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Effect of dietary sugars on dual-species biofilms of Streptococcus mutans and Streptococcus sobrinus – a pilot study

Efeito dos açucares da dieta em biofilme dupla espécie de Streptococcus mutans e Streptococcus sobrinus – um estudo piloto

Abstract

Introduction

Frequent consumption of sugars and the presence of Streptococcus mutans and Streptococcus sobrinus are correlated with higher caries experience.

Objective

The aim of this pilot study was to elucidate the effect of different fermentable carbohydrates on biomass formation and acidogenicity of S. mutans and S. sobrinus biofilms.

Material and method

Single and dual-species biofilms of S. mutans ATCC 25175 and S. sobrinus ATCC 27607 were grown at the bottom of microtiter plates at equal concentrations for 24 h at 37 °C under micro-aerobic atmosphere. Carbohydrates were added at 2% concentration: maltose, sucrose, glucose and lactose. BHI Broth (0.2% glucose) was used as negative control. Acidogenicity was assessed by measuring the pH of spent culture medium after 24 h, immediately after refreshing the culture medium and for the next 1 h and 2 h. Crystal violet staining was used as an indicator of the total attached biofilm biomass after 24 h incubation. Data were analyzed by two-way ANOVA followed by Bonferroni post hoc test. Significance level was set at 5%.

Result

All carbohydrates resulted in higher biomass formation in single- and dual-species biofilms when compared to the control group. Sucrose, lactose and maltose showed higher acidogenicity than the control group in both single- and dual-species biofilms after 24 h.

Conclusion

These findings indicate that the type of biofilm (single- or dual-species) and the carbohydrate used may influence the amount of biomass formed and rate of pH reduction.

Descriptors:
Biofilm; biomass; Streptococcus mutans; Streptococcus sobrinus

Resumo

Introdução

O consumo frequente de açucares e a presença de Streptococcus mutans e Streptococcus sobrinus estão correlacionados com maior experiência de cárie.

Objetivo

Elucidar o efeito de diferentes carboidratos fermentáveis na biomassa e acidogenicidade de biofilmes formados por S. mutans e S. sobrinus.

Material e método

Biofilmes única e dupla- espécie de S. mutans ATCC 25175 e S. sobrinus ATCC 27607 em concentrações iguais cresceram no fundo de placas de microtitulação por 24 h a 37 °C em microaerofilia. Maltose, sacarose, glicose e lactose foram adicionados a 2%. BHI caldo (0.2% glicose) foi usado como controle negativo. Acidogenicidade foi avaliada por meio da medição do pH do meio de cultura após 24 h, imediatamente após troca de meio e nas próximas 1 h e 2 h. Coloração por cristal violeta foi usada como indicador do total de biomassa aderida, após 24 h de incubação. Os dados foram analisados por teste ANOVA two way e Teste de Bonferroni. O nível de significância foi de 5%.

Resultado

Todos os carboidratos resultaram em maior formação de biomassa em ambos os tipos de biofilme (única ou dupla- espécie), quando comparado ao grupo controle. Sacarose, lactose e maltose mostraram maior acidogenicidade que o grupo controle após 24 h nos biofilmes única ou dupla-espécie, apenas após 24 h.

Conclusão

Os achados indicam que o tipo de biofilme (única ou dupla- espécie) e o tipo de carboidrato usado podem influenciar tanto na quantidade de biomassa formada quanto na taxa de redução do pH.

Descritores:
Biofilme; biomassa; Streptococcus mutans; Streptococcus sobrinus

INTRODUCTION

Epidemiological studies have shown a correlation between the presence of Streptococcus mutans and Streptococcus sobrinus with caries incidence11 Seki M, Yamashita Y, Shibata Y, Torigoe H, Tsuda H, Maeno M. Effect of mixed mutans streptococci colonization on caries development. Oral Microbiol Immunol. 2006 Feb;21(1):47-52. http://dx.doi.org/10.1111/j.1399-302X.2005.00253.x. PMid:16390341.
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2 Choi EJ, Lee SH, Kim YJ. Quantitative real-time polymerase chain reaction for Streptococcus mutans and Streptococcus sobrinus in dental plaque samples and its association with early childhood caries. Int J Paediatr Dent. 2009 Mar;19(2):141-7. http://dx.doi.org/10.1111/j.1365-263X.2008.00942.x. PMid:19250396.
http://dx.doi.org/10.1111/j.1365-263X.20...

