Correlation between salivary mutans streptococci, lactobacilli and the severity of early childhood caries.

BACKGROUND/
PURPOSE: Mutans streptococci (MS) and lactobacilli (LB) are associated with dental caries, but the acquisition of these organisms in young children with early childhood caries (ECC) and severe early childhood caries (S-ECC) has only been partly described. The purpose of this study was to evaluate the salivary MS and LB levels of preschool children with ECC and S-ECC, and the correlation of MS, LB levels with caries severity.
MATERIALS AND METHODS: The study population was comprised of children from 3 to 6 years of age who visited the Pediatric Dental clinic of TCVGH. Oral examinations were performed, and whole unstimulated salivary samples were collected for buffer capacity, MS and LB counts by using commercially available diagnostic kits (CRT bacteria kit). The participants were divided into mild (dmft < 6), moderate (dmft:6-9) and severe caries (dmft > 9) groups; the salivary microbiological tests and buffer capacity were compared.
RESULTS: A total of 72 children with a mean age of 4.5 years participated in this study. The salivary test showed that the higher the salivary MS level the more severe the caries status in children (P = 0.001). There was also a tendency toward a higher salivary LB level in children with more severe caries, but the difference was not significant (P = 0.088). There was no significant correlation between buffer capacity and caries status.
CONCLUSION: The salivary MS level in preschool children was significantly correlated with the severity of early childhood caries.

Introduction

Early childhood caries (ECC) is a chronic, transmissible, infectious disease whose etiology is complex and multifactorial. ECC exhibits a distinct caries pattern that is often associated with prolonged, frequent, and inappropriate consumption of cariogenic foodstuffs. Mutans streptococci colonization is regarded as one of the principal factors involved in ECC.1, 2, 3 Early childhood caries results in a considerable direct burden of pain and suffering as well as poorer general health than that of caries free children.

Despite efforts in restorative therapy, children who experience ECC continue to be at a higher risk for new lesions in both the primary and the permanent dentition. A retrospective study showed that despite engagement in preventive dental caries, children with too numerous counts MS were six times more likely to experience cavity increments after 5 years than preschoolers with no recoverable MS at first visit. Interventions that disrupt the pathobiology of caries are needed to prevent and treat this aggressive infectious disease.

In previous studies, conventional culturing methods have been used to show that well-known bacteria species, such as Streptococcus mutans and Lactobacilli (LB), are associated with dental caries., A delay in mutans streptococci colonization is likely to be beneficial as early detection was found to be coupled with a higher caries experience in children., The combined “mutans streptococci + lactobacilli + past caries” model is useful for identifying at-risk children reported in a one year prospective study.

According to the definition of American Academy of Pediatric Dentistry, early childhood caries (ECC) is the caries occurring in children below 71 months of age, and severe early childhood caries (S-ECC) is the severe form of early childhood caries. The overall prevalence of ECC in 5 years old children of Taiwan is estimated to be 89% or more. This creates very serious oral health problems in Taiwan. Therefore understanding the role of specific bacterial species is important for creating a complete model of caries etiology and caries prevention.

The purpose of this study was to evaluate the salivary MS and LB levels of preschool children with ECC and S-ECC and the correlation of MS, LB levels with caries severity.

Materials and methods

Participant selection

Children who visited the pediatric dentistry at Taichung Veterans General Hospital, Taiwan, from July 2009 to Dec 2009, participated in this study. Children suffering from systemic disease or taking antibiotics were excluded from the study. Written informed consent was obtained from all parents who agreed to allow their children to participate in this study. This study protocol was approved by the Institutional Review Board of the Taichung Veterans General Hospital, Taiwan.

Oral examination

A complete dental examination was performed in the dental operating room of the pediatric dental clinic by two examiners. The examiners were trained regarding diagnosis criteria and clinical examination procedures. The subjects were examined in a supine position in a dental chair. An overhead light, a plane mouth mirror and an explorer were used. The caries status was first assessed by visual inspection and then confirmed by tactile inspection. The caries status of each tooth surface was evaluated and coded as “s” (sound), “d” (decayed), “m” (missed), or “f” (filled) surface. After oral examination, the participants were divided into three groups according to the dmft score. Mild caries is dmft smaller than 6 (dmft < 6), moderate caries is dmft between 6 and 9 (dmft: 6–9) and severe caries is dmft greater than 9 (dmft > 9).

