Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Identification of anti-biofilm components in Withania somnifera and their effect on virulence of Streptococcus mutans biofilms S. Pandit1, J.N. Cai1, K.Y. Song2 and J.G. Jeon1,2 1 Department of Preventive Dentistry, School of Dentistry, Institute of Oral Bioscience and BK 21 Plus Program, Chonbuk National University, Jeonju, Korea 2 Research Institute of Clinical Medicine of Chonbuk National University, Biomedical Research Institute of Chonbuk National University Hospital, Jeonju, Korea

Keywords acidogenicity, aciduricity, dental caries, linoleic acid, Streptococcus mutans biofilm, Withania somnifera. Correspondence Jae-Gyu Jeon and Kwang-Yeob Song, Research Institute of Clinical Medicine of Chonbuk National University, Biomedical Research Institute of Chonbuk National University Hospital, Jeonju 561-756, Korea. E-mails: [email protected] and [email protected] 2014/2479: received 3 December 2014, revised 14 April 2015 and accepted 12 May 2015 doi:10.1111/jam.12851

Abstract Aims: The aim of this study was to identify components of the Withania somnifera that could show anti-virulence activity against Streptococcus mutans biofilms. Methods and Results: The anti-acidogenic activity of fractions separated from W. somnifera was compared, and then the most active anti-acidogenic fraction was chemically characterized using gas chromatography-mass spectroscopy. The effect of the identified components on the acidogenicity, aciduricity and extracellular polymeric substances (EPS) formation of S. mutans UA159 biofilms was evaluated. The change in accumulation and acidogenicity of S. mutans UA159 biofilms by periodic treatments (10 min per treatment) with the identified components was also investigated. Of the fractions, n-hexane fraction showed the strongest anti-acidogenic activity and was mainly composed of palmitic, linoleic and oleic acids. Of the identified components, linoleic and oleic acids strongly affected the acid production rate, F-ATPase activity and EPS formation of the biofilms. Periodic treatment with linoleic and oleic acids during biofilm formation also inhibited the biofilm accumulation and acid production rate of the biofilms without killing the biofilm bacteria. Conclusions: These results suggest that linoleic and oleic acids may be effective agents for restraining virulence of S. mutans biofilms. Significance and Impact of the Study: Linoleic and oleic acids may be promising agents for controlling virulence of cariogenic biofilms and subsequent dental caries formation.

Introduction Dental caries is a dental biofilm-related disease. If dental biofilms are allowed to remain on tooth surfaces with the frequent consumption of sugar, acidogenic bacteria in dental biofilms will metabolize the sugar to organic acids (Takahashi et al. 2010). The persistence of this acidic condition leads to selection and dominance of highly acid tolerant and acidogenic micro-organisms such as mutans streptococci and other acidogenic streptococci as well as

lactobacilli and bifidobacteria (Marsh 2010). The shift of microbial composition to aciduric and acidogenic bacteria in dental biofilms (formation of cariogenic biofilms) is principal importance in the pathogenesis of dental caries (Marsh 2003). Although the abundance of mutans streptococci is not closely related to the incidence of dental caries (van Houte et al. 1991; Sansone et al. 1993), many studies have nevertheless identified Streptococcus mutans is the primary pathogen of dental caries (Hamada and Slade 1980; Loesche 1986).

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Streptococcus mutans is a Gram-positive, facultative anaerobic bacteria commonly found in the oral cavity. The bacterium can degrade carbohydrates rapidly, resulting in the formation of large amounts of acid, and which is highly tolerant of low-pH environments (Svens€ater et al. 1997). Under acidic conditions, S. mutans alters its physiology in a variety of ways to survive, including increased glycolytic activity and increased activity of the proton-translocating ATPase regulating intracellular pH (Welin-Neilands and Svens€ater 2007). Furthermore, S. mutans effectively utilizes dietary sucrose to synthesize extracellular polymeric substances (EPSs) through the activity of glucosyltransferases (GTFs) (Koo et al. 2010). EPSs are crucial to the formation, physical integrity and stability of the biofilm matrix (Koo et al. 2010; Xiao and Koo 2010). The elevated amount of EPSs in biofilm matrix can affect the diffusion properties and serves to protect against environmental and antimicrobial assaults (Falsetta et al. 2012). It is widely accepted that EPSs, acidification of the biofilm matrix and acid tolerance mechanisms are critical for the development of dental caries (Koo and Jeon 2009). Thus, if virulence of S. mutans in cariogenic biofilms (acidogenicity, aciduricity and EPS formation) can be reduced, the risk for dental caries development could be decreased. A widely adopted approach for the control of cariogenic biofilms is the topical application of chemoprophylactic agents such as chlorhexidine and essential oils (Ribeiro et al. 2007). The action of the majority of chemoprophylactic agents is bactericidal (Koo and Jeon 2009). The focus on bactericidal effects is logical considering that cariogenic biofilms are principally composed of bacteria. However, the use of bactericidal agents can result in a disruption of the resident microflora, which may in turn lead to pathological changes (Marsh 2009). Recently, chemoprophylactic approaches aimed at decreasing virulence of S. mutans without necessarily killing the target bacterium have attracted considerable attention (Koo and Jeon 2009; Falsetta et al. 2012). Disarming pathogens by targeting their virulence without threatening their existence may offer a reduced selection pressure for drug-resistant mutations and may also avoid dramatic alterations of the resident microflora (Cegelski et al. 2008). Natural products are a possible alternative source of agents capable of controlling cariogenic biofilms (Jeon et al. 2011; Furiga et al. 2014). Over 70% of the therapeutic agents developed between 1981 and 2003 for infectious diseases were derived from natural products (Newman et al. 2003). Withania somnifera (Ashwagandha) is a plant of the Solanaceae family. It has been widely used to remedy a variety of ailments as well as a general tonic for overall health and longevity in Indian and Nepalese 572

