Rhamnolipids and surfactin inhibit the growth or formation of oral bacterial biofilm

Abstract

Background: Bacteria survive in various environments by forming biofilms. Bacterial biofilms often cause significant problems to medical instruments and industrial processes. Techniques to inhibit biofilm formation are essential and have wide applications. In this study, we evaluated the ability of two types of biosurfactants (rhamnolipids and surfactin) to inhibit growth and biofilm formation ability of oral pathogenic bacteria such as Aggregatibacter actinomycetemcomitans, Streptococcus mutans, and Streptococcus sanguinis.

Results: Rhamnolipids inhibited the growth and biofilm formation ability of all examined oral bacteria. Surfactin showed effective inhibition against S. sanguinis ATCC10556, but lower effects toward A. actinomycetemcomitans Y4 and S. mutans UA159. To corroborate these results, biofilms were observed by scanning electron microscopy (SEM) and confocal microscopy. The observations were largely in concordance with the biofilm assay results. We also attempted to determine the step in the biofilm formation process that was inhibited by biosurfactants. The results clearly demonstrated that rhamnolipids inhibit biofilm formation after the initiation process, however, they do not affect attachment or maturation.

Conclusions: Rhamnolipids inhibit oral bacterial growth and biofilm formation by A. actinomycetemcomitans Y4, and may serve as novel oral drug against localized invasive periodontitis.

Keywords: Biofilm inhibition; Oral bacteria; Rhamnolipids; Surfactin.

Fig. 1

Growth inhibition of oral pathogenic bacteria by biosurfactants. Total growth of (a, d) A. actinomycetemcomitans Y4, (b, e) S. mutans UA159, and (c, f) S. sanguinis ATCC10556 by rhamnolipids (a-c) or surfactin (d-f). Rhamnolipid was used at a final concentration ranging from 6.5 w/v% to 1.21 × 10^− 8 w/v% (a 2-fold serial dilution was applied) in BHI broth. Surfactin was used at a final concentration ranging from 10.36 w/v% to 1.93 × 10^− 8 w/v% (a 2-fold serial dilution was applied) in BHI broth. Error bars indicate standard deviations of at least three experiments

Fig. 2

Inhibition of biofilm formation by biosurfactants. Total biofilm formation of (a, d) A. actinomycetemcomitans Y4, (b, e) S. mutans UA159, and (c, f) S. sanguinis ATCC10556 by rhamnolipids (a-c) or surfactin (d-f). Rhamnolipids was used at a final concentration ranging from 6.5 w/v% to 1.21 × 10^− 8 w/v% (a 2-fold serial dilution was applied) in BHI broth. Surfactin was used at a final concentration ranging from 10.36 w/v% to 1.93 × 10^− 8 w/v% (a 2-fold serial dilution was applied) in BHI broth. Error bars indicate standard deviations of at least three experiments

Fig. 3

Biofilm observation. Representative images of A. actinomycetemcomitans Y4 (a, d, g), S. mutans UA159 (b, e, h), and S. sanguinis ATCC10556 (c, f, i) biofilms as visualized by SEM. The SEM images for the following samples is shown: untreated control (0 w/v%), rhamnolipid treatment (0.65 w/v%), and surfactin treatment (1.04 w/v%)

Fig. 4

Effect of rhamnolipids on each step of biofilm formation. a Graphical representation of the total biofilm formed by A. actinomycetemcomitans Y4 treated with rhamnolipids (same as Fig. 2a). The image on the right shows SEM image of A. actinomycetemcomitans Y4 biofilms after incubation with 0.013 w/v% rhamnolipid. b Graphical representation of the total A. actinomycetemcomitans Y4 biofilm formed on pre-treated rhamnolipid microtiter plate. This assay demonstrates inhibition of the attachment step of biofilm formation. The image on the right shows SEM image of A. actinomycetemcomitans Y4 biofilm upon pre-treatment with 0.013 w/v% rhamnolipid. c Graphical representation of the total A. actinomycetemcomitans Y4 biofilm formed after rhamnolipid dispersal. This assay demonstrates the effect of rhamnolipids against matured biofilm. The image on the right shows SEM images of A. actinomycetemcomitans Y4 biofilms after dispersal treatment with 0.013 w/v% rhamnolipid. Black arrows indicate the rhamnolipid concentration that exhibited highest inhibitory effect in (a) (0.013 w/v%). Error bars indicate standard deviations of at least three experiments. Scale bars indicate 3 μm