Alteration of oral microbial biofilms by sweeteners

Abstract

There is a growing interest in using sweeteners for taste improvement in the food and drink industry. Sweeteners were found to regulate the formation or dispersal of structural components of microbial biofilms. Dietary sugars may enhance biofilm formation and facilitate the development of antimicrobial resistance, which has become a major health issue worldwide. In contrast, bulk and non-nutritive sweeteners are also beneficial for managing microbial infections. This review discusses the clinical significance of oral biofilms formed upon the administration of nutritive and non-nutritive sweeteners. The underlying mechanism of action of sweeteners in the regulation of mono- or poly-microbial biofilm formation and destruction is comprehensively discussed. Bulk and non-nutritive sweeteners have also been used in conjunction with antimicrobial substances to reduce microbial biofilm formation. Formulations with bulk and non-nutritive sweeteners have been demonstrated to be particularly efficient in this regard. Finally, future perspectives with respect to advancing our understanding of mechanisms underlying biofilm regulation activities of sweeteners are presented as well. Several alternative strategies for the application of bulk sweeteners and non-nutritive sweeteners have been employed to control the biofilm-forming microbial pathogens. Gaining insight into the underlying mechanisms responsible for enhancing or inhibiting biofilm formation and virulence properties by both mono- and poly-microbial species in the presence of the sweetener is crucial for developing a therapeutic agent to manage microbial infections.

Highlights

• •Sweeteners regulate the formation or dispersal of microbial biofilms.
• •Dietary sugars can enhance oral biofilm formation.
• •Bulk and non-nutritive sweeteners can inhibit oral biofilm formation.
• •Sweeteners are used in combination with antimicrobials to reduce biofilm formation.
• •Applications of formulations of sweeteners are used to target biofilm formation.

1. Introduction

A carbohydrate-rich diet facilitates the colonization of microbial species in the gut and thereby protects gut microbiota from the host immune system and antimicrobials [1]. Nutritive and non-nutritive sweeteners were shown to affect the gut microbial community positively or negatively [[2], [3], [4]]. The ingestion of dietary sugars, such as glucose, sucrose, lactose, fructose, and maltose, which are metabolized by microbes into organic acids, may drive microbial communities to shift toward more aciduric and acidogenic profiles [5]. Such microbial communities predominantly inhabit the oral cavity, where they use sugars to form biofilms on tooth surfaces for survival [6]. The shift toward aciduric and acidogenic bacterial species lowers the pH level within the biofilm and ultimately causes demineralization of the hard tissue of the tooth and the development of dental caries [7].

Microbes have the ability to self-secrete exopolysaccharides with the assistance of intracellular enzymes that are self-produced by the eps gene cluster [8,9]. Oral bacteria such as Streptococcus mutans are capable of using sugars as substrates for metabolic activities that create extracellular polymeric substances (EPS) in biofilms that have a high level of physical stability [10]. In this case, the synthesis of EPS takes place as a result of the activity of secreted enzymes, such as glucosyltransferase (Gtf), which act on dietary sugars (e.g., sucrose) to generate extracellular polysaccharides (e.g., α-glucan), which in turn aid the attachment of bacterial cells to the surface of the tooth [11]. Gtf also strongly binds N- and O-linked mannans on Candida albicans cell surface [12]. Thus, the Gtf-assisted synthesis of bacterial EPS as well as the high affinity of Gtf for the mannan residues on C. albicans leads to a synergistic interaction as well as the formation of a mixed bacterial-fungal biofilm in the oral cavity [13]. The synergistic interaction between bacterial and fungal pathogens has resulted in enhanced poly-microbial biofilm formation and a tolerance mechanism against antibiotics or antimicrobials [[16], [17], [18]]. As a result, new treatment approaches are required to combat poly-microbial biofilms of bacterial and fungal pathogens [14,15].

The biofilm matrix also includes polysaccharides that facilitate biofilm formation and increase the expression of related genes [19]. EPS formation in the biofilm matrix in the presence of sweeteners further accelerates microbial colonization, and thus functions as a critical virulence factor and facilitates protection from the host immune system and host-derived antimicrobial peptides [11]. Biofilms also reduce the effectiveness of infectious disease treatment since they act as physical barriers between microbes and antimicrobial drugs [20]. To this purpose, it has been common practice to use bulk (sugar alcohols/polyols) and non-nutritive sweeteners with minimal calorie content to prevent the development of oral biofilms [21]. Bulk sweeteners, also known as nutritive sweeteners, are extensively employed in the food, medical, and pharmaceutical sectors because they have fewer calories and lower glycemic responses than sugar [22]. Aside from inhibiting biofilm formation, non-nutritive sweeteners have been frequently employed for a variety of reasons, including increased sweetening power relative to sucrose, inexpensive, and accessibility [23].