traditional medicine (Bhattarai et al. 2010). Withania somnifera has also been traditionally used to control dental diseases (Mirjalili et al. 2009). We previously reported that W. somnifera extract inhibits physiological ability of S. mutans biofilms (Pandit et al. 2014), suggesting that the main components of the plant might be useful for restraining virulence of cariogenic biofilms. However, the precise anti-virulence components of the plant and their effect on dental biofilms, especially cariogenic biofilms, have not been elucidated. In this study, we attempted to identify anti-virulence components of W. somnifera and evaluate their effect on acidogenicity, aciduricity and EPS formation of cariogenic biofilms. Specifically, we investigated the change in biofilm accumulation and acidogenicity following periodic treatments (10 min per treatment, a total of five times) with the identified components in S. mutans biofilms. Materials and methods Bacterial strains, plant materials and fractionation For this study, S. mutans UA159 (ATCC 700610; serotype c) was grown in buffered tryptone-yeast extract broth (TYE broth; 2
5% tryptone and 1
5% yeast extract, pH 7
0) supplemented with 1% glucose (for planktonic cells) or 1% sucrose (for biofilms). Prior to use, the medium had been filtered through a Prep/Scale 10 kDa molecularweight cut-off membrane (Millipore, Billerica, MA). Withania somnifera root was purchased from Dekha Herbals (Hattiban, Dhapakhel-1 Lalitpur, Nepal). The dried and finely-ground root (200 g) was extracted with methanol (2 l) at room temperature for 24 h and concentrated under vacuum (yield: 6%). A portion of methanol extract (10 g, ME) was resuspended in 70% aqueous methanol and serially fractioned with n-hexane, and ethyl acetate. All of the fractioned materials were concentrated under vacuum, lyophilized and stored at 20°C. The final yield of hexane fraction (HF), ethylacetate fraction (EF) and aqueous fraction was 0
67, 11
05 and 64% of ME respectively. Identification of anti-virulence components To identify anti-virulence components, the anti-acidogenic activity of the fractions against S. mutans was compared and the most active fraction was chemically characterized. Comparison of anti-acidogenic activity of natural products is recommended for screening natural products for anti-virulence components against cariogenic biofilms (Jeon et al. 2011). To compare the anti-acidogenic activity of the fractions, a glycolytic pH drop assay was carried out using planktonic S. mutans UA159 (Belli

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et al. 1995). Briefly, S. mutans was harvested, washed and resuspended in a salt solution containing vehicle control (4% dimethlysulfoxide, DMSO), ME (100 lg ml 1), or each fraction of ME (100 lg ml 1). The pH was adjusted to 7
2, and glucose was added to the mixture to a final concentration of 1%. The drop of pH in each suspension was measured at 5-min intervals. The effect of the test agents on acidogenicity of the bacterial suspension was determined by calculating the acid production rate using pH values in the linear range (0–10 min). To identify the main components of the most active anti-acidogenic fraction, gas chromatography-mass spectrometry (GC-MS) analysis was performed using an Agilent HP-6890 GC/5973 mass selective detector (Agilent Technologies, Palo Alto, CA) with an HP-DB5 MS capillary column (30 m 9 0
25 mm 9 0
25 lm). Sample was dissolved in n-hexane (injection volume: 1 ll), and the injector port was heated to 280°C. Helium at a constant flow of 1 ml min 1 served as the carrier gas. The oven temperature was initially set at 70°C for 3 min, increased to 300°C at a rate of 10°C min 1 and held for 5 min. Spectra were obtained in EI mode with 70 eV ionization energy. The sector mass analyzer was set to scan from 50 to 600 amu. Components were identified by comparing their mass spectra with those in a mass spectra library (The Wiley Registry of Mass Spectral Data, 7th Ed.). The identified components were purchased from Sigma-Aldrich (St Louis, MO). The experimental concentration of the components was 100 lg ml 1. The most active antiacidogenic fraction (100 lg ml 1), vehicle control (4% DMSO) and NaF (positive control, 100 lg ml 1) were also included for this study. Streptococcus mutans biofilm preparation Streptococcus mutans UA159 biofilms were formed on saliva-coated hydroxyapatite (sHA) discs (2
93 cm2) placed vertically in 24 well plates, as detailed elsewhere (Pandit et al. 2011). Briefly, the sHA discs were generated by incubation with sterilized saliva for 1 h at 37°C. The sHA discs were transferred to a 24-well plate containing 1% sucrose TYE broth (pH 7
0) with S. mutans UA159 (2– 5 9 106 CFU ml 1). The biofilms were grown in batch cultures at 37°C in 5% CO2 for 74 h. During the first 22 h, the biofilms grew undisturbed to allow initial biofilm formation. After 22 h, the culture medium was changed twice daily (9 a.m. and 6 p.m.) until 74 h. Finally, 74-h-old biofilms were used for virulence assays. To determine the effect of periodic treatments on biofilm accumulation and acidogenicity, the biofilms were treated twice daily (9 a.m. and 6 p.m.) with the test agents beginning at 22 h and which continued throughout the experimental period (74 h).