Saliva tests

Unstimulated saliva samples were collected for buffer capacity, mutans streptococci and lactobacilli counts. The subjects were instructed to spit a total of 3 ml of saliva into a vial. The whole saliva samples were collected for buffer capacity and bacterial counts immediately. CRT® Bacteria kit (Ivoclar Vivadent AG products) was used for evaluating buffer capacity and bacterial cultures. The salivary cultures were incubated at 37 °C for 48 h. Then, the bacteria counts were measured according to the standard chart of CRT® Bacteria kit. The counts of colony forming units per milliliter (CFU/ml) saliva were categorized into four levels by two calibrated examiners who were blinded to the subject's caries status. CRT scores 0 to 3 denote a MS level of “less than 104,”

“104–105,” “105–106,” and “greater than 106,” respectively, and CRT scores 1 to 4 denote a LB level of “less than 103,” “equal to or greater than 104,” “equal to or greater than 105,” and “greater than 106,” respectively. CRT scores 1 to 3 denotes a buffer capacity of “high”, “medium” and “low” respectively.

Statistical analysis

Data were analyzed using SPSS 24.0 soft-ware (SPSS Inc, Chicago, Ill., USA). The Pearson correlation coefficient was used to identify the correlation between age and dmft score. The correlation between bacteria counts and caries status were also analyzed by using Pearson correlation coefficient test. Differences in bacterial counts between the three caries severity groups were evaluated by using analysis of variance (ANOVA) and Tukey's test at a p value of 0.05. Chi-square test and Bonferroni test were used to analyze the correlation of caries severity in different bacteria concentration at a p value of 0.05.

Results

A total of 72 children (45 boys, 27 girls) with a mean age of 4.5 years (range 3.5–6.0years) participated in this study.

Caries status

The mean dmft was 8.0 for boys and 7.9 for girls. The mean dmfs was 17.6 for boys and 16.7 for girls. There were no significant differences between girls and boys in dmf scores. There was a tendency that older children had higher dmft score (Fig. 1), but the difference was not significant (Pearson correlation coefficient, P = 0.114, R = 0.188).

Figure 1

Correlation of age and dmft (P-value* = 0.114). *Pearson Correlation Coefficient (R = 0.188).

Bacteria counts

The salivary test showed that the higher the salivary MS level the higher the dmft score (Fig. 2). There were significant differences with p value <0.001 (Pearson correlation coefficient, R = 0.428). The participants were divided into three groups: mild, moderate and severe caries. A positive correlation was found between the severity of caries and MS level, the higher the MS score (CRT score) the more severe the caries status (Table 1). There was a significant difference in the salivary MS level between these three groups of children (ANOVA test, P = 0.001). Tukey's test showed that there was a significant difference (p < 0.05) between severe caries and mild caries, moderate caries and mild caries in MS counts (Table 1).

Figure 2

The correlation of salivary mutans streptococci (MS) and the caries status. (P-value* <0.001). *Pearson Correlation Coefficient(R = 0.428).

Table 1

Comparison of buffer capacity, MS and LB level in three caries severity groups with CRT score.

Characteristic	Mild caries	Moderate caries	Severe caries	P-valuea
	(n = 24)	(n = 23)	(n = 25)
	Mean ± SD	Mean ± SD	Mean ± SD
Buffer (CRT score)	1.54 ± 0.72	1.61 ± 0.72	1.64 ± 0.81	0.898
MS (CRT score)b	0.67 ± 0.92	1.52 ± 1.12	1.88 ± 1.27	0.001
LB (CRT score)	1.63 ± 0.71	1.78 ± 0.95	2.16 ± 0.90	0.088

One-way ANOVA.

Tukey's Test for MS (P-value <0.05): Mild vs Moderate, Mild vs Severe.

In severe caries group, 48% of children had a MS level higher than 106, whereas in mild caries group, only 8.3% of children had a MS level higher than 106 (Table 2). In the mild caries group, 54.2% of children had a MS level less than 104, but in severe caries group, only 24% of children showed a MS level less than 104 (Table 2). The salivary MS concentration was significant different between these three groups of children (Chi-square Test, P = 0.002). There was a significant difference (p < 0.05) between severe and mild caries group in MS concentration greater than 106.

Table 2

The salivary MS level in mild, moderate and severe caries groups.

MS	Mild caries	Moderate caries	Severe caries	P-valuea
	(n = 24)	(n = 23)	(n = 25)
	n (%)	n (%)	n (%)
<104	13 (54.2)	6 (26.1)	6 (24.0)	0.002
104–105	8 (33.3)	4 (17.4)	3 (12.0)
105–106b	1 (4.2)	8 (34.8)	4 (16.0)
>106c	2 (8.3)	5 (21.7)	12 (48.0)

Chi-square Test.