Despite these advantages, the intake of non-nutritive sweeteners was also shown to have significant adverse effects on health [24]. Although non-nutritive sweeteners cannot be digested to generate energy, they can be potentially toxic to cells following cellular absorption [25]. Nevertheless, non-nutritive sweeteners have also been shown recently to be beneficial in managing microbial infections [[26], [27], [28], [29], [30]]. Bulk sweeteners also exhibit antibacterial efficacy against a variety of drug-resistant pathogens [[31], [32], [33]]. These sweeteners have also been used in conjunction with antimicrobial substances to inhibit biofilm formation [[34], [35], [36]]. Formulations with bulk and non-nutritive sweeteners have been described to efficiently reduce biofilm-forming microbial pathogens [[37], [38], [39]].

The clinical significance of oral biofilms that are developed upon the use of nutritive and non-nutritive sweeteners is thoroughly examined here. The regulatory effects of sweeteners on microbial biofilms and the underlying mechanisms are discussed in detail. In addition, strategies involving the application of mixed formulations of nutritive and non-nutritive sweeteners are presented. Understanding the mechanisms involved in sweetener-regulated promotion or inhibition of mono- and poly-microbial biofilm formation will facilitate efforts in the discovery of therapeutics to control microbial infections.

2. Sweeteners

Sweeteners augment the taste of sweetness and are therefore used in a variety of foods and beverages. Sweeteners can be classified as nutritive or non-nutritive depending on whether they provide energy (Fig. 1) [40]. Nutritive sweeteners comprise dietary sugars such as mono- and di-saccharides as well as bulk sweeteners known as sugar alcohols/polyols [22]. Non-nutritive sweeteners are also known as ‘artificial sweeteners’, and are produced via chemical synthesis [21].

Fig. 1.

Chemical structures of nutritive and non-nutritive sweeteners. (A) The blue color chemical structures are dietary sugars, (B) the red color chemical structures are bulk sweeteners, and (C) the green color chemical structures are non-nutritive sweeteners. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Glucose, fructose, and galactose are the major natural monosaccharide types found in plants [41]. These monosaccharides are combined to form disaccharides such as lactose (glucose and galactose), sucrose (fructose and glucose), and maltose (glucose and glucose) [42]. Sucrose is widely and readily available in various plant species such as sugarcane (Saccharum spp.) and sugar beets (Beta vulgaris) [43]. Sucrose is widely used in dishes; however, excessive intake of sucrose may cause numerous health issues such as obesity, diabetes, and hypertension [44]. In particular, dietary sucrose is directly related to dental caries since EPS synthesis by dental bacteria requires sucrose [45]. Lactose has a sweetness of approximately 20–40 % that of sucrose [46]. Lactose also causes dental caries; however, it is less carcinogenic than sucrose since it induces a lower level of reduction in pH level in the oral environment compared to sucrose [47,48]. Caries formation due to maltose is also lower than that due to sucrose [49]. Hence, despite being a major energy source for human metabolism, sugar can also cause various diseases and metabolic abnormalities, including dental caries [50].

Bulk sweeteners (sugar alcohols/polyols) include erythritol, mannitol, xylitol, lactitol, and maltitol [51]. These sweeteners are either produced naturally or synthesized from monosaccharides or disaccharides [50]. Owing to the negative effects of excessive sugar consumption on health, bulk sweeteners play an important role as sugar substitutes in the food industry [52]. Compared to sugars, bulk sweeteners are poorly absorbed by the body, resulting in lower levels of calorie intake and glycemic responses [53]. In addition, they are also non-cariogenic since oral bacteria are unable to utilize them [54]. Among these sweeteners, xylitol is a particularly good sugar substitute because it promotes salivation and reduces plaque formation [55].

Non-nutritive sweeteners include aspartame, acesulfame-K, sucralose, saccharin, and steviol glycosides [24]. They have a sweet taste when consumed in very small amounts with little or no calories [23]. Unlike fermentable carbohydrates, non-nutritive sweeteners cannot be fermented by oral microorganisms, and are therefore considered non-cariogenic [46]. Because of these properties, non-nutritive sweeteners are increasingly being employed in a variety of food and hygiene products, as well as drug formulations [24]. However, evidence also continues to emerge regarding the effects of non-nutritive sweeteners on carcinogenesis and metabolism, requiring critical evaluation of their use [25].

