title: The impact of cellular debris on Pseudomonas aeruginosa adherence to silicone hydrogel contact lenses and contact lens storage cases.
abstract: OBJECTIVE
  To evaluate neutrophil-enhanced Pseudomonas aeruginosa (PA) biofilm formation on silicone hydrogel contact lenses and to determine the effect of epithelial biodebris on PA adherence in contact lens storage cases.
  
  
  METHODS
  A fully invasive PA corneal isolate stably conjugated to green fluorescent protein was used. Unworn lotrafilcon A contact lenses were incubated at various ratios of PA to polymorphonuclear neutrophil (PMN) for 24 hours at 37°C. Lens-associated PA was evaluated using laser scanning confocal microscopy and nonviable PA were visualized using propidium iodide. Viable bacteria were enumerated by colony-forming unit (CFU) analysis. For acute epithelial cell studies, PA viability was determined after coincubation with freeze-thaw epithelial cell lysates in 96-well polystyrene plates. Levels of residual cellular debris and bacterial viability were further assessed in used contact lens storage cases.
  
  
  RESULTS
  Laser scanning confocal microscopy demonstrated that cotreatment with PMA-stimulated neutrophils increased PA adherence over 24 hours to lens surfaces with a striking alteration of PA architecture. Propidium iodide staining showed that the adherent bacteria consisted of a mixture of viable and nonviable PA; a PMN-associated increase in viable PA was confirmed by CFU (PA:PMN 0.1:1, P = 0.025; PA:PMN 1:1, P = 0.005). Acute epithelial cell debris studies revealed a significant increase in viable PA in 96-well plates in the presence of epithelial freeze-thaw lysates (PA:debris 1:1, P = 0.002; PA:debris 100:1, P = 0.002). Crystal violet staining of used lens storage cases revealed residual cellular debris at all time points, which was independent of microbial contamination; all lens cases used for periods of 9 months or more were uniformly associated with high levels of viable microorganisms.
  
  
  CONCLUSIONS
  These results demonstrate that prolonged corneal inflammation with the presence of PMNs when confronted with simultaneous PA challenge in extended contact lens wear has the potential to stimulate biofilm formation on silicone hydrogel contact lenses. These findings further suggest that a persistent buildup of extracellular debris in lens storage cases may contribute to the heavy biofilms reported on these surfaces.
authors.affiliation: Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9057, USA.
authors.forename: Geoffrey W
authors.initials: GW
authors.lastname: Burnham
authors.forename: H Dwight
authors.initials: HD
authors.lastname: Cavanagh
authors.forename: Danielle M
authors.initials: DM
authors.lastname: Robertson
mesh_terms: D001422:Bacterial Adhesion / Q000502:physiology*; D018441:Biofilms / Q000254:growth & development; D002470:Cell Survival; D015169:Colony Count, Microbial; D003263:Contact Lenses, Hydrophilic / Q000382:microbiology*; D004209:Disposable Equipment / Q000382:microbiology; D004847:Epithelial Cells / Q000502:physiology; D004866:Equipment Contamination; D006801:Humans; D020100:Hydrogels; D009504:Neutrophils / Q000502:physiology; D011550:Pseudomonas aeruginosa / Q000502:physiology*; D012828:Silicones
chemical_list: D020100:Hydrogels; D012828:Silicones