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ORIGINAL ARTICLE
Year : 2018  |  Volume : 36  |  Issue : 4  |  Page : 386-390
 

Salivary levels of Streptococcus mutans and Streptococcus sanguinis in early childhood caries: An in vivo study


1 Department of Pediatric and Preventive Dentistry, Maharishi Markandeshwar College of Dental Sciences and Research, Ambala, Haryana, India
2 Department of Microbiology, Maharishi Markandeshwar Institute of Medical Sciences and Research, Ambala, Haryana, India
3 Department of Oral and Maxillofacial Surgery, Maharishi Markandeshwar College of Dental Sciences and Research, Ambala, Haryana, India

Date of Web Publication16-Oct-2018

Correspondence Address:
Dr. Suma Sogi
Department of Pediatric and Preventive Dentistry, Markandeshwar College of Dental Sciences and Research, Mullana, Ambala, Haryana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JISPPD.JISPPD_204_18

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   Abstract 


Background: The oral microflora primarily involved in early childhood caries (ECC) is Streptococcus mutans while Streptococcus sanguinis is related to healthy caries-free mouth. The antagonism between the two at biological level is well recognized. Aim: This study aims to compare the pre- and posttreatment salivary levels of S. mutans and S. sanguinis in ECC. Methods: This cross-sectional study was performed on 40 eligible participants. Clinical examination was done, and decayed teeth were recorded. Pre- and Posttreatment saliva sample was taken, processed and bacterial counts were compared. Results: A statistically significant posttreatment reduction was found in S. mutans counts; however, a significant posttreatment elevation was seen in S. sanguinis counts (P < 0.001) using Wilcoxon signed-rank test. A significant weak positive correlation was found between S. mutans and decayed teeth in the mouth (r = 0.366 and P = 0.028) using Spearman's correlation. No significant gender association was found between pre- and posttreatment S. mutans and S. sanguinis counts (P = 0.908) and also between decayed teeth (P = 0.321) using Mann–Whitney U-test. Conclusion: Complete dental treatment procedure showed significant reduction and elevation in S. mutans and S. sanguinis counts, respectively. A positive association was observed between S. mutans and ECC while S. sanguinis were positively associated with posttreatment oral environment.


Keywords: Dental caries, dental treatment, microflora, oral environment, saliva


How to cite this article:
Patidar D, Sogi S, Singh V, Shinu P, Loomba A, Patidar DC. Salivary levels of Streptococcus mutans and Streptococcus sanguinis in early childhood caries: An in vivo study. J Indian Soc Pedod Prev Dent 2018;36:386-90

How to cite this URL:
Patidar D, Sogi S, Singh V, Shinu P, Loomba A, Patidar DC. Salivary levels of Streptococcus mutans and Streptococcus sanguinis in early childhood caries: An in vivo study. J Indian Soc Pedod Prev Dent [serial online] 2018 [cited 2018 Dec 13];36:386-90. Available from: http://www.jisppd.com/text.asp?2018/36/4/386/243452





   Introduction Top


Oral cavity harbors complex, multispecies communities of microflora.[1] In a homeostatic environment, these indigenous microflorae play a significant role in defending host from invasions by various exogenous pathogens; although when this homeostasis gets disturbed, it might result in dental caries.[2]

Caries is a biofilm-induced; saliva-mediated acidic demineralization of enamel and dentin of the teeth.[3] Early childhood caries (ECC) may be a particular aggressive form of caries, initiating on the smooth surfaces, rapidly progressing and having an unfavorable long-lasting effect on the dentition.[4] The development of dental caries involves a sucrose-induced ecological change in the bacterial composition toward cariogenic species. This cariogenic atmosphere generates favorable condition for certain conditional pathogens to compete with the commensal microflora more efficiently for the tooth surface resulting in dental caries.[5]

The oral microbial flora mainly involved in ECC is Streptococcus mutans.[6] It is a Gram-positive, facultative anaerobic bacteria,[7],[8] usually persists as a normal member of oral bacterial community, while in the presence of fermentable carbohydrates, these acid-producing organisms cause damage by dissolving tooth structure.[9],[10]Streptococcus sanguinis, also a Gram-positive facultative anaerobic bacteria,[7],[8] is a normal tooth colonizer, and its existence is related to healthy biofilms and absence of dental caries.[11]S. sanguinis can inhibit S. mutans and compete for colonization on tooth surfaces.[12] A positive and inverse correlation of S. mutans and S. sanguinis respectively is seen with decayed teeth in the mouth.[13] As infants and young children with ECC are generally at increased risk for further caries development, preventive as well as therapeutic methods are often necessary for them.[14] Hence, the present study was done to compare the pre- and post-treatment salivary levels of S. mutans and S. sanguinis in ECC.


