Inhibition of Streptococcus mutans biofilm accumulation and polysaccharide production by apigenin and tt-farnesol

Transcription

1 Journal of Antimicrobial Chemotherapy (2003) 52, DOI: /jac/dkg449 Advance Access publication 16 October 2003 Inhibition of Streptococcus mutans biofilm accumulation and polysaccharide production by apigenin and tt-farnesol H. Koo 1 *, M. F. Hayacibara 2, B. D. Schobel, J. A. Cury 2, P. L. Rosalen 2, Y. K. Park 3, A. M. Vacca-Smith 1 and W. H. Bowen 1 1 Center for Oral Biology and Eastman Department of Dentistry, University of Rochester Medical Center, Rochester, NY, USA; 2 Department of Physiological Sciences, Faculty of Dentistry of Piracicaba and 3 Department of Food Science, College of Food Engineering, State University of Campinas, SP, Brazil Received 14 July 2003; returned 31 July 2003; revised 8 August 2003; accepted 15 August 2003 Objectives: Apigenin is a potent inhibitor of glucosyltransferases and tt-farnesol affects the membrane integrity of Streptococcus mutans. We investigated the influence of apigenin and tt-farnesol, alone and in combination, on the accumulation, polysaccharide composition and viability of S. mutans UA159 biofilms. Methods: Initially, biofilms were grown for 54 h; then, the early-formed biofilms were treated for 1 min twice daily with one of the following: (i) 1.33 mm tt-farnesol; (ii) 1.33 mm apigenin; (iii) apigenin + tt-farnesol (1.33 mm each); (iv) vehicle control (20% ethanol with 0.75% dimethyl sulphoxide); (v) 0.12% chlorhexidine (1.33 mm); or (vi) physiological saline (145 mm NaCl). The procedure was repeated at biofilm ages of 78 and 102 h, and biofilms were harvested at 126 h. The dry weight, protein concentration, number of cfu, and polysaccharide composition per biofilm were determined. Results: The dry weights of the biofilms treated with the test agents were significantly less (30 50%) than those treated with vehicle control (P < 0.05). Biofilms treated with the test agents also resulted in lower amounts of extracellular alkali-soluble glucans, intracellular iodophilic polysaccharides and, to a lesser extent, fructans. The fructosyltransferase activity was affected only by apigenin and apigenin + tt-farnesol. The recoverable viable counts of S. mutans were slightly lower (0.5 to 1 log 10 decrease in cfu/biofilm) after apigenin and tt-farnesol treatments compared with the vehicle control. Chlorhexidine displayed potent bactericidal activity, and virtually halted the further accumulation of early-formed (54 h old) biofilms. Conclusions: Apigenin and tt-farnesol affected the accumulation and polysaccharide content of S. mutans biofilms without major impact on the bacterial viability. Keywords: glucans, fructans, glucosyltransferases, flavonoids, propolis Introduction Streptococcus mutans has been implicated as a primary aetiological agent of dental caries in animals and humans, and it is also involved in plaque formation and accumulation. 1 The acidogenic and aciduric (associated with acid tolerance) properties of S. mutans, together with its ability to synthesize extracellular glucans, are the major factors for the development and establishment of cariogenic biofilms. 1,2 Glucans, synthesized from dietary sucrose (and possibly in combination with starch) by glucosyltransferases (GTFs), enhance the pathogenic potential of dental plaque by promoting the adherence and accumulation of cariogenic streptococci on the tooth surface, and by contributing to the bulk and structural integrity of plaque. 3 5 S. mutans produces at least three GTFs: GTF B, which synthesizes mostly insoluble glucan (α1,3-linked); GTF C, which synthesizes a mixture of insoluble and soluble glucan (α1,6-linked); and GTF D, which synthesizes soluble glucan. 1 Enzymatically active GTFs are present: (i) in the human whole saliva; 6 (ii) in the salivary pellicle that is formed on the tooth surface; 6 and (iii) on bacterial surfaces. 7,8 The glucans synthesized by these enzymes in distinct loci are critical for the adherence of the bacteria to the surface of teeth and to each other; GTF B and C have been shown to be essential virulence factors of mutans streptococci associated with the pathogenesis of dental caries. 9 S. mutans also produces a single fructosyltransferase (FTF), which... *Correspondence address. University of Rochester Medical Center, Center for Oral Biology, 601 Elmwood Avenue Box 611, Rochester, NY 14642, USA. Tel: ; Fax: ; Hyun_Koo@urmc.rochester.edu JAC vol.52 no.5 The British Society for Antimicrobial Chemotherapy 2003; all rights reserved.