3 Nurelhuda NM, Al-Haroni M, Trovik TA, Bakken V. Caries experience and quantification of and in saliva of Sudanese schoolchildren. Streptococcus mutansStreptococcus sobrinusCaries Res. 2010;44(4):402-7. http://dx.doi.org/10.1159/000316664. PMid:20714152.
http://dx.doi.org/10.1159/000316664...
-44 Okada M, Kawamura M, Oda Y, Yasuda R, Kojima T, Kurihara H. Caries prevalence associated with Streptococcus mutans and in Japanese schoolchildren. Streptococcus sobrinusInt J Paediatr Dent. 2012 Sep;22(5):342-8. http://dx.doi.org/10.1111/j.1365-263X.2011.01203.x. PMid:22225789.
http://dx.doi.org/10.1111/j.1365-263X.20...
. In early childhood caries, the presence of both microorganisms promoted a significantly higher caries increment than S. mutans alone55 Okada M, Soda Y, Hayashi F, Doi T, Suzuki J, Miura K, et al. Longitudinal study of dental caries incidence associated with Streptococcus mutans and Streptococcus sobrinus in pre-school children. J Med Microbiol. 2005 Jul;54(Pt 7):661-5. http://dx.doi.org/10.1099/jmm.0.46069-0. PMid:15947431.
http://dx.doi.org/10.1099/jmm.0.46069-0...
. Although S. sobrinus has been isolated less frequently from carious lesions, it has been associated with active dental caries and may be considered a determinant of caries experience33 Nurelhuda NM, Al-Haroni M, Trovik TA, Bakken V. Caries experience and quantification of and in saliva of Sudanese schoolchildren. Streptococcus mutansStreptococcus sobrinusCaries Res. 2010;44(4):402-7. http://dx.doi.org/10.1159/000316664. PMid:20714152.
http://dx.doi.org/10.1159/000316664...
, mainly early childhood caries22 Choi EJ, Lee SH, Kim YJ. Quantitative real-time polymerase chain reaction for Streptococcus mutans and Streptococcus sobrinus in dental plaque samples and its association with early childhood caries. Int J Paediatr Dent. 2009 Mar;19(2):141-7. http://dx.doi.org/10.1111/j.1365-263X.2008.00942.x. PMid:19250396.
http://dx.doi.org/10.1111/j.1365-263X.20...
.

It is believed that the cariogenic potential of these bacteria is directly related to their ability to generate acids and to tolerate acidic environments66 Nascimento MM, Lemos JA, Abranches J, Gonçalves RB, Burne RA. Adaptive acid tolerance response of Streptococcus sobrinus.J Bacteriol. 2004 Oct;186(19):6383-90. http://dx.doi.org/10.1128/JB.186.19.6383-6390.2004. PMid:15375118.
http://dx.doi.org/10.1128/JB.186.19.6383...
. These features provide a competitive advantage over other biofilm bacteria during the periods of acidification66 Nascimento MM, Lemos JA, Abranches J, Gonçalves RB, Burne RA. Adaptive acid tolerance response of Streptococcus sobrinus.J Bacteriol. 2004 Oct;186(19):6383-90. http://dx.doi.org/10.1128/JB.186.19.6383-6390.2004. PMid:15375118.
http://dx.doi.org/10.1128/JB.186.19.6383...
. S. mutans can grow and carry out glycolysis at pH values below 5.0 and can lower the pH to values below 4.077 Belli WA, Marquis RE. Adaptation of and to acid stress in continuous culture. Streptococcus mutansEnterococcus hiraeAppl Environ Microbiol. 1991 Apr;57(4):1134-8. PMid:1829347.. At low pH levels, S. sobrinus is capable of sustain acid production, whereas other species tend to discontinue or reduce this production88 de Soet JJ, Toors FA, de Graaff J. Acidogenesis by oral streptococci at different pH values. Caries Res. 1989;23(1):14-7. http://dx.doi.org/10.1159/000261148. PMid:2920379.
http://dx.doi.org/10.1159/000261148...
. S. sobrinus can produce acid more rapidly than S. mutans at pH values between 6.5 and 5.0. Thus, S. sobrinus may be considered the most acidogenic of the oral streptococci99 Beighton D. The complex oral microflora of high-risk individuals and groups and its role in the caries process. Community Dent Oral Epidemiol. 2005 Aug;33(4):248-55. http://dx.doi.org/10.1111/j.1600-0528.2005.00232.x. PMid:16008631.
http://dx.doi.org/10.1111/j.1600-0528.20...
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An association between the consumption of sugar-containing beverages and the presence of mutans streptococci in infants was found1010 Grindefjord M, Dahllof G, Wikner S, Hojer B, Modeer T. Prevalence of mutans streptococci in one-year-old children. Oral Microbiol Immunol. 1991 Oct;6(5):280-3. http://dx.doi.org/10.1111/j.1399-302X.1991.tb00492.x. PMid:1820564.
http://dx.doi.org/10.1111/j.1399-302X.19...
. Frequent consumption of sugar-sweetened snacks by school children was also correlated with higher caries experience and with higher isolation of S. mutans and S. sobrinus33 Nurelhuda NM, Al-Haroni M, Trovik TA, Bakken V. Caries experience and quantification of and in saliva of Sudanese schoolchildren. Streptococcus mutansStreptococcus sobrinusCaries Res. 2010;44(4):402-7. http://dx.doi.org/10.1159/000316664. PMid:20714152.
http://dx.doi.org/10.1159/000316664...
. Animals fed with carbohydrates exhibited formation of higher amounts of coronal plaque on the smooth surfaces of the tooth1111 Campbell RG, Zinner DD. Effect of certain dietary sugars on hamster caries. J Nutr. 1970 Jan;100(1):11-20. PMid:4904500.. The fermentation of sucrose produces large amounts of acids within biofilms and serves as a substrate for extracellular and intracellular polysaccharide synthesis1212 Cury JA, Marques AS, Tabchoury CPM, Del Bel Cury AA. Composition of dental plaque formed in the presence of sucrose and after its interruption. Braz Dent J. 2003;14(3):147-52. http://dx.doi.org/10.1590/S0103-64402003000300001. PMid:15057387.
http://dx.doi.org/10.1590/S0103-64402003...