Anti-biofilm components from W. somnifera

Effects on acidogenicity of Streptococcus mutans biofilms To evaluate the effect of the test agents on acidogenicity of S. mutans biofilms, a glycolytic pH drop assay was carried out as described previously (Pandit et al. 2011). Briefly, 74 h biofilms were transferred to a salt solution (50 mmol l 1 KCl plus 1 mmol l 1 MgCl2, pH 7
0) containing the test agents. The pH was adjusted to 7
2 with 0
2 mol l 1 KOH after which glucose (final concentration: 1%) was added. The decreases in pH were assessed over 120 min. The activity of the test agents against acidogenicity was determined according to the acid production rate, calculated by the change in pH values over the linear portion (0–50 min) of the pH drop curves. Effects on aciduricity of Streptococcus mutans biofilms To evaluate the effect of the test agents on aciduricity of S. mutans biofilms, acid killing, proton permeability and F-ATPase activity assays were performed. For the acid killing assay (Xiao and Koo 2010), 74 h biofilms were incubated in 0
1 mol l 1 glycine buffer (pH 2
5) with the test agents for 90 min. After 90 min incubation, biofilms were collected and homogenized. The homogenized suspensions were then serially diluted and plated on BHI agar plate to determine the number of the viable biofilm cells. Proton permeability assay was performed as described previously (Pandit et al. 2011). Briefly, 74-h-old biofilms that had been incubated in 20 mmol l 1 potassium phosphate buffer (pH 7
2) for 1 h to deplete endogenous catabolites were incubated initially at a constant pH of 4
6. Hydrochloric acid was then added to decrease the pH values by 0
4 units followed by the addition of the test agents. The subsequent increase in pH associated with proton movements across the cell membrane into the cytoplasm was monitored using a glass electrode. The activity of the test agents against proton permeability was determined using the proton permeability rate, calculated using pH values (0–10 min) of the pH curves. The F-ATPase activity of the biofilm cells were determined as described previously (Belli et al. 1995). Briefly, 74 h of biofilms were collected and homogenized by sonication at 7 W for three periods of 30 s each (VCX 130PB; Sonics and Materials Inc., Newtown, CT). Homogenized biofilm cells were then permeabilized with 10% toluene followed by the two freeze and thaw cycles. F-ATPase activity was measured in terms of the release of phosphate in the following reaction mixture: 75 mmol l 1 of Tris-maleate buffer (pH 7
0) containing 5 mmol l 1 adenosine triphosphate (ATP), 10 mmol l 1 magnesium chloride, permeabilized biofilm cells and test agents. The amount of phosphate released over the

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30 min reaction time was determined as described previously (Bencini et al. 1983). Effects on GTF activity and EPS formation of Streptococcus mutans biofilms To determine GTF activity and EPS formation of the biofilms, GTF activity assay and laser scanning confocal fluorescence microscopy (LSCM) analysis were performed respectively. The GTF activity assay is described previously (Song et al. 2006). Briefly, crude GTFs of S. mutans UA159 were precipitated from the culture supernatant by adding ammonium sulphate, and recovered. The reaction mixture (1 ml) consisted of 300 ll crude GTFs, 175 ll of 0
01 mol l 1 potassium phosphate buffer (pH 6
8), 500 ll of 0
01 mol l 1 potassium phosphate buffer (pH 6
8) containing 5% sucrose, and test agents, which was allowed to react for 24 h. For LSCM analysis, 74 h biofilms were transferred to new culture medium containing the test agents and 1 lmol l 1 Alexa Fluorâ 647-labelled dextran conjugate (10 000 MW; absorbance/fluorescence emission maxima 647/668 nm; Molecular Probes, Eugene, OR) and incubated for 2 h. The fluorescence-labelled dextran serves as a GTF primer and can be incorporated into newly formed EPS during synthesis of the EPS matrix (Koo et al. 2010). After incubation of 2 h, the bacterial cells were labelled with 2
5 lmol l 1 of SYTOâ 9 green-fluorescent nucleic acid stain (480/500 nm; Molecular Probes). LSCM imaging of the biofilms was performed using the LSM 510 META (Carl Zeiss, Jena, Germany) equipped with argon-ion and helium neon lasers. Two independent experiments were performed and image stacks (512 9 512 pixel) from 10 sites per experiment were collected (n = 20). EPS thickness and bio-volume were quantified from confocal stacks by COMSTAT (Heydorn et al. 2000). The bio-volume was defined as the volume of the biomass (lm3) divided by substratum (hydroxyapatite surface) area (lm2). Change in biofilm accumulation and acidogenicity after periodic treatments To determine the effect of periodic treatments of the test agents on accumulation and acidogenicity of S. mutans biofilms, the biofilms were treated twice daily (9 a.m. and 6 p.m.) with the test agents beginning at 22 h and continuing throughout the experimental period (74 h). Specifically, biofilms were exposed to the treatment for 10 min, dip-rinsed twice in autoclaved water and transferred to fresh culture medium. Biofilms were exposed to the treatment a total of five times. To analyse biofilm accumulation, the treated 74 h biofilms were removed 574

