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Acta Odontológica Latinoamericana

versão On-line ISSN 1852-4834

Acta odontol. latinoam. vol.25 no.1 Buenos Aires abr. 2012



Comparison of oral streptococci biofilm in caries-free and caries-affected preschool Mexican children


Rita E. Martinez-Martinez1, Taku Fujiwara2, Nuria Patiño-Marin1, Tomonori Hoshino2, Michael Wilson3, Juan P. Loyola-Rodríguez1

1 The Master's Degree in Dental Science Program, San Luis Potosi University, Mexico.
2 Division of Pediatric Dentistry, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki University, Japan
3 Department of Microbiology, University College London, Eastman Dental Institute, London, UK

CORRESPONDENCE Dra. Rita Elizabeth Martinez Martinez Fuente de la Primavera 119, Fracc. Balcones del Valle. San Luis Potosí, SLP México


Interaction of oral streptococci biofilm is the main etiological factor for dental caries. The aim of the study was to compare oral streptococci (OS) distribution in the biofilm of primary dentition from caries-free and caries-affected preschool Mexican children. This cross-sectional study involved 40 caries-free and 40 cariesaffected children with primary dentition. Each child was examined using the dmfs index, DNA was extracted from saliva and presence of OS was determined by PCR. Data obtained showed no statistical difference regarding age and gender (P>0.05). Streptococcus mutans (Smut), Streptococcus sobrinus (Ssob) and their combination showed significant statistical differences between groups (P<0.05). Smut, Streptococcus sanguinis and Streptococcus gordonii had an inverse relation with dmfs index and Ssob had a direct relation similar to combined with Smut. Smut-Ssob combined with other OS showed statistical differences (P<0.05). In free-caries group Streptococcus gordonii was more frequently identified than Smut. The ratio Smut/Streptococcus sanguinis could represent a high risk of dental caries development; this ratio was higher in the caries-affected (1.18) than in the caries-free group (0.32). In conclusion, OS play an important role in dental caries predisposition and severity, not only the presence of Smut and Ssob, but also the complexity and distribution of OS in the biofilm.

Key words: Dental Caries; Saliva; Streptococcus mutans.


Comparación de la composición de la biopelícula de estreptococos orales en niños preescolares Mexicanos con presencia y libres de caries dental

La interacción de los estreptococos orales en la biopelícula es el principal factor etiológico de la caries dental, por lo que objetivo del estudio fue comparar la distribución de los estreptococos orales en la biopelícula de niños preescolares con dentición temporal, afectados por caries y libres de esta enfermedad. Este estudio transversal incluyó 40 niños con caries y 40 niños libres de caries con dentición primaria. Cada sujeto fue examinado usando el índice ceo, y se tomó una muestra de saliva, de la cual se extrajo el DNA y se determinó la presencia de los estreptococos orales por medio de PCR. Comparando los dos grupos no se mostraron diferencias significativas en cuanto a edad y género (P>0.05). La presencia de Streptococcus mutans (Smut), Streptococcus sobrinus (Ssob) y su combinación mostraron diferencias estadísticas entre grupos (P<0.05). La presencia de Smut, Streptococcus sanguinis y Streptococcus gordonii mostró una relación inversa con el índice ceo, en contraste Ssob así como la combinación Smut- Ssob observaron una relación directa. Smut-Ssob combinados con otros estreptococos orales, mostraron diferencias estadísticas entre grupos (P<0.05). En el grupo libre de caries Streptococcus gordonii se identificó con mayor frecuencia que Smut. La proporción Smut/Streptococcus sanguinis podría representar un alto riesgo de desarrollo de caries dental, esta proporción fue mayor en los sujetos afectados por caries dental (1.18) en comparación con el grupo libre de caries (0.32). En conclusión, los estreptococos orales juegan un importante papel en el riesgo de caries dental así como en su severidad, no solo la presencia de Smut y Ssob, sino también la complejidad y distribución de los estreptococos orales en la biopelícula.

Key words: Caries dental; Saliva; Streptococcus mutans.



