Influence of culture heterogeneity in cell surface charge on adhesion and biofilm formation by Enterococcus faecalis

Abstract

Biofilm formation is an increasing problem in medicine, due to the intrinsic resistance of microorganisms in the biofilm mode of growth against the host immune system and antimicrobial therapy. Adhesion is an important step in biofilm formation, influenced, among other factors, by the surface hydrophobicities and charges of both the substratum and the adhering microorganisms. Enterococcus faecalis strains generally display subpopulations with different surface charges, expressed as bimodal zeta potential distributions. Two-thirds of E. faecalis strains isolated from clogged biliary stents displayed such heterogeneity of surface charges in culture. In this study, the influence of this culture heterogeneity on initial adhesion and subsequent biofilm formation was investigated. Heterogeneous strains were retained in higher numbers on polystyrene than homogeneous strains. Also, biofilm formation was much more pronounced for heterogeneous strains than for homogeneous strains. In a population enriched to display only one subpopulation, fewer bacteria were retained than in its original heterogeneous culture. Also, the enriched subpopulation formed less biofilm than its original heterogeneous culture. The presence of ox bile during adhesion resulted in fewer retained bacteria, although heterogeneous strains were still retained in significantly higher numbers than were homogeneous strains, and, in general, the presence of ox bile reduced biofilm formation. The initial adhesion and biofilm formation were independent of the presence of the gene encoding the enterococcal surface protein (esp) or the expression of gelatinase (GelE). It is concluded that heterogeneity in cell surface charge represents an advantage for bacteria in the colonization of surfaces.

FIG. 1.

Percentage distributions of subpopulations of the fresh clinical isolates E. faecalis BS4126, BS12297, BS385, BS1037, BS11297, and BS937. Distributions were determined with zeta potential measurements in 10 mM potassium phosphate (pH 7.0). In each case, the more-negative fraction is indicated in black and the less-negative fraction in white. The bars denote standard deviations for three experiments, with each experiment comprising 100 bacteria.

FIG. 2.

Biofilm formation by E. faecalis BS4126, BS12297, BS385, BS1037, BS11297, and BS937 after 2 h of adhesion in the microtiter plate wells. The ability of the strains to form biofilms was expressed relative to the biofilm-forming ability of E. faecalis BS4126 in TSB containing 0.5% glucose. Biofilms were grown for 24 h in TSB containing 0.5% glucose in the absence (A) and presence (B) of 50 mg/ml ox bile. Heterogeneous strains are indicated in black and homogeneous strains in white. All data represent the means ± standard deviations for 15 determinations (three independent experiments, each performed five times).

FIG. 3.

Biofilm formation by heterogeneous E. faecalis BS385 and its subpopulation enriched in the more negatively charged bacteria. Note that in this case, biofilm formation was expressed relative to the biofilm-forming ability of E. faecalis BS385 (A₅₇₅, 1.71). All data represent the means ± standard deviations for 15 determinations (three independent experiments, each performed five times).

FIG. 4.

Examples of CLSM images of biofilm growth of heterogeneous and homogeneous strains. The biofilm formed by the heterogeneous strain BS4126 (A) had a thickness of 42.9 μm, and the biofilm formed by the homogeneous strain BS11297 (B) had a thickness of 19.5 μm.