13 Leme AF, Koo H, Bellato CM, Bedi G, Cury JA. The role of sucrose in cariogenic dental biofilm formation - new insight. J Dent Res. 2006 Oct;85(10):878-87. http://dx.doi.org/10.1177/154405910608501002. PMid:16998125.
http://dx.doi.org/10.1177/15440591060850...
-1414 Azevedo MS, Van de Sande FH, Romano AR, Cenci MS. Microcosm biofilms originating from children with different caries experience have similar cariogenicity under successive sucrose challenges. Caries Res. 2011;45(6):510-7. http://dx.doi.org/10.1159/000331210. PMid:21967836.
http://dx.doi.org/10.1159/000331210...
. Extracellular polysaccharides increase porosity of the biofilm matrix, allowing carbohydrate diffusion through the biofilm. At the tooth-plaque interface these carbohydrates are fermented to acids resulting in pH decrease1414 Azevedo MS, Van de Sande FH, Romano AR, Cenci MS. Microcosm biofilms originating from children with different caries experience have similar cariogenicity under successive sucrose challenges. Caries Res. 2011;45(6):510-7. http://dx.doi.org/10.1159/000331210. PMid:21967836.
http://dx.doi.org/10.1159/000331210...
. Extracellular polysaccharides also increase microorganism adhesion and accumulation, mainly of S. mutans1313 Leme AF, Koo H, Bellato CM, Bedi G, Cury JA. The role of sucrose in cariogenic dental biofilm formation - new insight. J Dent Res. 2006 Oct;85(10):878-87. http://dx.doi.org/10.1177/154405910608501002. PMid:16998125.
http://dx.doi.org/10.1177/15440591060850...
,1515 Ma R, Sun M, Wang S, Kang Q, Huang L, Li T, et al. Effect of high-fructose corn syrup on the acidogenicity, adherence and biofilm formation of Streptococcus mutans.Aust Dent J. 2013 Jun;58(2):213-8. http://dx.doi.org/10.1111/adj.12074. PMid:23713642.
http://dx.doi.org/10.1111/adj.12074...
. Intracellular polysaccharides are reservoirs of carbohydrates that promote pH drop during nutrient deprivation, prolonging the exposure of tooth surfaces to organic acids1212 Cury JA, Marques AS, Tabchoury CPM, Del Bel Cury AA. Composition of dental plaque formed in the presence of sucrose and after its interruption. Braz Dent J. 2003;14(3):147-52. http://dx.doi.org/10.1590/S0103-64402003000300001. PMid:15057387.
http://dx.doi.org/10.1590/S0103-64402003...
.

Sucrose, which is considered the most cariogenic carbohydrate, needs to be catabolized into glucose and fructose by sucrase before it can be metabolized by S. mutans. On the other hand, glucose can be directly metabolized by this microorganism1515 Ma R, Sun M, Wang S, Kang Q, Huang L, Li T, et al. Effect of high-fructose corn syrup on the acidogenicity, adherence and biofilm formation of Streptococcus mutans.Aust Dent J. 2013 Jun;58(2):213-8. http://dx.doi.org/10.1111/adj.12074. PMid:23713642.
http://dx.doi.org/10.1111/adj.12074...
. Glucose also appears to be more efficiently metabolized by S. sobrinus, since a higher amount of acids is produced by this microorganism when compared to S. mutans1616 Homer KA, Patel R, Beighton D. Effects of N-acetylglucosamine on carbohydrate fermentation by Streptococcus mutans NCTC 10449 and Streptococcus sobrinus SL-1. Infect Immun. 1993 Jan;61(1):295-302. PMid:8418050.. Maltose, a starch derivative, is one of the most abundant carbohydrates in the human diet and is easily fermentable to potentially cariogenic acids by S. mutans1717 Kilic AO, Honeyman AL, Tao L. Overlapping substrate specificity for sucrose and maltose of two binding protein-dependent sugar uptake systems in Streptococcus mutans.FEMS Microbiol Lett. 2007 Jan;266(2):218-23. http://dx.doi.org/10.1111/j.1574-6968.2006.00522.x. PMid:17233733.
http://dx.doi.org/10.1111/j.1574-6968.20...
. Approximately one half of the total amount of acidic end products is produced from glucose and sucrose. However, a lower proportion of acid is produced from maltose by both S. mutans and S. sobrinus1818 Aizawa S, Miyasawa-Hori H, Nakajo K, Washio J, Mayanagi H, Fukumoto S, et al. Effects of alpha-amylase and its inhibitors on acid production from cooked starch by oral streptococci. Caries Res. 2009;43(1):17-24. http://dx.doi.org/10.1159/000189703. PMid:19136828.
http://dx.doi.org/10.1159/000189703...
. This fact suggests that glucose and sucrose may provide higher cariogenic potential to biofilms than maltose. In the same way, it has been suggested that lactose is less cariogenic than sucrose, glucose and maltose1919 Marshall TA, Eichenberger-Gilmore JM, Larson MA, Warren JJ, Levy SM. Comparison of the intakes of sugars by young children with and without dental caries experience. J Am Dent Assoc. 2007 Jan;138(1):39-46. http://dx.doi.org/10.14219/jada.archive.2007.0019. PMid:17197400.
http://dx.doi.org/10.14219/jada.archive....
.

