Contemporary paradigms of dental caries aetiology focus on the ecology of the dental plaque biofilm and how local environmental factors can modulate this to cause disease. The crucial role that a healthy oral microbiome plays in preventing caries and promoting oral health is also being increasingly recognized. Based on these concepts, several ecological preventive approaches have been developed that could potentially broaden the arsenal of currently available caries-preventive measures. Many of these ecological approaches aim for long-term caries control by either disrupting cariogenic virulence factors without affecting bacterial viability, or include measures that can enhance the growth of health-associated, microbially diverse communities in the oral microbiome. This paper argues for the need to develop ecological preventive measures that go beyond conventional caries-preventive methods, and discusses whether these ecological approaches can be effective in reducing the severity of caries by promoting stable, health-associated oral biofilm communities.
Oral health is integral to general well-being, with profound individual and societal implications that extend well beyond the functions of the craniofacial complex. Although largely preventable, diseases such as dental caries are major public health concerns, imposing a costly burden on health services. Traditional caries epidemiological measures do not adequately reflect the social impacts, economic costs, and health care system effects of the disease [Casamassimo et al., 2009].
At the individual level, control of dental caries remains largely dependent on twice-daily mechanical oral hygiene in the form of toothbrushing with fluoride dentifrices, a preventive approach that has been in place for over 50 years. Based on the current understanding of the dental caries process, several ecological preventive strategies have been developed or are currently under investigation, suggesting a future where caries prevention will not be narrowly focussed on fluoride therapies. This review will explore the rationale supporting the need for alternate methods of dental caries control, and will discuss the current status of some ecological approaches to biofilm modification and caries prevention.
Dental Caries: A Biofilm-Mediated Multifactorial Disease
Dental caries belongs to a group of diseases that are considered “complex” or “multifactorial,” with no single causation pathway, and therefore, are not amenable to simplistic preventive solutions such as the elimination of “one type of organism” or merely enhancing “tooth resistance” [Fejerskov, 2004]. Dental caries is now widely recognized to be an endogenous, biofilm-mediated disease that occurs when acidogenic/aciduric members of resident oral flora obtain a selective ecological advantage over other species, disrupting the homeostatic balance of the plaque biofilm and initiating the disease process [Marsh and Martin, 1999]. Modern molecular analyses and microbial culture techniques have demonstrated that an entire range of bacteria, not just mutans streptococci (MS) or lactobacilli (LB), can contribute to the caries process at different stages [Tanner et al., 2016], and that even fungi such as Candida albicans can significantly enhance the cariogenic virulence of plaque biofilms [Koo and Bowen, 2014]. An MS- or LB-dominated microbiome may be found only at the advanced stages of the disease, where the increased severity and frequency of biofilm acidification results in the oral microbiome becoming less microbially diverse [Takahashi and Nyvad, 2008]. Indeed, it is not the bacterial genotype per se but their shared phenotypic characteristics (being acidogenic and aciduric) that are more important for driving the microbial ecological shift that leads to dental caries [Takahashi and Nyvad, 2011].
While the ionic aspects of the dental caries process have been the focus of research for decades, new insights into the aetiology and microbial aspects of this biofilm-mediated disease have engendered novel concepts and approaches for its prevention and control [ten Cate and Cummins, 2013]. A consensus is now emerging that caries-preventive measures should aim to not only correct the environmental pressures responsible for the plaque biofilm dysbiosis, but also help to maintain a healthy, microbially diverse, resident microbiome [Marsh et al., 2015]. These new approaches acknowledge the possibility that bacteria yet to be identified and cultured may participate in the caries process, and aligns with engaging in a more comprehensive approach towards preventing dental caries as a disease at the patient level [Walsh, 2011].
Rationale for Newer Methods of Dental Caries Control
The historical focus in preventing and managing oral diseases was on eliminating dental plaque from teeth. However, contemporary evolving evidence is increasingly highlighting the beneficial aspects of a healthy oral microbiome [Kilian et al., 2016]. Commensal plaque microflora have a symbiotic relationship with the host, not only acting as a barrier to opportunistic pathogens, but also carrying out metabolic processes that benefit the host [He et al., 2011; Schlafer et al., 2017]. Acute infections of the oral mucosa are rare because of the interplay between the host immune system and microbial symbionts [Zaura et al., 2014]. Likewise, the pro- and anti-inflammatory activities of resident bacteria help maintain homeostasis in the oral cavity [Devine et al., 2015]. A moderate amount of healthy plaque has also been shown to prevent erosive enamel lesions and hypersensitivity [Honorio et al., 2010].
