molecular oral microbiology molecular oral microbiology

Inhibitory effect of Lactobacillus salivarius on Streptococcus mutans biofilm formation C.-C. Wu1, C.-T. Lin2, C.-Y. Wu3, W.-S. Peng1, M.-J. Lee1 and Y.-C. Tsai1 1 Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan, Republic of China 2 School of Chinese Medicine, China Medical University, Taichung, Taiwan, Republic of China 3 Institute of Oral Biology, National Yang-Ming University, Taipei, Taiwan, Republic of China

Correspondence: Ying-Chieh Tsai, Institute of Biochemistry and Molecular Biology, National Yang-Ming University, 155, Section 2, Linong Street, Taipei 11221, Taiwan, Republic of China Tel.: + 886 2 28267000 ext. 5641; fax: + 886 2 28264843; E-mail: [email protected] Keywords: exopolysaccharide; glucosyltransferase; plaque; probiotic Accepted 17 June 2014 DOI: 10.1111/omi.12063

SUMMARY Dental caries arises from an imbalance of metabolic activities in dental biofilms developed primarily by Streptococcus mutans. This study was conducted to isolate potential oral probiotics with antagonistic activities against S. mutans biofilm formation from Lactobacillus salivarius, frequently found in human saliva. We analysed 64 L. salivarius strains and found that two, K35 and K43, significantly inhibited S. mutans biofilm formation with inhibitory activities more pronounced than those of Lactobacillus rhamnosus GG (LGG), a prototypical probiotic that shows anti-caries activity. Scanning electron microscopy showed that co-culture of S. mutans with K35 or K43 resulted in significantly reduced amounts of attached bacteria and network-like structures, typically comprising exopolysaccharides. Spot assay for S. mutans indicated that K35 and K43 strains possessed a stronger bactericidal activity against S. mutans than LGG. Moreover, quantitative real-time polymerase chain reaction showed that the expression of genes encoding glucosyltransferases, gtfB, gtfC, and gtfD was reduced when S. mutans were co-cultured with K35 or K43. However, LGG activated the expression of gtfB and gtfC, but did not influence the expression of gtfD in the co-culture. A transwell-based biofilm assay indicated that these lactobacilli inhibited S. mutans biofilm formation in a contact-independent manner. In conclusion, we identified two L. salivarius

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

strains with inhibitory activities on the growth and expression of S. mutans virulence genes to reduce its biofilm formation. This is not a general characteristic of the species, so presents a potential strategy for in vivo alteration of plaque biofilm and caries.

INTRODUCTION The human mouth harbors approximately 1000 bacterial species, reaching homeostasis in the oral cavity (Dewhirst et al., 2010; Lazarevic et al., 2010). Oral bacteria are responsible for the two most common bacterial diseases in humans, i.e. dental caries and periodontal disease (Wade, 2013). Extensive investigation of dental caries has shown that Streptococcus mutans is the major pathogen (Bowen & Koo, 2011; Smith & Spatafora, 2012). Streptococcus mutans does not have a free-living lifestyle. Its natural habitat is the human mouth, specifically found in the dental plaque on the tooth surfaces (Bowen & Koo, 2011; Takahashi & Nyvad, 2011). Dental plaque is a biofilm that consists of a group of microorganisms embedded in a matrix composed mainly of insoluble polysaccharides. Bacterial or salivary proteins that are associated with bacterial adhesion or aggregation, lipid and nucleic acids can also be found (Bowen & Koo, 2011). The major virulence traits of S. mutans

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L. salivarius inhibits S. mutans biofilm formation

