Download Biofilm - SCIE501Nanobots

Creating an artificial immune system
to deal with Psuedomonas
aeruginosa’s biofilm
Mark Ly, Fahima Nakitende, Shannon
Wesley
Human cystic fibrosis
• Recessive genetic
disorder
• Excess secretion
– Mucous
– Sweat
• Bacterial infection
– Pseudomonas
aeruginosa
Fig. 1. Age distribution of the Canadian CF population
for 2008.
Psuedomonas aeruginosa
• Protective slime layer
– 200μm thick
• Transport
• Antibiotic resistance
– Genetic mutation
– Accumulation of
environmental genes
Fig. 2. Biofilm formation
Nanowire bundles
Wires with a diameter in
the nanometer scale
A group of nanowires
Fig. 3. Transmission electron microscopy of
Cu(OH)2 nanowires (Zhuang et al. 2007)
•Conductive
•Large surface area
•Used as detectors in
bioelectrochemistry
Fig. 4. Image of ordered nanowire in a
microarray.
Research Question
• Develop a new method to treat biofilms using
nanowire bundles
• Can copper oxide nanowires carrying
antibiotics diffuse through the porous
strucuture of Pseudomonas aeruginosa’s
biofilm?
Why copper oxide
• Copper was one of the effective metals in
previous experiments
• Works well in biological settings from glucose
and hemoglobin experiments.
Proposed experiment:
Artificial neutriphil net
•Emulate our immune
system with nanowire
bundles couple with
antibiotics:Ciprofloxacin
and Tobramycin
•Use this net on biofilms to
get through the slime layer
more effectively.
Fig. 5. Image of a neutriphil net trapping
bacteria
Methods
Biofilm
• Following the experimental
design done by Harrison et
al.
• Use of high throughput
MBEC assay
• Degrade
Nanowire synthesis
• Following experimental
design done by Li et al.
• Self assembled nanowire
bundles
Expected results
• We expect to see no growth if the antibiotics
are able to penetrate the biofilm layer
effectively
Previous studies on Pseudomonas
aeruginosa
Use of Heavy metals
Use of antibiotics
Metal cations (Harrison et al., 2005)
Cobalt, copper, nickel, silver.
Use of Ciprofloxacin and Tobramycin
antibiotics (Walters et al., 2003)
• Need high concentrations of
metal cations to kill populations
• Persister cells are killed at a
slower rate
• Slow diffusion of tobramycin due
to binding
• Ciprofloxacin ineffective
• Oxygen may be limiting factor.
Drawbacks of
metal cations
•High concentrations
needed
•Long continuous exposure
time
Fig. 6. Log killing of biofilm cultures with increasing
concentration of Copper ions over a 27 hour period. (Harrison
et al., 2005).
Why the antibiotics
didn’t work
Tobramycin slower than ciprofloxacin
•Lack of oxygen restricts
bacterial metabolic activity
•Took long to penetrate
through the biofilm
Fig. 7. Penetration of tobramycin (squares) and
ciprofloxacin (circles) in P. aeruginosa. Open
symbols are in sterile controls (Walters et al.,
2003)
Ineffective antibiotic experiment
Ciprofloxacin
Tobramycin
Time (h)
Fig. 8. Killing of P. aeruginosa in biofilms in exposure
to ciprofloxacin. Filled squares were the treatment
and the unfilled were the controls (Walter et al.)
Fig. 9. Killing of P. aeruginosa in biofilms in exposure to
tobramycin. Filled squares were the treatment and the
unfilled were the controls.
Types of antibiotics used
Ciprofloxacin
Tobramycin
Literature cited
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•
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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.
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.
Li Y, Zhang Q, Li J. 2010. Direct electrochemistry of hemoglobin immobilized in CuO
nanowire bundles. Talanta. 83: 162-66.
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.
Canadian Cystic Fibrosis Foundation. 2008. Canadian cystic fibrosis patient data
registry report. Pg: 11,24.
Take home message
• Interdisciplinary aspects
• Pseudomonas
aeruginosa most
common and increasing
• Possible other
applications
Fig. 10. Comparative percentage of the types
of bacterial infections in CF patients in 2007
and 2008.