The role of sugars in dental caries process has been discussed by numerous studies. However most of them have focused just on sucrose and/or glucose1212 Cury JA, Marques AS, Tabchoury CPM, Del Bel Cury AA. Composition of dental plaque formed in the presence of sucrose and after its interruption. Braz Dent J. 2003;14(3):147-52. http://dx.doi.org/10.1590/S0103-64402003000300001. PMid:15057387.
http://dx.doi.org/10.1590/S0103-64402003...

13 Leme AF, Koo H, Bellato CM, Bedi G, Cury JA. The role of sucrose in cariogenic dental biofilm formation - new insight. J Dent Res. 2006 Oct;85(10):878-87. http://dx.doi.org/10.1177/154405910608501002. PMid:16998125.
http://dx.doi.org/10.1177/15440591060850...
-1414 Azevedo MS, Van de Sande FH, Romano AR, Cenci MS. Microcosm biofilms originating from children with different caries experience have similar cariogenicity under successive sucrose challenges. Caries Res. 2011;45(6):510-7. http://dx.doi.org/10.1159/000331210. PMid:21967836.
http://dx.doi.org/10.1159/000331210...
,2020 Cury JA, Rebelo MA, Del Bel Cury AA, Derbyshire MT, Tabchoury CP. Biochemical composition and cariogenicity of dental plaque formed in the presence of sucrose or glucose and fructose. Caries Res. 2000 Nov-Dec;34(6):491-7. http://dx.doi.org/10.1159/000016629. PMid:11093024.
http://dx.doi.org/10.1159/000016629...

21 Bradshaw DJ, Marsh PD, Hodgson RJ, Visser JM. Effects of glucose and fluoride on competition and metabolism within in vitro dental bacterial communities and biofilms. Caries Res. 2002 Mar-Apr;36(2):81-6. http://dx.doi.org/10.1159/000057864. PMid:12037363.
http://dx.doi.org/10.1159/000057864...

22 Aires CP, Tabchoury CP, Del Bel Cury AA, Koo H, Cury JA. Effect of sucrose concentration on dental biofilm formed in situ and on enamel demineralization. Caries Res. 2006;40(1):28-32. http://dx.doi.org/10.1159/000088902. PMid:16352877.
http://dx.doi.org/10.1159/000088902...

23 Ccahuana-Vásquez RA, Tabchoury CP, Tenuta LM, Del Bel Cury AA, Vale GC, Cury JA. Effect of frequency of sucrose exposure on dental biofilm composition and enamel demineralization in the presence of fluoride. Caries Res. 2007;41(1):9-15. http://dx.doi.org/10.1159/000096100. PMid:17167254.
http://dx.doi.org/10.1159/000096100...
-2424 Arthur RA, Cury AADB, Graner RO, Rosalen PL, Vale GC, Leme AF, et al. Genotypic and phenotypic analysis of isolated from dental biofilms formed in vivo under high cariogenic conditions. S. mutansBraz Dent J. 2011;22(4):267-74. http://dx.doi.org/10.1590/S0103-64402011000400001. PMid:21861023.
http://dx.doi.org/10.1590/S0103-64402011...
. At present, little is known about the role of maltose and lactose. Since these fermentable carbohydrates are frequently present in children’s dietary in their natural form or in processed food and beverages, it is important to evaluate their acidogenic potential and the influence in biomass biofilm formation. Moreover, a contemporary approach is to study not only single-species but also dual-species biofilms, since co-existence of different species provides an advantage in surviving antimicrobial treatment2525 Luppens SB, Kara D, Bandounas L, Jonker MJ, Wittink FR, Bruning O, et al. Effect of Veillonella parvula on the antimicrobial resistance and gene expression of Streptococcus mutans grown in a dual-species biofilm. Oral Microbiol Immunol. 2008 Jun;23(3):183-9. http://dx.doi.org/10.1111/j.1399-302X.2007.00409.x. PMid:18402603.
http://dx.doi.org/10.1111/j.1399-302X.20...
. Therefore, this study was conducted to elucidate the effect of different fermentable carbohydrates on the amount of biomass and acidogenicity of biofilms formed by S. mutans and S. sobrinus.

MATERIAL AND METHOD

Bacterial Strains and Growth Conditions

An aliquot of 400 μL of frozen stocks of S. mutans ATCC 25175 and S. sobrinus ATCC 27607 were inoculated into 5 mL in Brain Heart Infusion (BHI) Broth (HIMEDIA Laboratories, Vadhani Industrial State, LBS MARG, India) and incubated at 37 °C for 24 h under micro-aerobic atmosphere (4-5% of CO2 and low O2 tension)2626 Maiti PK, Haldar J, Mukherjee P, Dey R. Anaerobic culture on growth efficient bi-layered culture plate in a modified candle jar using a rapid and slow combustion system. Indian J Med Microbiol. 2013 Apr-Jun;31(2):173-6. http://dx.doi.org/10.4103/0255-0857.115218. PMid:23867675.
http://dx.doi.org/10.4103/0255-0857.1152...
. Next, microorganisms were inoculated in BHI Agar (HIMEDIA Laboratories, Vadhani Industrial State, LBS MARG, India). After 48 h under micro-aerobic conditions (4-5% of CO2 and low O2 tension), one colony of each microorganism was transferred to individual tubes containing 5 mL of BHI Broth. After incubation under micro-aerobic atmosphere (4-5% of CO2 and low O2 tension) at 37 °C for an additional 18 h, bacterial suspensions were prepared using McFarland scale, which yielded a cell density of 1 × 108 CFU mL–1 for each inoculum. The bacterial suspension was used to prepare a 1% fresh inoculum in BHI Broth, which was transferred to microtiter plates. Initial medium pH before experiments was 7.4 ± 0.2 and no buffer was added. Biofilms of either S. mutans (single-species biofilms) or a combination of S. mutans and S. sobrinus (dual-species biofilms) were grown at the bottom of microtiter plates (n=2 wells) at equal concentrations. Stock solutions of carbohydrates were prepared at 20%: maltose, sucrose, glucose and lactose (Lab Synth, Diadema-SP, Brazil). The carbohydrates were added at 2% final concentration as negative control, BHI Broth (contains 0.2% glucose) was used.

