Inhibition of Streptococcus mutans and S. sobrinus biofilms by liamocins from Aureobasidium pullulans.

The aim of this study was to determine if the novel anti-streptococcal inhibitors, liamocins, also inhibit biofilm formation by S. mutans and S. sobrinus. S. mutans strain ATCC 25175 and S. sobrinus strain ATCC 33478 were tested for biofilm formation in a rapid microtiter plate (MTP) assay and the effects of added liamocins were determined. This assay measures relative biofilm growth on pin lids. Results were verified in a biofilm flow cell assay, using hydroxyapatite-coated coupons to simulate dental material. Planktonic cultures of S. mutans and S. sobrinus were inhibited by 0.1 mg liamocins/ml. When liamocins were added after the adhesion phase in a rapid microtiter plate assay, S. mutans was inhibited 53% by 5 mg liamocins/ml, while S. sobrinus was more sensitive, showing 100% inhibition at 0.5 mg liamocins/ml. When liamocins were added during the adhesion phase, biofilms of S. mutans showed 78% inhibition at 3.0 mg liamocins/ml. In a biofilm flow cell assay, liamocins added after the adhesion phase at 0.5 mg liamocins/ml inhibited biofilms of S. sobrinus, and appeared to remove biofilms over time. Liamocins were shown for the first time to inhibit biofilm formation by S. mutans and S. sobrinus. Since liamocins are specific for Streptococcus spp., they are potential new inhibitors of oral streptococcal biofilms that should not affect normal oral microflora.

Introduction

Dental caries is associated with biofilm formation by oral bacteria, particularly Streptococcus mutans and S. sobrinus [1,2]. Dental plaque is a type of biofilm, which provides a matrix for various cariogenic bacteria [3,4]. Thus, control of streptococcal biofilms has become a major target for management of dental caries and periodontic diseases. Antibiofilm agents currently under study include cetylpyridinium chloride [33], carolacton [5], pentose sugars and xylitol [6,7], green tea extracts [8], red propolis [9], anti-fungal azoles [10], lemon essential oil [11,12], and clove essential oil [13].

Liamocins are novel inhibitors of Streptococcus species produced by the fungus Aureobasidium pullulans [14]. A. pullulans is a polymorphic ascomycete that is best known as the source of the commercial polysaccharide pullulan, used in water-soluble films for food packaging and fast dissolving oral films [[15], [16], [17]]. Nagata et al. [18] first reported the production of unusual heavier-than-water “oil” by Aureobasidium spp. A partial structure showing a polyol lipid was proposed by Kurosawa et al. [19]. Price et al. [20] first deduced the complete structure of these oils, subsequently named “liamocins.” Liamocins are composed of a single polyol headgroup (usually mannitol), partially O-acylated with either three or four 3,5-dihydroxydecanoic ester groups, which may contain a single 3′-O-acetyl group [20]. Variations in the polyol headgroup and ratios of lipid tailgroups are possible, depending on the strain and culture conditions [[21], [22], [23]].

Liamocins are surface active [22,24,25] and inhibit mammalian cancer cells [22,26,27]. Bischoff et al. [14] discovered that liamocins have antibacterial activity with specificity for planktonic cultures of Streptococcus species, including S. mutans. However, liamocins have not previously been tested for inhibition of biofilms.

Materials and methods

Strains and culture conditions

Aureobasidium pullulans strain NRRL 50380 was from the ARS Culture Collection, Peoria, IL. Cultures were maintained on yeast malt extract agar (YMA). For oil production, 50 ml cultures in 250 ml flasks were grown in 5% (w/v) sucrose, 0.06% (w/v) peptone, 0.04% (w/v) yeast extract, 0.5% (w/v) K2HPO4, 0.04% (w/v) MgSO4·7H2O, and 0.1% (w/v) NaCl for 7 days at 28 °C with shaking at 150 rpm. Liamocins were extracted by the whole culture method of Manitchotpisit et al. [22], using 100 ml of methyl ethyl ketone (MEK), dried, and then resuspended in 10 ml of 1:1 MEK and DMSO.

Streptococcus mutans strain ATCC 25175 and S. sobrinus strain ATCC 33478 were from the American Type Culture Collection, Manassas, VA. Cultures were grown on a modified Todd-Hewitt broth (THB) (1.5% agar for plates) with 11 mM glucose for culture maintenance or 3 mM sucrose for biofilm production. Composition of modified THB per liter is 2% (w/v) trypticase peptone, 1% (w/v) beef extract powder, 0.2% (w/v) NaHCO3, 0.2% (w/v) NaCl, and 0.04% (w/v) Na2HPO4, plus carbon source.