include: (i) its acidogenicity that exacerbates the damage to dental hard tissues, (ii) its aciduricity that contributes to its survival in low pH environments or to its out-competition against other oral bacteria, and (iii) its ability to synthesize insoluble exopolysaccharides (EPS) from sucrose, which is involved in the initial attachment, colonization, and accumulation of dental plaque (Takahashi & Nyvad, 2011; Koo et al., 2013). Streptococcus mutans expresses three glucosyltran sferases (Gtfs) to synthesize EPS. GtfB and GtfC produce insoluble EPS, while GtfD forms a soluble, readily metabolized polysaccharide and acts as a primer for GtfB (Bowen & Koo, 2011; Koo et al., 2013). Probiotics are defined as living microorganisms that, when administered in adequate amounts, confer health benefits to the host (FAO/WHO, 2001). Most microorganisms identified to date as probiotics are grampositive and belong to the genera Lactobacillus or Bifidobacterium, which have been used for centuries because of their benefits to human health (Behnsen et al., 2013; Turroni et al., 2014). In recent years, the interest in probiotic therapies to prevent and control oral diseases has grown significantly (Tanzer et al., 2010; Twetman & Keller, 2012; Yanine et al., 2013). While the existing animal and clinical trials have apparent limitations and additional studies are required, several reports have demonstrated the therapeutic potential of probiotics as anti-caries treatments (Twetman & Keller, 2012). This concept is based on the idea of maintaining or restoring the natural microbiome in oral biofilm via interference and/or inhibition of pathogenic bacteria. Specific modes of bacterial interference include the secretion of anti-microbial substances, such as bacteriocins, in addition to competition for nutrients and adhesion. Protection against oral pathogen-induced inflammation by probiotic strains has also been described (Yanine et al., 2013). However, the manner by which probiotics exerts antagonistic activities against oral pathogens remains largely unknown. In this study, we focused on the identification of potential oral probiotics from Lactobacillus salivarius strains. These specific microorganisms are most frequently found on the tooth surface (26.7%) (Colloca et al., 2000), and are also isolated from human saliva (8.3–48%) (Colloca et al., 2000; Koll-Klais et al., 2005; Nelun Barfod et al., 2011) or the surface of tongue (5.6–9.5%) (Ahrne et al., 1998; Colloca et al., 2000). Persistent presence of a probiotic strain makes its interference of pathogens straightforward, L. sali2

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varius are widely studied as candidate probiotics in the oral cavity (Strahinic et al., 2007; Neville & O’Toole, 2010; Messaoudi et al., 2013). In this study, 64 L. salivarius strains were isolated and analysed for their inhibitory activities on S. mutans biofilm formation in a co-culture model. A prototypical probiotic strain, Lactobacillus rhamnosus GG (LGG), which has been reported to demonstrate anti-caries activities, was also included. Two L. salivarius strains were identified and their antagonistic mechanisms against S. mutans biofilm formation were analysed. METHODS Bacterial strains, media, and growth conditions Streptococcus mutans type strain ATCC 25175 (serotype c) isolated from carious dentine and the commercial probiotic strain LGG were obtained from the Bioresource Collection and Research Center (BCRC). The 64 L. salivarius strains were isolated from various sources, including fermented vegetables (five strains), human saliva (49 strains), and breast milk (10 strains). Identification of bacterial species was performed according to 16S rDNA sequencing, as previously described (Chao et al., 2008, 2013), and both K35 and K43 were isolated form human saliva. Lactobacilli and S. mutans were respectively cultured in deMan, Rogosa, and Sharpe (MRS) and brain–heart infusion (BHI) media (BD Difco, Franklin Lakes, NJ) at 37°C for 20 h under anaerobic atmospheric conditions (10% CO2, 10% H2, 80% N2). BHI agar plates containing 1.75 lg ml
1 polymyxin B, 0.3 U ml
1 bacitracin, and 0.005% crystal violet were used for the selective isolation of S. mutans. Biofilm formation Stationary-phase S. mutans and lactobacilli were respectively adjusted to an OD600 of approximately 1. Then, both lactobacilli and S. mutans were diluted 100-fold in BHI medium supplemented with 0.2% sucrose, mixed well, and then inoculated in polystyrene cell culture plates (Corning Inc., Corning, NY). Lactobacilli were also cultivated alone to establish mono-species biofilms. Biofilm cultures were grown in an anaerobic incubator at 37°C for 24 h, and the final pH of the biofilm cultures was measured with a pH meter (Ai-On Industrial Corp., Osaka, Japan). © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