Biofilm Acidogenicity

Microtiter plates with 12 wells were used. An aliquot of 4 mL of the 1% fresh inoculum were transferred to each well and 400 μL of each carbohydrate was added in duplicate. The plates were incubated at 37 °C under micro-aerobic atmosphere. Biofilm acidogenicity was assessed by pH measurements of culture medium using a microelectrode connected to a pH meter in combination with a glass reference electrode (Orion Res Inc., Cambridge, Mass., USA). The microelectrode was calibrated using standard pH buffers (pH 4.0 and 7.0) prior to and after each test as well as during tests if necessary. The pH determinations were made in duplicate for all carbohydrates studied and performed on two different days. The pH was measured after 24 h incubation, immediately after refreshing the culture medium and for the next 1 h and 2 h. To refresh the culture medium, 3 mL was removed from each well, then it was added 3 mL of fresh BHI Broth and 400 μL of each carbohydrate were added.

Biomass

Crystal violet assay was used as an indicator of the total attached biofilm biomass. The advantage of this analysis is that it can be used directly, without disrupting the biofilm. Microtiter plates with 24 wells were used. The 1% fresh inoculum was transferred to each well in a volume of 1.5 mL and 150 μL of each carbohydrate was added. Experiments were performed in two different days. Two wells with just BHI Broth were used as negative controls. The plates were incubated at 37 °C under micro-aerobic atmosphere (4-5% of CO2 and low O2 tension). After 24 h of biofilm growth, the supernatant was removed and biofilms were washed two times with 2 mL of sterile water, to remove loosely attached cells. The 2 mL of sterile water was removed carefully with the aid of pipettes and biofilms were immersed in 2 mL of ethanol for 15 min (to fix the biofilm). Ethanol was removed and the plates were dried at room temperature (approximately 20 min). A volume of 2 mL of 1% crystal violet were added to each well and incubated at room temperature. After 5 min, crystal violet was removed and 2 mL of sterile water were added. The water was removed and the biofilms were allowed to dry at room temperature. Then, 2 mL of 33% acetic acid were added to dilute the stain and 200 μL from each well was transferred in triplicate to a 96 wells microtiter plate. The absorbance of the crystal violet solution was measured at 590 nm wavelength (BioPhotometer Plus, Eppendorf, São Paulo, Brazil). Biomass assay was performed twice, in different moments.

Statistical Analysis

Statistical analysis was carried out using GraphPad Prism Version 3.02 (GraphPad Software Inc., San Diego, CA, USA). Data showed equality of variances (Bartlett's test) and normal distribution (Kolmogorov–Smirnov test). Data were analyzed by two-way ANOVA followed by Bonferroni post hoc test. The significance limit was set at 5%.

RESULT

The type of biofilm (single- or dual-species) and the carbohydrate used influenced the amount of biomass formed (Figure 1). All carbohydrates resulted in higher biomass formation in single- and dual-species biofilms when compared to the control group.

Figure 1
Biomass quantification (mean ± sd) in S. mutans single-species biofilms (white bars) or S. mutans and S. sobrinus dual-species biofilms (grey bars) after 24 h of incubation. Means followed by different uppercase letters show statistically significant differences for S. mutans single-species biofilms. Means followed by different lowercase letters show statistically significant differences for S. mutans and S. sobrinus dual-species biofilms (two-way ANOVA followed by Bonferroni pos roc test, p<0.05).

The rate of pH reduction was carbohydrate and time-dependent. For both single- and dual-species biofilm, statistically significant differences were observed only after 24 h, when sucrose, lactose and maltose showed higher acidogenicity when compared to the control group (BHI broth only) (Table 1).

Table 1
Acidogenicity (mean pH ± sd) of single-species (S. mutans) biofilms and dual-species (S. mutans + S. sobrinus) biofilms

DISCUSSION

There are a considerable number of studies suggesting that sucrose is the most cariogenic carbohydrate1111 Campbell RG, Zinner DD. Effect of certain dietary sugars on hamster caries. J Nutr. 1970 Jan;100(1):11-20. PMid:4904500.