and sonicated, and the resulting homogenized suspension was used to determine dry weight, CFU count and waterinsoluble EPS amount as previously described (Koo et al. 2005). To determine the cell viability, homogenized suspensions were serially diluted and plated onto BHI agar to count CFUs. To evaluate acidogenicity, the acid production rate was calculated using the 2 h pH change in culture medium after each treatment. LSCM analysis was also performed to confirm the change in the biofilm accumulation as described previously (Jeon et al. 2009; Koo et al. 2010). Briefly, 1 lmol l 1 Alexa Fluorâ 647labelled dextran conjugate was added to the culture medium during biofilm formation and development. After 74 h, treated biofilms were exposed to 2
5 lmol l 1 SYTOâ 9 green-fluorescent nucleic acid stain. Two independent experiments were performed and 10 image stacks (512 9 512 pixel) from 10 sites per experiment were collected (n = 20). Biofilms (bacterial bio-volume and thickness, and EPS bio-volume and thickness) were quantified from the confocal stacks by COMSTAT (Heydorn et al. 2000). The three-dimensional architecture of the biofilms was visualized using IMARIS software (Bitplane, Zurich, Switzerland). Statistical analysis All assays except for GC-MS and LSCM analysis were performed at least five times. Data are presented as the mean  standard deviation (SD). The intergroup differences were estimated by one-way ANOVA, followed by a post hoc multiple comparison (Tukey test). Values were considered statistically significant at P < 0
05. Results Identification of anti-virulence components The glycolytic pH drop assay was used to select the most active anti-acidogenic fraction from W. somnifera. As shown in Fig. 1a, ME and its fractions reduced acid production rate of S. mutans compared to the vehicle control (P < 0
05). Of the fractions separated, HF exhibited the strongest anti-acidogenic activity, reducing the acid production rate by up to 99
9% (Fig. 1a). Thus, HF was selected as the most active anti-acidogenic fraction. The identification of the main components of HF was performed by GC-MS analysis. As shown in Fig. 1b, approximately nine components were detected in HF. The main components were palmitic acid (31
14% of the total ion chromatogram (TIC)), linoleic acid (34
57% of the TIC) and oleic acid (24
31% of the TIC). The total relative area (RAP) of these components in the chromatogram was 90%, suggesting that HF principally consisted of

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Anti-biofilm components from W. somnifera

(b) 12E+06

(a) 7·5

Abundance

7·0 6·5

pH

6·0

2 3

8E+06 6E+06 4E+06 2E+06 The number of peak: 9 0

5·5

6

5·0 4·5

8 10 12 14 16 18 20 22 24 26 28 30 32 Time (min) RT (min) RAP (%)

Peak # Components

4·0 3·5

1

10E+06

1

Palmitic acid

O

19·24

31·14

20·90

34·57

20·95

24·31

HO 0

10

20

Acid production rate Vehicle

ME

(H+

40 30 Time (min)

50

concentration (µmol

l–1

AF

60

Linoleic acid 2

EF

min–1))

3

HF

3·09±0·12a 0·51±0·27b 1·10±0·18c 0·04±0·12d 0·002±0·00d

O

HO Oleic acid O OH

Figure 1 Selection and chemical characterization of the most active anti-acidogenic fraction from Withania somnifera. (a) Effect of methanol extract (ME, ●) and its fractions on acidogenicity of planktonic Streptococcus mutans; (b) Total ion chromatogram (TIC) of n-hexane fraction (HF, ▲) and its major components by gas chromatography-mass spectrometry. Vehicle control (Vehicle, ■) was 4% DMSO. All data represent mean  standard deviation. Values followed by the same superscripts are not significantly different from each other (P > 0
05). Abbreviations are: AF (□), Aqueous fraction; EF (○), Ethyl acetate fraction, RT, Retention time; RAP, Relative percentage of peak area (peak area relative to total area %).

