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; 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 (Harrison et al. 2005). 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 et al. 2005).
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 (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).
According to Brinkmann (2004), white blood cells in a
normal immune response 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. NETs bind to bacterial
cells, prevent the spread of infection, and kill the pathogen using
antimicrobial agents (toxins, chemicals, or antibodies) (Brinkmann
et al. 2004; Weinrauch et al. 2002). As a result of these above
2. Neutrophil
advantageous properties, we will use a bundle of nanowires to imitate a Figure
NET and
trap the extracellular
trap (NET).
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 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
Experimental synopsis
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). We will use a confocal laser
scanning microscope (CLSM) and its 3D imaging properties to examine the biofilm’s structure
and confirm the presence of pores.
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 follow Harrison et al. 2006 procedure on the creation of Pseudomonas
aeruginosa biofilm with the exception of using a scanning electron microscopy.
Synthesis of copper oxide nanowires
We will follow the methods outlined by Li et al. 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 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 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. We will look for successful assembly of the copper oxide nanowire bundles.
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.
EXPERIMENTAL
GROUP
EXPERIMENTAL TREATMENT
PARAMETERS
EXPECTED
RESULT
A
Control
B
Antibiotic only
C
Nanowire only
D
Nanowire + Antibiotic
No antibiotic
No copper-oxide nanowire
Antibiotic
No copper-oxide nanowire
No antibiotic
Copper-oxide nanowire
Antibiotic fused
Copper-oxide nanowire
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.
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
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 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). Each
treatment will consist of 24 wells out of the 96 (Ceri et al. 1999).
Sample Size
We expect little to no growth once we penetrate the biofilm layer of Pseudomonas
aeruginosa with our synthetic neutrophil net. To determine the sample size (N) we will a chisquared 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 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. We will need at least 13 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).
Conclusion
Positive outcomes of results
REFERENCES
Anwar H, Costerton JW. 1992. Effective use of antibiotics in the treatment of biofilm-associated
infections. American Society for Microbiology News. 58: 665–668.
Bao SJ, Li CM, Zang JF, Cui XQ, Qiao Y, Guo J. 2008. New nanostructured TiO2 for direct
electrochemistry and glucose sensor applications. Advanced Functional Materials. 18:
591-599.
Brinkmann V, Reichard U, Goosmann C, Fauler B, UhlemannY, Weiss SD, Weinrauch Y,
Zychlinsky A. 2004. Neutrophil extracellular traps kill bacteria. American Association
for the Advancement of Science. 303: 1532-1535.
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, Cheng KJ, Geesey GG, Ladd TI, Nickel JC, Dasgupta M, Marrie TJ. 1987.
Bacterial biofilms in nature and disease. Annual Reviews of Microbiology. 41: 435–464.
Costerton JW, Stewart PS, Greenberg EP. 1999. Bacterial biofilms: A common cause of
persistent infections. Science. 284: 1318-22.
Diaz F, Mosovich LL, Neter E. 1970. Serogroups of Pseudomonas aeruginosa and the immune
response of patients with cystic fibrosis. Journal of Infectious Diseases. 121: 269-274.
Diaz F, Neter E. 1970. Pseudomonas aeruginosa: Serogroups and antibody response in patients
with neoplastic diseases. American Journal of the Medical Sciences. 259: 340-345.
Govan JRW, Deretic V. 1996. Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas
aeruginosa and Burkholderia cepacia. Microbiological Reviews. 60: 539–574.
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.
Lu X, Zou G, Li J. 2007. Hemoglobin entrapped within a layered spongy Co3O4 based
nanocomposite featuring direct electron transfer and peroxidase activity. Journal of
Materials Chemistry. 17: 1427-1432.
Neter E. 1974. Pseudomonas aeruginosa infection and humoral antibody response of patients
with cystic fibrosis. The Journal of infectious diseases. 130: 132-133.
Nickel JC, Ruseska I, Wright JB, Costerton JW. 1985. Tobramycin resistance of Pseudomonas
aeruginosa cells growing as a biofilm on urinary catheter material. Journal of
Antimicrobial Agents and Chemotherapy. 27: 619-624.
Stewart PS. 1996. Theoretical aspects of antibiotic diffusion into microbial biofilms. Journal of
Antimicrobial Agents and Chemotherapy. 40: 2517-2522.
Tirouvanziam.R, Gernez Y, Conrad KC, Moss BR, Schrijver I, Dunn EC, Davies AZ,
Herzenberg AL, Herzenberg AL. 2007. Profound functional and signaling changes in
viable inflammatory neutrophills. National Academy of Science. 105: 4335-4339.
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.
Weinrauch Y, Drujan D, Shapiro SD, Weiss J, Zychlinsky A. 2002. Neutrophil elastase targets
virulence factors of enterobacteria. Nature. 417: 91-94.