Based on this changed understanding of the importance of a healthy oral microbiome, caries-preventive strategies should ideally take an ecological approach to the holobiont. Indiscriminate or “shotgun” suppression of almost the entire oral biota, without understanding the overall effects on plaque ecology, is unlikely to have long-term success in controlling the disease [Caufield et al., 2001]. Instead, preventive and curative products should either specifically target cariogenic bacteria without affecting other resident microflora, or they should inhibit virulence factors (e.g., glucan synthesis or acid production) rather than bacterial viability [ten Cate and Zaura, 2012]. Measures that enhance colonization of health-promoting microbial communities can also help in correcting the ecological imbalance in cariogenic biofilms. The advantage of such an approach is that the impacts of low pH environments generated by acidogenic organisms can be counterbalanced by ammonia production from other bacteria. Following an ecological approach to caries prevention can potentially preserve the favourable effects that the host derives from the resident oral microbiome, while reducing cariogenic virulence factors that are responsible for plaque biofilm dysbiosis.
The control and prevention of any disease should preferably focus on the aetiological factors involved. For dental caries, this would be the periodic disorganization of the oral plaque biofilm by mechanical oral hygiene, along with dietary modification to reduce exposure to fermentable carbohydrates [Cury and Tenuta, 2008]. Unfortunately, individual oral hygiene measures have only a limited impact in caries prevention [Bellini et al., 1981; Nyvad, 2003; Hujoel et al., 2006]. It has also been suggested that toothbrushing has been effective in preventing caries mainly because it brings fluoride into the oral cavity at regular intervals, rather than any particular efficiency in disrupting cariogenic plaque biofilms [ten Cate and Zaura, 2012]. Dietary modification, which requires individuals to restrict their exposure to sucrose and other fermentable substrates, is particularly difficult to achieve in present-day society where cariogenic foods are easily available [Duggal and Van Loveren, 2001]. Furthermore, restriction of sucrose intake alone is unlikely to completely prevent dental caries if frequent intake of other starches persisted [Bradshaw and Lynch, 2013]. Thus, while minimizing the aetiological factors contributing to the disease is critical, additional preventive measures commensurate with individual risk status may still be required in many segments of the population.
For a long time, dental caries was a pandemic that affected children and adults almost universally. The landmark discovery of fluoride as an agent that could prevent dental caries, and the widespread use of fluoride-based caries-preventive programmes, have been responsible for the significant reductions seen in caries prevalence of developed countries in the latter half of the 20th century [Fejerskov, 2004]. However, the latest Global Burden of Disease report revealed that untreated caries in permanent teeth still remains the most common human disease condition worldwide [Kassebaum et al., 2015]. Doubts have also been expressed on whether the earlier decline in caries prevalence has continued into this new century [Gimenez et al., 2016]. Recent caries prevalence studies in the USA and Australia indicate tooth decay may in fact be increasing again [Dye et al., 2007; Chrisopoulos et al., 2016]. A similar trend has been seen in countries such as Norway and Iceland, which had initially shown the biggest improvements, but are now registering increases in caries experience [Haugejorden and Birkeland, 2005; Agustsdottir et al., 2010]. The flattening of prevalence rates that has occurred even in dentally aware populations where individuals commonly brush their teeth with fluoridated dentifrices is concerning and underscores the need to develop additional caries-preventive measures that are synergistic with or complementary to fluoride.
Fluoride Alone May Not Be Sufficient
While fluoride is a highly effective and economical agent for dental caries prevention and will remain the mainstay of any caries-preventive programme, it must be recognized that in many situations fluoride alone may not be sufficient. Even with regular fluoride use, carious lesions can still develop when there are more than 6 dietary sugar exposures per day [Duggal et al., 2001; Ccahuana-Vasquez et al., 2007]. The ready availability of cariogenic snack foods and drinks in the modern consumer culture can overwhelm the benefits of community water fluoridation and daily use of a fluoride dentifrice. The limit to the repair potential of fluoride could partly explain the reversal of the caries decline being observed in contemporary epidemiological reports from developed countries [Agustsdottir et al., 2010; Dye et al., 2017].
The cariostatic actions of fluoride are largely attributed to its physiochemical ability to inhibit enamel demineralization and enhance remineralization [Cury and Tenuta, 2008]. Recent laboratory studies have confirmed the notion that under appropriate conditions fluoride ions can also influence critical MS virulence factors, significantly reducing acidogenicity, aciduricity, and glucan formation [Domon-Tawaraya et al., 2013; Pandit et al., 2013, 2015]. Metabolome analysis of plaque biofilms has demonstrated that fluoride can repress acid production in vivo too [Takahashi and Washio, 2011]. However, questions still remain on the extent to which these antibacterial mechanisms contribute towards caries-preventive effects. A recent study showed that the brief fluoride exposure from toothpastes or mouthwashes could not sustain anti-acid production activity, with the biofilms recovering acidogenicity over time regardless of the fluoride concentration used [Dang et al., 2016]. Concerns have also arisen regarding the emergence of micro-organisms that are more resistant to the effects of fluoride on microbial metabolism [Mitsuhata et al., 2014].
