Anti-Biofilm Activity of a Hyaluronan-like Exopolysaccharide from the Marine Vibrio MO245 against Pathogenic Bacteria

Abstract

Biofilms, responsible for many serious drawbacks in the medical and marine environment, can grow on abiotic and biotic surfaces. Commercial anti-biofilm solutions, based on the use of biocides, are available but their use increases the risk of antibiotic resistance and environmental pollution in marine industries. There is an urgent need to work on the development of ecofriendly solutions, formulated without biocidal agents, that rely on the anti-adhesive physico-chemical properties of their materials. In this context, exopolysaccharides (EPSs) are natural biopolymers with complex properties than may be used as anti-adhesive agents. This study is focused on the effect of the EPS MO245, a hyaluronic acid-like polysaccharide, on the growth, adhesion, biofilm maturation, and dispersion of two pathogenic model strains, Pseudomonas aeruginosa sp. PaO1 and Vibrio harveyi DSM19623. Our results demonstrated that MO245 may limit biofilm formation, with a biofilm inhibition between 20 and 50%, without any biocidal activity. Since EPSs have no significant impact on the bacterial motility and quorum sensing factors, our results indicate that physico-chemical interactions between the bacteria and the surfaces are modified due to the presence of an adsorbed EPS layer acting as a non-adsorbing layer.

Keywords: Pseudomonas aeruginosa; Vibrio harveyi; anti-adhesive activity; anti-biofilm activity; exopolysaccharide; marine bacteria; non biocidal agent.

Figure 1

Structure of (A) MO245 and (B) HA.

Figure 2

Impact of MO245 or HA (125 µg/mL) on the growth of (A) P. aeruginosa in LB medium at 37 °C and (B) V. harveyi in Zobell medium at 28 °C for 30 h under 125 rpm agitation. Average of three independent replicates ± standard deviation. Bactericidal effect and consumption of MO245 or HA (125 µg/mL) or glucose (4 g/L) as a carbon source in M9 medium for (C) P. aeruginosa at 37 °C and (D) V. harveyi at 28 °C for 26 h under 125 rpm agitation.

Figure 3

Conditions of use of MO and HA during adhesion. (A) Addition of MO245 or HA within the bacterial suspension and (B) glass slide conditioned with MO245 or HA.

Figure 4

Impact of MO245 and HA on a 24 h P. aeruginosa biofilm maturation. (A) Biomass and average thickness quantification after COMSTAT analysis of confocal laser microscopy observations. (B) Confocal laser microscopy observation (Syto9^®) without or with the addition of MO245 or HA at 125 µg/mL in the LB growth medium. Data represent the mean ± the standard deviation. * represents the significant difference at α 5%: p < 0.05, *** represents the significant difference at α 5%: p < 0.001.

Figure 5

Impact of MO245 and HA on a 24 h V. harveyi biofilm maturation. (A) Biomass and average thickness quantification after COMSTAT analysis of confocal laser microscopy observation. (B) Confocal laser microscopy observation (Syto9^®) without or with the addition of MO245 or HA at 125 µg/mL in the Zobell growth medium. Data represent the mean ± the standard deviation. ** represents the significant difference at α 5%: p < 0.01; *** represents the significant difference at α 5%: p < 0.001.

Figure 6

Impact of MO245 and HA on the degradation of a 24 h P. aeruginosa biofilm already formed. (A) Biomass and average thickness quantification after COMSTAT analysis of confocal laser microscopy observation. (B) Confocal laser microscopy observation (Syto9^®) without or with the addition of MO245 or HA at 125 µg/mL on a 24 h biofilm already formed. Data represent the mean ± the standard deviation. * represents the significant difference at α 5%: p < 0.05, ** represents the significant difference at α 5%: p < 0.01.

Figure 7

Impact of MO245 and HA on the degradation of a 24 h V. harveyi biofilm already formed. (A) Biomass and average thickness quantification after COMSTAT analysis of confocal laser microscopy observation. (B) Confocal laser microscopy observation (Syto9^®) without or with the addition of MO245 or HA at 125 µg/mL on a 24 h biofilm already formed. Data represent the mean ± the standard deviation. ** represents the significant difference at α 5%: p < 0.01, *** represents the significant difference at α 5%: p < 0.001.

Figure 8

Motility assay of (A) P. aeruginosa and (B) V. harveyi incubated with MO245 or HA at 125 µg/mL or nothing for 2 h at room temperature. Diameters were measured after the overnight incubation of agar plates at 37 °C for P. aeruginosa and 28 °C for V. harveyi. Data represent the mean ± the standard deviation. * represents the significant difference at α 5%: p < 0.05.

Figure 9

Anti-quorum sensing effect of MO245, HA, and kojic acid (KA) at 125 µg/mL on (A) the biosensor E. coli pSB401 and (B) V. harveyi for 9 h at 28 °C under 125 rpm agitation. Luminescence and O.D.₆₀₀ were measured every hour and RLU ratios were calculated.

Figure 10

Percentage hydrophobicity of P. aeruginosa and V. harveyi in the presence of MO245 or HA. A total of 10⁹ bacteria were put in contact with MO245 or HA at 125 µg/mL for 2 h and then in contact with toluene. At the appearance of a phase separation, the aqueous phase was recovered and the optical density was measured. The percentage of hydrophobicity was then calculated. * represents the significant difference at α 5%: p < 0.05.

Figure 11

Evolution of the frequency F3 versus time for MO245 and HA on silica-coated quartz crystal (pH 7, 150 mM NaCl). MO245 and HA were injected at t = 0 min. After the adsorption of MO245 on the surface of silica-coated quartz crystal, rinsing was performed to observe if desorption occurred.

Figure 12

The water contact angle of a surface conditioned with MO245 or HA at 125 µg/mL measured with a Digidrop. A total of 3 mL of MO245 and HA were deposited on a glass slide and allowed to evaporate in a sterile environment. Contact angles were measured at room temperature with a volume of 3 µL. *** represents the significant difference at α 5%: p < 0.001.

Figure 13

Emulsifying properties of MO245, HA, and Triton X-100 at 0.25% (w/v) in the oil-in-water phase over time. The emulsion index was calculated after 1 h, 24 h, 48 h, and 168 h at 20 °C.

Figure 14

Summary scheme of the potential anti-biofilm mode of action of MO245.