2 Influence of apigenin and tt-farnesol on biofilms catalyses the synthesis of fructans from sucrose. 10 Fructans are believed to function primarily as extracellular storage polysaccharides that can be metabolized during periods of nutrient deprivation. 11 These polysaccharides provide the organisms with a unique microenvironment for their growth, metabolism and survival. 9,12 Therefore, inhibition of formation and accumulation of cariogenic biofilm communities by affecting the synthesis of these polysaccharides is an attractive route for preventing both dental caries and plaque formation. Propolis, a natural non-toxic beehive product, has been shown to reduce the incidence of dental caries in rats, 13,14 and the accumulation of supragingival plaque in vivo. 15 Recently, we explored the effects in vitro of 30 compounds identified in propolis on the activity of GTFs and viability of mutans streptococci; two compounds, apigenin and tt-farnesol, each displaying distinct biological activities, were identified as potential novel anti-plaque/anti-caries agents. 16,17 We have found that apigenin (4,5,7-trihydroxylflavone) is a potent inhibitor of GTF B and C; it has virtually no antibacterial activity against S. mutans, 16,17 although it was selectively inhibitory to Staphylococcus aureus growth. 18 tt-farnesol, a natural sesquiterpene alcohol (3,7,11-trimethyl-2,6,10-dodecatrien-1-ol), displayed inhibition of growth and metabolism of mutans streptococci by disrupting the bacterial membrane; 16 agents that disrupt the properties of cell membranes may also affect glucan synthesis. 19 tt-farnesol also exhibited antimicrobial activity against Streptomyces tendae and Saccharomyces cerevisiae, but not against Escherichia coli. 20 In addition, apigenin and tt-farnesol, alone or in combination, showed cariostatic properties in rats without significant effects on the microbial viability in the animals mouths. 17 Apigenin and tt-farnesol have been shown to be non-mutagenic and non-toxic both in vitro and in vivo. 21,22 Considering the effects of apigenin and tt-farnesol on GTFs and bacterial membranes, the purpose of this study was to evaluate the influence of these agents, alone or in combination, on the accumulation, polysaccharide composition and population of viable cells of S. mutans UA159 biofilms in vitro. Materials and methods Test compounds Apigenin and tt-farnesol were obtained from Extrasynthese Co. (Genay, France); the compounds were verified by means of high-performance liquid chromatography and gas chromatography/mass spectrometry as standard procedures performed by the company for purity (99%) and authenticity. Chlorhexidine was purchased from Sigma Aldrich Co. (St Louis, MO, USA). A single concentration of 1.33 mm of the test agents (equivalent to 0.035% apigenin and 0.028% tt-farnesol, w/v) was chosen for this investigation based on our previous dose response data, 16,17 and also to compare with our positive control 0.12% chlorhexidine (equivalent to 1.33 mm), which is a clinically proven anti-plaque agent. 23 Apigenin and tt-farnesol were dissolved in 20% ethanol containing 0.75% dimethyl sulphoxide (DMSO) just prior to carrying out the assays. Appropriate solvent controls were always included. Preparation and treatment of biofilms Biofilms of S. mutans UA159 were formed on standard glass microscope slides (Micro slides; VWR Scientific, Inc., West Chester, PA, USA) in batch cultures for 5 days. 17 S. mutans UA159 is a proven virulent cariogenic pathogen and was the strain selected for genomic sequencing. 24 Cells of S. mutans were grown in ultrafiltered (10 kda molecular weight cut-off membrane; Amicon) tryptone-yeast extract broth with addition of 30 mm sucrose containing either [ 14 C]glucose-sucrose (0.02 µci/ml) or [ 3 H]fructose-sucrose (0.4 µci/ml) at 37 C and 5% CO 2. The culture medium was replaced daily; the biofilms were grown for 54 h to allow initial bacterial deposition. At this point (54 h old), the biofilms were treated twice daily until the fifth day of the experimental period (126 h old biofilms) with one of the following: (i) 1.33 mm tt-farnesol (0.028%, w/v); (ii) 1.33 mm apigenin (0.035%, w/v); (iii) 0.12% chlorhexidine (1.33 mm); (iv) tt-farnesol + apigenin (1.33 mm each); (v) vehicle control (20% ethanol containing 0.75% DMSO); or (vi) physiological saline (145 mm NaCl). The biofilms were exposed to the treatments for 1 min, double-dip rinsed in sterile saline solution and transferred to fresh culture medium. The treatments were repeated 6 h