12 Cury JA, Marques AS, Tabchoury CPM, Del Bel Cury AA. Composition of dental plaque formed in the presence of sucrose and after its interruption. Braz Dent J. 2003;14(3):147-52. http://dx.doi.org/10.1590/S0103-64402003000300001. PMid:15057387.
http://dx.doi.org/10.1590/S0103-64402003...
-1313 Leme AF, Koo H, Bellato CM, Bedi G, Cury JA. The role of sucrose in cariogenic dental biofilm formation - new insight. J Dent Res. 2006 Oct;85(10):878-87. http://dx.doi.org/10.1177/154405910608501002. PMid:16998125.
http://dx.doi.org/10.1177/15440591060850...
,1515 Ma R, Sun M, Wang S, Kang Q, Huang L, Li T, et al. Effect of high-fructose corn syrup on the acidogenicity, adherence and biofilm formation of Streptococcus mutans.Aust Dent J. 2013 Jun;58(2):213-8. http://dx.doi.org/10.1111/adj.12074. PMid:23713642.
http://dx.doi.org/10.1111/adj.12074...
. Nevertheless, this study shows that other fermentable carbohydrates may also influence biomass formation and biofilm acidogenicity. Glucose, lactose and maltose were able to produce the same amount of biomass than sucrose in both single- and dual- species biofilms. Moreover, despite glucose resulted in higher pH values than sucrose, lactose and maltose, all fermentable carbohydrates tested resulted in a pH drop below critical values (5.5) after 24 h in both types of biofilms.

The control group had ten-fold lower carbohydrate than other groups (0.2% glucose). Nutrient limitation contributed to the lower amount of biomass formed by the control group than by other carbohydrates, which is in agreement to Cury et al.2020 Cury JA, Rebelo MA, Del Bel Cury AA, Derbyshire MT, Tabchoury CP. Biochemical composition and cariogenicity of dental plaque formed in the presence of sucrose or glucose and fructose. Caries Res. 2000 Nov-Dec;34(6):491-7. http://dx.doi.org/10.1159/000016629. PMid:11093024.
http://dx.doi.org/10.1159/000016629...
Besides, under this low glucose condition, the cells tend to enter stationary-phase2727 Renye JA Jr, Piggot PJ, Daneo-Moore L, Buttaro BA. Persistence of in stationary-phase batch cultures and biofilms. Streptococcus mutansAppl Environ Microbiol. 2004 Oct;70(10):6181-7. http://dx.doi.org/10.1128/AEM.70.10.6181-6187.2004. PMid:15466565.
http://dx.doi.org/10.1128/AEM.70.10.6181...
. On the other hand, high carbohydrate availability influences the expression of physiologic and biochemical pathways of bacteria2828 Moye ZD, Zeng L, Burne RA. Modification of gene expression and virulence traits in in response to carbohydrate availability. Streptococcus mutansAppl Environ Microbiol. 2014 Feb;80(3):972-85. http://dx.doi.org/10.1128/AEM.03579-13. PMid:24271168.
http://dx.doi.org/10.1128/AEM.03579-13...
, allows bacterial growth and increases lactic acid production2727 Renye JA Jr, Piggot PJ, Daneo-Moore L, Buttaro BA. Persistence of in stationary-phase batch cultures and biofilms. Streptococcus mutansAppl Environ Microbiol. 2004 Oct;70(10):6181-7. http://dx.doi.org/10.1128/AEM.70.10.6181-6187.2004. PMid:15466565.
http://dx.doi.org/10.1128/AEM.70.10.6181...
. It is known that S. mutans produces elevated amounts of extracellular polysaccharides (EPS) when sucrose is available, which provides support to development and accumulation of microcolonies and increase the cohesiveness and structural integrity of the biofilm2929 Xiao J, Koo H. Structural organization and dynamics of exopolysaccharide matrix and microcolonies formation by Streptococcus mutans in biofilms. J Appl Microbiol. 2010 Jun;108(6):2103-13. http://dx.doi.org/10.1111/j.1365-2672.2009.04616.x. PMid:19941630.
http://dx.doi.org/10.1111/j.1365-2672.20...
. Intracellular polysaccharides (IPS) are also synthesized and provide bulk to biofilms1212 Cury JA, Marques AS, Tabchoury CPM, Del Bel Cury AA. Composition of dental plaque formed in the presence of sucrose and after its interruption. Braz Dent J. 2003;14(3):147-52. http://dx.doi.org/10.1590/S0103-64402003000300001. PMid:15057387.
http://dx.doi.org/10.1590/S0103-64402003...
,1313 Leme AF, Koo H, Bellato CM, Bedi G, Cury JA. The role of sucrose in cariogenic dental biofilm formation - new insight. J Dent Res. 2006 Oct;85(10):878-87. http://dx.doi.org/10.1177/154405910608501002. PMid:16998125.
http://dx.doi.org/10.1177/15440591060850...
.

