Broad-spectrum biofilm inhibition by a secreted bacterial polysaccharide

Abstract

The development of surface-attached biofilm bacterial communities is considered an important source of nosocomial infections. Recently, bacterial interference via signaling molecules and surface active compounds was shown to antagonize biofilm formation, suggesting that nonantibiotic molecules produced during competitive interactions between bacteria could be used for biofilm reduction. Hence, a better understanding of commensal/pathogen interactions within bacterial community could lead to an improved control of exogenous pathogens. To reveal adhesion or growth-related bacterial interference, we investigated interactions between uropathogenic and commensal Escherichia coli in mixed in vitro biofilms. We demonstrate here that the uropathogenic strain CFT073 and all E. coli expressing group II capsules release into their environment a soluble polysaccharide that induces physicochemical surface alterations, which prevent biofilm formation by a wide range of Gram-positive and Gram-negative bacteria. We show that the treatment of abiotic surfaces with group II capsular polysaccharides drastically reduces both initial adhesion and biofilm development by important nosocomial pathogens. These findings identify capsular polymers as antiadhesion bacterial interference molecules, which may prove to be of significance in the design of new strategies to limit biofilm formation on medical in dwelling devices.

Fig. 1.

Biofilm inhibitory effect of CFT073. (A) Biofilm formation of MG1655 F′ in microfermentors inoculated with 1 or 10 OD₆₀₀ equivalent of KS272 (gray bars) or CFT073 (black bars) cells. White bars indicate MG1655 F′ biofilm alone (Ø). Results are the average of six replicates ± SD. P < 0.001 compared with MG1655 F′ biofilm. (B) Microtiter plate MG1655 F′ biofilm alone (Ø) or in the presence of KS272 or CFT073 supernatant (S.KS272 and S.CFT073, respectively). (C) MG1655 F′ biofilm in microfermentors perfused with medium without supernatant (Ø) or with S.KS272 or S.CFT073. (D) Growth curves of MG1655 F′ alone (Ø) or with S.KS272 or S.CFT073. (E) MG1655 F′ cell viability alone (Ø) or with S.KS272 or S.CFT073 visualized with BacLight staining.

Fig. 2.

Effect of CFT073 supernatant on Gram-positive and Gram-negative bacterial biofilm formation. (A) Quantification of the microtiter plate biofilm formation of different bacteria alone (Ø) or with KS272 (S.KS) or CFT073 (S.CFT) supernatant. Levels of crystal violet retained were measured spectrophotometrically (OD₅₇₀). (B) Quantification of biofilm formed by several pathogenic bacteria in microfermentors by using media not supplemented (Ø) or supplemented with S.CFT or S.KS. Error bars represent SD of two independent experiments.

Fig. 3.

Relationship between capsule production and CFT073 anti-biofilm activity. (A) Genetic organization of the CFT073 capsule R1, R2, and R3 regions. Genes with transposon insertions are marked with an asterisk. (B) Biofilm formation of MG1655 F′ cultivated in the presence of capsule mutant supernatants. (C) Hexose levels in supernatants. kpsF, kpsU, c3692, and c3693 correspond to mutants that do not impair capsule production. (D) Stationary-phase CFT073 or CFT073ΔkpsD bacterial cell capsules stained with ferritin and examined by transmission electron microscopy (×100,000). (Scale bar: 0.2 μm.) (Left) A total of 125 and 105 cells were observed, respectively. Stained CFT073 capsule is indicated by an arrow. (Right) Scanning electron micrographs of stationary-phase CFT073 or CFT073ΔkpsD (×50,000). (Scale bar: 0.5 μm.) A total of 45 and 37 cells were observed, respectively.

Fig. 4.

Physicochemical properties of CFT073 supernatant. (A) ζ Potential of cationic colloids incubated with dialyzed supernatants from: CFT073 (S.CFT), U-9, (S.U9), IHE3034 (S.IHE), ECOR72 (S.E-72), and their respective capsule kpsD mutants when applicable. Ø corresponds to M63B1glu medium. (B) Propidium iodide adsorption onto cationic particles incubated with supernatants of S.CFT, S.U-9, S.IHE, S.E-72, and their respective capsule mutants when applicable. FR2:CFT073 supernatant purified fraction. The extent of adsorption is given by the fluorescent intensity (>670 nm). Error bars represent SD of the mean. (C) Fluorescence microscopy of cationic particles incubated with S.CFT, S.ΔkpsD, FR2, and M63B1 (Ø).

Fig. 5.

Biofilm inhibition effect of CFT073 supernatant on treated surfaces. Biofilm formation in microfermentors by several bacteria by using the following: untreated glass slides (Top), glass slides treated with CFT073 supernatant (Middle), and glass slides treated with CFT073ΔkpsD supernatant (Bottom).

Fig. 6.

CFT073 supernatant affects cell–cell interaction. (A) MG1655F′ biofilm formation in microfermentors with media supplemented with CFT073 supernatant (S.CFT) at times 0, 1, 6 (24 h of culture), and 24 h (48 h of culture). Ø, no addition of S.CFT. (B) GFP-tagged MG1655 F′ inoculated in a flow cell and monitored by confocal microscopy. CFT073 or KS272 supernatants were supplemented after 3 h of culture, and biofilms were grown for 12 h total. (C) Autoaggregation assay with strains that aggregate via different mechanisms: MG1655 F′ (F conjugative pilus expression); MG1655ompR234 (curli overexpression); MG1655ΔoxyR (Ag43 autotransporter adhesin overexpression); and 1094 (cellulose production). Cells were diluted to OD₆₀₀ = 2 in 3 ml of M63B1 (▴), CFT073 supernatant (·), and ΔkpsD supernatant ().