3. Clinical significance of oral biofilm

Oral microbes have a strong tendency to adhere onto tooth surfaces [56]. Oral microorganisms include bacteria that are beneficial to oral health as well as harmful species that cause various oral diseases [57]. Oral microbial communities adhere onto the surface of dental pellicles through adhesion-receptor interactions mediated by hydrophobic or electrostatic forces to initiate colonization and biofilm formation [58]. Dental caries is a biofilm-induced disease that causes damage to tooth enamel [59]. Uptake of sugars results in an acidic environment (pH 4.5–5.5) in the oral environment, and thus promotes the growth of cariogenic bacteria (S. mutans, Actinomyces, and Lactobacillus) that form oral biofilms, eventually leading to the development of oral diseases [5]. EPS in oral biofilms is mainly composed of S. mutans-derived glucans, in addition to soluble fructans and glucans produced by other oral microorganisms (Actinomyces, S. gordonii, and S. salivarius) [19]. S. mutans promotes carious biofilm formation by metabolizing various carbohydrates into organic acids on tooth surfaces [6]. Sucrose is a fermentable disaccharide that serves as a substrate for EPS-synthesizing bacterial enzymes [60]. S. mutans uses sugars, including sucrose, starch, glucose, and fructose as carbon sources to produce EPS and acids [10]. Thus, the pathogenicity of S. mutans in the oral microbiome is based on its ability to utilize extracellular insoluble glucans for EPS formation [61]. S. mutans secretes Gtf to degrade sucrose and produce extracellular glucan [62]. Gtf induces the production of glucan-rich exopolysaccharides to form a scaffold that provides protection against external stress and antimicrobial agents [11]. GtfB is responsible for the synthesis of insoluble glucans with primarily α-1,3 crosslinks, whereas GtfC is responsible for the synthesis of both insoluble and soluble glucans that are abundant in α-1,6 linkages [63,64]. GtfB and GtfC synthesize glucans that enhance the adhesion of bacterial cells onto the tooth surface, thereby improving the structural stability of the biofilm matrix [61]. GtfD produces soluble glucans that are easily metabolized and serve as primers for GtfB. In addition, S. mutans synthesizes fructans by secreting fructosyltransferase (Ftf), which uses sucrose as a substrate [65]. Fructans produced by Ftf are stored to be used as nutrients later on and also increase the virulence of oral biofilms by facilitating bacterial adhesion and colonization [66]. Glucan, produced in the oral environment, is bound by S. mutans cells via the glucan-binding protein (Gbp) [67]. Gbps, including GbpA, GbpB, GbpC, and GbpD, are considered caries-inducing factors due to their glucan-binding properties [68]. Moreover, S. mutans also regulates the expression of several genes that sense and adapt to external stressors through a two-component system [69]. In particular, the quorum sensing system encoded by comCDE is involved in biofilm formation and formation of the protective extracellular matrix in S. mutans [69]. Although different microorganisms are present in oral biofilms, most of them do not contribute to glucan synthesis until they are coated by Gtf produced by S. mutans [70]. Instead, GtfB binds to other oral microorganisms (A. viscosus, L. casei, and C. albicans) and non-Gtf-producing microorganisms, converting them into extracellular glucan producers [71,72]. Accordingly, S. mutans contributes to poly-microbial biofilm growth by building EPS together with other oral microorganisms [70]. In particular, C. albicans forms symbiotic relationships with oral bacteria in dental biofilms [73]. S. gordonii increases hyphae and biofilm formation of C. albicans by interacting with C. albicans [74]. In addition, C. albicans provides an adhesion site for S. mutans, which enhances the formation of poly-microbial biofilms [75].

The structural and genetic profile of S. mutans biofilms is affected by sugar availability as well [76]. Changes in the oral environment caused by sugar exposure can disturb the microbial balance within the biofilm, and promote the growth of pathogens such as S. mutans, Actinomyces spp., S. salivarius, and S. gordonii [77]. In contrast, bulk and non-nutritive sweeteners are mainly non-fermentable and, therefore, cannot be metabolized to acids by oral microorganisms and are therefore considered non-cariogenic [21,46,55].

4. Role of sweeteners in oral biofilm modulation

4.1. Enhancement of oral biofilm formation by dietary sugars

The cariogenic properties of S. mutans biofilms are mainly regulated by genes related to the extracellular polysaccharide, acid production, microbial adhesion, acid tolerance, and other biofilm-related genes (Fig. 2) [10,76,78]. Therefore, identification of the mechanisms used by S. mutans to adhere to the tooth surface may facilitate the development of new approaches for the treatment of dental caries [79]. Nutritive sweeteners, including sucrose, improve oral microbial biofilm properties through mechanisms shown in Table 1.

Fig. 2.

Enhancement of oral biofilm properties by sucrose. In representative oral bacteria Streptococcus mutans, dietary sugars upregulate the expression of extracellular polysaccharide synthesis (gtfB, gtfC, gtfD, and ftf), acid production (ldh), microbial adhesion (gbpB), acid tolerance (comD, comE, and aptD), and other biofilm-related (spP and brpA) genes. Additionally, S. mutans GtfB binds to other oral microorganisms, transforming them into extracellular glucan producers. In an oral environment rich in dietary sugar, S. mutans GtfB binds to N- and O-linked mannans on the surface of Candida albicans, inducing the conversion of sucrose to α-glucan in the host. Then, α-glucan provides a binding site for S. mutans, leading to the formation of C. albicans-S. mutans mixed biofilms, which causes severe dental caries. Information obtained from the literature [[11], [12], [13],70,71]. EPS, extracellular polymeric substances; Ftf, fructosyltransferase; Gbp, glucan-binding protein; Gtf, glucosyltransferase.

