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Introduction

 Xylitol is a naturally occuring five-carbon sugar alcohol and has been widely used as a sweetener. Xylitol is not fermented by oral microorganisms, such as Streptococcus mutans [1]. However, most of oral streptococci are able to transport xylitol into the cell via the fructose phosphotransferase system [2]. Once in the cell, xylitol is phosphorylated to xylitol-5-phosphate and subsequently expelled from the cell, which requires energy [3, 4]. The energy-consuming cycle is responsible for the inhibition of S. mutans growth observed both in vitro and in vivo by xylitol [3].

 Although the inhibitory mechanisms of xylitol are not fully understood, regular consumption of xylitol has been reported to reduce the incidence of caries [5-7]. The most significant effect of xylitol against S. mutans is to reduce the growth and acid production [8] and to decrease the adherence [9]. Research into caries has not shown any other major changes in oral flora, except occasionally a reduction in other oral streptococci [5, 10].

 Periodontitis is a chronic inflammatory disease induced by infection of major periodontopathogens, such as Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola [11, 12]. We have reported that xylitol inhibited inflammatory responses in macrophages infected with both live P. gingivalis (unpublished data) and P. gingivalis LPS [13]. However, direct inhibitory effects of xylitol against periodontopathogens are not studied well.

 In this study, we hypothesized that xylitol could also affect the growth of bacteria in oral cavity, which could further influence oral flora population. To test our hypothesis, we evaluated the growth of several oral bacteria mostly found in healthy subjects and periodontopathogens when xylitol was added to the bacterial culture medium.

Materials and Methods

Bacterial strains

 The bacterial strains used in this study and their culture conditions are listed in table 1.

Table 1. Bacteria species and their culture conditions

 S. vestibularis, G. morbillorum, A. naeslundii, N. mucosa, N. sicca and V. parvula were selected as normal oral bacteria mostly found in healthy subjects [14, 15]. P. gingivalis, P. intermedia, A. actinomycetemcomitans, F. nucleatum, C. ochracea, C. sputigena, T. denticola and P. denticola were selected as periodontotpathogens. The strains had been obtained from ATCC and KCTC. All the strains were freshly cultured for the experiments.

Media and cultivation of bacteria

 For growth inhibition measurements, each tested strain was cultured in the recommended media as described in Table 1. Brain heart infusion (BHI, Difco, Detroit, MI, USA), Gifu Anaerobic medium (GAM, Nissui Seiyaju, Tokyo, Japan) and new oral spirochete broth (NOS) were prepared for culture medium. Xylitol (Sigma, St. Louis, MO, USA) was added to the basic medium to the appropriate concentrations and sterilized by filtration (Saltorius, Goettingen, Germany). The test medium contained 5%, 10% or 15% (wt/vol) xylitol, while the corresponding control medium was free of xylitol. Each strain was cultured aerobically or aerobically in the appropriate basic medium at 37ŶC. The dilution ratios are also described in Table 1. Each strain was diluted into 96 well plate (Corning, Williamsburg, NY, USA) containing test medium with various xylitol concentrations. The test plates were incubated at 37 °C for 1 to 4 days. The optical density (OD) of each well was measured at a wavelength of 650 nm with an ELISA reader (Molecular device, Sunnyvale, CA, USA) against the standard medium, with the measurements being performed during the bacterial growth. The OD results were calculated as the mean of at least four measurements.

Results

Effect of xylitol on the growth of nonperiodontogenic oral bacteria

 First, the growth inhibitory effect of xylitol on oral bacteria frequently found in healthy subjects was tested. For oral bacteria found in healthy subjects, S. vestibularis, G. morbillorum, A. naeslundii, N. mucosa, N. sicca, and V. parvula were selected. They were grown to late log phase and 1x10⁷ CFU of each species were inoculated into fresh media with various xylitol concentration.

 In the presence of 5% xylitol, marked growth inhibition (e.g. more than 50% inhibition) was observed in S. vestibularis and G. morbillorum. A. anaeslundii growth was greatly inhibited in the presence of 10% xylitol. The growth of N. mucosa, N. sicca and V. parvula was inhibited depending on xylitol concentration. At 15% xylitol, only N. sicca showed mild growth while the others showed almost no bacterial growth (Fig. 1).

Figure 1. Growth of normal oral bacteria in terms of OD counts, in media, 5% xylitol, 10% xylitol, and 15% xylitol. The OD of each tube was measured at a wavelength of 650 nm with a spectrophotometer against the standard medium, with the measurements being performed during the bacterial growth. The OD results were calculated as the means of at least three measurements. Symboles :♦, no xylitol; ■, 5% xylitol; ▲, 10% xylitol; ✖, 15% xylitol.