Despite previous studies showing that lactose is the least cariogenic sugar1919 Marshall TA, Eichenberger-Gilmore JM, Larson MA, Warren JJ, Levy SM. Comparison of the intakes of sugars by young children with and without dental caries experience. J Am Dent Assoc. 2007 Jan;138(1):39-46. http://dx.doi.org/10.14219/jada.archive.2007.0019. PMid:17197400.
http://dx.doi.org/10.14219/jada.archive....
, the present study indicates that lactose produced the same amount of biomass and it was as acidogenic as the other carbohydrates. It was suggested that the regular consumption of carbohydrates may predispose to early colonization of mutans streptococci and influence caries risk in the primary dentition1010 Grindefjord M, Dahllof G, Wikner S, Hojer B, Modeer T. Prevalence of mutans streptococci in one-year-old children. Oral Microbiol Immunol. 1991 Oct;6(5):280-3. http://dx.doi.org/10.1111/j.1399-302X.1991.tb00492.x. PMid:1820564.
http://dx.doi.org/10.1111/j.1399-302X.19...
. Despite the recommendation of exclusive breast feeding for at least 6 months3030 World Health Organization. Exclusive Breastfeeding [Internet]. Geneva: WHO [cited 2013 Jul 14]. Available from: http://www.who.int/nutrition/topics/exclusive_breastfeeding/en/
http://www.who.int/nutrition/topics/excl...
,3131 American Academy of Pediatrics. Breastfeeding and the use of human milk. Pediatrics. 2012 Mar;129(3):e827-41. http://dx.doi.org/10.1542/peds.2011-3552. PMid:22371471.
http://dx.doi.org/10.1542/peds.2011-3552...
, an association between breast feeding for at least 6 or 7 months and early childhood caries was found3232 Kato T, Yorifuji T, Yamakawa M, Inoue S, Saito K, Doi H, et al. Association of breast feeding with early childhood dental caries: Japanese population-based study. BMJ Open. 2015 Mar;5(3):e006982. http://dx.doi.org/10.1136/bmjopen-2014-006982. PMid:25795694.
http://dx.doi.org/10.1136/bmjopen-2014-0...
. Perera et al.3333 Perera PJ, Fernando MP, Warnakulasooriya TD, Ranathunga N. Effect of feeding practices on dental caries among preschool children: a hospital based analytical cross sectional study. Asia Pac J Clin Nutr. 2014;23(2):272-7. http://dx.doi.org/10.6133/apjcn.2014.23.2.13. PMid:24901097.
http://dx.doi.org/10.6133/apjcn.2014.23....
also showed that ECC was present in children older than two years who were fed overnight any type of milk. Moreover, the incidence of ECC was higher in children who harbored both species studied (S. mutans and S. sobrinus)22 Choi EJ, Lee SH, Kim YJ. Quantitative real-time polymerase chain reaction for Streptococcus mutans and Streptococcus sobrinus in dental plaque samples and its association with early childhood caries. Int J Paediatr Dent. 2009 Mar;19(2):141-7. http://dx.doi.org/10.1111/j.1365-263X.2008.00942.x. PMid:19250396.
http://dx.doi.org/10.1111/j.1365-263X.20...
. Considering that human milk has a higher concentration of lactose (7%)3434 Hamrǽus L, Lönnerdal B. Nutritional aspects of milk proteins. In: Fox PF, McSweeney PLH, editors. Advanced dairy chemistry- 1 Proteins. New York: Kluwer Academic/Plenum Publishers; 2003. v. 1, p. 605-45. than that used in the present study, these findings may contribute to the understanding of the metabolism of carbohydrates in biofilms and the relationship between carbohydrates and dental caries. On the other hand, milk may also have caries-protective properties3535 Aimutis WR. Lactose cariogenicity with an emphasis on childhood dental caries. Int Dairy J. 2012 Feb;22(2):152-8. http://dx.doi.org/10.1016/j.idairyj.2011.10.007.
http://dx.doi.org/10.1016/j.idairyj.2011...
and its role in caries development should be further evaluated.

As previously stated, few studies investigated the metabolism of maltose. Kilic et al.1717 Kilic AO, Honeyman AL, Tao L. Overlapping substrate specificity for sucrose and maltose of two binding protein-dependent sugar uptake systems in Streptococcus mutans.FEMS Microbiol Lett. 2007 Jan;266(2):218-23. http://dx.doi.org/10.1111/j.1574-6968.2006.00522.x. PMid:17233733.
http://dx.doi.org/10.1111/j.1574-6968.20...
found that S. mutans cells were able to transport and metabolize maltose two-fold more efficiently than in the presence of glucose. Our findings (Table 1) support these data, since maltose was more acidogenic than glucose and as acidogenic as sucrose and lactose after 24 h, in either single- or dual-species biofilms. Maltose is a starch derivate and one of the most abundant fermentable carbohydrate in the human diet. It is suggested that this carbohydrate enhances S. mutans competitiveness3636 Webb AJ, Homer KA, Hosie AH. A phosphoenolpyruvate-dependent phosphotransferase system is the principal maltose transporter in Streptococcus mutans.J Bacteriol. 2007 Apr;189(8):3322-7. http://dx.doi.org/10.1128/JB.01633-06. PMid:17277067.
http://dx.doi.org/10.1128/JB.01633-06...
. However, it is still not known how maltose is taken up by S. mutans and S. sobrinus. Further studies are required to better understand the behavior of these bacteria in the presence of this carbohydrate.

Although 2% glucose fed biofilms formed similar amount of biomass compare to other carbohydrates, surprisingly, pH of the culture media after 24 h was higher for glucose than for other fermentable carbohydrates. Sucrose, lactose and maltose are disaccharides and have the same molecular weight (342.3 g/mol). On the other hand, glucose was the only monosaccharide studied and its molecular weight is about 50% the molecular weight of the disaccharides (180.2 g/mol). Thus, although culture media had 2 g carbohydrates per 100 mL, the number of glucose molecules present in the culture media was different than the number of molecules present in disaccharides (sucrose, lactose and maltose). This may explain why glucose showed a statistically significant lower acidogenicity than the other carbohydrates after 24 h in both biofilms.