these components and that the anti-acidogenic activity of the fraction may be due to these components. Thus, palmitic, linoleic and oleic acids were putatively identified as anti-virulence components. Effects on acidogenicity and aciduricity of Streptococcus mutans biofilms HF and its main components inhibited acidogenicity and aciduricity of S. mutans biofilms. HF and its main components reduced acid production rate of the biofilms by 77–89% compared to the vehicle control (P < 0
05) (Fig. 2a). The inhibitory activity of HF and its main components against the acid production rate was similar to that of NaF, although there were no significant differences among the individual test agents. HF, linoleic acid and oleic acid significantly reduced the number of surviving biofilm cells after 90 min exposure at pH 2
5 compared to the vehicle control (P < 0
05) (Fig. 2b). However, palmitic acid did not affect the number of surviving biofilm cells. The effect of HF and its main components on acid killing was lower than that of NaF. In addition to the acid killing assay, F-ATPase activity and proton permeability assays were performed to confirm the effect of HF and its main components on aciduricity of biofilm cells. As shown in Fig. 2c,d, although HF and

its main components did not affect the proton permeability rate, the test agents reduced F-ATPase activity of the biofilm cells by 13–65% compared to the vehicle control (P < 0
05). Of the test agents, linoleic and oleic acids had the strongest inhibitory effects on the F-ATPase activity, decreasing the enzyme activity by about 65%, which was stronger than that of NaF (P < 0
05). Effects on GTF activity and EPS formation of Streptococcus mutans biofilms Although HF and its main components did not affect GTF activity (Fig. 3a), the test agents decreased the extent of EPS synthesis by the biofilm cells. As shown in Fig. 3b,c, HF and its main components reduced bio-volume and thickness of EPS compared to the vehicle control (P < 0
05). Of the test agents, linoleic acid had the strongest inhibitory effect, decreasing the EPS bio-volume by about 95% and thickness by about 91% compared to the vehicle control (P < 0
05). The inhibitory effect of linoleic acid on EPS bio-volume was higher than that of NaF (P < 0
05), while the effects of HF, palmitic acid and oleic acid were similar to that of NaF. Fig. 3d-1, d-2, d-3 and d-4 show representative LSCM images of the EPS of biofilms treated with the vehicle control, HF, linoleic acid and NaF, respectively, in which the EPS amounts

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Anti-biofilm components from W. somnifera

0·25

(b) 1E6

a

0·20

b c

1E4

0·15 0·10

b

c

1E3 1E2 10

b

0·05

b

d

b

1 b

0 HF

PA

LA

OA

5

b

4 a

a

a a

a

3

2

1

0

0·1

NaF (d) F-ATPase activity (% of activity)

H+ permeability rate (µmol l–1 min–1))

Vehicle (c)

a

a

1E5

CFU disc–1

Acid production rate (H+ concentration (µmol l–1 min–1))

(a)

S. Pandit et al.

HF

PA

LA

OA

NaF

HF

PA

LA

OA

NaF

a 100

c

80

b

b 60 d d

40 20 0

Vehicle

Vehicle

Vehicle

HF

PA

LA

OA

NaF

Figure 2 Effect of HF and its main components on acidogenicity and aciduricity of Streptococcus mutans biofilms. (a) Acid production rate. (b) Number of viable cells during acid challenges. (c) Proton permeability rate. (d) F-ATPase activity. The acid production and proton permeability rates were calculated by the pH values in the linear portion (0–50 min) of the result of glycolytic pH drop assay and by pH values (0–10 min) of the result of proton permeability assay, respectively. The experimental concentration of the test agents was 100 lg ml 1. Vehicle control (Vehicle) was 4% DMSO. Abbreviations are: HF, n-Hexane fraction, PA, Palmitic acid; LA, Linoleic acid; OA, Oleic acid; NaF, Sodium fluoride. All data represent mean  standard deviation. Values followed by the same superscripts are not significantly different from each other (P > 0
05).

after treatment with HF, linoleic acid and NaF were reduced compared to the vehicle control. Change in biofilm accumulation and acidogenicity after periodic treatments Among the test agents, linoleic and oleic acids strongly diminished dry weight and water-insoluble EPS formation of S. mutans biofilms following the periodic treatments (Table 1). However, palmitic acid did not affect the dry weight and water-insoluble EPS formation. Linoleic acid was the most effective components and decreased the dry weight and water-insoluble EPS by 36 and 43%, respectively, compared to the vehicle control (P < 0
05). Oleic acid also reduced the dry weight and water-insoluble EPS formation by 25 and 31%, respectively, compared to the vehicle control (P < 0
05). Generally, the inhibitory effect of linoleic and oleic acids was 576

similar to that of NaF. Despite these results, none of the test agents exhibited bactericidal activity (Table 1). Linoleic acid also affected acid production rate of the biofilms after each treatment (30–50% reduction) (P < 0
05) (Table 2). Linoleic acid began to reduce the acid production rate after the first treatment and which continued throughout each treatment thereafter. However, the inhibitory effect of linoleic acid on the acid production rate was weaker than that of NaF. Interestingly, the other test agents did not begin to affect the acid production rate until the third treatment. As shown in Fig. 4, LSCM analysis confirmed the inhibitory effect of linoleic and oleic acids on EPS formation as a result of the periodic treatments. Specifically, linoleic acid reduced EPS bio-volume and thickness by 42 and 39% compared to the vehicle control, respectively (P < 0
05) (Fig. 4c,d), which was similar to that of NaF (P > 0
05). The inhibitory effect of oleic acid was lower

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a

a

a

EPS bio-volume (µm3 µm–2)

0·5 GTF avtivity (O.D)