   Methods Top


The present study was performed in subjects reporting to the Department of Pediatric and Preventive Dentistry, Maharishi Markandeshwar College of Dental Sciences and Research and in collaboration with the Department of Microbiology, Maharishi Markandeshwar Institute of Medical Sciences and Research. Approval for the study design, protocol, and methodology was obtained from the Institutional Research Committee and Ethics Committee, M.M.U with the project No. 488 dated on April 8, 2015. Informed written consent of parents and/or guardian was taken with a detailed case history, and preoperative clinical finding was recorded in patient assessment form.

Protocol for ECC was followed according to the American Academy of Pediatric Dentistry guidelines.[3] Procedures were performed by principal investigator.

Subjects reported to the outpatient department in the department of pediatric and preventive dentistry fulfilling the inclusion criteria was selected to participate in the study.

Children (both males and females) between 3 and 6 years of age with decayed teeth ≥2 and whose parents agreed to participate in the study were included in the study while children with special health-care needs/any systemic illness, taking antibiotics, or completed a course of antibiotic within the past 4 weeks and those with abscess due to dental caries were excluded from the study.

Saliva collection

After the initial parental counseling and diet counseling, parents were instructed neither to give food nor to rinse their child's mouth 2 h before saliva sample collection in the next visit. Resting whole saliva sample was collected on dental chair in the mid-morning time by the principal investigator. Subjects were instructed to accumulate saliva in the mouth for 1 min and then spit in a sterile disposable plastic container. A volume of 1 ml of saliva was transported on ice box to the microbiology laboratory and processed within 1 h.[15],[16]

Microbiological procedures

Bacterial culturing and identification were performed in microbiology laboratory. Mutans-Sanguis agar medium (M977) recommended for the differentiation of S. mutans and S. sanguinis was used.[17] The autoclaved media was then poured into sterile disposable  Petri dish More Detailses (90 mm) and allowed to dry. The pretreatment saliva samples were inoculated by using 4 mm diameter loop on Mutans Sanguis agar medium for S. mutans and S. sanguinis growth. Culture plate was then incubated at 37°C for 24 h for S. mutans and S. sanguinis growth in an incubator. Following incubation, colonies were identified by the morphological characteristics of S. mutans and S. sanguinis on Mutans Sanguis agar medium. S. sanguinis colonies appeared smooth or rough, hard, and rubbery in consistency, either gray, white, or colorless which adhere strongly to the agar medium making them difficult to remove with a inoculating loop and can be differentiated from S. mutans colonies which are irregular, rough, white or yellow in color, resembling granular frosted glass and can be easily picked off or easily detachable from the agar medium[17] [Figure 1].
Figure 1: Bacterial growth

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Various confirmatory tests such as Gram-staining, catalase test, mannitol, sorbitol fermentation, and bile esculin tests were done. Colony counting was done and expressed in CFU/ml.

After the above microbiological procedures, the dental treatment procedures (oral prophylaxis, restorations, endodontic treatment, and extractions) were carried out depending on their need base. Similar saliva sample collection method and microbiological procedures were applied after the dental treatment procedures. Both the bacterial colony counts (pre- and post-treatment) for S. mutans and S. sanguinis were then analyzed and compared [Figure 2].
Figure 2: Study methodology

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   Results Top


The present study included 40 eligible participants (19 males and 21 females), age group of 3–6 years with the mean age of 4.83 years [Table 1] and [Table 2]. A significant weak positive correlation was found between decayed teeth and pretreatment S. mutans count with r = 0.366 and P = 0.028 and a nonsignificant weak positive correlation between decayed teeth and pretreatment S. sanguinis count was seen with r = 0.137 and P = 0.425 using Spearman's correlation [Table 3]. A statistically significant difference was observed in posttreatment mean S. mutans and S. sanguinis counts as compared with mean pretreatment counts (P < 0.001) using Wilcoxon signed-rank test [Table 4]. No significant gender association was found between pre- and post-treatment S. mutans and S. sanguinis counts with P = 0.908 and also between decayed teeth with P = 0.321 using Mann–Whitney U-test.
Table 1: Distribution of subjects according to gender

