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

aeruginosaFig. 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

•Conductive•Large surface area•Used as detectors in bioelectrochemistry

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

• 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 metalsMetal 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

Use of antibioticsUse of Ciprofloxacin and Tobramycin

antibiotics (Walters et al., 2003)

• 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

•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)

Tobramycin slower than ciprofloxacin

Ineffective antibiotic experiment

Ciprofloxacin Tobramycin

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.

Time (h)

Types of antibiotics used

Ciprofloxacin Tobramycin

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 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.