(b)

a a

a

0·4 0·3 0·2 0·1 0 Vehicle HF

(d-1)

PA

OA

LA

90

0µ

20 15

90

m

0µ

m

b

b,c 5

b

c

120 100 b

80

b b,c

b,c

60 40

c

20

0

0 Vehicle HF

90 0µ

b

10

NaF

a

140

a

25

(d-2)

Vehicle

(c)

30

EPS thickness (µm)

(a)

PA

LA

(d-3)

HF

90

m

OA

0µ

90 0µ

m

NaF

Vehicle HF

PA

LA

(d-4)

LA

m

NaF

NaF

m

90

0µ

0µ

0µ

90

m

OA

90

m

Figure 3 Effect of HF and its main components on glucosyltransferase (GTF) activity and extracellular polymeric substances (EPS) formation by Streptococcus mutans biofilms. (a) GTF activity. (b) EPS bio-volume and (c) EPS thickness, calculated from laser scanning confocal fluorescence microscopy (LSCM) data. (d) Representative LSCM images of EPS, formed during 2 h treatment with vehicle control (d-1), HF (d-2), linoleic acid (d-3) and sodium fluoride (d-4). The experimental concentration of the test agents was 100 lg ml 1. Vehicle control (Vehicle) was 4% DMSO. Abbreviations are: HF, n-Hexane fraction; PA, Palmitic acid; LA, Linoleic acid; OA, Oleic acid; NaF, Sodium fluoride. All data represent mean  standard deviation. Values followed by the same superscripts are not significantly different from each other (P > 0
05). Table 1 Change in Streptococcus mutans biofilm composition after periodic treatment with hexane fraction and its main components

1

Dry weight (mg disc ) CFU per disc (9108) Water-insoluble extracellular polymeric substances (mg disc 1)

Vehicle control

Hexane fraction

Palmitic acid

5
20  0
40 3
78  1
93a 1
52  0
27a

4
33  0
50 1
68  0
90a 1
14  0
17a,b

4
73  0
47 3
46  1
62a 1
45  0
31a,c

a

b

Linoleic acid a,b

Oleic acid

3
33  0
35 2
86  2
77a 0
88  0
17b c

NaF

3
90  0
52 4
11  2
09a 1
06  0
15b,c b,c

4
00  0
33b,c 4
64  2
03a 0
91  0
31b

Each biofilm was twice daily treated with the respective agent (10 min per treatment) a total of five times during biofilm formation. All data represent mean  standard deviation. Values followed by the same superscripts (in the same raw) are not significantly different from each other (P > 0
05).

Table 2 Change in acid production rate (lmol H+ h 1) of Streptococcus mutans biofilms by periodic treatment with hexane fraction and its main components Vehicle control After After After After After

1st treatment 2nd treatment 3rd treatment 4th treatment 5th treatment

0
08 0
76 0
78 1
04 1
31

    

0
02a 0
10a 0
15a 0
04a 0
23a

Hexane fraction 0
06 0
52 0
61 0
83 1
09

    

0
02a,b 0
07b,c 0
16a,b 0
08b 0
39a,b

Palmitic acid 0
06 0
64 0
72 0
84 1
02

    

0
02a,b 0
04a,b 0
13a,b 0
03a,b 0
17a,c

Linoleic acid 0
04 0
41 0
45 0
74 0
70

    

0
01b 0
07c,d 0
04b,c 0
08b 0
12c

Oleic acid 0
05 0
61 0
63 0
73 0
90

    

0
02a,b 0
11a,b 0
17a,b 0
14b 0
11a,c

NaF 0
03 0
29 0
20 0
48 0
56

    

0
02b 0
08d 0
03c 0
10c 0
10c

Each biofilm was twice daily treated with the respective agent (10 min per treatment) a total of five times during biofilm formation. All data represent mean  standard deviation. Values followed by the same superscripts (in the same raw) are not significantly different from each other (P > 0
05).

than that of linoleic acid. Interestingly, HF also diminished EPS bio-volume and thickness by 41 and 45%, respectively, compared to the vehicle control (P < 0
05).

Nevertheless, none of the test agents affected bacterial bio-volume, though bacterial thickness changed in accordance with EPS thickness (Fig. 4a,b). Figure 4e-1, e-2,

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Figure 4 Laser scanning confocal fluorescence microscopy (LSCM) analysis of Streptococcus mutans biofilms after periodic treatment with HF and its main components. (a) Bacterial bio-volume. (b) Bacterial thickness. (c) Extracellular polymeric substances (EPS) bio-volume. (d) EPS thickness. (e) Representative LSCM images from S. mutans biofilms treated with vehicle control (e-1), HF (e-2), linoleic acid (e-3) and sodium fluoride (e-4). Vehicle control (Vehicle) was 4% DMSO. Abbreviations are as follows: HF, n-Hexane fraction; PA, Palmitic acid; LA, Linoleic acid; OA, Oleic acid; NaF, Sodium fluoride. The experimental concentration of the test agents was 100 lg ml 1. Each biofilm was treated twice daily with the respective agent (10 min per treatment) a total of five times during biofilm formation. All data represent mean  standard deviation. Values followed by the same superscripts are not significantly different from each other (P > 0
05). In the LSCM images, the green is bacteria and the red is EPSs.