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Table 2: Descriptive statistics of the study variables

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Table 3: Correlation between pretreatment decayed teeth and Streptococcus mutans and Streptococcus sanguinis counts

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Table 4: Pre- and post-treatment comparison of Streptococcus mutans and Streptococcus sanguinis count

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   Discussion Top


ECC is a particularly aggressive form of dental caries affecting the primary teeth of young children.[11] Although ECC has got multifactorial etiology, the role of diet and microorganisms is essential and well recognized.[18] In healthy environmental state, various microbial species in biofilms maintains a natural balance and restricts potentially harmful bacteria from colonizing. This balance could be disrupted by few ecological features and interspecies interactions, which facilitate the dominant growth of definite species, leading to dental caries.[19]

S. mutans is the principal bacteria isolated from children with ECC and plays a crucial role in the commencement and progression of the disease.[20],[21] However, S. sanguinis has been associated with good oral health and caries-free environment.[11]

The antagonism between S. mutans and S. sanguinis at the biological level is well recognized. Various studies have shown that earlier colonization and increased S. sanguinis level in oral cavity associates significantly with delayed S. mutans colonization on tooth surfaces, whereas, increased S. mutans levels in the oral cavity associated with decreased S. sanguinis levels.[22],[23]

Similarly, Carlsson et al. in their study found that S. sanguinis compete with S. mutans for tooth colonization sites, since both microorganism need the existence of teeth for their colonization process.[24]

In the present study, a significant weak positive correlation was found between pretreatment S. mutans counts and decayed teeth; however, a nonsignificant weak positive correlation was found between pretreatment S. sanguinis and decayed teeth [Table 3], similar to the study done by Loesche et al. in which children with higher number of decayed teeth surfaces had significantly greater proportions of S. mutans than caries-free children.[25]

However, in the present study, no significant relationship was observed between gender and decayed teeth, which was also shown by Ge et al.[11] The present study was performed with the main objective of comparing the pre- and posttreatment salivary levels of S. mutans and S. sanguinis in ECC. The result of the study demonstrated a significant reduction in S. mutans count and significant elevation in S. sanguinis count after the treatment procedures (“P” <0.001) [Table 4]. In a comparable study done by Roshan et al., significant decrease in S. mutans levels in saliva was observed after treatment with glass ionomer cement.[26] Similarly, lower levels of S. mutans were noticed posttreatment as compared with pretreatment by Tanner et al.[27] Becker et al. and Li et al. found higher S. sanguinis level in caries-free healthy children as compared with children with ECC.[28],[29]

In the present study, full mouth dental treatment procedure was performed for ECC. This may cause ecological shift toward more beneficial oral microbial species such as S. sanguinis and hence maintaining a proper balance and homogeneous state among various microbial species in the oral environment. Apart from this, there is competition, and mutual antagonism exist between these two organisms by Mutacin IV and Hydrogen Peroxide (H2O2) production by S. mutans and S. sanguinis respectively as shown by Kreth et al. and these could be the most promising explanation for the significant elevation of S. sanguinis counts after the treatment in this study.[30]


   Conclusion Top


Complete dental treatment procedures showed a significant reduction in the salivary levels of S. mutans and a significant elevation in salivary levels of S. sangunis. A positive association is observed between S. mutans and ECC while S. sanguinis is positively associated with posttreatment oral environment.

Clinical significance of the study

Based on the results of the study, assessment of relative salivary levels of S. mutans and S. sanguinis is helpful in identifying children at high risk for ECC and also creating novel preventive and treatment protocol. Ecological balanced and homogeneous environment were re-established by performing complete dental treatment for ECC as a significant decrease in the bacterial counts of cariogenic species S. mutans and significant increase in the bacterial counts of beneficial species S. sanguinis were reported after the treatment.

