title: Corneal Biofilms: From Planktonic to Microcolony Formation in an Experimental Keratitis Infection with Pseudomonas Aeruginosa.
abstract: OBJECTIVE
  Microbial biofilms commonly comprise part of the infectious scenario, complicating the therapeutic approach. The purpose of this study was to determine in a mouse model of corneal infection if mature biofilms formed and to visualize the stages of biofilm formation.
  
  
  METHODS
  A bacterial keratitis model was established using Pseudomonas aeruginosa ATCC 9027 (1 × 10(8) CFU/ml) to infect the cornea of C57BL/6 black mouse. Eyes were examined post-infection (PI) on days 1, 2, 3, 5, and 7, and imaged by slit lamp microscopy, and light, confocal, and electron microscopy to identify the stages of biofilm formation and the time of appearance.
  
  
  RESULTS
  On PI day 1, Gram staining showed rod-shaped bacteria adherent on the corneal surface. On PI days 2 and 3, bacteria were seen within webs of extracellular polymeric substance (EPS) and glycocalyx secretion, imaged by confocal microscopy. Scanning electron microscopy demonstrated microcolonies of active infectious cells bound with thick fibrous material. Transmission electron microscopy substantiated the formation of classical biofilm architecture with P. aeruginosa densely packed within the extracellular polymeric substances on PI days 5 and 7.
  
  
  CONCLUSIONS
  Direct visual evidence showed that biofilms routinely developed on the biotic surface of the mouse cornea. The mouse model can be used to develop new approaches to deal therapeutically with biofilms in corneal infections.
authors.affiliation: Singapore Eye Research Institute (SERI), Singapore.
authors.forename: Padmanabhan
authors.initials: P
authors.lastname: Saraswathi
authors.affiliation: Singapore Eye Research Institute (SERI), Singapore; Duke-NUS SRP Neuroscience and Behavioural Disorders and Emerging Infectious Diseases, Singapore; Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. Electronic address: rwbeuerman@gmail.com.
authors.forename: Roger W
authors.initials: RW
authors.lastname: Beuerman
mesh_terms: D000818:Animals; D018441:Biofilms; D015169:Colony Count, Microbial; D003315:Cornea / Q000382:microbiology* / Q000648:ultrastructure; D004195:Disease Models, Animal; D015818:Eye Infections, Bacterial / Q000382:microbiology* / Q000473:pathology; D007634:Keratitis / Q000382:microbiology* / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D017348:Microscopy, Electron, Scanning Transmission; D011552:Pseudomonas Infections / Q000382:microbiology* / Q000473:pathology; D011550:Pseudomonas aeruginosa / Q000502:physiology*
keywords: Pseudomonas aeruginosa; biofilm; imaging; keratitis; microscopy; polymeric extracellular substance