e-3 and e-4 show representative LSCM images of the biofilms treated with the vehicle control, HF, linoleic acid and NaF, respectively, in which the bio-volume and EPS thickness were reduced by the test agents compared to the vehicle control. Discussion Dental caries is one of the most common infectious oral diseases. Although several chemoprophylatic agents are available for the prevention of dental caries, the search for an effective agent continues due to the undesirable side effects associated with these agents (Newman et al. 2003; Khan et al. 2010; Giacaman et al. 2014a,b). In this context, this study was conducted to estimate the potential of W. somnifera as a new anti-dental biofilm agent. Furthermore, since the majority of the chemoprophylactic agents involve bactericidal activity, this study focused especially on an approach aimed at reducing the virulence properties of bacteria without necessarily killing the bacteria. In this study, HF and its main components were not bactericidal on 74 h biofilm cells at the test concentration (100 lg ml 1) over 2 h (data not shown), suggesting that false-positive results due to bactericidal activity of the test agents could be eliminated. In addition, anti-biofilm activity of the test agents were evaluated at the concentration of 100 lg ml 1, as medicinal 578

plants that exhibit antimicrobial activity at the concentration lower than 100 lg ml 1 could show a great antimicrobial potential (Yatsuda et al. 2005; Song et al. 2007). Strategies for the identification of bio-active components from natural products have evolved quite significantly over the last two decades (Sarker et al. 2006). Of the strategies, bioassay-guided approaches are widely and typically applied (Sarker et al. 2006). In this study, the glycolytic pH drop assay was used as a bio-assay guide for the identification of anti-virulence components against S. mutans biofilms, as the assay can evaluate acidogenicity and aciduricity of S. mutans (Gregoire et al. 2007). In addition, the data from the glycolytic pH drop assay may reflect to some extent EPS formation by the bacterium, as the enzyme secretion for EPS synthesis is generally coupled to the DpH across the cell membrane. Therefore, the glycolytic pH drop assay can be appropriate to screen bio-active components against virulence associated with the pathogenesis of dental caries. Furthermore, as shown in Figs 2 and 3, the identified components inhibited virulence of S. mutans biofilms, suggesting that this assay may be useful for the identification of anti-cariogenic components in future studies. In this study, unsaturated fatty acids such as linoleic and oleic acids were identified as anti-virulence components (Fig. 1), and reduced acidogenicity and aciduricity of S. mutans biofilms (Fig. 2). The reduction in the

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acidogenicity and aciduricity of the biofilms may be due to the inhibitory effect of linoleic and oleic acids on FATPase activity (Fig. 2d). Protons from the extracellular environment diffuse across cell membranes, but are then extruded by the membrane-associated F-ATPase enzyme (Quivey et al. 2001). The proton-translocating F-ATPase protects S. mutans against environmental acid stress by regulating pH homeostasis, which is critical for optimum function of glycolysis in S. mutans (Sturr and Marquis 1992). Therefore, the inhibitory effect of linoleic and oleic acids on F-ATPase activity may lead to the disturbance of aciduricity and then inhibit glycolysis by the biofilm cells. The inhibitory effect of linoleic and oleic acids on F-ATPase activity may be associated with their detergent properties. The detergent properties of free fatty acids can affect bacterial membranes to such an extent that the functions of various membrane proteins are impaired (Desbois and Smith 2010; Huang et al. 2010). In addition to F-ATPase activity, the changes in proton permeability are also important in maintenance of aciduricity of S. mutans (Quivey et al. 2001). In this study, linoleic and oleic acids did not disturb proton permeability of the biofilm cells (Fig. 2c); however, NaF strongly increased proton permeability. This result may indicate that fatty acids and NaF have, at least in part, different mechanisms of action with respect to modulating the aciduricity of the biofilm cells. Furthermore, this result may explain the effect of NaF on acid killing (Fig. 2b), whereby NaF affected the survival of acid challenged cells to a greater degree than linoleic and oleic acids. Unsaturated fatty acids, such as linoleic and oleic acids, are well known to have antibacterial activity and their in vitro as well as in vivo anti-cariogenic potential, although it remains unclear exactly how these acids exert their activity (Kabara et al. 1972; Schuster et al. 1980). In the dental field, previous studies also demonstrated that linoleic and oleic acids have strong bactericidal activity against planktonic S. mutans (Osawa et al. 2001; Huang and Ebersole 2010), suggesting the potential of these fatty acids as anti-biofilm agents. Furthermore, linoleic and oleic acids showed inhibitory effects on sucrose induced demineralization (Giacaman et al. 2014a,b). However, little information is available on effects of the fatty acids on the viability and physiology of pathogenic bacteria in biofilms, although bacteria in biofilms are generally understood to be physiologically and functionally distinct from planktonic cells (Hall-Stoodley et al. 2004). Linoleic and oleic acids also reduced new EPS formation by mature S. mutans biofilms (Fig. 3b,c), suggesting that these components can modulate the development and accumulation of cariogenic biofilms. As shown in Fig. 3a, the activity of the components against EPS formation was not due to inhibition of GTF activity. Thus, the possible mechanisms