Table 1.

Enhancement of oral biofilm properties by dietary sugars.

Product	Studied microorganism	Testing method	Active concentration	Surface material	Mechanism	References
Sucrose	Streptococcus mutans	In vitro	10, 40 sucrose g/L in TY media (1.4 % tryptone, 0.8 % yeast extract)	Polystyrene multi dishes	Increased expression of genes that positively regulate biofilm formation (gtfB, gtfC, comDE, ftf, gpbB, and spP)	[78]
Sucrose	S. mutans	In vitro	1 % sucrose in tryptone yeast-extract broth containing 1 % starch	Saliva-coated hydroxyapatite (sHA) discs	Increased expression of genes involved in extracellular polysaccharide matrix formation (gtfB)	[80]
Sucrose	S. mutans	In situ	5 % sucrose in Schaedler broth	Methacrylate-based mounting material embedded with intact human third molars	Increased expression of gtfB, gtfD, and ftf genes	[76]
Sucrose	S. mutans	In vitro	0.45–2.40 % (w/v)	sHA discs	Increased the accumulation and virulence of biofilms	[81]
Sucrose + raffinose	S. mutans	In vitro	0.002 % (w/v) sucrose + 0.25 % raffinose (w/v)	sHA discs	Increased cell surface hydrophobicity by the reaction of Gtf-I (sucrose) Contributed to the aggregation of extracellular DNA in the biofilm (raffinose)	[82]
Sucrose	Candida albicnas + C. tropicalis	In vitro	5 %	Microtiter plates	Promoted the production of a floccular and fibrillar layer, which mediated adhesion and subsequent biofilm formation	[83]
Sucrose	S. mutans	In vitro	1–5 %	sHA discs	Upregulated expressions of genes related to exopolysaccharide formation (gtfB, gtfC, and gtfD), glycolysis (ldh), and acid tolerance (atpD)	[10]
Sucrose	Actinomyces israelii S. sanguinis S. mitis Fusobacterium periodonticum Tannerella forsythia Prevotella melaninogenica Eubacterium saburreum S. mutans	In situ	–	Titanium surfaces	–	[84]
Sucrose	S. mutans	In vitro	37.5, 75.0, 375, 750 mM	Titanium surfaces	–	[85]
Lactose	S. mutans	In vitro	2–5 % (w/v)	Polystyrene plates	Increased expression of biofilm-related genes (gtfB, gtfC, gtfD, ftf, brpA, and SMU.1039)	[86]

In an early study examining the correlation between the use of sweeteners and biofilm formation, sucrose (10 and 40 g/L) was found to enhance the properties of S. mutans biofilms formed in TY growth medium containing 1.4 % tryptone and 0.8 % yeast extract [78]. The expression levels of gtfB, gtfC, comDE, ftf, gpbB, and spaP, which promote biofilm formation, were found to increase in the presence of sucrose. In contrast, the expressions of these genes were suppressed when sucrose was introduced into the BHI medium. These results were attributed to the fact that the glucose in the BHI medium neutralized the effect of sucrose since microorganisms prefer simple sugars as their main carbon source. In a study investigating the effect of a combination of starch and sucrose on biofilms, a mixture including 1 % starch and 1 % sucrose was found to affect the exopolysaccharide composition and the expression of genes involved in exopolysaccharide formation in S. mutans biofilms [80]. S. mutans formed a tightly attached biofilm composed mostly of water-insoluble polysaccharides derived from a combination of starch and sucrose. Moreover, biofilms formed from the combination of starch and sucrose included higher levels of gtfB than those grown with sucrose alone or a combination of sucrose and glucose. In another study, the presence of 5 % sucrose in the growth medium was found to result in a higher percentage of vital cells in S. mutans biofilms compared to xylitol [76]. In addition, sucrose addition led to increased expression of genes associated with biofilm formation (gtfB, gtfD, and ftf). In another study that assessed the impact of sucrose on S. mutans biofilms, the effects of sucrose on bacterial adhesion, biofilm composition, and acidogenicity of S. mutans followed a second-order polynomial curve with sucrose concentration dependency [81]. The adhesion and biofilm development of S. mutans increased and subsequently decreased as sucrose concentration increased, with a turning concentration range of 0.45–2.40 %. Raffinose, together with sucrose, induced biofilm formation at concentrations lower than that required to induce S. mutans biofilm formation [82]. Sucrose increased bacterial cell-surface hydrophobicity and raffinose-induced fructan synthesis via Ftf, which enhances extracellular DNA-dependent cell aggregation. In mono- and co-culture studies of C. albicans and C. tropicalis, the application of 5 % sucrose enhanced the growth, adhesion, and biofilm formation of Candida spp. [83]. These findings were attributed to sucrose promoting the formation of aggregates and fibrillar layers and the subsequent biofilm formation by Candida spp.. In a study imitating human meal patterns, the application of 1–5 % sucrose increased acid production and accumulation in S. mutans biofilms [10]. However, biofilm development and acid production were found to decrease with increasing sucrose concentration at concentrations beyond 5 %, suggesting that the effect of sucrose on S. mutans biofilm formation follows a second-order polynomial curve. In contrast, EPS formation, acid production, and acid tolerance-related gene expression were upregulated with increasing sucrose concentrations. Hence, high sucrose concentrations could stimulate the expression of related genes to compensate for the EPS reduction. In an in situ study of humans wearing palatal devices containing titanium specimens, daily sucrose exposure was found to increase biofilm biomass and negatively affect the biochemical and microbiological composition of the biofilm formed [84]. In addition, the adhesion of S. mutans biofilms on titanium substrates was found to be induced by sucrose levels ranging from 0–750 mM [85]. In particular, S. mutans biofilms formed in the presence of 75 mM sucrose were found to show the maximum adhesion and mounds. In addition to these studies, 2–5 % lactose was also found to significantly enhance S. mutans biofilm formation [86]. Lactose was found to lead to increased expression of biofilm formation-related genes, such as gtfB, gtfC, gtfD, ftf, brpA, and SMU.1039. Furthermore, the biomass of S. mutans biofilms was also increased to a level similar to that obtained upon application of sucrose, yet with a different polysaccharide composition.

