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Curr Microbiol (2008) 56:382385 DOI 10.

1007/s00284-007-9076-6

Growth Inhibition of Streptococcus mutans with Low Xylitol Concentrations


derling Tiina C. Ekman Eva M. So Teemu J. Taipale

Revised: 28 August 2007 / Accepted: 17 October 2007 / Published online: 5 January 2008 Springer Science+Business Media, LLC 2008

Abstract No studies on the concentration dependency of the inhibition of Streptococcus mutans with xylitol are available. We studied xylitol-induced growth inhibition of two type strains, S. mutans NCTC 10449 and Ingbritt, and three clinical isolates of S. mutans. The strains were grown in Brain Hearth Infusion Medium in the presence of 0.001% (0.066 mM), 0.005% (0.33 mM), 0.01% (0.66 mM), 0.1% (6.6 mM), and 1% (66 mM) xylitol. Growth was followed by measuring the absorbance at a wavelength of 660 nm. The highest xylitol concentration tested in this study, 1%, showed mean inhibition percentages ranging from 61% to 76% when the growth inhibition of the ve strains was compared to the control without xylitol at logphase. For 0.1% xylitol, the inhibition percentages ranged from 22% to 59%. A concentration dependency was seen in the growth inhibition, with 0.01% xylitol being the lowest xylitol concentration inhibiting all ve strains signicantly (p \ 0.001). The growth inhibition percentages determined for 0.01% xylitol, however, were low, and the inhibition was signicantly weaker as compared to 0.1% and 1% xylitol. Our results suggest that low xylitol concentrations of 0.1% (6.6 mM) could inhibit mutans streptococci in vivo but even lower xylitol concentrations may be inhibitory.

Introduction In clinical studies, xylitol has decreased levels of mutans streptococci [10, 11], and habitual consumption of xylitol has reduced motherchild transmission of mutans streptococci [13, 17]. These effects were mainly attributed to the fact that xylitol selectively inhibits growth of mutans streptococci [for review, see 7]. The rst study to report that xylitol inhibits growth of Streptococcus mutans was published in 1975 [5]. Since then, the mechanism of this inhibition has been studied by several research groups [for review, see 7], and new reports on it have been published recently [8]. The growth inhibition has been studied in various liquid growth media [3, 18], but growth is also inhibited when S. mutans is grown in saliva [14]. Five percent, that is, 33 mM, xylitol has been shown to inhibit both type strains and clinical isolates of S. mutans [18]. Xylitol concentrations ranging from 0.5% to 2% inhibited growth of S. mutans LG-1 [3], while S. sobrinus OMZ 176 appeared to be inhibited by xylitol concentrations ranging from 0.05% to 1%; however, in this study, growth was measured only at 16 h [12]. Growth inhibition has also been demonstrated for some otopathogenic bacteria with 1% and 5% xylitol [6], but no data on growth inhibition of S. mutans with xylitol concentrations below 0.5% have been published. Xylitol is released from various vehicles, resulting in maximal salivary xylitol concentrations ranging from 0.8% to 9% [4, 15, 16]. We have studied the release of xylitol from a novel slow-release pacier into saliva [15]. The xylitol tablet inserted into the pouch of the pacier dissolved within 7.5 min, resulting in salivary xylitol concentrations of 12% with no concentration peak [15]. Recently, the development of products resulting in slow release of xylitol has been proposed [1]. With such

derling (&) T. C. Ekman E. M. So Institute of Dentistry, University of Turku, FI-20520 Turku, Finland e-mail: eva.soderling@utu. T. J. Taipale Korpilahti-Muurame Health Care Center, FI-40950 Muurame, Finland

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vehicles, the salivary xylitol concentrations reached may be much lower. The aim of this study was to determine how even low xylitol concentrations can signicantly inhibit the growth of S. mutans.

Materials and Methods Microorganisms Two type strains of Streptococcus mutans were used in the study: S. mutans NCTC 10449 and S. mutans Ingbritt (kind n, University of Go teborg, Sweden). The gift from A. Carle three clinical isolates of S. mutans195-s-2, 2366-s-1, and 267-s-4were isolated from the salivas of three nonconsumers of xylitol who participated in the motherchild study conducted in Finland during the 1990s [13]. S. mutans colonies were identied on the basis of colony morphology on Mitis Salivarius (MS; Difco, MI) Bacitracin plates [2]. The colonies were picked from the plates and sequentially cultured at 37C on blood (Orion Diagnostica, Espoo, Finland) and Mitis Salivarius plates to produce pure clinical strains. Purity was veried by growing the strains overnight in Brain Heart Infusion Medium (BHI, Difco), followed by plateculturing on both MS and blood agar. The identication of the S. mutans isolates has been described earlier [13].