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After the incubation, supernatants were removed from the cultures, and the plates were washed once with deionized water. To determine biofilm mass, wells were stained with 0.1% safranin for 30 min, washed three times with deionized water, and airdried. The dye was solubilized in 33% acetic acid, and the absorbance at 492 nm was determined using €nnea microtiter plate absorbance reader (Tecan, Ma dorf, Switzerland). For the transwell-based biofilm assay, a 24-well Millicell plate with a PET membrane insert (Millipore, Billerica, MA) was used. The pore size of the membrane was 0.4 lm because bacteria were unable to traverse filters with pore size < 0.4 lm (Wu et al., 2001). Lactobacilli were inoculated in the upper insert, while S. mutans was inoculated in the bottom. Scanning electron microscopy Both mono-species and dual-species biofilms were grown on glass slides placed in a 24-well plate for scanning electron microscopic (SEM) observation. Slides were gently washed with phosphate-buffered saline (PBS) once, fixed with PBS containing 2.5% glutaraldehyde and 4% paraformaldehyde, dehydrated in graded ethanol solutions, dried in liquid CO2, and finally sputter-coated with gold before SEM observation (JSM-7600F, JEOL). Growth inhibition on agar plate The inhibitory effect of lactobacilli against S. mutans was assessed using an agar diffusion method with minor modifications (Teanpaisan et al., 2011). Briefly, BHI agar plates were seeded with overnight-grown S. mutans using an aseptic cotton swab. After S. mutans was swabbed, 3 ll of stationary-phase lactobacilli cultures were dropped onto the BHI plates. The plates were then anaerobically incubated at 37°C for 24 h to generate an inhibitory zone. Quantitative real-time polymerase chain reaction Bacterial RNA was prepared and analysed as previously described (Lin et al., 2013). In brief, total RNA was isolated from the lactobacilli S. mutans co-culture using RNeasy midi-columns (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The RNA was DNase treated with RNase-free DNase I © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

L. salivarius inhibits S. mutans biofilm formation

(MoBioPlus) to eliminate DNA contamination. A 100-ng sample of RNA was reverse-transcribed with the Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland) using random primers. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed with a Roche LightCyclerâ 1.5 Instrument using Light Cycler TaqMan Master (Roche) to detect gtfB (primers gtfB-F: 50 -CTT CTG ATC GCG TGG TTG T-30 and gtfB-R: 50 -AAG GTC GGT AAG CTT GGT TCT-30 ; probe: 60), gtfC (primers gtfC-F: 50 -GGC TAA TTC CAA CTA CCG TAT CTT-30 and gtfC-R: 50 -GGT AAG TGG GGC CTT AGC TC-30 ; probe: 82), gtfD (primers gtfD-F: 50 -CCA ATA TTC CGA CAG CCT ATG-30 and gtfD-R: 50 -TCA CCA TAA TAA AGA CGT GTA ATT GAA-30 ; probe: 56), and 23S rRNA (primers 23S-F: 50 -GCG ATC AGC TGT ATA CCT TGG-30 and 23S-R: 50 -GAT CGA ACC GCT GAC CTC-30 ; probe: 67). Primers and probes were designed for selected target sequences using the Universal Probe Library Assay Design Center (RocheApplied Science). Data were analysed using the realtime PCR software of the Roche LightCyclerâ 1.5 Instrument. Relative gene expression was quantified using the comparative threshold cycle 2-DDCt method with 23S rRNA of S. mutans as the endogenous reference. Analytical Profile Index (API) typing Sugar utilization by K35 and K43 was investigated rieux, Marcy using the API 50CHL system (bioMe l’Etoile, France) according to the manufacturer’s instructions. The biochemical profile for the strain was identified using the APIWEBTM identification software with database (V5.1). Peripheral blood mononuclear cells assay Isolation of human peripheral blood mononuclear cells (hPBMCs) from healthy volunteers and the treatment of lactobacilli were performed as previously described (Liu et al., 2011) with slight modifications. The hPBMCs were collected and resuspended in RPMI-1640 medium containing 10% fetal bovine serum, 100 IU ml
1 penicillin, 0.1 mg ml
1 streptomycin, 0.25 lg ml
1 amphotericin, and 1% L-glutamate. Human PBMCs (2 9 105 cells/well) were seeded in 96-well tissue culture plates and treated with 2 9 106 colony-forming units of heat-killed lactobacilli 3

