https://orcid.org/0000-0003-0469-1788
Abstract
Human saliva contains natural antimicrobial enzymes. In this in-vitro study, we evaluate the antimicrobial activity of a dentifrice containing a salivary enzyme complex (SEC) with xylitol versus a standard 0.12% chlorhexidine (CHX) dentifrice. Adherent cells of Streptococcus gordonii, Strep. mutans, Actinomyces naeslundii, Fusobacterium nucleatum subsp polymorphum, and Corynebacterium matruchotii were exposed to SEC-xylitol and CHX dentifrices for 2 min and viable CFUs were enumerated. Exposure to the SEC-xylitol dentifrice resulted in a significant reduction in bacterial viability, which was greater than that shown by the CHX dentifrice, against all organisms tested. The SEC-xylitol dentifrice also exhibited greater antimicrobial activity against all organsims in well diffusion assays compared to CHX. Dentifrice activity was also evaluated against a three species community of Strep. gordonii, Strep. mutans, and Coryne. matruchotii using bacterial live/dead stain. The SEC-xylitol dentifrice was at least as effective as CHX in removal of the multispecies community. The combination of SEC and xylitol generates a highly effective antimicrobial dentifrice with greater antibacterial activity than a standard 0.12% CHX formulations. SEC and xylitol combinations are worthy of further investigation for routine use and in the management of gingivitis and periodontal disease.
Antimicrobial oral hygiene products have an important role in controlling oral diseases such as dental caries and periodontal disease. Chlorhexidine is the gold standard antimicrobial in such products but is often associated with staining. In order to find improved antimicrobials with greater antiplaque activity, we investigated a combined salivary enzyme complex (SEC)-xylitol dentifrice for antimicrobial activity. Our SEC-xylitol formulation shows enhanced antimicrobial activity compared to chlorhexidine dentifrice, including higher activity against adherent cells of the cariogenic bacterium Streptococcus mutans. SEC-xylitol combination dentifrices show promise as natural antimicrobial alternatives to chemical antimicrobials for the control of oral diseases.
Introduction
Many mammalian secretions, including milk, saliva, and respiratory mucous, contain active antimicrobial proteins that exhibit antibacterial, antifungal, or antiviral activity (Vasstrand and Jensen 1984, Tenovuo 2002, Cawley et al. 2019, Magacz et al. 2019, Nakano et al. 2020). These antimicrobial proteins include lysozyme, lactoferrin, lactoperoxidase, and a variety of small antimicrobial peptides (Vasstrand and Jensen 1984, Roger et al. 1994, Pinheiro et al. 2020). These enzymes and proteins have varied mechanisms of action including hydrolysis of bacterial cell walls (lysozyme), iron sequestration (lactoferrin), and oxidative attack on microbial cell surfaces (lactoperoxidase). Lactoperoxidase (LPO) catalyzes the oxidation of many inorganic substrates by H2O2 to generate reactive oxygenated derivatives. The most significant of these substrates in the oral cavity is thiocyanate (SCN−), which is oxidized to form hypothiocyanite (OSCN−). LPO in saliva has significant antimicrobial activity largely mediated through the production of hypothiocyanite (OSCN−) ions, which are thought to oxidize microbial surface proteins and exhibit a microbicidal effect (Bafort et al. 2014). This reaction involves the initial oxidative activation of the native LPO enzyme by H2O2 in saliva to form compound I (Magacz et al. 2019). The active compound I can then carry out the oxidation of thiocyanate (SCN−) to hypothiocyanite (OSCN−). Thiocyanate ions are the preferred substrate of the active enzyme and these are naturally found in saliva. The oxidized hypothiocyanite is highly reactive and can react with thiol groups on bacterial proteins and this has a bactericidal effect (Thomas and Aune 1978). In saliva, LPO works in synergy with other enzymes including lactoferrin and lysozyme to regulate the oral microbiome and prevent oral disease. LPO has been shown to have activity against planktonic and biofilm growing oral bacteria and may inhibit bacterial biofilm formation on tooth surfaces due to its ability to adhere to the salivary pellicle (Roger et al. 1994).