Dental caries has been one of the most prevalent and expensive diseases of humans. It is a multifactorial disease, so in order to understand the role of different bacterial species, it is important to promote a complete model of caries etiology which should include specific species of oral streptococci1. Mutans streptococci (MS) [Streptococcus mutans (Smut), Streptococcus sobrinus Ssob)] have been considered to be the principal cariogenic microorganisms2. Together with Mitis streptococci [Streptococcus mitis, Streptococcus sanguinis (Ssan), Streptococcus oralis (Sora), Streptococcus gordonii (Sgor)] and Salivarius streptococci [Streptococcus salivarius (Ssal), Streptococcus vestibularis], they are the most important constituents of human oral flora3. Although mitis and salivarius groups have a low cariogenicity, their interactions could be important for the establishment and maintenance of pathogenic plaque4. It has been reported that interactions between cariogenic bacteria such as Smut with other dental plaque microorganisms could modulate its cariogenic potential in a biofilm community5. Therefore, detection and identification of oral streptococci is considered to be important to understand biofilm organization and to develop treatment alternatives for dental caries prevention.
Oral Streptococci are early colonizers in neonates but the establishment of Smut and Ssob occurs after dental eruption. Studies have shown that when pits and fissures in occlusal surfaces are initially colonized by a non-cariogenic bacterial flora, they may confer host protection by physically occupying the space and blocking the colonization by cariogenic microorganisms, preventing the onset of dental caries6. Therefore, competition between pioneer colonizing bacteria may determine the dental plaque composition after its organization; for example, Sgor competes with Ssan to adhere to saliva coated hydroxyapatite7. Ssan promotes aggregation with other oral bacteria modeling the process of maturation of dental plaque8. It can also compete with Smut, and both species appear after tooth eruption and show direct antagonism in situ9.
Saliva participates in different functions such as tissue protection, swallowing, dental remineralization and antimicrobial action. Its quantity, pH and buffer capacity play a significant role in caries development10. Saliva samples have been a valuable, non-invasive, simple method used for the diagnosis, treatment and prevention of some diseases11. Several methods have been used to detect the presence of oral streptococci, such as identification of colony morphologies on Mitis Salivarius Bacitracin (MSB) agar plates from saliva samples, but the classification is slow and difficult. A PCR detection method based on amplification of glucosyltransferase genes (gtf) has been reported12, this approach having been used to detect the oral streptococci from whole saliva samples as a proposal for epidemiological studies. The aim of this study was to use a molecular identification method (PCR) to compare the distribution of oral streptococci from whole saliva samples of caries-free and caries-affected preschool Mexican children, to apply this knowledge in future to prevention strategies against dental caries, such as replacement therapy.


This cross-sectional prospective study involved 80 healthy and cooperative children with primary dentition, all of them living in San Luis Potosi (northern- central), Mexico. Child recruitment was undertaken from the Department of Pediatric Dentistry of the Advanced General Dentistry Program. Parents of children completed a health questionnaire that included information about pediatric and oral evaluations, and the last course of antibiotics. Informed and voluntary written consent from parents was obtained prior to clinical examination according to the ethical principles of the world medical association declaration of Helsinki (version 2002). The pediatric sample involved two groups of 40 children each selected by non-probabilistic consecutive sampling: A) without clinical presence of dental caries and restorations (caries-free) and B) affected by dental caries without restorations (caries-affected). Inclusion criteria were: age between 3 to 6 years and either gender. Exclusion criterion was children who had received antibiotics during the last three months. The outcome variable was the identification by PCR of Smut, Ssob, Ssan, Sgor, Ssal and Sora. The explanatory variable studied was dental caries (dmfs index).

Dental caries
The World Health Organization (WHO) caries diagnostic criteria were used for determining the dmfs (decayed, missing, and filled tooth surfaces of primary teeth) index13.

Saliva sampling
Paraffin-stimulated whole saliva from children was sampled with the method reported previously14. Samples were transported on ice and stored at -40°C until PCR evaluations were performed.

DNA extraction and PCR
DNA from saliva was extracted and quantified as described in a previous report14. The presence of gtf genes in the extracted DNA was determined using species-specific gtf primers3. PCR assay was carried out in 25 µl of a reaction mixture with the conditions reported previously3,14. Positive and negative controls were included in each PCR set by using DNA of the following bacterial strains: S. mutans (ATCC 35665, MT8148 (c) and OMZ 175 (f), S. sobrinus (6715 (g), B13 (d), and ATCC 27351), S. sanguinis (ATCC 10556, ST3, ST202, B220), S. gordonii (ATCC 10558), S. salivarius (NCTC 8618, HHT) and S. oralis (NCTC 11427, ATCC 10557). The DNA of the abovementioned bacterial strains were kindly donated by Dr. Taku Fujiwara of the Department of Pediatric Dentistry from Nagasaki University, Japan. The PCR products were analyzed by electrophoresis in 2% agarose gel using Tris-acetate-EDTA buffer, using a 100-bp DNA ladder marker (New England Biolab, Beverly, MA, USA) to estimate the molecular size. Each gel was stained with ethidium bromide (0.5 µg/ml) and photographed under UV illumination (Chemi Doc, BIO-RAD laboratories, Hercules, CA, USA).