Limitations of the study

Other risk factors such as diet, frequency, and type of sugar consumption, oral hygiene measures, and fluoride exposure could have been a significant impact on the salivary bacterial counts have not been included in the present study and the mechanism by which oral microorganisms compete with each other requires further detail investigations.

Acknowledgment

G.M. Sogi, Prof/HOD, Department of Community Dentistry, Maharishi Markandeshwar College of Dental Sciences and Research, for review in the article.

Prof. Meera Sandhu, Department of Pediatric and Preventive Dentistry, Maharishi Markandeshwar College of Dental Sciences and Research, for suggestions.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Marsh PD. Microbiology of dental plaque biofilms and their role in oral health and caries. Dent Clin North Am 2010;54:441-54.  Back to cited text no. 5
    
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Barrow GI. Manual for the Identification of Medical Bacteria. 3rd ed. New York, U.S.A: Cambridge University Press; 1993. p. 58-9.  Back to cited text no. 8
    
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Marsh PD. Are dental diseases examples of ecological catastrophes? Microbiology 2003;149:279-94.  Back to cited text no. 9
    
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Caufield PW, Griffen AL. Dental caries. An infectious and transmissible disease. Pediatr Clin North Am 2000;47:1001-19, 5.  Back to cited text no. 10
    
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Ge Y, Caufield PW, Fisch GS, Li Y. Streptococcus mutans and Streptococcus sanguinis colonization correlated with caries experience in children. Caries Res 2008;42:444-8.  Back to cited text no. 11
    
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Giacaman RA, Torres S, Gómez Y, Muñoz-Sandoval C, Kreth J. Correlation of Streptococcus mutans and Streptococcus sanguinis colonization and ex vivo hydrogen peroxide production in carious lesion-free and high caries adults. Arch Oral Biol 2015;60:154-9.  Back to cited text no. 12
    
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Mitrakul K, Vongsawan K, Sriutai A, Thosathan W. Association between S mutans and S sanguinis in severe early childhood caries and caries-free children a quantitative real-time PCR analysis. J Clin Pediatr Dent 2016;40:281-9.  Back to cited text no. 13
    
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Priyadarshini HR, Fernandes BH, Rohini M, Nagarathnamma T. Relationship between severe-early childhood caries, salivary Mutans streptococci, and Lactobacilli in preschool children of low socioeconomic status in Bengaluru city. J Indian Soc Pedod Prev Dent 2014;32:44-7.  Back to cited text no. 15
    
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Kaur A, Kwatra KS, Kamboj P. Evaluation of non-microbial salivary caries activity parameters and salivary biochemical indicators in predicting dental caries. J Indian Soc Pedod Prev Dent 2012;30:212-7.  Back to cited text no. 16
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Mutans-Sanguinis Agar Media M977. Technical Data. Mumbai: Himedia Laboratories Pvt. Ltd. Avaialble from: http://www.himedialabs.com. [Last accessed on 2018 Sep 25].  Back to cited text no. 17
    
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Palmer CA, Kent R Jr., Loo CY, Hughes CV, Stutius E, Pradhan N, et al. Diet and caries-associated bacteria in severe early childhood caries. J Dent Res 2010;89:1224-9.  Back to cited text no. 18
    
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Kreth J, Zhang Y, Herzberg MC. Streptococcal antagonism in oral biofilms: Streptococcus sanguinis and Streptococcus gordonii interference with Streptococcus mutans. J Bacteriol 2008;190:4632-40.  Back to cited text no. 19
    
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Loesche WJ, Eklund S, Earnest R, Burt B. Longitudinal investigation of bacteriology of human fissure decay: E: Epidemiological studies in molars shortly after eruption. Infect Immun 1984;46:765-72.  Back to cited text no. 25
    
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Tanner AC, Kent RL Jr., Holgerson PL, Hughes CV, Loo CY, Kanasi E, et al. Microbiota of severe early childhood caries before and after therapy. J Dent Res 2011;90:1298-305.  Back to cited text no. 27
    
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Becker MR, Paster BJ, Leys EJ, Moeschberger ML, Kenyon SG, Galvin JL, et al. Molecular analysis of bacterial species associated with childhood caries. J Clin Microbiol 2002;40:1001-9.  Back to cited text no. 28
    
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