Diffusion of copper-oxide nanowire bundles carrying antibiotics through the biofilm pores
produced by Pseudomonas aeruginosa
INTRODUCTION AND BACKGROUND
We propose to study control of Pseudomonas aeruginosa biofilms using nanoparticles for
the treatment of human chronic infections and cystic fibrosis. Pseudomonas aeruginosa is a
human pathogen that causes major problems in a number of clinical settings, particularly in
patients that have wounds or burns, dwelling medical devices or cystic fibrosis (Hanlon et
al.,2001). This bacterium can grow as a biofilm, which is its best defense against human
immunity and conventional antibiotic therapy (Costerton et al., 1999). Pseudomonas
aeruginosa’s biofilm is important because it secretes a large amount of exopolysaccrides that
surrounds the cells, and forms a glycocalyx (extracellular polymeric material) which act as a
barrier to prevent antibiotics from reaching the pathogens (Hanlon et al., 2001). Therefore we
will develop a method using copper-oxide nanoparticles to target the biofilms produced by
Pseudomonas aeruginosa’s to help deliver antibiotics through the biofilm’s protective layer to
eradicate the bacteria residing inside the biofilm.
A nanoparticle is any microscopic particle approximately less than 100 nanometers.
Nanoparticles can be made to self-assemble into nanowires which are nanometer scale wires
made of materials that conduct electricity (Tang et al., 2002). Nanomedicine is a field of science
that combines nanotechnology and medical treatments is starting to become more prevalent in
the treatment and diagnosis of patients (Roy et al., 2005). The use of nanoparticles in
nanomedicine has been increasing; especially those made from metal oxides such as copper
oxide nanoparticles or nanowires (Roy et al., 2005). Metal oxide nanowires have been used for:
their outstanding optical, magnetic and electrical properties, their large surface area, their ease of
preparation and their high stability (Li et al., 2010). Nanomedicine has used nanoparticles as
platforms for diagnostic probes, and for effective targeted therapy (Roy et al., 2005). The
involvement of nanoparticles in drug delivery, suggests that it’s possible to use copper oxide
nanowires to carry antibodies through its biofilm to eliminate Pseudomonas aeruginosa.
In attempts to kill the Pseudomonas aeruginosa pathogen, the biofilm seemed to be a
major barrier to their success. Many 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 has an extracellular matrix that consists of numerous pores
and channels, which can be used for the diffusion of oxygen and other ions (Costerton et al.,
1999).
Although we propose to use diffusion to go through the Pseudomonas aeruginos’s
biofilm, there is a contradiction in the way agents or treatments are transported through this
biofilm layer. Previous studies have targeted Pseudomonas aeruginos’s biofilm with metal ions,
bacteriophages, and direct use of antibiotics whereas this proposed study will use copper oxide
nanowires to carry antibiotics through the biofilm. Metal ions have been used to kill the pathogen
by diffusing metal cations such as copper, lead, nickel and others individually through the
Pseudomonas aeruginos’s biofilm (Harrison et al., 2005). High exposure of specific metals such
as copper to the biofilm helped kill biofilm bacterial populations, although the method was
ineffective when exposed with other metal cations (Harrison et al., 2005). This is because metal
cations were observed to ionically interact with the negatively charged phosphodiester and other
groups outside the biofilm, retarding their diffusion through the biofilm(Harrison et al., 2005).
The use of bacteriophages as a method of treatment was successful in treating many infections,
even those proved to be resistant to antibiotics initially such as Pseudomonas aeruginosa biofilm
associated infections (Hanlon et al., 2001). This treatment diffused phages or viruses into the
biofilm to directly kill the pathogen inside this biofilm. However phages were problematic due to
the presence of an extracellular polymeric material that acted as a barrier to the virus’s entry
through the biofilm (Hanlon et al., 2001). Finally in another study, there was direct use of
antibiotics most susceptible to Pseudomonas aeruginosa pathogen such as Ciproflaxin and
Tombramycin antibiotics for the treatment of the bacterium (Walters et al., 2003). Ciproflaxin
diffused more readily through the biofilm than Tombramyacin. The slower penetration of
Tombramycin was due to antibiotic binding to the extracellular matrix of Pseudomonas
aeruginosa’s biofilm creating a diffusion barrier (Walters et al., 2003).