L. salivarius inhibits S. mutans biofilm formation

(hPBMCs : lactobacilli ratio of 1 : 10) at 37°C and 5% CO2 for 48 h. The use of phytohemagglutinin (2 lg ml
1), Escherichia coli lipopolysaccharide (1 lg ml
1), or medium only were performed as control experiments. The cultured plates were centrifuged (1000 g, 10 min, 4°C), and the supernatants were collected for cytokine [interferon-c (IFN-c) and interleukin-10 (IL-10)] determination. The concentrations of IFN-c and IL-10 were determined using an enzyme-linked immunosorbent assay procedure according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN). Repetitive sequence-based PCR genomic fingerprinting Repetitive sequence-based (rep)-PCR analysis is considered a convenient tool for the discrimination of bacterial strains. To discriminate the two L. salivarius strains, three rep-PCR genomic fingerprinting methods, including enterobacterial repetitive intergenic consensus (ERIC)-PCR, BOX-PCR, and (GTG)5-PCR, were used. Purification of the bacterial genomic DNA and PCR were performed as previously described (Mohapatra et al., 2007; Chao et al., 2008). PCR primers: ERIC1R (50 -ATG TAA GCT CCT GGG GAT TCA C-30 ) as well as ERIC2 (50 -AAG TAA GTG ACT GGG GTG AGC G-30 ), BOXA1R (50 -CTA CGG CAA GGC GAC GCT GAC G-30 ), and (GTG)5 (50 -GTG GTG GTG GTG GTG-30 ) were used for the PCR. The PCR products were separated by electrophoresis on a 1% agarose gel. Statistical analysis Experimental results were analysed for statistical significance with PRISM5 software (GraphPad, San Diego, CA) using one-way analysis of variance followed by Bonferroni post hoc correction. A P-value < 0.01 was used as significant in all cases. The results of the biofilm formation assay, qRT-PCR, and cytokine production assay were confirmed with at least three independent experiments. Each sample was assayed in triplicate and the mean activity and standard deviation are presented. Ethics statement For isolation of normal human peripheral blood from healthy volunteers, the procedure and the respective 4

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consent documents were approved by the Ethics Committee of the National Yang-Ming University, Taipei, Taiwan. All healthy volunteers provided written informed consent. RESULTS Co-culture with lactobacilli reduces S. mutans biofilm formation A total of 64 L. salivarius strains isolated from fermented vegetables, human saliva, or breast milk were analysed to determine if these strains possess potential inhibitory effects on S. mutans biofilm formation. A biofilm formation assay was performed as described with LGG as a control, because this probiotic strain has been described to have anti-caries activity (Nase et al., 2001). We found that S. mutans co-cultured with most of the L. salivarius strains, respectively, resulted in 0–30% reduction of total biofilm mass compared with that of the S. mutans only (data not shown). However, two particular strains, K35 and K43, which did not form strong biofilms in a mono-species model (Fig. 1A), appeared to cause evident reductions (P < 0.01) in biofilm formation, approximately 64.3% and 69.6%, respectively. The control species, LGG, caused an approximately 44% reduction of the biofilm mass (Fig. 1B). Besides, LGG presented relatively strong biofilm forming activity in a mono-species model (Fig. 1A). Heat treatment (80°C for 20 min) of K35 or K43 cultures before the inoculation in the biofilm co-culture resulted in loss of the inhibitory activities. Nevertheless, heat-killed LGG still caused a slight reduction (~24%) in biofilm formation compared with that with S. mutans only (Fig. 1C). Co-culture with lactobacilli influence the morphology of S. mutans biofilms Biofilm formation of the co-cultures, as observed by SEM, is shown in Fig. 2. The S. mutans appeared to form a compact and island-like biofilm covered by large amounts of slime or network-like structures, suggested to be EPS. Co-culture of S. mutans with K35 or K43 resulted in visibly fewer bacteria and smaller microcolonies attached to the surface. Moreover, the amount of EPS appeared to be decreased in the co-culture with the K35 or K43 strains. LGG, the long bacilli observed in the photograph, was © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

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L. salivarius inhibits S. mutans biofilm formation

found to occupy most of the exterior of the biofilm, and network-like EPS was still observed in the co-culture. Additionally, S. mutans co-cultured with heatkilled LGG also resulted in an island-like biofilm covered with EPS; however, the biofilms appeared to have larger spaces between the bacterial cells, which were not as compact as that of the S. mutans only. L. salivarius inhibits the growth and expression of gtf of S. mutans

B

C

Figure 1 Lactobacilli inhibit Streptococcus mutans biofilm formation. (A) Mono-species biofilm formation of lactobacilli (Lb). *P < 0.01 compared with the medium-only control group. (B) Biofilm formation of S. mutans (Sm) co-cultured with live lactobacilli. (C) Biofilm formation of S. mutans co-cultured with heat-killed lactobacilli (hk-Lb). Both the biomass produced by lactobacilli and S. mutans were stained and quantified. *P < 0.01 compared with the S. mutans-only control groups. Experiments were performed as described in the Methods. CON, control group of medium only.

