Diffusion of copper-oxide nanowire bundles carrying antibiotics through the biofilm pores
produced by Pseudomonas aeruginosa
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). 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). This is important because
a Pseudomonas aeruginosa’s biofilm forms a barrier of sugars and carbohydrates to prevent
antibiotics from reaching the pathogens (Hanlon et al., 2001).
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). Nanowires have been used in various biological
applications due to their size, and selectivity (Li et al., 2010). These wires have a diameter of the
nanometer scale, which can have different shapes and properties depending on the material they
are made of. In a previous study by Li et al. (2010), CuO NWBs have been used as a biosensor to
detect and interact with hemoglobin. Research performed by Li et al. (2010) using CuO NWBs
revealed these bundles interacted with the hemoglobin redox center deep inside a large protein
shell. Thus, we suggest CuO NWBs could interact with antibiotics and penetrate through
Pseudomonas aeruginosa’s biofilm layer.
Metal ions have been used to kill the pathogen by diffusing metal ions such as copper,
lead, nickel and others individually through the Pseudomonas aeruginos’s biofilm (Harrison et
al., 2005). In addition, the results from Harrison’s (2005) experiment showed that a high
concentration and long exposure time of various metal ions were able to completely eliminate the
biofilm. The lengthy exposure time is a result of cationic binding of the metal ions to the biofilm,
which restricted the rate of penetration (Harrison et al., 2005).
We will use CuO NWBs as a carrier for our antibiotics. Copper was shown to be
effective in dealing with the pathogen and biofilm of Pseudomonas aeruginosa (Harrison et al.,
2005). We plan on attaching antibiotics electrostatically to the surface of the CuO NWB to
increase the rate of diffusion into the biofilm. We suggest the antibiotics will occupy the
positive charge on the CuO NWBs so that they do not adhere to the surface of the biofilm.
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 (Walters et al., 2002). 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 (Walters et al., 2002). 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 (NET).
In a normal immune response, white blood cells form a
neutrophil net by secreting proteins and chromatin. This net traps
the pathogen and prevents damage to the adjacent tissues at the site
of infection. According to Brinkmann (2004), NETs bind to
bacterial cells, prevent the spread of infection, and kill the pathogen
using antimicrobial agents (toxins, chemicals, or antibodies). As a
result of these above advantageous properties, we will use a bundle
of nanowires to imitate a NET and trap the pathogen.
Figure 2. Neutrophil extracellular
trap (NET).
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 synthetic NET? We hypothesize that CuO NWBs will imitate
a synthetic NET and carry antibiotics through the biofilm of Pseudomonas aeruginosa to kill the
pathogen from the inside. If this synthetic NET carrying antibiotics diffuses through the biofilm
successfully and if the antibiotics detach from the NET inside the biofilm, we predict no growth
on the antibiotic/CuO NWB agar plates.
PROPOSED RESEARCH
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) and examine the biofilm’s
structure using a confocal laser scanning microscope (CLSM).
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).
We will grow our Pseudomonas aeruginosa biofilms at the optimal temperature of 35℃
(Harrison et al., 2006). We will prepare a pure culture of Pseudomonas aeruginosa by streaking
two LB agar plates incubated for 24 hours each: one from the stock culture and the other from a
single colony on the previous plate (Fig. 1A). To grow the biofilms, a 30-fold diluted McFarland
inoculum will be prepared using the Pseudomonas aeruginosa pure culture (Fig. 1B), and a CBD
peg lid will be inserted into a 96-well corrugated trough containing 22 mL of inoculum in each
well. Then, the corrugated trough will be placed in a humidified incubator on a rocking table
(set at 3.5 rocks per min.) and incubated for 24 hours (Fig. 1C).
To examine Pseudomonas aeruginosa biofilm growth, we will place a CBD peg lid into a
96-well microtiter plate containing 200 µL of 0.9% NaCl in each well for 2 min. (Fig. 1D). Next,
CBD pegs are removed with flamed pliers (Fig. 1E) and placed into acridine orange stained wells
of a 96-well microtiter plate. Then, each peg will be mounted on a glass coverslip with 2 drops of
0.9% NaCl (Fig. 1I) and examined under a confocal laser scanning microscope (Fig. 1J).
Synthesis of copper oxide nanowires
We will follow the methods outlined by Li (2010) to synthesize the copper oxide
nanowire bundles. We will create a one dimensional nanowire and combine it with an aqueous
solution of CuCl2. This composite solution will be vacuum filteredand 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 copper
nanowire carriers takes 24 hours to complete. We will get a visual representation of our carriers
by using transmission electron microscopy (TEM) to generate an image. X-ray diffraction
(XRD) analysis will be used to determine the structure and correct chemical composition of our
carriers.