Statistical analysis
Before starting the study, two examiners were calibrated in all variables with an expert in pediatric dentistry and a microbiologist, through the Kappa test. All variables included were blind-analyzed. All data are expressed as mean ± standard deviation and range. Shapiro-Wilks, Levene and Brown Forsythe tests were used to test the distribution of variables. The non-parametric Mann Whitney U test was used to compare continuous variables; X2 of Mantel-Haenszel test was used to compare categorical variables. JMP version 5.1 and Stat View 4.0 (SAS Institute, Cary, NC, USA) were used for statistical analysis; statistical significance was set at P< 0.05.


The inter-observer and intra-observer reproducibility regarding the diagnosis of dental caries reached by two examiners showed a Kappa of 1.0.

Comparison of morphometric traits
The mean of the age of the children in the caries-free group was 4.58 years (22 males and 18 females). The mean age of the children in the caries-affected group was 4.25 years (23 males and 17 females). There was no statistical difference in age and gender (P>0.05) between groups. The homogeneity of these variables allows us to compare the groups in relation to the presence or absence of dental caries.

Identification of oral streptococci from saliva by PCR (Table 1)
The OS most frequently detected in the caries-free group were Ssal (75%), Ssan (62.5), Sora (60%); the species least frequently detected was Ssob (10%). For the caries-affected group, the OS most frequently detected were Smut (80%) and Ssob (70%); the species least frequently detected was Sgor (40%). S. mutans was identified in 80% of the caries-affected and only 20% of the caries-free children. The frequency of detection of Ssob was only 10% in the caries-free children and 70% in the caries-affected group. There was a significant statistical difference between groups (P<0.05) for both bacterial species. For Ssan, Sgor, Ssal and Sora there was no significant statistical difference (P>0.05) between groups.

Table 1: Identification of oral streptococci from saliva by PCR.

Frequency of combinations of different oral streptococci (Table 2) In the caries-free group there was a mean of 2.62 ±1.42 bacterial species detected and in the caries-affected group the mean was 3.85 ±1.43; there was a significant statistical difference between groups (P<0.05). The positive combination of Smut and Ssob was found in 4 (10%) of caries-free children and in 22 (55%) of caries-affected children. There was a statistical difference between the groups (P>0.05). The combination of Smut-Ssob-Ssan; Smut-Ssob-Sgor; Smut-Ssob- Ssal; Smut-Ssob-Sora showed a significant statistical difference between the groups (P>0.05). In 7 caries-free children most of the bacterial species were absent, but only Ssal was detected, while in 8 children in the caries-affected group, most bacterial species were detected but only Sgor was absent (data not shown).

Table 2: Frequency of combination of different oral streptococci.

Detection of oral streptococci and their interaction with caries experience (Table 3)
The mean dmfs index in the caries-free group was 0, there were no decayed, missing or filled teeth. For the caries-affected group the mean was 7.35 ± 3.38 with a range of 2-15. Ssob was present in 100% of patients with a dmfs index of >10 and was detected in only 58.3% of children with dmfs index 1-5, being the only bacterial strain that increased according to caries experience. The frequency of detection of Smut (91.6%, 76.2%, 71%), Ssan (75%, 71.4%, 42.8%) and Sgor (58.3%, 33.3%, 28.5%) decreased when the three levels of dmfs increased. In patients with a dmfs index of 1-5, the microorganism most frequently detected was Smut (91.6%). In children with dmfs index of 6-10 it was Sora (80.9%) and in patients with dmfs index >10 it was Ssob (100%).

Table 3: Frequency of detection of S. mutans, S. sobrinus, S. sanguinis, S. gordonii, S. salivarius, S. oralis and their relation to caries experience.

Combination of different oral streptococci and their interaction with caries experience (Table 4)
The combination of Smut- Ssob-Sgor was found more frequently in children with a dmfs index of 1-5. The combinations of Smut- Ssob-Ssan; Smut-Ssob- Ssal; Smut-Ssob-Sora were found more frequently in children with a dmfs index of 6-10 (42.8%, 47% and 46%, respectively). The frequency of detection of the combination Smut-Ssob (50%, 52.3%, 71.4%) increased when the dmfs index increased.

Table 4: Frequency of combinations of oral streptococci and their relation to caries experience.