There now exists an extensive literature showing the effectiveness of dentifrices containing natural enzymes, including LPO, in terms of antimicrobial activity and oral healthy promoting properties (reviewed by Magacz et al. 2019). In vitro, antimicrobial activity of LPO has been demonstrated against cariogenic Streptococcus mutans and also Gram-negative periodontal pathogens such as Porphyromonas gingivalis and also multispecies biofilms (Roger et al. 1994, Welk et al. 2009, Cawley et al. 2019). In human trials, LPO dentifrices have been shown to reduce plaque scores, reduce gingival bleeding, and remission of symptoms of dry mouth (Kirstilä et al. 1996, Epstein 1999, Tenovuo 2002, Jyoti et al. 2009, Nakano et al. 2019, Pinheiro et al. 2020, Welk et al. 2021). In human trials, LPO has been associated with increased levels of hypothiocyanite (Lenander-Lumikari et al. 1993) and reduced levels of Strep. mutans and periodontal pathogens such as P. gingivalis and Fusobacterium nucleatum (Jyoti et al. 2009, Nakano et al. 2020, Rabe et al. 2022).
Xylitol has also been incorporated in dentifrices and has been proposed to have several antibacterial mechanisms of action, including disruption of bacterial energy metabolism and direct antimicrobial activity (Benahmed et al. 2020, Teng et al. 2022). Interestingly, some studies have indicated that xylitol may enhance LPO activity in the oral cavity and in vitro (Mäkinen et al. 1976, Kim et al. 2015). Mäkinen et al. provided evidence that ingestion of xylitol increased LPO activity in vivo in volunteers who ingested xylitol sweeteners (Mäkinen et al. 1976). Similarly, in-vitro studies by Kim et al. showed that xylitol enhanced the enzymatic activity of salivary LPO (Kim et al. 2015).
Activity of LPO against oral biofilms has been demonstrated with in-vivo and in-vitro studies (Modesto et al. 2000, Rabe et al. 2022). Attempts to replicate dental biofilm growth in the laboratory often involves growth of multiple bacterial species on solid surfaces coated with saliva (Paqué et al. 2022). In the current study, we use bacteria grown on saliva-coated surfaces to examine the antibacterial activity of a dentifrice containing a salivary enzyme complex (SEC) combined with xylitol. As a control, we use the gold-standard antimicrobial agent, chlorhexidine (CHX). We examine activity against adherent communities of a variety of common pathobionts including Strep. mutans. We also examine activity against organisms known to be important for plaque maturation and development, including Fus. nucleatum, which acts as a bridge species to allow incorporation of many Gram-negative periodontal pathogens into plaque biofilms (Zijnge et al. 2010). In addition, we examine activity against Corynebacterium matruchotii. Recent studies of plaque architecture on human teeth have shown that Corynebacterium species play an important role as a scaffold for other bacteria to bind to in plaque biofilms (Welch et al. 2016). We examine single species Coryne. matruchotii cultures and a simple multispecies community incorporating Strep. gordonii, Strep. mutans, and Coryne. matruchotii.
Materials and methods
Bacterial strains and culture conditions
Bacterial strains were obtained from the DSMZ German Collection of Microorganisms and Cell Cultures GmbH or the UK National Collection of Type Cultures (NCTC). These included Strep. gordonii DL-1, Strep. mutans NCTC10449, Actinomyces naeslundii DSM43013, Fus. nucleatum subsp polymorphum NCTC10953, and Coryne. matruchotii DSM20635.
Fusobacterium nucleatum and A. naeslundii were cultured anaerobically in brain heart infusion (BHI) broth in 2.5 l anaerobic jars (Oxoid) using the AnaeroGen gas generating system (Oxoid).
All other bacteria were cultured aerobically at 37°C in BHI broth in 250 ml Erlenmyer flasks with shaking at 250 rpm. Aerobic and anaerobic plate culture was carried out with BHI agar at 37°C.
Dentifrices
Two dentifrice preparations were compared. The base formulations of both preparations were identical and exhibit limited antimicrobial activity. One contained chlorhexidine (0.12% w/v) as the active antimicrobial ingredient. The second preparation consisted of a lactoperoxidase containing SEC supplemented with xylitol (5.4%). Samples of both preparations are available by request from the authors. Dentifrices were tested as 30% v/v suspensions to improve handling and represent dilution that occurs with saliva in the oral cavity. Test suspensions were prepared by vigorous mixing with Dulbecco’s modified Eagle’s medium (DMEM).