Preventive approaches that combine fluoride with other protective agents have been advocated to enhance the ability of fluoride to modify biofilms and diminish the cariogenic bacterial challenge [Li et al., 2015]. Fluoride-antimicrobial combinations have been recommended based on the 2-part rationale that fluoride can reduce the critical pH at which dissolution starts, while effective antimicrobial agents can decrease the depth of the Stephan curve pH drop following consumption of fermentable carbohydrates [Øgaard, 2000]. Randomized controlled trials (RCTs) have shown that fluoride-chlorhexidine interventions significantly reduced bacterial load and pH drop from glucose metabolism [Giertsen and Scheie, 1995], and most importantly, lowered caries increment in high-risk groups [Featherstone et al., 2012]. Sytematic reviews have also found high-quality evidence that fluoride-triclosan toothpastes give a small improvement in reducing coronal caries compared to conventional fluoride dentifrices [Blinkhorn et al., 2009; Riley and Lamont, 2013]. However, the use of such broad-spectrum antimicrobial agents may not be ideal, as the aim of antimicrobial treatment in caries prevention should preferably be to modify the plaque biofilm ecology by weakening cariogenic virulence factors rather than eliminating microflora.
An alternative to the fluoride-antimicrobial approach is combining fluoride with agents that promote an overall community-wide microbial shift by encouraging the growth of health-associated bacteria, thereby beneficially rebalancing the biofilm ecology and potentially resulting in better long-term dental caries control. Among such biofilm-modifying oral products, there is now sufficient evidence supporting fluoride-arginine combinations as a new standard of care for caries prevention [ten Cate and Cummins, 2013; Zheng et al., 2015]. Likewise, a dentifrice containing enzymes and proteins was shown to significantly shift the ecology of the oral microbiome resulting in a community with a stronger association to health [Adams et al., 2017].
Over time, the distribution of caries in the community has changed from a disease that was pandemic in society, to now being endemic in specific risk groups. A cross-sectional study concluded that 75% of the caries-risk burden tends to reside in 25-40% of the population [Macek et al., 2004]. Likewise, there is evidence for genetic differences that exist with respect to caries susceptibility [Bretz et al., 2005; Opal et al., 2015], with not everyone in a population group benefiting to the same extent from traditional caries-preventive programmes [ten Cate, 2013]. The pattern of dental caries has also changed over the years, from a rapidly progressing disease of childhood, to a slowly progressing disease that can occur or persist throughout adulthood and old age [Lagerweij and van Loveren, 2015]. Data from contemporary population studies have revealed that carious lesions are also increasingly localized to specific tooth sites [Anderson, 2002]. In children and adolescents, occlusal surfaces are the sites most likely to have experienced caries [Carvalho, 2014; Carvalho et al., 2016]. The increasing life expectancy of dentition also means that older adults with exposed root surfaces are at greater risk of experiencing root caries during their lifetime [Gati and Vieira, 2011]. With fluoride known to exert its cariostatic actions primarily on smooth enamel surfaces, susceptible occlusal/root surfaces remain relatively predisposed to acid dissolution [ten Cate, 2009]. Caries-preventive strategies must therefore go beyond conventional methods (personal oral hygiene, fluoride dentifrices, and limiting sugar exposures) to better protect at-risk surfaces in at-risk patients.
The US Centers for Disease Control and Prevention ranked community water fluoridation as one of the 10 great public health achievements of the 20th century. Despite continued broad support within the dental profession worldwide, the polarized debate on the safety of fluorides used for caries prevention still continues in sections of the popular and scientific press. Recent reports can only add to the alarmist picture for sections of the general public who are not informed readers of the scientific literature on this topic. For example, a 2012 systematic review and meta-analysis concluded that high fluoride exposure may lower IQ levels in children [Choi et al., 2012], a finding which attracted media attention and triggered the inclusion of fluoride amongst chemicals classified as developmental neurotoxicants [Grandjean and Landrigan, 2014]. There are however a number of concerns with how the 2012 review was conducted - all selected studies were from China, fluoride exposure came from multiple sources not just drinking water, the definition of “high” fluoride levels varied widely, and the probability of confounding as covariates was not controlled. The authors themselves were extremely conservative, only concluding that “our results support the possibility of adverse effects” advocating future research to evaluate dose-response relations based on individual-level measures of exposure over time. However, these caveats are universally overlooked when studies are reported in the media, and this fans extremist views on the safety of fluoride used for caries prevention.
Additionally, dental fluorosis from excessive intake of fluoride during the period of tooth formation is a persisting concern that is raised by antifluoride lobbyists. While emphasizing that fluoride is safe when used at the recommended levels for community water fluoridation or dentifrices, behaviours such as swallowing large amounts of dentifrice are not. In modern times there are also now opportunities for “halo” exposure from various other sources of fluoride, which has triggered downward revision of recommended levels for community water fluoridation (from 0.7-1.2 to 0.7 mg/L), to ensure that the risk for dental fluorosis does not increase [EPA, 2011].