These three past studies are significant to ours because they all faced a diffusion barrier
presented by Pseudomonas aeruginosa’s biofilm. But none of the treatments used nanowires,
which brings in the importance of the use of nanowires in our proposed study hoping to be more
successful than the above treatments. The challenges found in past studies and the new
discoveries in nanomedicine has led us to a new scientific question that: Can copper oxide
nanowires carrying antibiotics diffuse through the porous structure of Pseudomonas
aeruginosa’s biofilm? This question hypothesizes that copper oxide nanowires will help carry
antibiotics susceptible to Pseudomonas aeruginosa’s pathogen through the biofilm to kill the
pathogen from the inside. Therefore, the use of copper oxide nanowires in this study is proposed
to reduce or remove the diffusion barrier through the biofilm found in previous studies. If these
copper oxide nanowires diffuse through the biofilm successfully, then this study predicts the
antibodies attached to the nanowires will be released to kill the pathogen inside the biofilm. One
alternate hypothesis to this proposed research question could be: can copper oxide nanowires
alone without antibiotics kill Pseudomonas aeruginosa pathogen when it successfully diffuses
through the biofilm? This hypothesis predicts that there is no need to attach antibiotics to the
nanowires if nanowires themselves are able to get rid of the infection. An alternate hypothesis
could be: do copper oxide nanowires attached to antibiotics diffuse right through the
Pseudomonas aeruginosa’s biofilm without killing the pathogen inside? This assumption could
be possible because of the nature of the biofilm’s extracellular layer containing numerous in and
out end pores, and also because infected bacterial cells mostly reside between these pores
(Hanlon et al., 2001). Therefore, this hypothesis predicts these copper oxide nanowires carrying
antibiotics might diffuse right through the biofilm pores before releasing the antibiotics to kill the
pathogen. The above alternate hypotheses will be answered when the proposed research question
is applied and successfully carried out as below. And if successful, we will have found a solution
or treatment to many biofilm associated infections in all species such as cystic fibrosis.
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 magnesium-oxide nanowire
Antibiotic
No magnesium-oxide nanowire
No antibiotic
Magnesium-oxide nanowire
Antibiotic fused
Magnesium-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
REFERENCES
Ceri H, Olson ME, Stremick C, Read RR, Morck D, Buret A. 1999. The Calgary biofilm device:
New technology for rapid determination of antibiotic susceptibilities of bacterial
biofilms. Journal of Clinical Microbiology. 37:1771-1776.
Costerton JW, Stewart PS, Greenberg EP. 1999. Bacterial biofilms: A common cause of
persistent infections. Science. 284: 1318-22.
Hanlon WG, Denyer Ps, Olliff JC, Ibrahim JL. 2001. Reduction in exopolysaccharide viscosity
as an aid to bacteriophage penetration through Pseudomonas aeruginosa biofilms.
American Society for Microbiology. 67: 2746-53.
Harrison JJ, Turner RJ, Ceri H. 2005. Persister cells, the biofilm matrix and tolerance to metal
cations in biofilm and planktonic Pseudomonas aeruginosa. Biofilm Research Group.
University of Calgary. 7: 981-94.
Harrison JJ, Ceri H, Yerly J, Stremick CA, Hu Y, Martinuzzi R, Turner RJ. 2006. The use of
microscopy and three-dimensional visualization to evaluate the structure of microbial
biofilms cultivated in the Calgary biofilm device. Biological Procedures Online. 8:194215.
Herrmann G, Yang L, Wu H, Song Z, Wang H, Hoiby N, Ulrich M, Molin S, Riethmuller J,
Doring G. 2010. Colistin-tobramycin combinations are superior to monotherapy
concerning the killing of biofilm Pseudomonas aeruginosa. The Journal of Infectious
Diseases. 202:1585-1592.
Li Y, Zhang Q, Li J. 2010. Direct electrochemistry of hemoglobin immobilized in CuO nanowire
bundles. Talanta. 83: 162-66.
Tang Z, Kotov AN, Giersig M. 2002. Spontaneous organization of single ctde nanoparticles into
luminescent nanowires. Science, new series. 297: 237-40.
Roy I, Ohulchanskyy YT, Bharali JD, Pudavar EH, Mistretta AR, Kaur N, Prasad NP, Rentzepis
MP. 2005. Optical tracking of organically modified silica nanoparticles as DNA carriers.
A nonviral, nanomedicine approach for gene delivery. National Academy of Science of
the United States of America. 102: 279-84.
Walters CM, Roe F, Bugnicourt A, Franklin MJ, Stewart SP. 2003. Contributions of
antibiotic penetration, oxygen limitation, and low metabolic activity to tolerance of
Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramyacin. American Society
for Microbiology. 47: 317-23.