later, except that the culture medium was not replaced. Following the second 1 min exposure, the biofilms were incubated undisturbed for 18 h. The experimental procedure was performed on the third (at the biofilm age of 78 h) and fourth (102 h) day. The final exposure to the test agents occurred when the biofilms reached 108 h; they were incubated an additional 18 h and harvested at the age of 126 h (fifth day of the experimental period). Each biofilm was exposed to the respective treatment six times. Our preliminary data showed that the vehicle control (1 min exposure, twice daily) allowed the continued formation of biofilm. Biofilm assays were performed in quadruplicate in at least three different experiments. Biofilm analyses At the end of the experimental period, the biofilms were dip-washed three times, and then gently swirled in physiological saline to remove loosely adherent material. The biofilms were placed in 30 ml of sterile saline solution, and the glass surfaces were gently scraped with a sterile spatula to harvest adherent cells. The removed biofilms were subjected to sonication by a Branson Sonifier 450 (twice, each time consisting of three 10 s pulses with 5 s intervals at 50 W) as detailed elsewhere. 17 The homogenized suspension was used for dry weight, total protein, bacterial viability and polysaccharide analyses. For the dry weight determination, three volumes of cold ethanol ( 20 C) were added to 5 ml of the cell suspension, and the resulting precipitate collected (10 000g for 10 min, 4 C). The supernatant was discarded, and the cell pellet was washed twice with cold ethanol, and then lyophilized and weighed. Total protein of the cell suspension was determined by acid digestion followed by ninhydrin assay. 25 Bacterial viability An aliquot (0.1 ml) of the homogenized suspension was serially diluted ( ) and plated on tryptic soy agar or blood agar by means of a spiral plater (Autoplate model 3000; Spiral Biotech, Inc., Bethesda, MD, USA). The plates were incubated in 5% CO 2 at 37 C for 48 h, and then the number of cfu was determined. The sonication procedure provided the maximum recoverable counts as determined experimentally. 17 Polysaccharide analyses An aliquot (10 ml) of the cell suspension was centrifuged at g for 10 min at 4 C. The supernatant was collected and the cell pellet resuspended and washed in the same volume of water; this procedure was repeated twice. All the supernatants were pooled and three volumes of cold ethanol were added, and the resulting precipitate collected. The precipitate, or water-soluble polysaccharides, were collected by centrifugation and washed three times with cold ethanol and dried in a Speed Vac concentrator. The water-soluble glucan (labelled with [ 14 C]glucose) and water-soluble fructan (labelled with [ 3 H]fructose) were determined by means of scintillation counting. 16,26 The cell pellet was dried in a Speed Vac concentrator and used for determination of: (i) extracellular alkalisoluble polysaccharides; and (ii) intracellular iodophilic polysaccharides (IPS). The alkali-soluble polysaccharides were extracted using 1 N NaOH (1 mg of biofilm dry weight/0.3 ml of 1 N NaOH) as detailed elsewhere. 27 The extract was precipitated with three volumes of cold eth- 783

3 anol. The precipitate was collected by centrifugation and washed three times with cold ethanol and dried in a Speed Vac concentrator. The alkali-soluble glucan (labelled with [ 14 C]glucose) and fructan (labelled with [ 3 H]fructose) were determined by means of scintillation counting. 16,26 The IPS were extracted with hot 5.3 M KOH (0.8 mg of biofilm dry weight/ml of KOH) and quantified using 0.2% I 2 /2% KI solution as described by DiPersio et al. 28 Total polysaccharide was defined as the sum of the extracellular (water-soluble glucan, water-soluble fructan, alkali-soluble glucan, alkali-soluble fructan) and the intracellular (IPS) polysaccharides. In addition, the amount of carbohydrates in each of the polysaccharide fractions (with the exception of IPS) was measured using colorimetric assays (anthrone method 29 with glucose as standard; and resorcinol method 30 with fructose as standard). Total carbohydrates in the biofilms were also determined by measuring the amount of sugars in the wholecell suspension; determination of sugars by colorimetric assays has been widely used to estimate the polysaccharide content in dental plaque and S. mutans biofilms. 