Confocal imaging of biofilm formation process using fluoroprobed Escherichia coli and fluoro-stained exopolysaccharide.
We developed a novel method of evaluating biofilm architecture on a synthetic material using green fluorescent protein-expressing Escherichia coli and red fluorescence staining of exopolysaccharides. Confocal laser scanning microscopy observation revealed the time course of the change in the in situ three-dimensional structural features of biofilm on a polyurethane film without structural destruction: initially adhered cells are grown to form cellular aggregates and secrete exopolysaccharides. These cells were spottily distributed on the surface at an early incubation time but fused to form a vertically grown biofilm with incubation time. Fluorescence intensity, which is a measure of the number of cells, determined using a fluorometer and biofilm thickness determined from confocal laser scanning microscopy vertical images were found to be effective for quantification of time-dependent growth of biofilms. The curli (surface-located fibers specifically binding to fibronectin and laminin)-producing Escherichia coli strain, YMel, significantly proliferated on fibronectin-coated polyurethane, whereas the curli-deficient isogenic mutant, YMel-1, did not. The understanding of biofilm architecture in molecular and morphological events and new fluorescence microscopic techniques may help in the logical surface design of biomaterials with a high antibacterial potential.
Maeyama
Ryo
R
Department of Biomedical Engineering, Faculty of Medical Sciences, Kyushu University, Maidashi, Fukuoka 812-8582, Japan.
Mizunoe
Yoshimitsu
Y
Anderson
James M
JM
Tanaka
Masao
M
Matsuda
Takehisa
T
D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000235:genetics; D018441:Biofilms / Q000254:growth & development*; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D008422:Materials Testing; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis; D011140:Polyurethanes; D011994:Recombinant Proteins / Q000235:genetics; D013499:Surface Properties
D001426:Bacterial Proteins; D011135:Polysaccharides, Bacterial; D011140:Polyurethanes; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; C077481:Crl protein, Bacteria