Ecological caries-preventive approaches synergistic with fluoride can potentially allow dental products to be designed with lower concentrations of fluoride and these could be useful for caries prevention in infants and young children, as well as for patients reluctant to use oral care products with high fluoride concentrations.
Ecological Approaches to Caries Prevention
Antimicrobial peptides (AMPs) are a heterogeneous group of molecules with unique antimicrobial characteristics that have great potential for controlling bacterial infections and modifying biofilms. AMPs have a broad range of antibacterial, antiviral, and antifungal activity mediated by selectively interacting electrostatically with negatively charged components of cell membrane phospholipids, resulting in membrane permeabilization and disruption, leading to cell death [Koczulla and Bals, 2003]. To escape the actions of bilayer-disruptive AMPs would entail changing membrane composition and organization, a “costly” process in evolutionary terms, meaning that AMPs have very low resistance rates compared to common antibiotics [Zasloff, 2002]. Besides naturally secreted salivary AMPs (lactoferrin, cathelicidins, histatins, defensins), a number of AMPs have been synthesized in the laboratory, and these include specific anticaries peptides that have shown the potential to modify plaque biofilms and inhibit dental caries.
The specifically targeted antimicrobial peptide (STAMP) is a synthetic fusion peptide with 2 independent functional domains, consisting of a Streptococcus · mutans-selective “targeting domain” designated as C16, and a “killing domain” designated as G2. C16 is derived from a fragment of the S. mutans competence stimulating peptide (CSP), while G2 is derived from a broad-spectrum antimicrobial peptide [Eckert et al., 2012]. C16G2 had antimicrobial mechanisms similar to traditional AMPs, and critically, its membrane-disrupting activity specifically targets S. mutans from multispecies biofilms without affecting closely related non-cariogenic oral streptococci [Eckert et al., 2006; Kaplan et al., 2011]. More recently, an in vitro study on human saliva-derived polymicrobial biofilms was able to demonstrate that treatment with C16G2 not only eliminated S. mutans, but also resulted in a more benign oral microbial community with increased populations of health-associated bacteria and fewer harmful Gram-negative bacteria [Guo et al., 2015]. C16G2 is recognized by the US Food and Drug Administration as an investigational new drug for dental caries prevention and has successfully completed phase II clinical trials, where it was delivered to patients in the form of a dental gel loaded in trays.
Another promising anticaries AMP is a synthetic α-helical antimicrobial decapeptide designated KSL-W, which can selectively destabilize the cell membranes of cariogenic bacteria including S. mutans, S. sobrinus, and L. acidophilus [Na et al., 2007; Leung et al., 2009]. This peptide resits enzymatic degradation in human saliva for 1 h, and the potential use of KSL-W as an antibiofilm agent in a chewing gum formulation has been suggested [Faraj et al., 2007; Na et al., 2007]. More recently, a hydroxyapatite-binding AMP was designed, based on the fusion of specific hydroxyapatite-binding heptapeptide (HBP7) with KSL-W, and this bioconjugate was shown to have improved oral retention and antibacterial efficacy [Huang et al., 2016b]. Other AMPs that have shown in vitro antimicrobial activity against cariogenic bacteria and inhibited oral biofilm formation include a synthetic peptide called L-K6 (derived from the naturally occurring peptide temporin-1CEb), a short synthetic amphiphilic peptide known as 1018, and an amphipathic α-helical peptide containing only 12 amino acids named GH12 [Shang et al., 2014; Wang et al., 2015; Tu et al., 2016].
Limitations of AMPs include their potential toxicity, susceptibility to proteases, high cost of peptide production, and the reduced cationic activity of most AMPs in physiological fluids like saliva. With regard to their use for dental caries prevention, questions still remain on whether anticaries AMPs will be able to function against a background of excessive acid production often seen in high caries-risk individuals [Maltz and Beighton, 2012]. Before any clinical recommendations can be made, it will be essential to test such agents in a caries-conducive oral environment, and to evaluate whether the desired outcome of reduced caries increment in at-risk population groups can be achieved.