27,31 33 H. Koo et al. FTF assay The FTF activity in the S. mutans UA159 culture supernatant was measured by the incorporation of [ 3 H]fructose from labelled sucrose (New England Nuclear Research Products, Boston, MA, USA) into fructans as described elsewhere. 26,34 Briefly, the culture supernatant of S. mutans UA159 was mixed with the test compounds (final concentration of 1.33 mm) and incubated with 100 mm sucrose supplemented with [ 3 H]fructose-sucrose (0.4 µci/ml) at 37 C with rocking for 2 h. For the control, the same reaction was carried out, but with 20% ethanol containing 0.75% DMSO replacing the test agent solution. Radiolabelled fructan was determined by scintillation counting. 26,34 Statistical analyses The data were analysed using ANOVA, and the F-test was used to test any difference between the groups. When significant differences were detected, pairwise comparison was made between all the groups using Tukey s method to adjust for multiple comparisons. Statistical software JMP version 3.1 (SAS Institute, Cary, NC, USA) 35 was used to perform the analyses. The level of significance was set at 5%. Results Effects of test agents on biofilm accumulation S. mutans UA159 (a proven virulent cariogenic pathogen 24 ) biofilms at an age of 54 h were treated with tt-farnesol and apigenin, either alone or in combination, for 1 min twice daily to determine whether these agents at 1.33 mm could adversely affect further biofilm development and accumulation. The concentration of 1.33 mm was chosen based on our previous dose response data, 16 and has been shown to be effective against dental caries in vivo. 17 The population of viable cells recovered from the biofilms before and after treatment is shown in Figure 1. The biofilms treated with the test agents showed slightly lower numbers of recoverable viable cells compared with the vehicle control (0.5 1 log 10 decrease in cfu/biofilm); however, none of them appeared to be bactericidal for S. mutans in the biofilms. In contrast, the positive control chlorhexidine dramatically affected the viability of S. mutans, showing a >4 log 10 decrease in cfu/biofilm. Both test agents significantly diminished the further accumulation of S. mutans biofilms compared with the vehicle control (P < 0.05), as shown in Table 1. tt-farnesol and apigenin treatments resulted in 32% and 43% less biomass (dry weight) than the vehicle control Figure 1. Average number of cfu recovered before (54 h old biofilm) and after treatments (126 h old biofilms) per S. mutans UA159 biofilm. *Values linked by lines are not significantly different from each other (P > 0.05; ANOVA, comparison for all pairs using Tukey test). treatment; the combination of agents showed slightly better results (50% less biomass). Chlorhexidine virtually halted the further accumulation of S. mutans biofilms, showing a similar dry weight of biofilms at an age of 54 h (before treatment). Effects of test agents on protein and polysaccharide composition of the biofilms The data from polysaccharide, carbohydrate and protein analyses are presented in terms of: (i) total amount (Tables 1 3); and (ii) percentage of the biofilm dry weight (Figures 2 and 3). Table 1 shows the total amount of polysaccharides, carbohydrates and protein in the biofilms before (54 h old biofilms) and after treatment (126 h old biofilms); 70 80% of the biofilms dry weight was comprised of proteins and carbohydrates (Figure 2). The biofilms treated with tt-farnesol, apigenin and apigenin + tt-farnesol exhibited 40 60% less polysaccharides than those treated with the vehicle control (P < 0.05; Table 1), and resulted in a significantly lower percentage of polysaccharides in the biofilms (P < 0.05; Figure 2). The total amount and percentage of polysaccharides in the biofilms treated with chlorhexidine were similar to those of early-formed untreated biofilms (54 h old); chlorhexidine treatment effectively stopped further development of biofilm. Furthermore, the total amount of protein in the biofilms treated with the test agents and chlorhexidine was significantly less than those treated with the vehicle control (P < 0.05; Table 1). However, in terms of percentage of the biofilm dry weight comprised of protein, only apigenin, apigenin + tt-farnesol and chlorhexidine treatments resulted in significant reduction of the protein content (P < 0.05; Figure 2). The total amount of carbohydrates (based on anthrone and resorcinol methods) was equivalent to that found for polysaccharides (based on scintillation counting and IPS determination assays), although the values obtained from carbohydrate determination, which includes monosaccharide and other sugars from the cell membrane, were slightly higher, as might be expected (Table 1). The total amount and percentage of the different polysaccharides in the biofilms were affected by treatment with apigenin and 784