4.2. Inhibition of oral biofilm formation by bulk and non-nutritive sweeteners

Sugar substitutes are often used to prevent caries. However, the mechanisms underlying the inhibition of oral biofilm formation and plaque reduction due to the use of sugar substitutes have not been fully elucidated. Bulk and non-nutritive sweeteners have been shown to reduce oral biofilm formation and activity by suppressing the expression of biofilm- and virulence-related genes (Fig. 3) [[26], [27], [28],87]. In addition, unlike sucrose, bulk and non-nutritive sweeteners cannot be utilized by S. mutans as a substrate for biofilm matrix synthesis, resulting in reduced biofilm formation [32]. The details of mechanisms involved in biofilm inhibition by sweeteners, especially bulk and non-nutritive sweeteners, are listed in Table 2.

Fig. 3.

Regulation of oral biofilms by sweeteners. (A) Enhancement of oral biofilm formation by sucrose. The Gtf of Streptococcus mutans degrades sucrose to produce glucan, which contributes to the accumulation of polysaccharides. Additionally, organic acids produced during fermentation metabolism contribute to reducing the pH of the oral environment, which leads to demineralization and the dental caries process. (B) Inhibition of oral biofilm formation by bulk and non-nutritive sweeteners. Non-fermentable sugars, including bulk sweeteners and non-nutritive sweeteners, cannot be utilized by S. mutans as a substrate for biofilm matrix synthesis. In addition, bulk sweeteners and non-nutritive sweeteners inhibit biofilm- and virulence-related gene expression, leading to a non-cariogenic biofilm with low levels of polysaccharide. Information obtained from the literature [6,[26], [27], [28],32,87]. EPS, extracellular polymeric substances; Gtf, glucosyltransferase.

Table 2.

Inhibition of oral biofilm formation by bulk and non-nutritive sweeteners.

Product	Studied microorganism	Testing method	Active concentration	Surface material	Mechanism	References
Xylitol	Streptococcus mutans S. sanguinis Actinomyces naeslundii	In situ	5 %	Hydroxyapatite discs	–	[31]
d-tagatose	S. mutans	In vitro	1, 4 %	Plastic discs	Interfered with glucosyltransferase (Gtf) activity	[26]
Sorbitol Xylitol	Candida albicans + S. mutans	In vitro	10 %	96-well plates	–	[32]
Erythritol Xylitol	S. mutans	In vitro	1, 2, 5 %	16-well electronic microtiter plates	Modified the expression levels of gbpB, gtfB, gtfC, and gtfD genes that were important in polysaccharide-mediated adherence of S. mutans	[33]
Rubusoside	S. mutans	In vitro	1 %	24-well microtiter plates	Downregulated virulence gene expression (gtfB, gtfC, gbpB, ldh, and comD)	[27]
Raffinose	Staphylococcus aureus + Pseudomonas aeruginosa	In vitro	1–1000 μM	Polystyrene microtiter plates	–	[87]
Raffinose	S. mutans	In vitro	1000 μM	Saliva-coated hydroxyapatite discs	Inhibited Gtf-related gene expression	[28]
Acesulfame-K Sucralose	Porphyromonas gingivalis	In vitro	10 % acesulfame-K 7.5 % sucralose	96-well plates	–	[88]
Stevioside	C. albicans + S. mutans	In vitro	1 %	24-well microplate plates	Decreased sucrose metabolism and increased galactose and intracellular polysaccharide metabolism in S. mutans Decreased genes related to glycosylphosphatidylinositol-modified proteins and secreted aspartyl proteinase family in C. albicans	[29]
Acesulfame-K	Acinetobacter baumannii	In vitro Ex vivo	8.85 %	Glass microscope slides Porcine skins	Disabled virulence behaviors such as biofilm formation, motility, and the ability to acquire exogenous antibiotic-resistant genes	[30]