Cultivation of the Microorganisms The cells were cultured in 5 ml BHI overnight at 37C from stocks kept frozen to produce log-phase cells, and then transferred to fresh BHI in the morning. The growth media contained xylitol (Oriola, Espoo, Finland), with the concentration ranging from 0.001% (0.066 mM) to 1% (66 mM). Xylitol was added to the sterile medium using lter sterilization. The control medium contained no added xylitol. The cells were cultured in a shaking waterbath at 37C. Growth was followed by measuring the absorbance at a wavelength of 660 nm. All experiments were performed as quadruplicates and were repeated at least twice.

products with special emphasis on slow-release vehicles. Our results suggest that there is a concentration dependency in the growth inhibition of S. mutans with xylitol. The highest xylitol concentration tested in this study, 1% (66 mM), signicantly inhibited all ve S. mutans strains when the growth inhibitions were compared to the control at late log-phase (p \ 0.001; Table 1, Fig. 1). The clinical isolates showed growth inhibition of a similar magnitude, ranging from 65% to 76%. The inhibition percentages found for the type strains S. mutans 10449 and Ingbritt were 61% and 66%, respectively. These percentages are lower than those reported for the same type strains earlier; however, in the earlier studies the growth medium was different from ours and the xylitol concentration used was as high as 5% [18]. Also, the growth of S. pneumoniae has earlier shown higher growth inhibition for 5% than for 1% xylitol [6]. On the other hand, S. mutans LG-1 showed similar, approximately 70%, growth inhibition for the three xylitol concentrations, 0.5%, 1%, and 2% [3]. The growth of the three clinical isolates and S. mutans Ingbritt was inhibited by 0.1% (6.6 mM) xylitol, with inhibition percentages ranging from 40% to 59% (Table 1). S. mutans 10449 showed a lower inhibition percentage of 22%, for 0.1% xylitol. When compared to the control, the inhibition at log phase was statistically signicant for all strains tested (p \ 0.001; Fig. 1). The growth inhibition with 0.1% xylitol was signicantly lower than with 1% xylitol (Table 1). Of the lower xylitol concentrations tested, 0.01% (0.66 mM) xylitol signicantly inhibited all ve strains at least at one examination point, at intermediate log-phase, when compared to the control (p \ 0.001; Fig. 1). The inhibition percentages, however, were low, ranging from 6.5% to 23% (Table 1). The growth inhibition for 0.01% xylitol was signicantly lower as compared to 0.1% xylitol (Table 1). Even 0.005% (0.33 mM) xylitol signicantly
Table 1 Inhibition percentages of the ve Streptococcus mutans strains in the presence of 0.01%, 0.1%, and 1% xylitola S. mutans strain Inhibition percentages (mean SD, n = 4) at log-phase 0.01% XYL 0.1% XYL 1% XYL

Statistics The growth was compared at each examination point using independent samples t-test (SPSS 14.0 for Windows). The level of statistical signicance was set at p \ 0.05. Results and Discussion We wanted to study xylitol concentrations found in connection with the consumption of xylitol-containing

1. Type strains NCTC 10449 Ingbritt 2. Clinical isolates 267-s-4 2366-s-1 195-s-2 19.8 1.7 23.1 4.3 18.3 2.1 40.0 1.1 44.3 1.0 59.4 3.9 72.5 0.6 76.4 1.4 65.3 1.8 10.9 0.6 6.5 0.9 21.5 1.2 46.6 0.8 61.1 0.5 65.8 4.1

For all strains, 0.1% XYL vs. 1% XYL: p \ 0.001; for 0.01% XYL vs. 0.1% XYL: p \ 0.001
a

Time of sampling shown by an arrow in Figure 1

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inhibited all strains except S. mutans Ingbritt (Fig. 1), but the lowest xylitol concentration tested, 0.001% (0.066 mM), did not inhibit any of the ve strains tested. According to clinical studies, a daily dose of 57 g of xylitol with a daily consumption frequency of at least three is needed to reduce counts of mutans streptococci [10, 11]. The Belize study [9] suggested that a concentration peak of xylitol in saliva is important for the benecial dental effects of xylitol. Xylitol was dissolved from xylitolcoated, high-concentration chewing gum, resulting in a salivary xylitol peak of 9%, and elevated salivary xylitol concentrations of 34% for up to 5 min [16]. With other xylitol vehicles, such as sucking tablets and candy, the peaks observed in the saliva were lower, approximately 2%

[4]. After consumption of the products, xylitol showed fast clearance from the oral cavity with only low salivary xylitol levels detected at 810 min [4, 16]. According to the present and earlier results [3], these xylitol vehicles show salivary xylitol concentrations that effectively inhibit mutans streptococci. Recently, the development of products resulting in slow release of xylitol has been proposed [1]. With such vehicles, the salivary xylitol concentrations will be much lower compared with, for example, consumption of chewing gum. On the other hand, with such vehicles, the salivary xylitol concentrations may be elevated for longer periods of time. According to the present results, a xylitol concentration of 0.1% (6.6 mM) should be high enough to inhibit mutans streptococci in vivo.