4 Influence of apigenin and tt-farnesol on biofilms Table 1. Dry weight, and total amount of polysaccharides, carbohydrates and protein in the biofilms before and after treatments Treatment Dry weight (mg) Polysaccharide (scintillation + IPS assays) (mg) Carbohydrates (anthrone + resorcinol methods) (mg) Protein (mg) 54 h old biofilm (before treatment) 5.6 ± 2.3 a 2.3 ± 0.1 a 2.7 ± 0.2 a 1.3 ± 0.2 a 126 h old biofilm (after treatment)* saline (145 mm NaCl) 40.0 ± 3.3 b 19.3 ± 1.9 b 22.9 ± 1.3 b 10.9 ± 1.2 b vehicle control (20% ethanol and 34.2 ± 2.9 b 16.7 ± 1.2 b 19.8 ± 1.7 b 9.0 ± 2.1 b 0.75% DMSO) tt-farnesol 1.33 mm 23.3 ± 1.7 c 10.1 ± 1.1 c 12.1 ± 1.5 c 5.8 ± 1.0 c apigenin 1.33 mm 19.5 ± 1.8 d 8.3 ± 0.6 d 9.7 ± 0.8 c,d 4.5 ± 1.2 c,d apigenin + tt-farnesol (1.33 mm each) 17.5 ± 0.7 d 7.0 ± 1.0 d 8.5 ± 1.3 d 3.9 ± 0.7 d 0.12% chlorhexidine (1.33 mm) 5.8 ± 2.1 a 2.4 ± 0.6 a 2.9 ± 0.5 a 1.4 ± 0.5 a Values in the same column followed by the same superscripts are not significantly different from each other (P > 0.05; ANOVA, comparison for all pairs using Tukey test). *Twice daily with 1 min exposure for each treatment. Table 2. Total amount of extracellular glucans (and the respective glucose) and IPS in the biofilms before and after treatments Treatment Alkali-soluble glucan (scintillation counting) (mg) Glucose (anthrone method) (mg) Soluble glucan (scintillation counting) (mg) Glucose (anthrone method) (mg) IPS (I 2 -polysaccharide assay) (mg) 54 h old biofilm (before treatment) 1.2 ± 0.4 a 1.5 ± 0.3 a 0.4 ± 0.1 a 0.6 ± 0.2 a 0.3 ± 0.1 a 126 h old biofilm (after treatment)* saline (145 mm NaCl) 10.3 ± 1.2 b 11.7 ± 2.3 b 2.5 ± 0.7 b 3.2 ± 0.4 b 3.4 ± 0.2 b vehicle control (20% ethanol and 8.9 ± 0.5 b 10.3 ± 1.4 b 2.2 ± 0.6 b 3.0 ± 0.5 b,c 2.9 ± 0.2 c 0.75% DMSO) tt-farnesol 1.33 mm 5.3 ± 0.7 c 6.0 ± 0.9 c 1.4 ± 0.3 b 1.9 ± 0.7 c 1.6 ± 0.1 d apigenin 1.33 mm 4.3 ± 0.6 c,d 5.0 ± 1.3 c,d 1.6 ± 0.4 b 2.1 ± 0.5 c 1.1 ± 0.1 e apigenin + tt-farnesol (1.33 mm each) 3.5 ± 0.4 d 4.3 ± 0.6 d 1.5 ± 0.3 b 2.0 ± 0.6 c 0.9 ± 0.2 e 0.12 % chlorhexidine (1.33 mm) 1.3 ± 0.6 a 1.5 ± 0.6 a 0.5 ± 0.0 a 0.7 ± 0.4 a 0.3 ± 0.0 a Values in the same column followed by the same superscripts are not significantly different from each other (P > 0.05; ANOVA, comparison for all pairs using Tukey test). *Twice daily with 1 min exposure for each treatment. tt-farnesol, alone or in combination; the results are summarized in Tables 2 and 3 and Figure 3. The total amount of alkali-soluble glucan in the biofilms treated with the test agents and chlorhexidine was significantly less than in the vehicle control (P < 0.05; Table 2), although only apigenin + tt-farnesol treatment resulted in a significantly lower percentage of alkali-soluble glucan in the biofilms (P < 0.05; Figure 3). The total amount of water-soluble glucans in the biofilms was unaffected by the test agents compared with the vehicle control (Table 2). However, the biofilms treated with apigenin, apigenin + tt-farnesol and chlorhexidine exhibited significantly higher percentages of water-soluble glucans than the vehicle control (P < 0.05; Figure 3). The total amount and the percentage of IPS were significantly lower in the biofilms treated with the test agents and chlorhexidine compared with the vehicle control (Table 2 and Figure 3; P < 0.05). In addition, only biofilms treated with apigenin and apigenin + tt-farnesol showed significantly lower concentrations of IPS per population of viable cells compared with the control treatment (P < 0.05) (data not shown). The biofilms treated with apigenin, apigenin + tt-farnesol and chlorhexidine displayed significantly less alkali-soluble fructan than the vehicle control (P < 0.05); however, its percentage in the biofilms was not affected significantly (Table 3 and Figure 3). In general, the biofilms treated with chlorhexidine displayed similar amounts and proportions of polysaccharides to those found in early-formed untreated biofilms (54 h old). The total amounts of glucose (based on the anthrone method) and fructose (resorcinol method) in the biofilms were similar to their respective glucans and fructans (scintillation counting). Among all the agents tested, only apigenin and apigenin + tt-farnesol inhibited the activity of the fructosyltransferase from S. mutans UA159 in solution (60 70% inhibition at 1.33 mm). Discussion Dental caries is one of the most common oral diseases and continues to inflict a large burden on many segments of the population in the USA and other Western countries. 19,36 Unlike many conditions, the 785