Xylitol at 5 % was found to inhibit early-stage (8 h) biofilms (formed in a medium containing 0.3 % sucrose) of S. mutans, S. sanguinis, and A. naeslundii [31]. This result was attributed to reduced polysaccharide-mediated cell adhesion upon xylitol usage without a reduction in exopolysaccharide production. Both 1 and 4 % d-tagatose were found to inhibit biofilm formation in S. mutans exposed to sucrose at a level much higher than that of xylitol [26]. d-tagatose inhibited Gtf, and thus reduced the production of water-insoluble glucans from sucrose. Hence, the formation of S. mutans biofilms was inhibited by restricting the access to released free d-fructose. In another study, xylitol and sorbitol were found to inhibit mono- and poly-microbial biofilm formation by S. mutans and C. albicans under sucrose-free conditions [32]. In contrast, the inclusion of 1 % sucrose was found to attenuate the inhibitory effects of xylitol and sorbitol on biofilm development. This was attributed to the fact that oral bacteria prefer hexose sugars over sugar alcohols such as xylitol and sorbitol. In a real-time monitoring study of biofilm formation, xylitol and erythritol were found to inhibit S. mutans biofilm formation in media containing 1 % sucrose [33]. Xylitol and erythrol strongly inhibited the initial biofilm formation, but not the biofilm at 10 h. However, the quantity of RNA in the biofilm after 10 h was much lower than that in the control group. This was due to the sugar alcohol starving bacterial cells, resulting in lower RNA levels in the biofilm. Rubusoside is another non-nutritive sweetener that was found to affect cariogenic characteristics and expression of virulence-related genes in S. mutans biofilms [27]. When S. mutans was exposed to each medium supplemented with 1 % rubusoside, 1 % xylitol, and 1 % sucrose, the presence of rubusoside was found to result in a lower level of acid production compared to the presence of sucrose and xylitol and reduce the level of biofilm accumulation and viability. Rubusoside also inhibited the expression of virulence-related genes such as atpF, spaP, gbpB, gtfB, gtfC, gtfD, ftf, ldh, comD, and vicR. Raffinose inhibited the biofilm formation of non-oral pathogens such as Escherichia coli, Staphylococcus aureus, Rhodococcus qingshengi, Clostridium tropical, and Bacillus amyloliquefaciens [87]. In particular, raffinose at 1 μM or higher was also shown to inhibit the formation of mixed biofilms of S. aureus and P. aeruginosa. In particular, raffinose was found to inhibit S. mutans biofilm (formed in a medium supplemented with 10 μM sucrose) formation and Gtf-related gene expression [28]. In addition, 10 % acesulfame-K and 7.5 % sucralose inhibited the biofilm formation of Porphyromonas gingivalis, an anaerobic periodontal pathogen, and showed bactericidal activity against bacteria within the biofilm [88]. Stevioside also inhibited the formation of mixed biofilms of C. albicans and S. mutans and acid production [29]. Stevioside facilitated the metabolic utilization of galactose and intracellular polysaccharides while reducing that of sucrose. In addition, the presence of stevioside was also found to inhibit the transformation of C. albicans, which reduces pathogenicity. The artificial sweetener acesulfame-K reduced the expression of genes encoding Bap (biofilm-associated protein) as well as genes encoding Csu pili (related to twitching motility) in Acinetobacter baumannii [30]. In particular, acesulfame-K decreased the twitching motility of A. baumannii in a dose-dependent manner. These results showed that acesulfame-K could be employed as a therapeutic agent by reducing A. baumannii biofilm development and twitching motility.

