Osteopontin reduces biofilm formation in a multi-species model of dental biofilm

Abstract

Background: Combating dental biofilm formation is the most effective means for the prevention of caries, one of the most widespread human diseases. Among the chemical supplements to mechanical tooth cleaning procedures, non-bactericidal adjuncts that target the mechanisms of bacterial biofilm formation have gained increasing interest in recent years. Milk proteins, such as lactoferrin, have been shown to interfere with bacterial colonization of saliva-coated surfaces. We here study the effect of bovine milk osteopontin (OPN), a highly phosphorylated whey glycoprotein, on a multispecies in vitro model of dental biofilm. While considerable research effort focuses on the interaction of OPN with mammalian cells, there are no data investigating the influence of OPN on bacterial biofilms.

Methodology/principal findings: Biofilms consisting of Streptococcus oralis, Actinomyces naeslundii, Streptococcus mitis, Streptococcus downei and Streptococcus sanguinis were grown in a flow cell system that permitted in situ microscopic analysis. Crystal violet staining showed significantly less biofilm formation in the presence of OPN, as compared to biofilms grown without OPN or biofilms grown in the presence of caseinoglycomacropeptide, another phosphorylated milk protein. Confocal microscopy revealed that OPN bound to the surface of bacterial cells and reduced mechanical stability of the biofilms without affecting cell viability. The bacterial composition of the biofilms, determined by fluorescence in situ hybridization, changed considerably in the presence of OPN. In particular, colonization of S. mitis, the best biofilm former in the model, was reduced dramatically.

Conclusions/significance: OPN strongly reduces the amount of biofilm formed in a well-defined laboratory model of acidogenic dental biofilm. If a similar effect can be observed in vivo, OPN might serve as a valuable adjunct to mechanical tooth cleaning procedures.

Figure 1. Quantification of biofilm formation by crystal violet staining.

Biofilms were grown in flow channels for 30 h on 1/10 diluted THB containing 26.5 µmol/L OPN, 26.5 µmol/L CGMP or none of the two proteins. A. Photograph showing biofilms grown with (right channel) and without OPN (left channel) after crystal violet staining. When OPN was present in the medium, less biofilm formed in the flow channels. B. Quantification of the biofilm biomass by spectrophotometry. OD₅₈₅ was significantly lower when biofilms were grown in the presence of OPN (+OPN), as compared to biofilms grown on THB only (−OPN). No such effect was observed when CGMP was present in the medium (CGMP). Error bars indicate standard deviations.

Figure 2. Viability of the organisms in the biofilms.

Biofilms grown without OPN (A) and with OPN in the medium (B) were stained with BacLight. Viable bacteria appear green and membrane-compromised bacteria red. The presence of OPN in the medium did not affect bacterial viability in the biofilms. Bars  = 20 µm.

Figure 3. Binding of OPN to bacteria in the biofilms.

After growth phase, a biofilm was incubated with fluorescently labelled OPN for 45 min at 35°C. OPN (green) bound to bacterial cell surfaces. Note that chains of streptococci can be recognized, although no bacterial stain was used. Bar  = 10 µm.

Figure 4. Biofilms grown in the presence of OPN, hybridized with EUB338 and species-specific probes SMIT, SSAN, ANAES, SDOW or SORA2.

EUB338 targets all organisms in the biofilms and was labelled with Atto633 (red). Species-specific probes were labelled with Cy3 (green). A. S. mitis SK24, the dominant organism in biofilms grown without OPN, accounted for 14% of the bacterial biovolume. B–F. The relative biovolumes of all other organisms increased in biofilms grown with OPN, as compared to biofilms grown without OPN. S. sanguinis SK150 (B) was the most abundant organism in the biofilms (48% of the biovolume). A. naeslundii AK6 was a prominent colonizer in basal layers of the biofilms (C, 22% of the biovolume in the basal layer), but was detected less frequently in upper layers of the biofilm (D, 9% of the total biovolume). S. downei HG594 (E, 11% of the biovolume) and S. oralis SK248 (F, 3% of the biovolume) represented smaller fractions of the bacterial biofilm.

Figure 5. Bacterial composition of biofilms grown in the presence and absence of OPN.

In biofilms grown without OPN (−OPN), S. mitis SK24 was the predominant organism. When OPN was present in the medium (+OPN), the abundance of S. mitis was dramatically lower, and the relative abundance of all other organisms increased. S. sanguinis SK150 became the predominant organism.