Antibiotic Coupled Nanowire Synthesis
We will use electrostatic interactions to carry our two types of antibiotics, Ciproflaxin
and Tombramycin, with our copper oxide nanowire carrier. We will synthesize the nanowire
carriers following the methods outlined in Li (2010), but we will reduce the copper oxide to form
a charged Cu2+ nanowire carrier. Our antibiotics will carry an opposite charge by salvation and
interact with the charged carriers by the method of electrostatics. We will use Fourier transformIR (FT-IR) to get spectra information on our copper-oxide nanowires, antibiotic, and the
combination of nanowires and antibiotics.
Controls and Test Samples
Pseudomonas aeruginosa biofilms will be exposed to each parameter listed in Table 1 below:
Table 1. Experimental group parameters tested with Pseudomonas aeruginosa biofilms.
A
EXPERIMENTAL
GROUP
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
No growth
(magnesium 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.
TEST GROUP
PREPARATION PROCEDURE
Control
Add 200 µL of 0.9% NaCl to 24 wells of a 96-well microtiter plate
of a randomly selected region (1, 2, 3, or 4) (Fig. 2)
Antibiotic only
Add 200 µL of antibiotic to 24 wells of a 96-well microtiter plate of
a randomly selected region (1, 2, 3, or 4) (Fig. 2)
Nanowire only
Add copper-oxide nanowires to 24 wells of a 96-well microtiter
plate containing 200 µL of 0.9% NaCl of a randomly selected region
(1, 2, 3, or 4) (Fig. 2)
Nanowire + Antibiotic
Prepare 6 dilutions of antibiotic fused copper-oxide nanowires by
factors of 10 using 200 µL of 0.9% NaCl.
Add 200 µL of diluted antibiotic fused copper-oxide nanowires to 4
wells of a 96-well microtiter plate of randomly selected regions (1,
2, 3, or 4) and (I, II, III, IV, V, VI) (Fig. 2)
Figure 2. A microtiter plate illustrating the regions of the treatment parameters control (A),
antibiotic only (B), nanowire only (C), and antibiotic fused nanowire (D) (1, 2, 3, and 4), and the
antibiotic fused nanowire dilution 10-1, 10-2, 10-3, 10-4, 10-5, and 10-6 aliquots (I, II, III, IV, V,
and VI) (Ceri, 1999).
Sample Size
We expect little to no growth once we penetrate the biofilm layer of Pseudomonas
aeruginosa with our nanowires carriers. To determine the sample size (N) we will 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 interesting in detecting. With our growth and no growth model
we expect to see a high effect size of 0.5. With 9 degrees of freedom in our experimental design,
we obtained a noncentrality parameter of 23.5894357. The sample size that is required for our
experiment is 95. We will need at least 4 MBEC plates to get the required sample size.
Analysis and Interpretation
We will use Harrison’s (2006) viable cell counting procedure to determine Pseudomonas
aeruginosa growth following each treatment parameter discussed previously. To collect our
results after conducting each treatment parameter, we will follow the procedure outlined in
Figure 3 below. First, we will rinse the biofilms by placing the CBD peg lid into a 96-well
microtiter plate containing 200 µL of 0.9% NaCl in each well for 2 min (Fig. 1D). Then, the
CBD pegs will be removed using flamed pliers and placed in a microtiter plate containing 200
µL of 0.9% NaCl in each well (Fig. 1E). This will be followed by sonication: using an
Aquasonic 250HT ultrasonic cleaner (60 Hz for 5 min.) to remove the bacterial cells from the
peg surface. The bacterial cells will be serially diluted in 0.9% NaCl, plated on LB agar
medium, and incubated at 35℃ for 24 hours. The following day, we will count the number of
colony forming units (CFUs) on each plate and record the results.
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).
Experimental Design
We propose to follow the experimental procedure outlined below:
Day 0
•Grow biofilms and make copper
oxide nanowires
Day 1
•1: Test for copper on nanowire and
combine copper nanowire with biofilm
•2: Couple antibiotics to copper oxide
nanowires, tests coupling, dilutes
antibiotics, and combines coupled
nanowires with biofilm
•3: Test for biofilm growth and do
antibiotic test
Day 3
Day 2
•1: Records results of toxicity test
•2: Records results of test route
•3: Grow Biofilms and make copper
oxide nanowires
•1: sonicates for toxicity test
•2: sonicates for test route
•3: Record results of antibiotic test
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