Effect of xylitol on the growth of periodontopathogens

 Next, the inhibitory effects of xylitol on periodontopathogens were measured. P. gingivalis, P. intermedia, A. actinomycetemcomitans, F. nucleatum subsp. vincentii, F. nucleatum subsp. nucleatum, C. ochracea, C. sputigena, T. denticola and P. denticola were selected as periodontopathogens. In the presence of 5% xylitol, marked growth inhibition was detected only in P. intermedia, while other periodontopathogens tested showed mild growth inhibition (e.g. less than 50% inhibition). However, most of bacteria tested showed marked growth inhibition in 10% xylitol. P. gingivalis, A. actinomycetemcomitans, F. nucleatum subsp. vincentii, and P. denticola showed marked bacterial growth inhibition in 10% xylitol. Bacterial growth was nearly completely inhibited by 10% xylitol for F. nucleatum subsp. nucleatum, C. ochracea, C. sputigena, and T. denticola. At 15% xylitol, all the periodontopathogens tested showed complete bacterial growth inhibition (Fig. 2).

Figure 2. Growth of periodontopathogens in terms of OD counts, in media, 5% xylitol, 10% xylitol, and 15% xylitol. The OD of each tube was measured at a wavelength of 650 nm with a spectrophotometer against the standard medium, with the measurements being performed during the bacterial growth. The OD results were calculated as the means of at least three measurements. Symboles :♦, no xylitol; ■, 5% xylitol; ▲, 10% xylitol; ✖, 15% xylitol.

Comparison of bacterial growth inhibition at late log phase

 To compare the growth inhibitory effect of xylitol at various concentrations, each bacterial growth inhibition was determined at the late log phase (Fig. 3). In the presence of 5 % xylitol, G. morbillorum, S. vestibularis and P. intermedia showed bacteria growth inhibition more than 60%, suggesting high susceptibility to xylitol. In the presence of 10% xylitol, A. naeslundii and all the periodontopathogens tested showed marked bacterial growth inhibition, suggesting moderate susceptibility to xylitol.

Figure 3. (A) Inhibition of the normal oral bacterial growth was determined at the late log phage. (B) Inhibition of the periodontopathogen growth was determined at the late log phage. Bacterial growth percentage was calculated by (OD 650 nm without xylitol- OD 650nm with xylitol) x100 / (OD 650 nm read without xylitol).

Discussion

 While most of studies focus on the inhibitory effect of xylitol on S. mutans which is the major cariogenic bacteria in oral cavity, there are only limited number of reports that include other oral bacteria [8, 16]. Thus, in this study, we examined the inhibitory effect of xylitol on both oral bacteria frequently found in healthy subject and periodontopathogens.

 Our results indicate that xylitol markedly reduced the growth of various oral bacteria in a dose dependent manner and these inhibitory effects were statistically significant throughout the exponential growth phase. Among oral bacteria mostly found in healthy subjects, S. vestibularis and G. morbillorum showed marked growth inhibition (more than 50%) with 5% xylitol during logarithmic phase. A. naeslundii showed marked growth inhibition with 10% xylitol and mild growth inhibition (20~ 50%) with 5% xylitol. N. mucosa, N. sicca, and V. parvula showed resistance to 5% xylitol and mild growth inhibition with 10% xylitol suggesting high resistance to xylitol.

 Among periodontopathogens which frequently found in periodontitis, P. intermedia showed almost complete growth inhibition with 5% xylitol, suggesting high sensitivity to xylitol. With 5% xylitol, the growth inhibition of other periodontopathogens tested was less than 50%. However, with 10% xylitol, marked growth inhibition was observed in all the periodontopathogens tested. With 10% xylitol, more selective growth inhibitory effect on periodontopathogens is expected, which indicates that long-lasting xylitol delivery influences oral microbiota population.

Xylitol is widely used in candy industry. Chewing gum and hard candy are considered ideal vehicles for providing a long-lasting effect in oral cavity. In our preliminary study, xylitol concentration up to 15% was well tolerated by volunteers (unpublished data). The xylitol concentrations of 5, 10, and 15% used in this study can be easily achieved, at least temporarily, in the saliva and on the mucous membranes. 

 The inhibitory mechanisms of xylitol on the growth of oral bacteria are unclear. Comparing the growth curves and bacterial growth inhibition at late log phase suggests the possibility of some common futile xylitol cycle between the bacterial species. The bacterial growth rate or bacterial genus seems to be of minor importance for the xylitol sensitivity.

 During the transition from periodontal healthy state to periodontal disease, a well characterized oral microbial shift is frequently described in periodontitis [17]. Several bacterial complexes associated with periodontitis have been identified. Three bacterial species which are designated as ‘red-complex’ are P. gingivalis, T. forsythus, and T. denticola. They have been well recognized for the strong association with periodontitis [11]. Other bacteria which have been also suggested to be involved in periodontitis include A. actinomytemcomitans, C. ochracea, C. sputigena, P. denticola, P. intermedia, and F. nucleatum [12, 18]. The most effective and most widely used treatment is physical removal of pathogenic dental plaque biofilm by scaling and root planning. However, restoring the microbial shift to healthy microbial state or preventing the pathogenic microbial transition should be better than the treatment after the occurrence of the disease.

 In this study, we have only tested the effect of xylitol against single bacterial species at each experiment. Several non-periodontopathogenic oral bacteria showed relatively higher resistance 10% xylitol suggesting the possibility of selectively restoring healthy oral microbial population.

Acknowledgements

 This work was supported by a 2-Year Research Grant of Pusan National University