4.3. Combined use of sweeteners to target microbial biofilm formation

Due to the biofilm-forming capabilities of some bacteria, such as Enterococcus faecium, S. aureus, Klebsiella pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter spp., it may be challenging to treat infections caused by these ESKAPE bacterial strains [89]. These pathogenic biofilms are enclosed by EPS, which protects cells from antimicrobial agents [20]. Limited penetration of drugs due to the presence of EPS within the biofilm contributes to high levels of tolerance to conventional antibiotics [90]. To address this issue, a treatment strategy has been developed in which biofilm-dispersing and antimicrobial agents are co-administered [91]. The biofilm-dispersing agent effectively dispersed the biofilm and thus facilitated the penetration of antimicrobial agents into bacterial cells [92]. Moreover, this combination therapy not only produces a greater antibacterial effect at a lower dose but also reduces the possibility of the emergence of multidrug-resistant bacteria [7]. The combination of sweeteners and antimicrobial agents this way was also effective in controlling several other pathogenic biofilms as well, including oral biofilms (Table 3).

Table 3.

Combinatorial applications of sweeteners with antimicrobials to target pathogenic biofilm.

Sweeteners	Other product	Studied microorganism	Mechanism	References
Xylitol	Lactoferrin	Pseudomonas aeruginosa	Inhibited the ability of biofilms to respond to environmental iron restriction	[34]
Xylitol	Ursolic acid	Streptococcus mutans S. sobrinus	Exhibited antibiofilm activity while preventing tooth demineralization by raising the pH above the threshold of 5.5	[7]
Ribose	Xylitol	S. mutans S. sobrinus	Inhibited the expression of dextran-dependent aggregation-responsible genes	[35]
Xylitol	Bacteriophages	Klebsiella pneumoniae + P. aeruginosa	Promoted bacteriophage host penetration by xylitol	[93]
Erythritol	Betaine	S. mutans	Reduced adhesive forces of the biofilms due to an increase in solubility of exopolysaccharides	[36]
Xylitol	Isothiazolone	P. aeruginosa S. aureus	–	[94]
Erythritol	Xylitol	S. mutans S. sobrinus	–	[95]

The combination of xylitol and lactoferrin exhibited a cooperative inhibitory effect on P. aeruginosa biofilms [34]. Here, lactoferrin treatment destabilized P. aeruginosa cell membrane through iron chelation. The combination with xylitol produced a synergistic effect, as membrane destabilization by lactoferrin enhanced the penetration of xylitol into bacterial cells. The combination of xylitol and ursolic acid was also found to significantly inhibit biofilm formation by S. mutans and S. sobrinus via synergistic interactions [7]. This combination also prevented the pH from falling below 5.5, effectively preventing tooth demineralization. The combination treatment with ribose and xylitol inhibited S. mutans and S. sobrinus biofilms more effectively than treatment with ribose or xylitol treatment alone [35]. Moreover, ribose-xylitol combination treatment also significantly downregulated biofilm formation and expression of dextran-dependent aggregation-related genes (gpbC and dblB) compared to treatment with ribose or xylitol alone. The combination of xylitol and bacteriophages inhibited biofilm formation as well through the release of DNA and proteins from a mixed biofilm of P. aeruginosa and Klebsiella pneumoniae [93]. This finding was attributed to the bacteriophages facilitating the penetration of xylitol by destroying the cell layers. In combination with the zwitterionic molecule betaine and sugar alcohol erythritol, the betaine-erythritol complex was found to induce spontaneous detachment of S. mutans biofilm from the surface [36]. The anionic site of betaine binding erythritol, and the remaining cationic site allowed the complex to be transferred to the negatively charged exopolysaccharide of S. mutans biofilm. In addition, the hydroxyl group of the sugar alcohol interfered with hydrogen bonding between the hydroxyl groups of the exopolysaccharide, which was promoted by the formation of a complex with the zwitterion. Combined treatment with xylitol and isothiazolones showed a synergistic inhibitory effect on early biofilm formation by S. aureus and P. aeruginosa [94]. Furthermore, a combination of xylitol and erythritol showed cooperative inhibitory effects against cariogenic biofilms of S. mutans, S. sobrinus, and Scardovia wiggsiae [95]. The combination with a high xylitol ratio effectively inhibited the growth of S. sobrinus and S. wiggsiae, whereas the combination with a high erythritol ratio effectively inhibited the growth of S. mutans.

4.4. Applications of sweeteners as formulation forms to target microbial biofilms

New types of antibacterial substances in forms such as conjugates and nanoparticles have been developed to combat biofilm infections. Nanoparticles can easily interact with microorganisms owing to their small sizes and high surface-area-to-volume ratios [96,97]. In addition, they can also serve as carriers of antibacterial agents for drug administration [39,98]. Nanoparticles loaded with sweeteners have been developed to improve biofilm penetration of xylitol [37]. These sweetener formulations were found to effectively control mono- or poly-microbial biofilms, the mechanisms of which are described in Table 4.

Table 4.

Applications of sweeteners as formulation forms to target pathogenic biofilm.