The term probiotics refers to “live micro-organisms, which, when administered in adequate amounts, confer a health benefit on the host” [Teughels et al., 2008], and is based on the Noble-prize-winning pioneering work of Metchnikoff  for maintaining a healthy gut flora. This concept of implanting a harmless effector strain into the host's microflora to maintain or restore a natural microbiome by inhibition of pathogenic micro-organisms is attractive and has also been used to support health-associated microbes or restore diversity in the oral plaque biofilm. The mechanisms by which probiotics re-establish ecological balance in oral biofilms are not fully understood, but probiotic bacteria are believed to have both local and systemic effects [Meurman, 2005]. The local anticaries effects may include competitive inhibition with cariogenic bacteria for nutrition or adhesive surfaces [Terai et al., 2015], selective co-aggregation of MS without disturbing other oral flora [Twetman et al., 2009; Lang et al., 2010], and bacteriocin-producing probiotics targeting MS [Burton et al., 2013]. The most commonly used and studied probiotics belong to the Lactobacillus and Bifidobacterium bacterial genera, although not all their strains have the same efficacy in the inhibition of S. mutans growth or biofilm formation [Schwendicke et al., 2017].
Evidence supporting the application of probiotics for preventing dental caries is controversial, with recent reports suggesting potential harmful effects for some probiotic bacterial strains [Gruner et al., 2016]. One of the problems identified in using probiotics for caries prevention is that the commonly available Lactobacillus and Bifidobacterium probiotic bacteria are themselves acidogenic and aciduric, and could contribute to the caries process if such bacteria are allowed to colonize the oral cavity [Maltz and Beighton, 2012]. Recent in vitro biofilm studies have confirmed this apprehension with different strains such as Lactobacillus salivarius W24 [Pham et al., 2009], Lactobacillus rhamnosus GG [Schwendicke et al., 2014a], Bifidobacteria animalis lactis BB12 [Schwendicke et al., 2014b], Lactobacillus rhamnosus LB21 [Fernández et al., 2015], and Lactobacillus acidophilus LA-5 [Schwendicke et al., 2017] which have all been shown to lower biofilm pH. In fact, some Lactobacillus and Bifidobacterium strains have greater cariogenic attributes than even MS [Beighton et al., 2010]. Thus, while displacing S. mutans from plaque biofilms may in principle be desirable, substituting them for even more cariogenic bacteria will not be useful [Schwendicke et al., 2014a]. Some of the non-acidogenic alternatives to Lactobacillus and Bifidobacterium that have shown promising early results include S. salivarius M18 [Burton et al., 2013], heat-inactivated BB12 [Schwendicke et al., 2014b], and Weissella cibaria CMU [Jang et al., 2016].
Another limitation with the traditional use of gut-associated Lactobacillus and Bifidobacterium probiotic species to promote oral health is that these non-oral bacterial strains may not efficiently colonize the oral niche, which is vital for the long-term success of probiotics [López-López et al., 2017]. Even the use of a bacteriocin-producing strain of S. salivarius may not succeed, as S. salivarius, while a typical member of the oral soft tissue flora, has limited ability to colonize tooth surfaces. However, very recently 2 natural oral commensal species, Streptococcus dentisani and Streptococcus A12 that were isolated from the supragingival plaque of caries-free individuals, have demonstrated promising probiotic effects against dental caries. Both these “active colonizers” have a double probiotic action, as they can not only inhibit the growth of MS, but also moderate plaque pH through their arginolytic actions [Huang et al., 2016a; López-López et al., 2017].
The current decade has seen a number systematic reviews and meta-analyses evaluating the effectiveness of using traditional gut-associated probiotics for caries prevention. A meta-analysis of studies with surrogate caries markers (MS and/or LB counts) concluded that these probiotics significantly decreased MS counts, but there were insufficient data on whether caries increment was reduced as well [Laleman et al., 2014]. A similar inference was also reached by a qualitative systematic review of probiotic caries studies, concluding that clinical recommendations would be premature without more comprehensive RCTs showing an actual reduction in individual caries experience [Twetman and Keller, 2012]. Relatively few RCTs of oral probiotics have used clinical dental caries indicators to prove the efficacy of probiotics in preventing or treating dental caries [Näse et al., 2001; Stecksén-Blicks et al., 2009; Burton et al., 2013; Taipale et al., 2013]. A comprehensive systematic review utilizing evidence from these RCTs concluded that current evidence is insufficient for recommending probiotics in controlling dental caries [Gruner et al., 2016].
Taken together, currently available data indicate that while traditional probiotic bacteria may have a beneficial effect on the gut flora and systemic health, a beneficial and clinically significant effect on the oral flora is yet to be demonstrated with sufficient rigor. However, observations from 2 major clinical trials in children [Näse et al., 2001; Stecksén-Blicks et al., 2009] support the intriguing concept of a “metabolic domino effect,” with reductions in caries risk seen to be accompanied by improvements in general health. Particularly promising for caries prevention is the move away from gut-associated probiotic bacteria to resident oral probiotic strains, such as the 2 S. mutans-antagonistic bacterial species S. dentisani and A12. Further evidence on the ability of these oral probiotics to inhibit dental caries is awaited with interest.
