Tp. Fulcher et al., Demonstration of biofilm in infections crystalline keratopathy using ruthenium red and electron microscopy, OPHTHALMOL, 108(6), 2001, pp. 1088-1092
Objective: Bacterial biofilm formation has been implicated in the pathogene
sis of infectious crystalline keratopathy. Biofilm cannot be visualized by
electron microscopy without the addition of a fixative that stabilizes the
polysaccharide-rich bacterial extracellular matrix that surrounds the bacte
rial colonies in a biofilm. We used ruthenium red as a fixative to evaluate
corneal biopsy specimens for the presence of bacterial biofilm in three ca
ses of infectious crystalline keratopathy (ICK) and five cases of chronic m
icrobial keratitis without crystalline changes.
Design: Case series with clinicopathologic correlation.
Participants: Eight patients underwent corneal biopsy or therapeutic kerato
plasty as part of their management for chronic unresponsive microbial kerat
itis.
Methods: The corneal specimens removed were trisected for microbiology, pat
hology, and transmission electron microscopy (TEM). The TEM specimens were
fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer with 0.05% r
uthenium red.
Main Outcome Measures: Demonstration of bacterial biofilm with TEM.
Results: TEM demonstrated organisms with a surrounding extracellular matrix
consistent with a bacterial biofilm in the three cases of ICK but not in t
he five other cases of chronic microbial keratitis.
Conclusions: The presence of biofilm in ICK can be demonstrated with TEM wi
th appropriate fixation techniques that stabilize the bacterial extracellul
ar matrix. Biofilm stains intensely with periodic acid-Schiff because of th
e polysaccharide-rich extracellular matrix and weakly with Gram stain becau
se of the high proportion of nonviable organisms. Biofilm formation occurs
in ICK but probably not in chronic bacterial keratitis without crystalline
changes. Secretion of an extracellular matrix by bacteria to form a biofilm
is a response to a nutrient-deprived environment in which growth and repli
cation is depressed. The extracellular matrix of the biofilm may mask bacte
rial antigens, explaining the relative lack of inflammatory response in the
se infections. It may also be one of the mechanisms explaining the resistan
ce to in vivo antimicrobial therapy when in vitro sensitivities have been p
roven. Ophthalmology 2001;108:1088-1092 (C) 2001 by the American Academy of
Ophthalmology.