Assays of growth inhibition of planktonic cultures

Growth inhibition in liquid planktonic cultures was measured by growing S. mutans strain ATCC 25175 and S. sobrinus strain ATCC 33478 in 10 ml liquid cultures of THB-glucose containing liamocins at 0–5 mg/ml for 24 h at 37 °C aerobically. In preliminary experiments, it was determined that S. sobrinus required a higher level of aeration than S. mutans for comparable growth, so S. sobrinus cultures were shaken at 200 rpm while S. mutans cultures were not shaken. Test cultures were inoculated to 0.1 OD600 and incubated with or without potential inhibitors under the same conditions for 24 h. Maximal solvent concentrations were 1.0% (v/v). Culture growth was measured by optical density, verified by viable cell counts.

Assays of biofilm formation in 96-well plates

S. mutans strain ATCC 25175 and S. sobrinus strain ATCC 33478 were tested for production of biofilms using a rapid microtiter plate assay as modified from Rich et al. [28]. Inocula were grown in 10 ml liquid cultures of THB-glucose for 48 h at 37 °C with (for S. sobrinus) or without (for S. mutans) shaking at 200 rpm. For both strains, these saturated cultures contained >107 CFU/ml. Twenty-five μl of cells were added per well of a 96-well microtiter plate (Nunc, Denmark), containing 125 μl of THB-sucrose per well. A sterile 96-pin lid (Nunc) was added and plates were incubated for 48 h to correspond to the adhesion phase of a conventional biofilm reactor. Pin lids then were transferred to fresh plates (time point zero) and subsequently transferred every 48 h until the assay endpoint at 144 h. Again, preliminary studies indicated that S. sobrinus required a higher level of aeration than S. mutans for comparable growth, so plates containing S. mutans strain ATCC 25175 were incubated in an anaerobic chamber at 37 °C, while plates containing S. sobrinus strain ATCC 33478 were incubated under static, aerobic conditions at 37 °C. Stocks of liamocins in 1:1 DMSO/MEK were diluted into medium at final concentrations of 0.1 to 5 mg/ml in 1.0% (v/v) solvent. Alternatively, xylitol was added to aqueous medium at a final concentration of 5 mg/ml. Replicator lid pins were stained according to Rich et al. [28] with a 0.1% crystal violet solution for 30 min at room temperature, and destained with 95% ethanol. The amount of crystal violet staining, representing biofilm growth, was measured at 600 nm using a SpectraMax M5 microtiter plate reader (Molecular Devices, Sunnyvale, CA). Viability was tested by direct plating of culture dilutions onto THB plates without liamocins.

Assays of biofilm formation in biofilm flow cells

S. mutans strain ATCC 25175 and S. sobrinus strain ATCC 33478 were tested for production of biofilms in biofilm flow cells by a modification of methods previously described [29]. Preinocula were grown in 20 ml of THB medium in 50 ml flasks at 37 °C for 48 h with shaking at either 200 rpm (for S. sobrinus) or 25 rpm (for S. mutans). For both strains, these saturated cultures contained >107 CFU/ml. Biofilm cultures were grown in a BST FC 271 flow cell (BioSurface Technologies Corp, Bozeman, MT, USA) equipped with hydroxyapatite-coated stainless steel sampling coupons. Preinocula were diluted 1:25 into 500 ml of THB-sucrose and flow cells were inoculated with this dilution at a flow rate of 0.2 ml/min for 16 h. This corresponds to the static adhesion phase of a conventional biofilm reactor. For post-adhesion (continuous phase) biofilm growth, flow cells were provided with a continuous flow of fresh medium at 0.2 ml/min. Flow cells are constructed with parallel dual channels, one of which received medium with liamocins at 0.5 mg/ml. Flow cells were maintained at 37 °C on heated pads (Model TP-3E temperature therapy pads, Stryker Medical, Portage MI). Biofilm coupons were sampled as previously described [29] and assayed on THB solid medium.

Results and discussion

Effect of liamocins on planktonic cultures of S. mutans and S. sobrinus

Bischoff et al. [14] reported that liamocins possess antibacterial activity with specificity for a number of species of Streptococcus, including S. mutans. MICs for tested strains were less than 0.1 mg liamocins/ml [14]. In the current study, the effect of liamocins was assessed on planktonic cultures of S. mutans strain ATCC 25175 and S. sobrinus strain ATCC 33478 (Fig. 1). Cultures were inhibited by 0.1 mg liamocins/ml, and little or no growth was observed at 0.5 mg liamocins/ml. S. sobrinus appeared to be more sensitive than S. mutans to liamocins. Solvent controls had no effect on cultures (Fig. 1). Thus, liamocins inhibit the planktonic growth of S. mutans and S. sobrinus. Viability assays showed that control cultures of both species (without liamocins) contained >107 CFU/ml, while cultures containing 1 mg liamocins/ml contained less than 300 CFU/ml (the lower limit of detection for this assay). These results are consistent with previous studies that showed that Liamocins are bacteriocidal rather than bacteriostatic for Streptococcus species [14,34].