It is important to note that the carbohydrates used in this study present different composition and thus yield different substrates after hydrolysis: sucrose, lactose and maltose are disaccharides composed by, respectively, glucose + fructose; glucose + galactose and glucose + glucose. These disaccharides also differ in the glycosidic bond between the monomers: while the monomers from sucrose and maltose are alpha-linked, lactose has a beta-link between them. Glucosyltransferases (Gtfs) are related to the synthesis of extracellular polysaccharides (EPS), which promote adhesion, microorganisms accumulation and biofilm matrix establishment, that is responsible for the structural integrity of dental biofilms1515 Ma R, Sun M, Wang S, Kang Q, Huang L, Li T, et al. Effect of high-fructose corn syrup on the acidogenicity, adherence and biofilm formation of Streptococcus mutans.Aust Dent J. 2013 Jun;58(2):213-8. http://dx.doi.org/10.1111/adj.12074. PMid:23713642.
http://dx.doi.org/10.1111/adj.12074...
,3737 Li Y, Burne RA. Regulation of the gtfBC and ftf genes of Streptococcus mutans in biofilms in response to pH and carbohydrate. Microbiology. 2001 Oct;147(Pt 10):2841-8. http://dx.doi.org/10.1099/00221287-147-10-2841. PMid:11577162.
http://dx.doi.org/10.1099/00221287-147-1...
,3838 Devulapalle KS, Gómez de Segura A, Ferrer M, Alcalde M, Mooser G, Plou FJ. Effect of carbohydrate fatty acid esters on and glucosyltransferase activity. Streptococcus sobrinusCarbohydr Res. 2004 Apr;339(6):1029-34. http://dx.doi.org/10.1016/j.carres.2004.01.007. PMid:15063188.
http://dx.doi.org/10.1016/j.carres.2004....
. S. mutans has been indicated as the main source of Gtfs in biofilms3939 Koo H, Falsetta ML, Klein MI. The exopolysaccharide matrix: a virulence determinant of cariogenic biofilm. J Dent Res. 2013 Dec;92(12):1065-73. http://dx.doi.org/10.1177/0022034513504218. PMid:24045647.
http://dx.doi.org/10.1177/00220345135042...
. However, S. sobrinus also produces glucosyltransferases for the production of water-soluble glucans and water-insoluble glucans4040 Nishimura J, Saito T, Yoneyama H, Bai LL, Okumura K, Isogai E. Biofilm formation by Streptococcus mutans and related bacteria. Adv Microbiol. 2012;2(03):208-15. http://dx.doi.org/10.4236/aim.2012.23025.
http://dx.doi.org/10.4236/aim.2012.23025...
,4141 Hashizume-Takizawa T, Shinozaki-Kuwahara N, Tomita N, Kurita-Ochiai T. Establishment of a convenient sandwich-ELISA for direct quantification of glucosyltransferase-I: application for dual diagnosis of dental caries. Monoclon Antib Immunodiagn Immunother. 2014;33(2):89-93. http://dx.doi.org/10.1089/mab.2013.0076. PMid:24746149.
http://dx.doi.org/10.1089/mab.2013.0076...
. Thus, both species are able to cleave the glycoside bond between disaccharides’ monomers.

It was suggested that the presence of bacteria other than S. mutans in biofilms may influence acid production4242 Kneist S, Kubieziel H, Willershausen B, Küpper H, Callaway A. Modeling of and . S. mutansA. naeslundii acid production in vitro with caries incidence of low- and high-risk childrenQuintessence Int. 2012;43(5):413-20. PMid:22536593.. Of particular interest, S. sobrinus is able to produce large amounts of acid end products from sugar metabolism88 de Soet JJ, Toors FA, de Graaff J. Acidogenesis by oral streptococci at different pH values. Caries Res. 1989;23(1):14-7. http://dx.doi.org/10.1159/000261148. PMid:2920379.
http://dx.doi.org/10.1159/000261148...
. In addition, the higher isolation of S. sobrinus from caries-active children indicates that this microorganism may be actively associated with dental caries and may be considered a determinant of caries experience33 Nurelhuda NM, Al-Haroni M, Trovik TA, Bakken V. Caries experience and quantification of and in saliva of Sudanese schoolchildren. Streptococcus mutansStreptococcus sobrinusCaries Res. 2010;44(4):402-7. http://dx.doi.org/10.1159/000316664. PMid:20714152.
http://dx.doi.org/10.1159/000316664...
. Thus, it was decided to study this species in association with S. mutans.

The idea of this pilot study was to select fermentable carbohydrates of interest using not only single-species biofilms but also a contemporary approach with dual-species biofilms. The results of the present study will be the basis to further analyze other parameters, such as metabolic activity, susceptibility to chlorhexidine of the biofilms, cell viability and matrix composition.

In this context, this study offers valuable insights about interactions among cariogenic bacteria and contributes to our general understanding of carbohydrate metabolism in biofilms and the relationship of carbohydrates to dental caries.

CONCLUSION

These findings of this pilot study indicate that the type of biofilm (single- or dual-species) and the carbohydrate used may influence the amount of biomass formed and rate of the pH reduction.

ACKNOWLEDGEMENTS

The authors thank São Paulo Research Foundation - FAPESP (grants: 2012/17236-4 and 2013/12326-8) for financial support. They are also grateful to Laura Boegli and Elinor Pulcini for the English reviewing.

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Publication Dates

  • Publication in this collection
    12 Feb 2016
  • Date of issue
    Mar-Apr 2016

History

  • Received
    29 Jan 2015
  • Accepted
    27 Aug 2015
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