Ray
Diffraction microscope ($15/hour) in the department of Geology at the University of Calgary. Chemicals
for creating the nanowires bundles will be provided by the Department of Chemistry at the University of
Calgary. We will use the Nicolet Avatar FT-IR Mass spectrometer at the University of Calgary Chemical
instrumentation facility. We will require an 3D Gyratory Rocker ($1114.99), an incubator ($2291.94),
and a refrigerator ($150.00).
Personal: We plan to hire a lab technician to set up and maintain our equipment during the 2-year period
costing $40K/year for a total cost of $80K.
Traveling Costs: We expect to present our findings at the Alberta Nanotech showcase ($25/registration).
We plan to send 2 people to the international conference on Nanotechnology, which should be a total of
($3K).
The total cost over 2 years will be $129 198.03.
TIMELINE
We expect to take 8 months to do our experimental proceed which consists of making,
observing, and examining our nanowires bundles, biofilms, and our synthetic net. Testing of our
hypothesis will also be done in this 8 month period. The next 4 months will be decided to data
collection and analysis of our results. The following year, we will spend 4-5 months interpreting
our results. The remainder of our time will be spent writing our paper and attending conferences
to promote our findings.