The prebiotic approach involves feeding resident microbiota with specific nutrients to create conditions that favour the growth and dominance of healthy bacteria in the biofilm. The nutritional stimulation of endogenous beneficial oral flora to restore microbial balance and promote oral health has been validated in mixed species models [Slomka et al., 2017]. Oral prebiotic substrates that are especially valuable to prevent caries include arginine, arginine-rich peptides, and urea, as these foods when metabolized create alkalizing effects that counteract the acidogenic environment created by cariogenic bacteria. Many commensal bacteria are able to use arginine or urea to generate ammonia by the arginine deaminase system or urease enzymes, respectively [Bradshaw and Marsh, 1998; Nascimento et al., 2009]. Multiple studies have shown that bacterial production of alkaline metabolites such as ammonia can play a major role in biofilm pH homeostasis and beneficially alter the de-/remineralization equilibrium [Burne and Marquis, 2000; Burne et al., 2012; Nascimento et al., 2014]. A substantial body of evidence from microbiological, genetic, biochemical analyses, and clinical studies has now accumulated confirming that the modulation of the alkalinogenic potential of dental biofilms is a promising strategy for caries control [Liu et al., 2012].
The preventive potential of oral alkali production has resulted in the development of commercial oral care products that utilize arginine to promote a healthy resident oral microbiome. In vitro biofilm experiments found that fluoride-arginine combinations synergistically inhibited S. mutans but enriched S. sanguinis growth within multispecies biofilms, while maintaining a “streptococcal pressure” against the potential growth of oral anaerobe Porphyromonas gingivalis in the alkalized biofilm [Zheng et al., 2015]. Fluoride-arginine combinations can also suppress exopolysaccharide production, thus targeting another critical virulence factor for cariogenic biofilms [Zheng et al., 2015]. Human in situ studies and several double-blinded RCTs using a fluoride dentifrice containing 1.5% arginine and an insoluble calcium compound have shown significantly greater protection against carious lesions than a fluoride dentifrice alone [Cantore et al., 2013; Kraivaphan et al., 2013; Srisilapanan et al., 2013; Yin et al., 2013; Petersen et al., 2015]. A systematic review and meta-analysis of the anticaries effects of arginine-containing dentifrice formulations concluded that arginine products provided a superior preventive effect over matched formulations containing fluoride alone [Li et al., 2015]. Other authors have been more conservative in their conclusions, either citing insufficient evidence in support of a caries-preventive effect for arginine, or expressing concerns over the higher cost of arginine-fluoride dentifrices versus any additional caries-preventive effect these may provide [Ástvaldsdóttir et al., 2016]. However, the preponderance of evidence does seem to suggest that the arginolysis is an effective approach to improve oral health and balance the microbial ecology.
Sucrose has been designated as the “arch criminal” in the caries process for a long time [Newbrun, 1967], and the search for alternative non-fermentable sweeteners has attracted much attention. Data collected from in vitro and in vivo studies indicate that such sugar substitutes can exhibit potential anticaries effects through a number of different mechanisms [Matsukubo and Takazoe, 2006]. Xylitol, a naturally occurring 5-carbon sugar polyol, is the non-nutritive sweetener that has been most extensively researched over the past 4 decades for its potential cariostatic effects. Xylitol inhibits MS growth by disrupting their energy production processes, leading to a futile energy cycle and cell death [Marttinen et al., 2012]. Although not all MS strains were inhibited by xylitol in this manner, even xylitol-resistant bacteria were found to be less virulent after xylitol treatment [Trahan, 1995]. The predominant delivery vehicle for xylitol has been chewing gums, with xylitol dentifrices, candies, lozenges, and mouthrinses also having been used with varying degrees of success. A substantial body of evidence suggests that 5-6 g of xylitol per day delivered over 3 exposures are needed for worthwhile anticaries effects [Milgrom et al., 2009]. Among other sugar polyols, erythritol has been attracting increasing attention as it is has been shown to be more effective than xylitol and sorbitol, and importantly, its anticaries effects were shown to persist for up to 3 years [Honkala et al., 2014; de Cock et al., 2016; Falony et al., 2016].
Despite an immense body of literature, the caries-preventive effects of xylitol products remain inconclusive because of inconsistent study outcomes. While numerous studies have indicated xylitol has beneficial effects on surrogate end points (MS levels, plaque pH, acid production), evidence for worthwhile reductions in caries experience remains equivocal, and there is a need for more double-blind placebo-controlled RCTs, focussing on optimal dosage, delivery vehicle, and possible synergism with other preventive agents [Twetman, 2009; Milgrom et al., 2012]. In fact, the more recent data conclude that there is limited evidence to show xylitol is effective in the fight against dental caries. A double-blind cluster RCT using xylitol gummy bears found that polyol consumption did not provide any additional benefit over other caries-preventive measures [Lee et al., 2015]. Recent Cochrane systematic reviews also found only very-low- to low-quality evidence on xylitol effectiveness which was not sufficient to determine whether xylitol-containing products can prevent caries in infants, children or adults [Duane, 2015; Riley et al., 2015]. Other issues that limit the usefulness of sugar polyols include their high costs and low compliance in high-risk patients because of the need for daily long-term use [Gold, 2016].