5 H. Koo et al. disease of dental caries continues throughout life, with new lesions in older people, an observation consistent with the prevalence of caries in adults where up to 95% have experienced one or more carious lesions. 19,36 In order to make continued progress towards eliminating dental caries, novel strategies should be developed aiming at the virulence factors involved in the pathogenesis of this common oral disease. The glucans synthesized by GTFs are of central importance in expression of virulence by S. mutans. 4,5,9 Furthermore, glucan-rich plaque matrix appears to increase the porosity 37 and the adhesive interactions, 38 and to decrease its inorganic concentration. 27 By aiming to prevent glucan production, therapeutic approaches to the prevention of the formation or virulence of dental plaque related to caries would be precise: unlike broad-spectrum antimicrobials, the oral flora would not necessarily be suppressed. However, most chemotherapeutic strategies are based on suppressing the levels of cariogenic bacteria by using antimicrobials (which are not selective), despite growing evidence that the ability of these organisms to synthesize glucans might be more important than their population in the mouth. 39,40 Figure 2. Percentage (per biofilm dry weight) of protein, polysaccharides and other carbohydrates in the biofilms before and after treatments. Values marked with asterisks are significantly different from vehicle control (P < 0.05; ANOVA, comparison for all pairs using Tukey test). Figure 3. Percentage (per biofilm dry weight) of glucans and fructans in the biofilms before and after treatments. Values marked with symbols are significantly different from vehicle control (P < 0.05; ANOVA, comparison for all pairs using Tukey test). ASG, alkali-soluble glucan; WSG, water-soluble glucan; IPS, intracellular iodophilic polysaccharides; ASF, alkali-soluble fructan; WSF, watersoluble fructan. Table 3. Total amount of fructans (and the respective fructose) in the biofilms before and after treatments Treatment Alkali-soluble fructan (scintillation counting) (mg) Total fructose (resorcinol method) (mg) Soluble fructan (scintillation counting) (mg) Total fructose (resorcinol method) (mg) 54 h old biofilm (before treatment) 0.3 ± 0.1 a 0.4 ± 0.1 a 0.06 ± 0.0 a 0.06 ± 0.0 a 126 h old biofilm (after treatment)* saline (145 mm NaCl) 2.5 ± 0.5 b 2.8 ± 0.2 b 0.6 ± 0.2 b 0.8 ± 0.2 b vehicle control (20% ethanol and 2.2 ± 0.3 b 2.5 ± 0.4 b 0.5 ± 0.2 b 0.8 ± 0.3 b 0.75% DMSO) tt-farnesol 1.33 mm 1.5 ± 0.4 b,c 1.6 ± 0.3 b,c 0.3 ± 0.2 b 0.5 ± 0.2 b,c apigenin 1.33 mm 1.1 ± 0.3 c 1.4 ± 0.4 c 0.2 ± 0.1 b 0.3 ± 0.2 c apigenin + tt-farnesol (1.33 mm each) 0.9 ± 0.4 c 1.1 ± 0.2 c 0.2 ± 0.1 b 0.4 ± 0.2 b,c 0.12% chlorhexidine (1.33 mm) 0.3 ± 0.2 a 0.4 ± 0.2 a 0.06 ± 0.0 a 0.08 ± 0.1 a Values in the same column followed by the same superscripts are not significantly different from each other (P > 0.05; ANOVA, comparison for all pairs using Tukey test). * Twice daily with 1 min exposure for each treatment. 786

6 Influence of apigenin and tt-farnesol on biofilms Recently, we demonstrated that apigenin and tt-farnesol reduced the incidence of dental caries in rats without displaying reduction of either total population of viable cells or the percentage of mutans streptococci in the animals plaque. 17 Therefore, we hypothesized that one of the mechanisms through which these two novel anti-caries/ anti-plaque agents expressed their effects on dental caries was by reducing the synthesis of glucans in the plaque, consistent with their effects on GTF activity and bacterial membranes in vitro. 16 Here, we investigated the influence of these agents on biofilms using an experimental S. mutans UA159 biofilm model with a brief treatment exposure (1 min) twice daily, in an attempt to mimic likely exposure in humans. The present model is the first attempt to determine simultaneously the polysaccharide composition and population of viable cells of S. mutans biofilms. In this study, we have found that topical application of apigenin and tt-farnesol, either alone or in combination, twice daily (total of six applications) resulted in lower amounts of polysaccharides in the S. mutans biofilms. Among the different polysaccharides affected by the test agents, alkali-soluble glucans are particularly important for biofilm development and accumulation. The alkali-soluble glucans are extracellular glucans adhered to the cells surface comprised of mainly insoluble α1,3-linked glucans, and also significant amounts of soluble α1,6-linked glucans. 