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

To determine how lactobacilli influence S. mutans biofilm formation, S. mutans growth in the co-culture was analysed using a spot assay. Biofilm from the lactobacilli and S. mutans co-culture was scraped and rigorously mixed with the planktonic fraction. Subsequently, the bacterial suspension was serially diluted with PBS, and a 5-ll aliquot was spotted onto the S. mutans selection plate, which inhibited the growth of lactobacilli. As shown in Fig. 3A, co-culture with the K35 or K43 strains apparently reduced the growth of S. mutans. Compared with K35 and K43, LGG appeared to have weaker inhibition on the growth of S. mutans, whereas heat-killed LGG did not exhibit bactericidal activity. To further verify the bactericidal activity, we analysed the inhibitory activity of lactobacilli against S. mutans growth on agar plates. As shown in Fig. 3B, clear zones with similar diameters, ranging from 18 to 19 mm, surrounding the macrocolonies of strains K35, K43, LGG, and G01, which did not influence biofilm formation in the co-culture experiment, could be observed. This result confirmed that these lactobacilli possess bactericidal activities to S. mutans, possibly through secreted factors. On the other hand, we used qRT-PCR to analyse the expression of S. mutans gtfB, gtfC, and gtfD genes, which encode glucosyltransferases and play a crucial role in biofilm formation. As shown in Fig. 4, co-culture with the K35 or K43 strains significantly decreased the mRNA levels of gtfB, gtfC, and gtfD of S. mutans, suggesting reduced EPS production. Interestingly, we also found that LGG evidently increased the expression of gtfB and gtfC and did not regulate the expression of gtfD. In addition, heat-killed LGG did not cause an apparent effect. Our results suggest that L. salivarius K35 and K43 strains not only inhibit the growth, but also decrease the expression of glucosyltransferase-encoding genes of S. mutans to reduce its biofilm formation. 5

L. salivarius inhibits S. mutans biofilm formation

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Figure 2 Scanning electron microscopy (SEM) observation of the biofilms. Streptococcus mutans (Sm) was co-cultured with lactobacilli, as indicated in the margin, and the resulting biofilms were observed by SEM at 3000 9 magnification. hk-LGG indicates heat-killed LGG.

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Figure 3 Bactericidal activity of lactobacilli against Streptococcus mutans. (A) S. mutans (Sm) was co-cultured with lactobacilli, as indicated in the margin, to form biofilms as described in the Methods. After 24 h of incubation, the co-cultures were vigorously mixed, and the bacterial suspensions were serially diluted with phosphate-buffered saline for the spot assay performed using selective plates for S. mutans. hk-LGG indicates heat-killed LGG. (B) Lactobacilli inhibit the growth of S. mutans on brain–heart infusion agar plates. The clear inhibitory zones surrounding the macrocolonies of lactobacilli, as indicated, can be observed. G01 is a Lactobacillus salivarius strain without an evident inhibitory activity against S. mutans biofilm formation.

L. salivarius reduces S. mutans biofilm formation in a contact-independent manner To determine if the inhibitory activity of lactobacilli on S. mutans biofilm formation required direct cell–cell interactions, a transwell-based biofilm formation assay 6

was performed. Lactobacilli were inoculated in the upper insert, and they could not enter the bottom of the culture well. As shown in Fig. 5, we found that the presence of lactobacilli in the upper insert apparently reduced the S. mutans biofilm formation in the bottom.

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

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Figure 4 Co-cultures with lactobacilli regulate the gene expression of Streptococcus mutans glucosyltransferases. The expression of gtfB, gtfC, and gtfD, encoding S. mutans glucosyltransferases, was analysed by quantitative real-time reverse transcription–polymerase chain reaction. The S. mutans was co-cultured with lactobacilli, as indicated at the bottom. CON designates S. mutans only; hk-LGG indicates heat-killed LGG. *P < 0.01 compared with the control group.