Another approach that may maintain and support a healthy oral plaque ecology is to interfere with the fundamental cell-cell communications system between biofilm bacteria. This process of quorum sensing (QS) is mediated through small diffusible hormone-like molecules (pheromones) and their specific receptors. For MS, the stress-dependent QS system is primarily comprised of the CSP and its ComD/ComE 2-component signal transduction system for communication between biofilm cells of the same species, while interspecies signalling is mediated via the autoinducer-2 molecule produced by LuxS [Senadheera and Cvitkovitch, 2008]. The CSP-mediated QS system in S. mutans affects biofilm formation, acidogenicity, aciduricity, genetic transformation, bacteriocin production, stress response, and the ability to produce persister phenotypes [Leung et al., 2015]. Targeting QS signalling pathways could provide a promising avenue in the development of novel therapeutics to alter cariogenic biofilms. As QS is not directly involved in processes essential for bacterial growth, targeting QS will allow less virulent bacteria to remain in the biofilm and will also not impose selective pressures that can lead to the development of antibiotic resistance [Rasmussen and Givskov, 2006].
Interfering with CSP signalling systems has been shown to inactivate a wide range of bacteriocins and mutacins that play an important role in the sustained existence of S. mutans in the dental plaque [Cvitkovitch et al., 2003; Qi et al., 2004]. The addition of an exogenous CSP can disrupt signalling events of S. mutans and induce cell death [Qi et al., 2005]. Another novel QS-modifying compound, 3-Oxo-N, was seen to significantly minimize lactic acid accumulation without affecting biofilm growth even in the presence of fermentable sugars, representing a promising agent for maintaining a healthy, non-cariogenic microbial ecology in dental plaque [Janus et al., 2016].
Natural products include secondary metabolites or phytochemicals derived from plants, fruits, herbs, or spices. They offer a rich source of structurally diverse molecules with a wide range of biological activities and could prove useful as alternative or adjunctive anticaries agents [Jeon et al., 2011]. Potential cariostatic mechanisms identified include inhibition of bacterial growth or acid production, inhibition of glucan synthesis by interfering with glucosyltransferase (Gtf) activity, and inhibition of bacterial adhesion [Ferrazzano et al., 2011; Jeon et al., 2011].
Polyphenols from propolis (apigenin and tt-farnesol) and cranberry proanthocyanidins have been shown to exert useful ecological effects on the plaque biofilm. Apigenin is a potent inhibitor of water-insoluble glucan synthesis, while tt-farnesol disrupts S. mutans membrane permeability and acid production [Koo and Jeon, 2009]. An animal study found a combination of these 2 phytochemicals with fluoride suppressed dental caries without affecting the viability of normal oral flora, being as potent as a fluoride-chlorhexidine control in caries inhibition, but without the broad antibacterial action of the control [Koo et al., 2005]. Similarly, cranberry proanthocyanidins, which lack significant biocidal activity, can modify plaque biofilms by reducing acidogenicity and glucan synthesis, and these surrogate end points were also translated into cariostatic effects in vivo [Koo et al., 2010]. A number of other polyphenol compounds have been found to be effective in killing S. mutans, with the minimal inhibitory concentrations of some bioactive molecules like xanthorrhizol (from Curcuma xanthorrhiza) or macelignan (from Myristica fragrans) almost comparable to chlorhexidine [Hwang et al., 2000; Chung et al., 2006]. While most of the tested anticaries phytochemicals showed growth-inhibitory or anti-adhesive effects, a potentially interesting natural agent in caries prevention is Galla chinensis, which was able to beneficially regulate the de-/remineralization balance of dental hard tissues [Cheng et al., 2015].
An analysis of how phytochemical research has impacted oral care in the period from 2000 to 2015 found that despite many in vitro, in vivo, and clinical studies testing natural products derived from plants, only 11% of studies were in phase IV clinical trials [Freires and Rosalen, 2016] Similar conclusions were reached in a systematic review of the anticaries effects of essential oils and their isolated constituents, which found that most studies were conducted in the laboratory, and did not provide botanical characterization or compositional data on the natural product being tested [Freires et al., 2015]. Natural products remain a largely unexplored source of effective and non-toxic antibiofilm molecules that could potentially be used in combination with fluoride as useful alternatives to traditional microbiocides like chlorhexidine or triclosan [Jeon et al., 2011]. However, future research needs to focus on translational approaches to advance the development of effective anticaries products containing phytochemicals or essential oils.
