Creating an artificial immune system to deal with psuedomonas aeruginosa s biofilm
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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.

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Creating an artificial immune system to deal with psuedomonas aeruginosa s biofilm

Creating an artificial immune system to deal with Psuedomonasaeruginosa’sbiofilm

Mark Ly, FahimaNakitende, Shannon Wesley


Human cystic fibrosis
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
Psuedomonasaeruginosa

  • Protective slime layer

    • 200μm thick

  • Transport

  • Antibiotic resistance

    • Genetic mutation

    • Accumulation of environmental genes

Fig. 2. Biofilm formation


Nanowire bundles
Nanowire bundles

Wires with a diameter in the nanometer scale

A group of nanowires

Conductive

Large surface area

Used as detectors inbioelectrochemistry

Fig. 3. Transmission electron microscopy of Cu(OH)2 nanowires (Zhuang et al. 2007)

Fig. 4. Image of ordered nanowire in a microarray.


Research question
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’sbiofilm?


Why copper oxide
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
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
Methods

Biofilm

Nanowire synthesis

Following experimental design done by Li et al.

Self assembled nanowire bundles

  • Following the experimental design done by Harrison et al.

  • Use of high throughput MBEC assay

  • Degrade


Expected results
Expected results

  • We expect to see no growth if the antibiotics are able to penetrate the biofilm layer effectively


Previous studies on pseudomonas aeruginosa
Previous studies on Pseudomonas aeruginosa

Use of Heavy metals

Use of antibiotics

Use of Ciprofloxacin and Tobramycin antibiotics (Walters et al., 2003)

Slow diffusion of tobramycin due to binding

Ciprofloxacin ineffective

Oxygen may be limiting factor.

Metal cations (Harrison et al., 2005)

Cobalt, copper, nickel, silver.

  • Need high concentrations of metal cations to kill populations

  • Persister cells are killed at a slower rate


Drawbacks of metal cations
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
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
Ineffective antibiotic experiment

Ciprofloxacin

Tobramycin

Time (h)

Fig. 8. Killing of P. aeruginosain biofilms in exposure to ciprofloxacin. Filled squares were the treatment and the unfilled were the controls (Walter et al.)

Fig. 9. Killing of P. aeruginosain biofilms in exposure to tobramycin. Filled squares were the treatment and the unfilled were the controls.


Types of antibiotics used
Types of antibiotics used

Ciprofloxacin

Tobramycin


Literature cited
Literature cited

  • 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 aeruginosabiofilms. 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 CuOnanowire 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 aeruginosabiofilms 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
Take home message

  • Interdisciplinary aspects

  • Pseudomonas aeruginosamost common and increasing

  • Possible other applications

Fig. 10. Comparative percentage of the types of bacterial infections in CF patients in 2007 and 2008.