Anti-biofilm components from W. somnifera

by which the components reduce EPS formation may involve their inhibitory effect on F-ATPase activity of S. mutans biofilm cells (Fig. 2d). As enzyme secretion by bacterial cells is generally coupled to DpH across the cell membrane (Marquis et al. 2003), it is possible that the components, which can affect DpH by disrupting F-ATPase activity of the biofilm cells, could affect the secretion of GTFs and thereby reduce the synthesis of EPS. When delivering topical chemoprophylatic agents to the oral cavity, a primary requirement is to deliver a sufficient concentration of the agents to have an effect on dental biofilms during a short period time (Marsh 2012). Thus, our experimental protocol employed a 10 min treatment schedule to model the potential use of the identified components as an ingredient of mouth rinses or dentifrices. In this study, our results showed that linoleic and oleic acids had inhibitory activity against biofilm accumulation and acidogenicity without killing the biofilm cells after periodic 10 min treatment (Tables 1 and 2). The reduction in biofilm accumulation (dry weight) was likely due to the inhibition of water-insoluble EPS formation by the treatment (Table 1), since 40% of the dry weight of biofilm composed of EPS (Xiao and Koo 2010). The treatment neither exerted bactericidal activity (Table 1) nor affected bacterial bio-volume (Fig. 4a). The inhibition of EPS formation by the treatment was confirmed by LSCM analysis (Fig. 4c–e). Taken together, our results suggest that linoleic and oleic acids may be useful to modulate the accumulation of and acidogenicity of human cariogenic biofilms without disruption of the resident microflora when delivered to the oral cavity via mouth rinse or dentifrice. In this study, NaF was used as a positive control for three reasons: (i) many previous studies have demonstrated NaF can affect the physiology of microbes, including cariogenic streptococci (Hamilton 1990; Marquis et al. 2003); (ii) NaF is generally used as an ingredient of mouth rinse or dentifrice; and (iii) there is a lack of other appropriate positive controls with the ability to modulate the virulence and accumulation of cariogenic biofilms. In this study, it was noteworthy that the potency of linoleic and oleic acids was comparable with that of NaF at the same concentration (Figs 2–4), suggesting that the components may be useful to enhance the anti-cariogenic activity of fluoride without increasing its exposure. Furthermore, the components may have some advantage for use as an ingredient of anti-biofilm agents because of its role in the human diet. It has been widely reported that linoleic acid is the most abundant polyunsaturated fatty acid in human nutrition, representing about 14 g per day in the American diet. Furthermore, the recommended intake for linoleic acid in the US ranges from 5–10% of energy (Choque et al. 2014).

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These reports suggest that linoleic acid may have little toxicity and few side effects when used as an ingredient of anti-biofilm agents for topical application. In conclusion, free fatty acids, especially linoleic and oleic acids, which were identified from W. somnifera, reduced acid production rate, F-ATPase activity and EPS formation of S. mutans biofilm cells. Furthermore, when S. mutans biofilms were briefly treated with linoleic acid (10 min, a total of five times), EPS accumulation and acid production rate of the biofilms were reduced without affecting bacterial viability. Importantly, the potency of linoleic acid was comparable with that of NaF. Taken together, these results suggest that linoleic and oleic acids may be effective agents for restraining virulence of cariogenic biofilms. However, the anti-biofilm activity of the fatty acids should be evaluated in vivo, because S. mutans does not occur in a monoculture in vivo and fatty acids may interact with the various components of saliva. Acknowledgements This work was supported by Fund of Biomedical Research Institute, Chonbuk National University Hospital. Conflict of Interest The authors have no conflict of interest to declare. References Belli, W., Buckley, D. and Marquis, R.E. (1995) Weak acid effects and fluoride inhibition of glycolysis by Streptococcus mutans GS-5. Can J Microbiol 41, 785–791. Bencini, D.A., Shanley, M.S., Wild, J.R. and O’Donovan, G.A. (1983) New assay for enzymatic phosphate release: application to aspartate transcarbamylase and other enzymes. Anal Biochem 132, 259–264. Bhattarai, J.P., Park, S.A. and Han, S.K. (2010) The methanolic extract of Withania somnifera acts on GABAA receptors in gonadotropin releasing hormone (GnRH) neurons in mice. Phytother Res 24, 1147–1150. Cegelski, L., Marshall, G.R., Eldridge, G.R. and Hultgren, S.J. (2008) The biology and future prospects of antivirulence therapies. Nat Rev Microbiol 6, 17–27. Choque, B., Catheline, D., Rioux, V. and Legrand, P. (2014) Linoleic acid: between doubts and certainties. Biochimie 96, 14–21. Desbois, A.P. and Smith, V.J. (2010) Antibacterial free fatty acids: activities, mechanisms of action and biotechnological potential. Appl Microbiol Biotechnol 85, 1629–1642. Falsetta, M.L., Klein, M.I., Lemos, J.A., Silva, B.B., Agidi, S., Scott-Anne, K.K. and Koo, H. (2012) Novel antibiofilm

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