Replacement Therapy with “Designer” Bacteria
Modifying dental plaque by replacing S. mutans, a member of the normal microbiota, with a less virulent effector strain has been an established concept for many years [Hillman, 1978]. The rationale for bacterial replacement therapy against dental caries is that relatively avirulent strains of MS are most likely to occupy the same ecological niche in plaque as their more cariogenic counterparts thereby reducing the overall cariogenicity of the plaque biofilm [Sun et al., 2009].
A number of “designer” bacteria have been studied for bacteriotherapy against cariogenic biofilms including a glucan synthesis-defective mutant of S. mutans, variants of S. salivarius (TOVE-R), and a recombinant alkali-generating ureolytic S. mutans strain [Tanzer et al., 1974, 1985; Clancy et al., 2000]. The most extensive research in using genetically modified bacteria for preventing dental caries used a wild-type S. mutans strain that naturally produces an antibiotic called mutacin 1140 capable of killing all other strains of S. mutans [Hillman, 2002]. This strain was genetically modified by deleting the open reading frame for lactate dehydrogenase, to yield a viable strain called BCS3-L1 that still produced wild-type levels of mutacin 1140, but notably produced no lactic acid [Hillman et al., 2000]. In laboratory and animal models, the BCS3-L1 strain proved to have significantly reduced cariogenic potential. It persistently and pre-emptively colonized tooth sites normally occupied by wild-type S. mutans strains, with no reported adverse effects [Hillman et al., 2000]. To overcome safety issues and to enable the altered strain to be implanted into human oral biofilms for clinical trials, additional genetic modifications of BCS3-L1 were made, which could facilitate its rapid elimination should an adverse event manifest itself. This strain was designated as A2JM and was extensively tested to assure its safe use in phase I clinical trials [Hillman et al., 2007].
It has also been suggested that hypocariogenic strains exhibiting only defects in acid production are unlikely to compete successfully with wild-type strains for initial plaque locations, and on this principle, an S. mutans strain that was deficient in the gcrR gene was genetically engineered for bacterial replacement therapy [Sun et al., 2009; Pan et al., 2013]. The gcrR gene functions as a negative transcriptional regulator of the gbpC gene, which encodes the glucan-binding lectin, an adhesin that is ubiquitous on S. mutans surfaces and plays an important role in initial bacterial aggregation and adhesion to tooth surfaces. The MS-gcr R-def mutant bacteria showed reduced acid production, out-competed wild S. mutans strains with its strong early colonization ability, and lowered caries incidence in vivo [Pan et al., 2013].
Modulation of oral plaque biofilms with genetically engineered “designer” bacteria has great potential through fostering a healthy oral environment, which prevents the dominance of cariogenic bacteria. A single treatment regimen could lead to persistent colonization by the effector strain affording lifelong protection, with minimal need for patient compliance. Whilst there have been encouraging results with genetically modified strains, the concept of replacement therapy needs to be tested for effectiveness in highly cariogenic environments. Even if successful, the widespread acceptance of genetically engineered “designer” bacteria may prove to be difficult for emotional, ethical, and legal reasons.
In addition to the strategies discussed above, several alternative approaches for modifying plaque biofilm ecology are under investigation including using compounds that specifically affect bacterial virulence proteins [Horst et al., 2012], calcium phosphate-osteopontin particles that can inhibit biofilm formation and reduce the fall in pH without affecting bacterial viability [Schlafer et al., 2017], nanoparticles [Allaker and Douglas, 2015], graphene oxide [He et al., 2015], and ceramic water [Nomura et al., 2017].
There is no doubt that fluoride will continue to be the mainstay of any caries prevention protocol as it still remains the most effective and economical protective agent against dental caries. However, fluoride alone may not offer complete protection against the disease, and it is generally recognized that the effectiveness of fluoride could be enhanced when combined with additional cariostatic agents [NIH, 2001]. Moreover, current paradigms emphasize the importance of maintaining a healthy and stable oral plaque biofilm for long-term disease control. One way to do this is to limit or exclude refined sugars from the diet; however, within the constraints of present-day consumer culture, behavioural dietary changes are difficult to achieve and sustain. Adopting ecological preventive measures can help in correcting the disturbed plaque ecology and drive the advent and persistence of a symbiotic oral microbiome. These could be valuable tools in achieving long-term dental caries control, allowing the clinician to shift to a biological model for the management of the disease.
It is imperative that the effectiveness of ecological preventive approaches be evaluated for success in individuals who consume a conventional diet containing a fairly high level of sugars before any clinical recommendations are made [Beighton, 2009]. Furthermore, rather than surrogate end points like lower MS levels or reduced acid production, the critically important outcome for all new caries-preventive measures will be whether they can ensure a significant reduction in individual caries experience.
The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
N.P. conceived and drafted the manuscript with support and input from B.S. L.J.W. critically reviewed and revised the manuscript. N.P., B.S., and L.J.W. approved the final manuscript as submitted.