Antimicrobial assay
Adherent layers of bacteria were grown on the surface of plastic 12-well dishes (Greiner Bio-one). The surface of the dish was pretreated with human saliva for 24 h prior to the assay. Saliva was unstimulated and was recovered from healthy adult volunteers by sampling in a sterile 50-ml tube. Collected samples were pooled and centrifuged at 4000 × g for 20 min at 4°C. Samples were then UV sterilized for 30 min and aliquoted in 1.5 ml Eppendorf tubes. Sterility was checked prior to use by direct aerobic culture. Samples were stored at −80°C.
Bacteria were grown in BHI broth to the late logarithmic phase of growth. Bacteria were collected by centrifugation at 10 000 × g and washed twice in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco). The OD600 nm of the bacterial suspension was measured and the density was adjusted to yield a suspension of ∼1 × 107 bacteria/ml. A 500 μl aliquot of this suspension was added to nine individual saliva-coated wells and allowed to adhere for 2 h in a humid incubator set at 37°C with 5% CO2. Following incubation, the liquid was removed and the adherent bacteria were washed with 500 μl of fresh DMEM. Three wells supplemented with 500 μl of fresh DMEM and acted as controls, three wells were supplemented with 500 μl of the chlorhexidine dentifrice and three wells were supplemented with a 500 μl volume of the SEC-xylitol dentifrice. Bacteria were exposed for 2 or 10 min. Following this incubation, the antimicrobial or control suspension was removed and the well was washed with 500 μl of PBS. A 1 ml volume of PBS was then added to each well and the remaining bacteria were removed by vigorous pipetting. Each sample was vortexed and serially diluted 10-fold and triplicate plate counts were performed to assess bacterial viability on BHI agar. Data were analysed and plotted using Prism GraphPad (San Diego, CA, USA). Data were analysed using a Kruskal–Wallis tests with Dunn’s test for pairwise comparisons.
Well diffusion assay
Bacterial suspensions were prepared in DMEM as described for biofilm assays and were diluted 1/10 in DMEM. A BHI agar plate containing 25 ml agar was prepared for each assay by punching a 10-mm diameter hole in the agar. Using a cotton swab, the bacterial suspension was spread evenly over the surface of the plate and allowed to dry. A 100 μl aliquot of DMEM or dentifrice suspension (30% v/v in DMEM) was then added to the well and the plate was incubated at 37°C aerobically (or anaerobically for Fus. nucleatum and A. naeslundii) until sufficient growth was achieved to discern a halo (24–48 h). Halo size was recorded using a Flash n’ Go plate visualizer (IUL Instruments) and each experiment was done triplicate.
Visualization of bacterial layer removal
In order to visualize bacterial removal and killing, a qualitative assay was carried out using the LIVE/DEAD Baclight bacterial viability kit (Thermofisher). A suspension of 2 × 107 cells of each of Strep. gordonii, Strep. mutans, and Coryne. matruchotii was used to generate a multispecies community in the same fashion as described above. The bacteria were exposed to DMEM (control) or dentifrice for 2 min. The suspension was removed and the bacteria were washed (3 ×) with 1 ml of PBS. The adherent bacteria were then stained with the LIVE/DEAD Baclight stain and visualized using a Zoe-inverted fluorescent microscope (BioRad).