27 It is well established that insoluble glucans synthesized from sucrose by GTFs play a critical role in the adherence and colonization of S. mutans on the tooth surface. 4,5,9 By diminishing the synthesis of alkali-soluble glucans, apigenin and tt-farnesol have had significant impact on the further development and accumulation of the biofilms. It is noteworthy that biofilms treated with the test agents displayed lower numbers of recoverable viable cells compared with the vehicle control. However, apigenin (at 1.33 mm) is devoid of any detectable antibacterial activity against S. mutans, in either planktonic or biofilm state. 16,17 Although tt-farnesol exhibited antibacterial activity against planktonic cells of mutans streptococci, it failed to kill S. mutans biofilms at 1.33 mm. 17 Our data indicate that by affecting the synthesis of extracellular insoluble glucans, the adherence and further accumulation of S. mutans (biofilm) on to a hard surface were reduced. Apigenin and tt-farnesol appear to have distinct mechanisms of action in reducing glucan synthesis. The primary target for apigenin is the GTF enzymes, especially GTF B and C, which are responsible for insoluble and, to a lesser extent, soluble glucan synthesis. Apigenin is a potent non-competitive inhibitor of the activity of GTFs B and C whether the enzymes are in solution (90 95% at 1.33 mm) or adsorbed on a saliva-coated hydroxyapatite surface (60 65% inhibition at 1.33 mm); 17 this level of inhibition has not been observed previously with any other natural or synthetic agents, including currently commercially available anti-plaque agents. 42 We also observed that apigenin had little effect on salivary enzymes, such as amylase and lysozyme (H. Koo & W. H. Bowen, unpublished results). Interestingly, apigenin appears to increase the proportion of water-soluble glucans in the biofilms compared with the vehicle control. A possible explanation for this phenomenon may be related to the effects of apigenin in inducing the expression of GTF D enzyme by S. mutans (study in progress); in addition, apigenin was not as effective in inhibiting the activity of surface-adsorbed GTF D as it was against GTF B and C. 16,17 The inhibition of glucan synthesis by tt-farnesol might be a result of its effect on the cell membrane rather that a direct action on enzymatic activity, since tt-farnesol is a poor inhibitor of GTFs. 16,17 The chemical structure and the lipophilic properties of tt-farnesol favour its membrane localization, causing changes in the permeability and fluidity of the cell membrane, an observation consistent with our findings that tt-farnesol disrupts the bacterial membrane of planktonic cells of mutans streptococci. 16 It has been recognized that agents which inflict damage to the cell membrane not only reduce the bacterial metabolism, but also affect the glucan synthesis by S. mutans. 19,46 This compound has been recently identified as a naturally occurring quorum-sensing molecule that inhibits Candida albicans biofilm formation without affecting the viability of this opportunistic pathogen. 44,45 The effect of the test agents on the amount of fructans in the biofilms was less dramatic than that observed on glucans. Biofilms treated with apigenin exhibited lower amounts of alkali-soluble fructans, probably due to moderate inhibitory effects on the activity of fructosyltransferase by apigenin (60% inhibition at 1.33 mm). The amount and percentage of fructans in the biofilms were unaffected by tt-farnesol. Furthermore, the amount and percentage of IPS were lower in the biofilms treated with apigenin and tt-farnesol; IPS are glycogen-like storage polymers with α1,4 and α1,6 linkages, and are an important source for acid production under environmental conditions in which exogenous carbohydrates are absent. 28,47 The precise role that such biological activity plays in caries prevention has not been fully explored, but nevertheless it has been shown that IPS is associated with the cariogenicity of S. mutans. 