Journal of Histochemistry and Cytochemistry, Vol. 47, 1648d-1649, December 1999, Copyright © 1999, The Histochemical Society, Inc.


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27 Three-dimensional analysis of Streptococcus mutans biofilms: the relationship between biofilm structure and sensitivity to a biocide.

J. E. Mazurkiewicza, K. R. O. Hazletta, and J. A. Banasa
a Department of Microbiology, Immunology and Molecular Genetics, Albany Medical College, Albany, NY, 12208

Biofilms are matrix-enclosed bacterial populations adherent to each other and to surfaces or interfaces. Microcolonies that develop within a biofilm change their shape and size in respose to environmental conditions. Biofilm-grown bacteria also appear to possess an increased resistance to the action of antimicrobials. The gbpA gene of Streptococcus mutans codes for a non-enzymatic glucan-binding protein that contributes to the plaque-forming ability of these bacteria thus contributing to biofilm structure. In a gnotobiotic rat model, inactivation of the gbpA gene increased cariogenicity and promoted the accumulation of virulence-attenuating gtfBC recombinant bacteria. The gtfBC recombination occurred between two contiguous glucosyltransferase genes encoding enzymes that synthesize the glucan polysaccharide that is necessary for plaque formation and resuted in a reduction in the total amount produced. The change in cariogenicity was hypothesized to result from changes in the biofilm structure (1). To study the relationship between biofilm structure and antimicrobial resistance, confocal laser scanning microscopy was used to analyze biofilms generated by wild type (wt) and gbpA mutant strains of S. mutans, and a gbpA strain containing 25% gtfBC recombinant organisms. Optical sections of biofilms stained with LIVE Baclight fluorescent dye were collected at 1 um steps to a sample depth of ~130 um. Maximum intensity projection images were constructed, TIFF images of Y-Z slices were made, and dimensions of the microcolonies were measured. Wt biofilms consisted of large microcolonies that incompletely coat the hydroxyapatite surface on which they grew; gbpA mutant biofilms consisted of a uniform layer of smaller microcolonies that almost entirely coated the surface, and biofilms formed by the gbpA/25% gtfBC strain were similar to wt. The biofilms displayed differential susceptibility when challenged with ampicillin: gbpA mutant biofilm organisms were more susceptible than organisms in either the wt or gbpA/gtfBC biofilms. As the percentage of gtfBC organisms within a mixed biofilm of gbpA and gbpA/gtfBC organisms was increased, both the morphology and the sensitivity to ampicillin more closely resembled that of the wt. These data present direct evidence that both polysaccharide polymers and the proteins that bind then are essential for optimal biofilm development and can also influence virulence. (1) Infect. Immun. 66:218-85, 1998





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