Bonferroni Test for severity (P-value <0.05): Mild vs Moderate.

Bonferroni Test for severity (P-value <0.05): Mild vs Severe.

There was also a tendency toward a higher salivary LB level in children with more severe caries, but the difference was not significant (Table 1, ANOVA test, P = 0.088). Table 3 showed that in different salivary LB concentrations, there was also no significant difference between these three groups of children (Chi-square Test, P = 0.279). Although it revealed that in high LB concentration (LB = 105–106, LB > 106), only 12.5% mild caries children had LB greater than 105, however 26.1% moderate caries and 32% severe caries children had LB level greater than 105. In low LB concentration (LB < 104), it also revealed less severe caries children (24%) than mild caries (50%) and moderate caries (52%) children (Table 3).

Table 3

The salivary LB level in mild, moderate and severe caries groups.

LB	Mild caries	Moderate caries	Severe caries	P-valuea
	(n = 24)	(n = 23)	(n = 25)
	n (%)	n (%)	n (%)
<104	12 (50.0)	12 (52.2)	6 (24.0)	0.279
104–105	9 (37.5)	5 (21.7)	11 (44.0)
105–106	3 (12.5)	5 (21.7)	6 (24.0)
>106	0 (0.0)	1 (4.4)	2 (8.0)

Chi-square Test.

Buffer capacity

The salivary buffer capacity was not associated with caries severity. There were no significant differences of buffer capacity between mild, moderate and severe caries groups (Table 1, ANOVA test, P = 0.898).

Discussion

Since salivary micro flora reflects the gross composition of the microbial deposits on the various oral surfaces, salivary counts of cariogenic bacteria can be used as an indicator of dental plaque cariogenicity. Theoretically plaque is more appropriate for estimating MS infection, and several studies have shown that MS levels in stimulated saliva correctly reflect those in plaque. In this study unstimulated saliva was used because stimulated saliva sampling by paraffin is often impossible in preschool children. According to the study done by Seki et al., the unstimulated saliva could replace stimulated saliva for testing of preschool children.

Previous studies have shown that MS and LB are associated with dental caries., These bacteria species have been reported as potential contributors to caries onset and development., Therefore in this study, the salivary MS and LB levels in preschool children were evaluated in an attempt to determine the correlation between MS, LB level and the severity of early childhood caries.

A study done by Ramos-Gomez and associates showed that salivary MS levels among young children with ECC were higher than those in dentally healthy children. In other studies, which compared children with S-ECC and caries free children, the S-ECC group had higher salivary MS levels than the caries free group., A systematic review article showed that MS level is a strong risk indicator for ECC. In present study, a positive correlation was found between severity of ECC (measured by the dmft index) and high levels of MS counts, indicating that as number of MS colonies increased, the number of teeth affected by caries also increased. Therefore, not only caries occurrence but also caries severity was significantly associated with salivary MS counts in these children. Therefore preventing early colonization of MS in young children may play an important role in reducing the risk of early childhood caries.

On the other hand, the association between lactobacilli and caries remain equivocal.20, 21, 22 In present study, there was also a tendency toward a higher salivary LB level in children with more severe caries, but the difference was not significant.

Our study population comprised of patients who visited Pediatric Dentistry of Taichung Veterans General Hospital, Taiwan. Most of them were referral patients with a high caries rate; it was not easy to find caries free children in this population. In our study population, approximately one third of children had early childhood caries, and two thirds of them were severe early childhood caries. Therefore we divided participants into mild, moderate and severe caries groups, and significant difference between the salivary MS levels and the severity of early childhood caries was found in the present study.

A systemic review in 2015 revealed that salivary markers have generally proved unhelpful in the formal assessment of caries risk in 0–5 year old age group. The present study confirmed that buffer capacity was not correlated with the severity of early childhood caries in preschool children.

ECC is a chronic, transmissible, infectious disease whose etiology is complex and multifactorial. Therefore, control of other risk factors is also import in preventing early childhood caries. In conclusion, the salivary MS levels in preschool children were significantly correlated with the caries severity. The higher the salivary MS level was, the more severe the caries was in preschool children. The levels of MS in the oral cavity play an important role in caries development of preschool children. There was also a tendency toward a higher salivary LB level in children with more sever caries. The salivary buffer capacity was not associated with caries severity.

Conflicts of interest

The authors have no conflicts of interest relevant to this article.