Fig. 1 Growth curves of Streptococcus mutans strains in the presence of various concentrations of xylitol (X). Signicant differences compared to the control curve (C): ***p \ 0.001; **p \ 0.01; *p \ 0.05. The arrows indicate maximal growth inhibition during log-phase compared to control. N = 4

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385 8. Miyasawa-Hori H, Aizawa S, Takahashi N (2006) Difference in the xylitol sensitivity of acid production among Streptococcus mutans strains and the biochemical mechanism. Oral Microbiol Immunol 21:201205 kinen KK, Bennett CA, Hujoel PP, Isokangas PJ, Isotupa KP, 9. Ma Pape HR (1995) Xylitol chewing gums and caries rates: a 40month cohort study. J Dent Res 74:19041913 kinen KK, So derling E, Isokangas P, Tenovuo J, Tiekso J 10. Ma (1989) Oral biochemical status and depression of Streptococcus mutans in children during 24- to 36-month use of xylitol chewing gum. Caries Res 23:261267 11. Milgrom P, Ly KA, Roberts MC, Rothen M, Mueller G, Yamaguchi DK (2006) Mutans streptococci dose response to xylitol chewing gum. J Dent Res 85:177181 lla G (1988) Combined effect of xylitol, 12. Scheie AA, Assev S, Ro NaF and ZnCl2 on growth and metabolism of Streptococcus sobrinus OMZ 176. APMIS 96:761767 derling E, Isokangas P, Pieniha kkinen K, Tenovuo J (2000) 13. So Inuence of maternal xylitol consumption on acquisition of mutans streptococci by infants. J Dent Res 79:882887 derling E, Trahan L, Lenander-Lumikari M (1998) Growth of 14. So xylitol-resistant versus xylitol-sensitive Streptococcus mutans strains in saliva. Acta Odontol Scand 56: 116121 kkinen K, Alanen P, Jokela J, So derling E 15. Taipale T, Pieniha (2007) Dissolution of xylitol from a food supplement administered with a novel slow-release pacier. Eur Arch Ped Dent 8:123125 16. Tapiainen T, Renko M, Kontiokari T, Uhari M (2002) Xylitol concentrations in the saliva of children after chewing xylitol gum or consuming a xylitol mixture. Eur J Microbiol Infect Dis 21:53 55 17. Thorild I, Lindau B, Twetman S (2003) Effect of maternal use of chewing gums containing xylitol, chlorhexidine or uoride on mutans streptococci colonization in the mothers infant children. Oral Health Prev Dent 1:5357 18. Vadeboncoeur C, Trahan L, Mouton C, Mayrand D (1983) Effect of xylitol growth and glycolysis of acidogenic oral bacteria. J Dent Res 62:882884

In summary, a concentration dependency was seen in the growth inhibition of S. mutans, with 0.01% (0.66 mM) being the lowest xylitol concentration signicantly inhibiting all ve S. mutans strains. Our results suggest that a low xylitol concentration of 0.1% (6.6 mM) could inhibit mutans streptococci in vivo, but that even lower xylitol concentrations may be inhibitory.
Acknowledgments Dr. Anna Haukioja is thanked for valuable comments on the manuscript. The excellent technical assistance of llfors is gratefully acknowlbiomedical research technician Oona Ha edged. The Finnish Dental Society Apollonia and the Finnish Dental Association have supported the study nancially.

References
1. Featherstone JDB (2006) Delivery challenges for uoride, chlorhexidine and xylitol. BMC Oral Health 6(suppl 1):S8 2. Gold OG, Jordan HV, van Houte J (1973) A selective medium for Streptococcus mutans. Arch Oral Biol 18:13571364 3. Gauthier L, Vadeboncoeur C, Mayrand D (1984) Loss of sensitivity to xylitol by Streptococcus mutans LG-1. Caries Res 18:289295 berg M, Twetman stro m I, O 4. Holgerson PF, Stecksen-Blicks C, Sjo S (2006) Xylitol concentration in saliva and dental plaque after use of various xylitol-containing products. Caries Res 40:393397 kinen KK (1975) Effect of xylitol on the 5. Knuuttila ML, Ma growth and metabolism of Streptococcus mutans. Caries Res 9:177189 6. Kontiokari T, Uhari M, Koskela M (1995) Effect on xylitol on growth of nasopharyngeal bacteria in vitro. Antimicrob Agents Chemother 39:18201823 7. Maguire A, Rugg-Gunn AJ (2003) Xylitol and caries preventionis it a magic bullet? Brit Dent J 194:429436

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