Biofilm Surface Density Determines Biocide Effectiveness

Abstract

High resistance of biofilms for chemical challenges is a serious industrial and medical problem. In this work a gradient of surface covered with biofilm has been produced and correlated to the effectiveness of different commercially available oxidative biocides. The results for thin Escherichia coli biofilms grown in rich media supplemented with glucose or lactose on glass or poly methyl methacrylate surfaces indicate that the effectiveness of hydrogen peroxide or chlorine dioxide and quaternary ammonium compounds is inversely proportional to the fraction of the surface covered with the biofilm. In areas where biofilm covered more than 90% of the available surface the biocide treatment was inefficient after 60 min of incubation. The combined effect of oxidant and surfactant increased the effectiveness of the biocide. On the other hand, the increased biofilm viscoelasticity reduced biocide effectiveness. The results emphasize differential biocide effectiveness depending on the fraction of the attached bacterial cells. The results suggest that biofilm biocide resistance is an acquired property that increases with biofilm maturation. The more dense sessile structures present lower log reductions compared to less dense ones.

Keywords: E. coli; antimicrobial; biocide; biofilms; surface coverage; viscoelasticity.

FIGURE 1

Biofilms grown on glass surface in the rich medium supplemented with glucose (A), biofilms grown on PMMA surface in the rich medium supplemented with lactose (B) after 48 h of incubation. Zone I – arbitrarily designed high-density region where biofilm covered 90% or more of the available surface, Zone II – biofilm covered between 10 and 90% of the available surface, Zone III – biofilm surface coverage was less than 10%. Columns represent a low magnification DIC micrographs taken every 0.8 mm in the vertical direction from the water–air interphase (total depth 20 mm). Three representative higher magnification micrographs for different zones are shown on the right of the columns. Scale bar on micrographs represents 20 μm.

FIGURE 2

The live/dead assay on cells in Zone I (A,B), Zone II (C,D), and Zone III (E,F) biofilms. Biofilms were grown for 48 h in rich growth medium supplemented with glucose on glass surfaces and treated with Klercide B for 20 min. Scale bars in (A) and (B) represent 100 μm, in other panels they represent 50 μm.

FIGURE 3

Fraction of the dead cells in different biofilm zones. Biofilms of E. coli were grown in the rich medium with glucose on glass surface for 48 h. Different biocides were added to for 2, 20, and 60 min. The average values and standard errors are given (n = 9).

FIGURE 4

Effectiveness of different biocides. Biofilms of E. coli grown in the rich medium supplemented with lactose on PMMA surface for 48 h. Biocides were added for 2, 20, and 60 min. The average values and standard errors are given (n = 9).

FIGURE 5

Fractions of the dead cells in planktonic culture of E. coli grown in the rich medium with glucose. Cells were grown to cell density of 10⁷ cells/mL and treated with different biocides (A). Cells treated with Klercide B at different cell densities (B). Bacterial suspensions with different cell density were treated with biocides for 20 min. The average values and standard errors are given (n = 9).

FIGURE 6

Viscosity curves for the confluent biofilm of E. coli grown in the rich medium with glucose or lactose after 24 h of incubation on agar plates.

FIGURE 7

Viscoelastic moduli G′ and G″ as a function of strain amplitude for the confluent E. coli biofilms grown in the rich medium supplemented with glucose or lactose after 24 h of incubation on agar plates.