47 The combination of agents was significantly more effective in reducing the biofilm accumulation than tt-farnesol alone (P < 0.05). The combination of agents also displayed better results than did apigenin alone, although the differences did not reach statistical significance (P > 0.05). Furthermore, the combination of apigenin and tt-farnesol was the only treatment to significantly diminish the percentage of alkali-soluble glucan in the biofilms compared with the vehicle control (P < 0.05). The greater inhibitory effects of apigenin + tt-farnesol on biofilm accumulation and on alkali-soluble glucan concentration may explain our observation that the combination of agents was more effective in reducing the incidence of smooth surface caries in animals than either compound alone. 17 In contrast to apigenin and tt-farnesol, chlorhexidine inhibited the biofilm accumulation primarily due to its potent antibacterial properties; chlorhexidine is a broad-spectrum antimicrobial agent and exhibits negligible inhibitory activities against surface-adsorbed GTFs (10 20% inhibition at 1.33 mm). 17 It appears that by reducing the viability of the early-formed biofilms (54 h old), chlorhexidine virtually halted the further accumulation of S. mutans on the hard surface; the polysaccharide and protein content of the chlorhexidinetreated biofilms showed similar profiles to those from early-formed untreated biofilms. Clearly, the influence of apigenin and tt-farnesol on this monospecies biofilm model has many implications on their therapeutic potential in the context of the complex biochemical composition of human dental plaque. First and foremost, these agents remarkably affected the synthesis of glucan, the main polysaccharide in the plaque matrix; by inhibiting GTF activity and glucan synthesis, the further bacterial accumulation, including oral microorganisms that do not form polysaccharides, would be disrupted. Furthermore, it has been shown that a glucan matrix is the main factor for a high cariogenicity of dental plaque (as a microbiologically and biochemically heterogeneous biofilm) formed in the presence of sucrose in vivo. 27 In this biofilm model (monospecies), the percentage of polysaccharides ( 45% of biofilm dry weight) appears to be higher than that found in human dental plaque (multispecies) (15 25% of the plaque dry weight). 3,27,31 However, the concentration of polysaccharides in 787

7 H. Koo et al. human plaque depends on duration since the last intake of the dietary sugars, and, in addition, there are no degradative processes occurring on the polysaccharides in our model, in contrast to what occurs in dental plaque. The higher polysaccharide concentration may also be related to the constant exposure of S. mutans to sucrose in our biofilm model; a similar concentration of total carbohydrates was found in other models of S. mutans biofilms growing, in the presence of sucrose, in either batch or continuous mode. 32,33 Collectively, the data obtained in this study showed that a brief exposure (1 min) twice daily of apigenin and tt-farnesol caused a significant impact on the accumulation and the polysaccharide content of S. mutans biofilms. In general, apigenin and tt-farnesol had a more dramatic effect on the biomass and the total amount of polysaccharides than on the population of viable cells of S. mutans and the proportions of polysaccharides in the biofilms. The topical application of the test agents was sufficient to significantly affect the rate of glucan synthesis, and, consequently, reduce the accumulation and biomass of the biofilms. We are currently pursuing the molecular mechanisms involved in the inhibition of the biofilms by these agents. Although details of the toxicology of these compounds were not investigated here, we did not observe any adverse reactions in our recent animal study. 17 Apigenin is a naturally occurring, nonmutagenic, non-toxic bioflavonoid ubiquitously found in vegetables and fruits, and it is part of the human daily diet (1 4 mg/day). 21,48 tt-farnesol is a natural sesquiterpene alcohol found in essential oils of citrus fruits, and is also devoid of toxic effects in vivo. 22 These two natural compounds may represent a novel approach to the prevention of dental caries, since they will not necessarily have a major effect on the viability of the oral flora population (in contrast to chlorhexidine), but rather they will disrupt the accumulation and the polysaccharide content of dental plaque related to this common oral disease. Acknowledgements We are grateful to Dr R. E. Marquis for critical reading of the manuscript prior to submission and to Kathleen Scott-Anne for technical assistance. 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