Results and discussion
Antibacterial activity
Streptococcus gordonii adhering to saliva-coated plastic wells were exposed to either a chlorhexidine dentifrice (CHX) or a salivary enzyme complex dentifrice (SEC-xylitol) for 2 or 10 min (Fig. 1). Viable counts showed a ∼3-log reduction in CFUs following 2-min exposure to the SEC-xylitol formulation, which was highly significant (P = .0073) compared to the CHX treatment (P = .18). Following a 10-min exposure to SEC-xylitol, we observed a greater reduction in viability of the Strep. gordonii cells (P = .007). Additional assays were carried out to compare the antimicrobial effects on other plaque forming organisms including Fus. nucleatum, Coryne. matruchotii, Strep. mutans, and A. naeslundii (Fig. 1). In the case of the Gram-positive organisms, a ∼3-log reduction in viability was observed after 2-min exposure to SEC-xylitol, which was significant (all P < .05). In the case of the Gram-negative organism Fus. nucleatum, we observed a ∼2-log reduction in viability (P = .015). In each case, the effective drop in viable CFUs was significantly greater with the SEC-xylitol formulation compared to the CHX dentifrice. In the case of Fus. nucleatum, Coryne. matruchotii, Strep. mutans, and A. naeslundii, a 10-min exposure yielded similar results to the 2-min exposure (data not show).
(a) Viable counts of adherent Strep. gordonii following exposure to CHX and SEC-xylitol dentifrices for 2 or 10 min. (b) Viable counts of adherent Fus. nucleatum, Coryne. matruchotii, Strep. mutans, and A. naeslundii following 2-min exposure to CHX and SEC-xylitol dentifrices. *P < .05 and **P < .01 in Kruskal–Wallis test with Dunn’s test for multiple comparisons.
Well diffusion assays
We examined the capacity of CHX and SEC-xylitol dentifrices to inhibit the growth of agar adherent bacteria in well diffusion assays (Fig. 2). In this assay format, each organism tested yielded a larger halo of inhibition with the SEC-xylitol dentifrice compared to the CHX formulation. Streptococcus mutans appeared to exhibit the least susceptibility to SEC-xylitol; however, the activity of SEC-xylitol against Strep. mutans was reproducibly greater than the CHX formulation. Fusobacterium nucleatum exhibited the greatest susceptibility to SEC-xylitol in this format.
Well diffusion assay to assess susceptibility to CHX and SEC-xylitol dentifrices. (a) Representative images showing halos of inhibition for Coryne. matruchotii and Strep. mutans. (b) Halo sizes from three replicate experiments showing average diameter in mm +/− variance. The more intense red colour indicates larger halo size.
Biofilm visualization
A qualitative assessment of multispecies community viability and removal was carried out using the LIVE/DEAD Baclight bacterial viability kit (Thermofisher). A tri-species community of Strep. gordonii, Strep. mutans, and Coryne. matruchotii was grown and exposed to DMEM (control) or to each dentifrice for 2 min. The LIVE/DEAD Baclight stain allowed visualization of viable (green) and dead (red) fluorescing bacteria (Fig. 3). Without treatment, we could observe microcolonies of bacteria, which exhibited green fluorescence only, indicating high levels of viability. Treatment with CHX dentifrice for 2 min resulted in decreased levels of adherent bacteria and an increase in red fluorescence indicating loss of bacterial viability. Treatment with SEC-xylitol dentifrice (30% v/v) also resulted in increased red fluorescence and removal of biofilm at a level comparable to the CHX treatment. These observations were consistent in replicate experiments.
Qualitative assessment of adherent bacterial removal and killing was carried out by staining adherent bacterial communities with the LIVE/DEAD Baclight bacterial viability kit (Thermofisher). A tri-species community of Strep. gordonii, Strep. mutans, and Coryne. matruchotii was grown and exposed to DMEM (control) or to a 30% v/v suspension of CHX or SEC dentifrice for 2 min. Bacteria were observed using a Zoe fluorescence microscope (BioRad). White bar corresponds to 100 μM.
Conclusions
In conclusion, our study shows that a novel SEC-xylitol dentifrice formulation exhibits greater antimicrobial activity in comparison to the gold-standard antimicrobial CHX. CHX is used in many commercially available dentifrices used to treat gingivitis and periodontal disease (Brookes et al. 2021). However, use of CHX is commonly associated with staining of teeth and prostheses and in rare cases, it can cause irritation or allergic responses (Pałka et al. 2022). As antimicrobials, salivary enzymes offer some advantages over chemical biocides. As they are naturally occurring proteins, they exhibit excellent biocompatibility (Magacz et al. 2019). In addition, as microbes are naturally exposed to salivary enzymes in vivo, they should not result in increased selection of organisms resistant to clinically used antibiotics. LPO containing dentifrices have been shown to have good antimicrobial activity against biofilms of oral microorganisms (Modesto et al. 2000, Jones et al. 2018, Rabe et al. 2022). Clinical trials have also shown that regular use of LPO containing dentifrices can reduced plaque levels and improve gingival health (Nakano et al. 2019, Nakano et al. 2020).