To date there are few molecular and epidemiological studies where the distribution of oral streptococci has been associated with cariogenicity and biofilm composition in caries-free and caries-affected populations3. Such information could lead to a better understanding of the roles of different bacterial species associated with the severity of dental caries.
In this study, the main outcome that showed significant statistical difference between the two groups was the detection of Smut and Ssob. The species least commonly identified (10%) in the caries-free group was Ssob; the opposite scenario occurred in the caries-affected group where Ssob was identified in 28 children (70%). In the cases of Ssan, Sgor, Ssal and Sora, there were no statistical difference between groups, but in combination with Smut and Ssob they showed statistical differences between groups. The most frequently isolated species in caries-free children were Ssal, Ssan and Sora. These species could play a significant role in the caries process because they can interact to preserve the biofilm integrity. Sgor was identified in 35% of the caries-free group. A recent study reported the absence of Sgor in caries-free children, these differences possibly being explained by sample size3. Some reports indicate that Sgor can attenuate some of the virulence properties of Smut and inhibit sucrose-dependent biofilm formation5. In this study in the caries-free group, Sgor was more frequently isolated than Smut, which suggests that Sgor can compete with Smut and Ssan to adhere to enamel surface7. Some reports say that Ssan may also compete with mutans streptococci for colonization sites on tooth surfaces, and may exhibit direct biochemical antagonism in situ. Because the cariogenic potential of Ssan is lower than that of Smut, several investigators have suggested that the Smut/Ssan ratio may serve as an indicator for caries risk9. In our study, we observed a Smut/Ssan ratio in the caries-free group of 0.32 and in the caries-affected group of 1.18; although there was not a significant difference, it suggests that the caries-affected group presents a higher risk factor. Another study reported similar data but collected plaque samples as opposed to saliva samples as done in our study4. The early introduction of Ssan into the oral cavity of children, or increasing its relative proportions by artificial means, could affect mutans streptococci colonization because it could be deferred. Delayed colonization of Smut has been associated with lower caries scores15. It could be a treatment alternative for dental caries in children but has not yet been substantiated. It has been reported that Sora produces GTF, it has an agonist effect with Smut in caries development, because there is evidence that contributes to the establishment of oral biofilms and plays an important role in the subsequent colonization of mutans streptococci16. In our study, Sora, was the second species most frequently identified in the caries-affected group.
There was no significant statistical difference between the groups in the detection of Sora, but this species was one of the two species most frequently isolated in both groups. Smut has been implicated in pits and fissures caries and seems to participate in the early stages of the caries process while Ssob is associated with caries on smooth surfaces and is closely related to the severity of dental caries2,17,18. However, in this study, the positive combination of Smut-Ssob was associated with the caries-affected group, these data agree with some reports that indicate that people who harbour both species have a higher frequency and severity of caries18,19. The combination more frequently identified in both groups was Smut-Ssob-Sora. In the caries-free children, 7 of them were negative to most bacterial species except Ssal; therefore, this finding suggests that Ssal is the bacterial species least related to dental caries. On the other hand, in 8 caries-affected children, most bacterial species were present but only Sgor was absent, which agrees with some reports where it was shown that Smut produces antibacterial substances termed mutacins that exhibit different degrees of inhibition against other bacteria, including Sgor20.
The frequency of detection of Smut showed an inverse relationship with the dmfs index, identified in 91.6% of patients with a low (1-5) dmfs index and 70% of patients with a high (>10) dmfs index. The same pattern was observed with Ssan and Sgor. On the other hand, Ssob was the only species that presented a direct relationship with the dmfs index, it was detected in 100% of patients with severe caries, and only in 58.3% of patients with a low (1-5) dmfs index. This outcome suggests that Smut participates in the initial and Ssob in the advanced stages of dental caries. The combination of Smut-Ssob was increased in close correlation with the dmfs index. The importance of Smut in the etiology of dental caries has been well documented, but there is growing recognition that the cariogenic potential may be determined by complex interactions in dental plaque biofilm rather than solely the virulence properties of a single organism. In this study the cariesaffected group showed higher number of bacterial species than the caries-free group, supporting the theory that the presence of different species modulates the severity of dental caries20. Probably the best way to study dental caries is to consider the interaction among several bacterial species in biofilm to provide new information on its pathogenesis by molecular epidemiology studies. Recently, bacteria associated with dental caries have been seen to play an important role in systemic diseases, since they have been isolated from blood of patients affected by infective endocarditis and cardiovascular diseases21,22.


In conclusion, there was a different distribution of oral streptococci between caries-free and cariesaffected groups of children. Ssob alone and its combinations are closely related to the severity of caries. Smut is likely to participate at the beginning of the caries process. Ssan, Sgor, Ssal and Sora could interact with Smut and Ssob in the formation of a cariogenic biofilm because their combination with Smut and Ssob showed statistical differences between the two groups. The identification of oral streptococci by PCR is a reliable, useful and rapid molecular method that can contribute greatly to our knowledge on the composition of caries biofilms.


This study was supported by CONACYT Grant Number 33650, UASLP-FAI and CONACYT (student scholarship No.173644). The authors thank the personnel of the Master's Degree in Dental Science with Specialization in Advanced General Dentistry Program at The University of San Luis Potosi, Mexico.


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