Fig. 1

Planktonic growth of Streptococcus mutans and S. sobrinus in the presence of liamocins at the indicated concentrations. Controls include growth medium with and without solvent at 1.0% (v/v).

Effect of liamocins on biofilm formation by S. mutans and S. sobrinus

Liamocins previously were reported to have no effect on biofilm formation by species of Lactobacillus [30]. Although liamocins inhibited planktonic cultures of S. mutans and S. sobrinus (Fig. 1), biofilms are generally known for their resistance to antibiotics [31]. Biofilm growth of S. mutans strain ATCC 25175 and S. sobrinus strain ATCC 33478 was assessed in a rapid microtiter plate assay modified from Rich et al. [28]. In the absence of added liamocins, strains showed crystal violet staining of about 0.8 OD600, representing good biofilm growth [28]. When liamocins were added after the adhesion phase, biofilm growth of S. mutans was inhibited at liamocins concentrations of 0.5–5.0 mg/ml, showing 53% inhibition at 5 mg liamocins/ml (Fig. 2A). S. sobrinus was more sensitive, showing 72% inhibition at only 0.1 mg liamocins/ml, and 100% inhibition at 0.5 mg liamocins/ml (Fig. 2A). When liamocins were added during the adhesion phase, S. mutans biofilms were somewhat more sensitive, showing 78% inhibition at 3.0 mg liamocins/ml (Fig. 2B). These results compare favorably with those for xylitol inhibition of S. sobrinus biofilms. In our MTP assay, S. sobrinus was inhibited 75% by 5 mg xylitol/ml added after the adhesion phase, and 91% by xylitol added during the adhesion phase.

Fig. 2

Biofilm formation by Streptococcus mutans and S. sobrinus, determined by rapid microtiter plate bioassay [28]. Liamocins at the indicated concentrations were added (A) after the biofilm adhesion phase or (B) during the adhesion phase.

Thus, biofilms of S. mutans and S. sobrinus are inhibited by liamocins, particularly during the adhesion phase of biofilm formation. It has been suggested that liamocins may have application in mouthwashes against oral Streptococcus species [32]. Results suggest that liamocins might be most effective immediately after dental cleanings, before biofilms are established.

Biofilm formation in biofilm flow cells

S. mutans strain ATCC 25175 and S. sobrinus strain ATCC 33478 were cultured in commercial biofilm flow cells, using stainless steel coupons coated with hydroxyapatite to simulate dental material [33]. To confirm the differential effects of liamocins on S. mutans and S. sobrinus, 0.5 mg liamocins/ml was added after the initial adhesion phase. Under these conditions liamocins had little effect on S. mutans biofilms (Fig. 3A), but inhibited S. sobrinus biofilms at 4.5 h after the start of continuous flow phase (Fig. 3B). These results are consistent with those obtained from the rapid microtiter assays. Interestingly, liamocins appear to not only prevent growth of S. sobrinus biofilms, but actually to remove biofilms over time.

Fig. 3

Biofilm formation by Streptococcus mutans and S. sobrinus, determined by biofilm flow cell assay [29]. Liamocins at 0.5 mg/ml were added after the biofilm adhesion phase to (A) S. mutans or (B) S. sobrinus.

The mechanism of action for liamocins is thus far unknown, although it appears to kill cells by disrupting the cell membrane [14,34]. The basis for its selective action against species of Streptococcus is not understood. Interestingly, liamocins appear to be more active against S. sobrinus strain ATCC 33478 than against S. mutans strain ATCC 25175. Song et al. [35] found diversity among the genomes of strains in the mutans streptococci. Conrads et al. [36] concluded that S. mutans and S. sobrinus should be considered equally virulent with regard to dental caries, although they found that S. sobrinus has fewer two-component signaling systems and bacteriocin-related genes, and lacks central competence genes found in S. mutans. Further studies are needed to clarify the selectivity and mechanism of action of liamocins. In previously published studies on the inhibition of mammalian cancer cells, liamocins were generally inactive against control cell lines, usually Vero monkey cells [22,26,27]. However, further testing would be necessary to determine the clinical safety and appropriate applications of liamocins. Using an optimized medium, liamocins have been produced at up to 22 g/l [37]. However, further research into formulations for clinical applications would be required.

In conclusion, liamocins were tested for the first time as potential inhibitors of biofilm formation by S. mutans and S. sobrinus. Biofilms were sensitive to liamocins in a rapid microtiter plate assay, particularly during the adhesion phase of biofilm formation. Biofilm flow cell assays using hydroxyapatite-coated coupons validated the rapid microtiter plate assay. Since liamocins specifically inhibit species of Streptococcus, they would be expected to reduce the development of plaque biofilms and thus dental caries, without disrupting the normal oral microflora.

Funding

None.

Conflicts of interest

The authors have no competing interests.

Ethical approval

Not required.