L. salivarius inhibits S. mutans biofilm formation

were added. LGG caused a modest reduction (~30.2%), whereas heat-killed LGG did not exhibit an evident influence. As lactobacilli in the upper insert could not pass through the membrane, their modulation of S. mutans biofilm formation is suggested to be dependent on secretory factors, such as bacteriocins, hydrogen peroxide, and lactic acid, which remain to be investigated. Additionally, the measurement of the final pH values of the biofilm co-cultures revealed that, compared with that of S. mutans only (pH ~4.97), the presence of K35 or K43 resulted in decreased pH values to approximately 4.84 and 4.86, respectively; LGG also caused a slight reduction (pH ~4.92), whereas heatkilled LGG did not have an evident effect (pH ~4.96) (Fig. 5). The change in pH values seemed obscure, implying the involvement of other factors in this phenomenon, which have yet to be elucidated. Our results showed that K35, K43, and LGG could inhibit S. mutans biofilm formation in a contact-independent manner and suggested that heat-killed LGG reduced biofilm formation via physical interference. Characterization of the L. salivarius strains

Figure 5 Transwell-based biofilm formation assay. Inhibitory effects of lactobacilli on Streptococcus mutans biofilm formation were assessed using a transwell-based assay, as shown in the margin. Lactobacilli [2 9 107 colony-forming units (CFU) per 0.5 ml] and S. mutans (2 9 107 CFUper 1.5 ml) were respectively inoculated in the upper insert and the bottom of the transwell plate. The membrane of the insert, with a 0.4-lm pore size, impeded the bacterial translocation and S. mutans biofilm formation in the bottom was determined as described in the Methods. *P < 0.01 compared with the control group.

Compared with the biofilm formed by only S. mutans, biofilm formation was reduced by approximately 52.5% and 46.3%, when K35 or K43 strains, respectively, © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

To further characterize the L. salivarius strains, in vitro sugar utilization and immunomodulatory activities of K35 and K43 were analysed. A sugar fermentation test was performed by the API 50CHL system, and the results showed that both K35 and K43 harbored a biochemical property highly similar to L. salivarius (see Table S1). However, K35 was able to produce acid from arbutin, esculin ferric citrate, and salicin, whereas K43 was not. These three sugars were fermented by approximately 28% of L. salivarius strains (according to the user manual of API 50CHL), and our result indicated that K35 and K43 harbored different biochemical properties. Many probiotic strains have been described as possessing immunomodulatory activities that provide health benefits (Dongarra et al., 2013; Ashraf & Shah, 2014). The in vitro capacity of probiotics to induce higher levels of the T-helper type 1 cytokines, such as IFN-c and IL-12, and the anti-inflammatory cytokine IL-10, has been correlated to protection from allergic and inflammatory diseases (Foligne et al., 2007; Borchers et al., 2009; Mei et al., 2013). Hence, an hPBMC experimental model was used to determine if the selected L. salivarius strains possess immunomodulatory activities. Heat-killed lactobacilli 7

L. salivarius inhibits S. mutans biofilm formation

were incubated with hPBMCs isolated from healthy volunteers for 48 h, and the production of IFN-c and IL-10 cytokines was measured. In addition, lipopolysaccharide and phytohemagglutinin mitogens, as well as two well-studied probiotic strains with immunomodulatory activities, LGG and L. casei Shirota (LcS), were also included as controls. The results showed that K35 and K43 harbor in vitro immunomodulatory activity that is similar to that of LcS, which is prone to activate IFN-c production (Fig. 6). Based on the results described above, the L. salivarius strains K35 and K43 were found to share some characteristics. To determine if these L. salivarius strains had a similar genetic background, three

Figure 6 The effects of heat-killed lactobacilli on interferon-c (IFN-c) and interleukin-10 (IL-10) production in human peripheral blood mononuclear cells (hPBMCs). Cells were cultured with medium as a control (CON), phytohemagglutinin (2 lg ml
1), lipopolysaccharide (1 lg ml
1), or heat-killed lactobacilli. After 48 h of incubation, culture supernatants were collected for cytokine determination using enzyme-linked immunosorbent assay.