In the current study, we directly compare the antibiofilm activity of a novel SEC-xylitol combination versus a standard 0.12% chlorhexidine dentifrice. In order to maximize SEC activity, xylitol was included in the formulation. Although not extensively investigated, there is some evidence that LPO activity is enhanced in the presence of xylitol; however, the exact mechanism for this has not been elucidated (Mäkinen et al. 1976, Kim et al. 2015). We initiated our investigations against biofilms composed of organisms considered to be early colonizers of human teeth, namely Strep. gordonii and A. naeslundii. A 2-min exposure to SEC-xylitol dentifrice was sufficient to cause a ∼3-log reduction in viability compared to controls, which was statistically significant compared to the effects of a chlorhexidine dentifrice. Superior activity was also demonstrated against the Gram-negative anaerobe Fus. nucleatum and the Gram-positive organism Coryne. matruchtii. Both of these species were selected for investigation due to their important role in plaque maturation (Zijnge et al. 2010, Welch et al. 2016). Corynebacterium species have been shown to act as scaffold in supragingival plaque and Fus. nucleatum has been shown to act as bridge between supragingival and subgingival plaque, allowing biofilm incorporation of late colonizers such as P. gingivalis (Kolenbrander and Andersen 2006). The activity against these species supports a mechanism whereby SEC-xylitol can disrupt plaque maturation. We also observed a significant 3-log reduction in the viability of Strep. mutans, an organism with a major role in the development of dental caries, suggesting a caries protective role. This is in agreement with numerous studies that have shown activity of LPO against Strep. mutans (Roger et al. 1994, Modesto et al. 2000, Jyoti et al. 2009, Welk et al. 2009). These data were supported by well diffusion assays, which also demonstrated the enhanced activity of SEC-xylitol dentifrice compared to chlorhexidine formulations. We also examined a combination of organisms in a mixed species community, namely Strep. gordonii, Coryne. matruchotii, and Strep. mutans. Although this analysis was qualitative in nature, we observed that SEC-xylitol was at least as effective as chlorhexidine formulations in removal of the multispecies biofilm and in reducing bacterial viability, as indicated by the level of red fluorescence.
Although our study shows excellent antimicrobial activity by the SEC-xylitol dentifrice, we have not specifically addressed if the incorporation of xylitol enhances the activity of the enzyme complex, as suggested by some previous studies. Future studies comparing the SEC dentifrice with and without the xylitol addition will be required to address this.
Overall, our data indicate that an SEC-xylitol dentifrice formulation can exhibit antimicrobial activity greater than chlorhexidine formulations. This activity supports a role for SEC-xylitol formulations as excellent choices for individuals at high risk of caries or periodontal disease, or those with reduced manual dexterity who require extra antimicrobial support. Further research is required to determine the mechanistic nature of this antimicrobial combination.
Conflict of interest
D.L. is the Director of LA Research labs (manufacturer of oral healthcare products). The other authors have no conflict of interest to declare.
Funding
This work was supported by a grant from the Enterprise Ireland (IV20220129).
Author contributions
Mackenzie O'Connor (Data curation [Equal], Formal analysis [Equal], Investigation [Equal], Methodology [Equal], Writing – original draft [Supporting]), Grant Harrison (Data curation [Equal], Formal analysis [Equal], Investigation [Equal], Methodology [Equal], Writing – original draft [Supporting]), Declan Lenahan (Conceptualization [Equal], Funding acquisition [Equal], Methodology [Equal], Writing – review & editing [Supporting]), and Gary P. Moran (Conceptualization [Equal], Data curation [Equal], Formal analysis [Equal], Funding acquisition [Equal], Methodology [Equal], Project administration [Equal], Supervision [Lead], Writing – original draft [Lead], Writing – review & editing [Lead])
Data availability
All data and materials are available on request from the authors.