Formulation type	Studied microorganism	Mechanism	References
Zinc-erythritol complex	Streptococcus mutans	Removed mature biofilms due to metal ions and the coordination properties of sugar alcohols	[38]
PLGA/xylitol nanoparticles	Pseudomonas aeruginosa + Staphylococcus aureus	Penetrated the biofilm matrix as compared to the xylitol solution and hence facilitated the release of the drug inside the biofilm matrix	[37]
Aspartame-gold nanoparticles	Carbapenem-resistant Enterobacteriaceae	Inhibited biofilm formation from the decoration of aspartame	[39]

A coordination compound composed of zinc chloride and erythritol effectively eliminated mature S. mutans biofilms [38]. The antibiofilm activity of the zinc-erythritol complex was attributed to the facilitation of zinc penetration into mature biofilms by erythritol. Polylactic-co-glycolic acid (PLGA) nanoparticles incorporating xylitol have also been shown to exert antibiofilm activities against the poly-microbial biofilms of S. aureus and P. aeruginosa [37]. PLGA nanoparticles containing xylitol showed high levels of biofilm activity by enhancing the penetration of S. aureus and P. aeruginosa biofilms into EPS. In a study synthesizing non-nutritive decorated gold nanoparticles, aspartame-decorated gold nanoparticles showed stronger antibacterial and antibiofilm effects against Carbapenem-resistant Enterobacteriaceae than gold nanoparticles decorated with saccharin, sucralose, and acesulfame [39]. Gold nanoparticles decorated with aspartame showed an antibacterial effect by accumulating reactive oxygen species in bacteria and improving internal membrane permeability. In addition, the inhibition of biofilm formation by aspartame was also observed, indicating that the antibiofilm activity of aspartame-gold nanoparticles may be due to the decoration of aspartame.

5. Conclusion and future perspectives

The use of nutritive as well as non-nutritive sweeteners has increased in food, beverage, and pharmaceutical industries in recent years to enhance palatability. On the other hand, with technological advances, evidence suggests that excessive dietary sugar consumption, in conjunction with unhealthy nutrition and physical activity habits, can have detrimental health consequences [99,100]. In particular, dietary sugars promote the growth of aciduric and acidogenic bacteria, which form acids from sugars and thus lead to tooth demineralization and dental cavities when used regularly. Additionally, dietary sugars facilitate the synthesis of EPS by bacteria, thereby contributing to biofilm antimicrobial tolerance. Therefore, bulk and non-nutritive sweeteners have gained popularity as alternatives to dietary sugars in this regard. Furthermore, numerous studies have also indicated that bulk and non-nutritive sweeteners show antibacterial effects that reduce virulence and inhibit biofilm formation. With a growing number of studies on antibiofilm activities of bulk and non-nutritive sweeteners, a variety of alternative formulations for use in industry have also been developed. Solutions include combining bulk sweeteners or non-nutritive sweeteners with antimicrobials as well as formulations, all of which were found to boost antibiofilm activities against microbial pathogens. Future perspectives that will assist in improving our understanding of the mechanism of action of sweeteners on microbial biofilms are summarized as follows.

• •To determine the mechanism of actions involved, it will be necessary to establish structure-activity relationships for sweetener-mediated regulation of biofilms and virulence-regulating proteins.
• •Encapsulating sweeteners in nanomaterials may allow for targeted applications for the elimination of microbial biofilms.
• •Most of the studies on sweetener-enhanced biofilm development have been conducted in vitro. However, in vivo research is also required to imitate the host environment and elucidate the influence of host factors on biofilm activity.
• •Because the majority of nutritive and non-nutritive sweeteners contain hydroxyl groups, which act as a good reducing and capping agent [99,101,102], nanoparticle production utilizing these sweeteners as a reducing and capping agent is required to broaden their antibiofilm action against oral and non-oral biofilm-forming microbial pathogens.
• •Due to the presence of poly-microbial interactions in the host system, antibiofilm and antivirulence activities must be performed utilizing poly-microbial pathogens in vitro or in vivo for future applications in the host system.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

Not applicable.

Authorship contribution statement

Geum-Jae Jeong: Literature Search, Writing-original Draft & Editing, Fazlurrahman Khan: Conceptualization, Funding, Supervising, Literature Search, Writing-review & Editing, Nazia Tabassum: Literature search, Writing & Editing, and Young-Mog Kim: Supervision, Funding, Writing-review & Editing.

Funding

This research was supported by the Basic Science Research Program through the National Research Foundation (NRF) of Korea, funded by the Ministry of Education (RS-2023-00241461 to F. Khan). This research was also supported by the Basic Science Research Program through the NRF of Korea grant funded by the Ministry of Education (2021R1A6A1A03039211 and 2022R1A2B5B01001998).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Abbreviations

EPS

extracellular polymeric substances

Ftf

fructosyltransferase

Gbp

glucan-binding protein

Gtf

glucosyltransferase

PGLA

polylactic-co-glycolic acid

Data availability

No data was used for the research described in the article.