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types of rep-PCR (ERIC, BOX, and GTG5) were performed and the PCR products were resolved by DNA electrophoresis. As shown in Fig. 7, distinguishable PCR-fingerprinting profiles for K35 and K43 could be observed, especially in the ERIC-PCR profiles, suggesting different genetic backgrounds for these strains, which may result in their identified strain-specific activities. DISCUSSION Oral microbiota contains various microorganisms that reach homeostasis in the mouth and inhibit the colonization of non-oral bacteria and pathogens via synergistic and antagonistic interactions (Jenkinson, 2011); furthermore, immune exclusion may also be involved (Wade, 2013). Some clinical and animal experiments have demonstrated that lactobacilli appear to have beneficial roles for caries prevention and control (Twetman & Keller, 2012). LGG is a prototypical and well-studied probiotic strain that has been shown to have beneficial effects on children’s dental health, both in the reduction of the risk of caries and the number of mutans streptococci present (Nase et al., 2001; Lebeer et al., 2010). We found that LGG formed a relatively strong biofilm (Fig. 1A), compared with most of the L. salivarius tested (data not shown), which may be advantageous in competing with S. mutans in the oral cavity. In addition, K35 and K43 were shown to have profound effects on the reduction of S. mutans biofilm (Fig. 1B), even stronger than that of LGG, albeit they did not form strong mono-species biofilms (Fig. 1A). An intestinal probiotic L. salivarius,

Figure 7 Repetitive sequence-based polym erase chain reaction (rep-PCR) genomic fingerprinting profiles of Lactobacillus sali varius strains. Enterobacterial repetitive intergenic consensus (ERIC-), BOX-, and (GTG)5-PCR analyses were performed to discriminate L. salivarius strains. K35 and K43 are indicated, and other lanes represent individual L. salivarius isolates, respectively. Apparent differences between the two strains are indicated by arrows. M denotes the 100-bp ladder (Bioman DM-100).

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W24, has also been shown to affect the compositional stability and cariogenicity of oral microbial communities; moreover, W24 did not permit the formation of a mono-species biofilm (Pham et al., 2009). Our results indicated that K35, K43, and LGG have inhibitory effects on biofilm formation of S. mutans ATCC 25175T (Fig. 1B), the strain used throughout the study presented here. Similar results were obtained when other extensively studied virulent S. mutans strains GS5 and UA159 were used (see Fig. S1), and K35 showed the strongest inhibitory effects. Besides, heat-killed K35 and K43 lost their inhibitory activities on S. mutans biofilm formation, whereas heat-killed LGG surprisingly still had a slight effect (Fig. 1C). Although the mechanism remains unknown, we have noted that heat-killed LGG could tightly adhere to the bottom of the well (data not shown) and may, therefore, impede S. mutans biofilm formation. In addition, we found that biofilm formed by S. mutans with heatkilled LGG is more easily removed in the lavage process, suggesting a loose biofilm structure, compared with that of S. mutans alone, as observed by SEM analysis (Fig. 2). These findings suggested that heatkilled LGG may alter biofilm structure in the co-culture through physical interference, such as the competition for binding sites on the solid surface and bacterial coagglutination. It has been demonstrated that an oral probiotic strain, Lactobacillus paracasei DSMZ16671, even if heat-killed, sensitively co-aggregates mutans streptococci specifically (Tanzer et al., 2010). As shown in Fig. 3B, K35, K43, and LGG were found to possess bactericidal activities against S. mutans. We also tested all of the 64 L. salivarius strains using this assay and found that most of the strains possessed strong bactericidal activities (inhibition zones ranging from 17 to 19 mm) (data not shown). A previous report has also indicated that strong bactericidal effects, as assessed by an agar diffusion assay, were associated with six Lactobacillus species (L. salivarius, L. casei, L. paracasei, L. plantarum, and L. rhamnosus) (Teanpaisan et al., 2011). These findings suggested that the bactericidal activity of the lactobacilli is one of the mechanisms for inhibiting S. mutans biofilm formation. Nevertheless, although most of the L. salivarius strains have bactericidal activities against S. mutans, only a minority of the strains (such as K35 and K43) inhibited the S. mutans biofilm formation. Besides, many L. salivarius strains are able to produce bacteriocins of sub-classes IIa, IIb, and IId © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

L. salivarius inhibits S. mutans biofilm formation

(Messaoudi et al., 2013). Moreover, a new type of cationic bacteriocin, bactofencin A, has been reported (O’Shea et al., 2013). These bacteriocins can inhibit the growth of competing strains or pathogens, modulate the composition of the microbiota, and influence the host immune system (Messaoudi et al., 2013). However, whether K35 and K43 are bacteriocin producers awaits further investigations. Many natural products with potential uses as alternative or adjunctive anti-caries therapeutics have been reported to have inhibitory activity on the growth and biosynthesis of EPS of S. mutans (Jeon et al., 2011). As shown in Fig. 2, the S. mutans-only group, but not the co-culture groups with K35 or K43, was covered with network-like structures, which are suggested to be EPS. Hence, we used qRT-PCR to analyse the expression of gtfB, gtfC, and gtfD, encoding major S. mutans Gtfs that are responsible for the biosynthesis of glucans and play crucial roles in biofilm formation (Bowen & Koo, 2011; Koo et al., 2013). As shown in Fig. 4, the presence of K35 or K43 reduced the expression of gtfB, gtfC, and gtfD, supporting our SEM observations (Fig. 2). It is interesting to note that LGG appeared to significantly increase gtfB and gtfC expression, whereas heat-killed LGG did not have a clear effect, as expected. Although the underlying mechanism of how lactobacilli regulate the expression of genes encoding glucosyltransferases remains unknown, it is conceivable that in a multispecies oral biofilm, bacteria could influence the gene expression profiles of each other via complicated pathways, such as quorum sensing (Kuramitsu et al., 2007; Redanz et al., 2011). Our results suggested that L. salivarius K35 and K43, but not LGG, reduce the EPS production of S. mutans to inhibit biofilm formation. Although lactobacilli have also been reported to be involved in the process of caries formation (Chhour et al., 2005; Corby et al., 2005; Yang et al., 2010), it is unclear whether lactobacilli act as causative agents or accomplices in dental caries formation (O’Callaghan & O’Toole, 2013). In the biofilm co-cultures of S. mutans and lactobacilli (Fig. 5), the presence of live lactobacilli slightly decreased the final pH values, which may exacerbate the damage to dental hard tissues in vivo. An L. salivarius strain LS1952R has also been shown to possess an inherent cariogenic activity in rats (Matsumoto et al., 2005). In contrast, previous studies reported that probiotic strains and fermented milk beverages did not promote erosion of 9

L. salivarius inhibits S. mutans biofilm formation

the dental enamel or only resulted in a superficial mineral loss (Nase et al., 2001; Nikawa et al., 2004; Lodi et al., 2010). Hence, it is necessary to evaluate the safety of K35 and K43 before using them in commercial applications. In this study, we identified two L. salivarius strains, K35 and K43, with inhibitory effects on S. mutans biofilm formation. Such specific inhibition, however, was not a general characteristic of this species or the genus Lactobacillus. The presence of K35 or K43 in dual-species biofilm cultures not only reduced the growth, but also the EPS production of S. mutans. Hence, our results present a potential strategy for in vivo alteration of plaque biofilm and caries formation. ACKNOWLEDGEMENTS This work was supported by the Asian Probiotics and Prebiotics Ltd. and F.B.W Bio-Medicine Services Ltd. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors report no conflicts of interest related to this study. REFERENCES Ahrne, S., Nobaek, S., Jeppsson, B., Adlerberth, I., Wold, A.E. and Molin, G. (1998) The normal Lactobacillus flora of healthy human rectal and oral mucosa. J Appl Microbiol 85: 88–94. Ashraf, R. and Shah, N.P. (2014) Immune system stimulation by probiotic microorganisms. Crit Rev Food Sci Nutr 54: 938–956. Behnsen, J., Deriu, E., Sassone-Corsi, M. and Raffatellu, M. (2013) Probiotics: properties, examples, and specific applications. Cold Spring Harb Perspect Med 3: a010074. Borchers, A.T., Selmi, C., Meyers, F.J., Keen, C.L. and Gershwin, M.E. (2009) Probiotics and immunity. J Gastroenterol 44: 26–46. Bowen, W.H. and Koo, H. (2011) Biology of Streptococcus mutans-derived glucosyltransferases: role in extracellular matrix formation of cariogenic biofilms. Caries Res 45: 69–86. Chao, S.H., Tomii, Y., Watanabe, K. and Tsai, Y.C. (2008) Diversity of lactic acid bacteria in fermented brines used to make stinky tofu. Int J Food Microbiol 123: 134–141. Chao, S.H., Huang, H.Y., Chang, C.H. et al. (2013) Microbial diversity analysis of fermented mung beans

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