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. 2011 Nov;90(11):1298–1305. doi: 10.1177/0022034511421201

Microbiota of Severe Early Childhood Caries before and after Therapy

ACR Tanner 1,3,*, RL Kent Jr 2,4, P Lif Holgerson 1,5, CV Hughes 6, CY Loo 7, E Kanasi 1,4,a, NI Chalmers 1,b, I Johansson 5
PMCID: PMC3188461  PMID: 21868693

Abstract

Severe early childhood caries (ECC) is difficult to treat successfully. This study aimed to characterize the microbiota of severe ECC and evaluate whether baseline or follow-up microbiotas are associated with new lesions post-treatment. Plaque samples from 2- to 6-year-old children were analyzed by a 16S rRNA-based microarray and by PCR for selected taxa. Severe-ECC children were monitored for 12 months post-therapy. By microarray, species associated with severe-ECC (n = 53) compared with caries-free (n = 32) children included Slackia exigua (p = 0.002), Streptococcus parasanguinis (p = 0.013), and Prevotella species (p < 0.02). By PCR, severe-ECC-associated taxa included Bifidobacteriaceae (p < 0.001), Scardovia wiggsiae (p = 0.003), Streptococcus mutans with bifidobacteria (p < 0.001), and S. mutans with S. wiggsiae (p = 0.001). In follow-up, children without new lesions (n = 36) showed lower detection of taxa including S. mutans, changes not observed in children with follow-up lesions (n = 17). Partial least-squares modeling separated the children into caries-free and two severe-ECC groups with either a stronger bacterial or a stronger dietary component. We conclude that several species, including S. wiggsiae and S. exigua, are associated with the ecology of advanced caries, that successful treatment is accompanied by a change in the microbiota, and that severe ECC is diverse, with influences from selected bacteria or from diet.

Keywords: pediatric dentistry, clinical outcomes, microbial ecology, Streptococcus mutans, Scardovia wiggsiae

Introduction

Severe early childhood caries can devastate the primary dentition (Hughes et al., 2011), is a risk factor for caries in permanent teeth (Li and Wang, 2002), and is difficult to treat successfully (Almeida et al., 2000; Graves et al., 2004). In the US, while 28% of children have ECC (Beltran-Aguilar et al., 2005), only a subset of children have advanced disease, mainly concentrated in populations of low socio-economic status (Beighton et al., 2004; Edelstein and Chinn, 2009). Treatment is difficult because of the young age of children and is frequently performed while the children are under general anesthesia. Further treatment may be of limited benefit, because up to 50% of children have new caries lesions after therapy (Almeida et al., 2000; Graves et al., 2004).

The etiology of severe ECC includes infection with selected bacteria, and dietary and host factors, which are influenced by environment and socio- economic status (Beighton et al., 2004). Differences in the plaque microbiota were observed in children from different socio-economic backgrounds, suggesting diversity within ECC (Beighton et al., 2004). We recently confirmed that Streptococcus mutans was strongly associated with severe ECC, although at a low proportion of the carious plaque microbiota, using molecular (Kanasi et al., 2010a) and culture-based (Hughes et al., 2011) assays. Other species significantly associated with severe ECC included Scardovia wiggsiae, by culture (Tanner et al., 2011), and Bifidobacteriaceae, by PCR (Palmer et al., 2010).

To investigate the reasons for new lesions post-treatment, this study aimed to compare the microbiota of caries-free with that of severe-ECC children, and to compare pre-treatment and follow-up microbiotas in children with and without new caries lesions. We modeled microbial, clinical, and dietary characteristics to evaluate whether subgroups existed within severe ECC, to improve our understanding of this complex infectious disease.

Materials & Methods

Clinical Methods

Children aged 2 to 6 yrs were recruited as previously described (Palmer et al., 2010). Children were medically healthy, had not used antibiotics within the preceding 3 mos, and the parent or guardian provided informed consent for the child’s participation. Severe-ECC children (n = 53) had extensive lesions in the primary dentition (Table), and 32 children were caries-free (Hughes et al., 2011). The study design, protocol, and informed consent were approved by the Institutional Review Boards of participating institutions. The children’s parents or caregivers completed a demographic and dietary survey (Palmer et al., 2010).

Table.

Demographic and Clinical Characteristics of Study Population

Caries-free n = 32 Severe ECC n = 53 p value
Mean age (yrs) ± SEM a 3.66 ± 0.17 3.97 ± 0.13 0.160 b
Gender: Male 15 (47%) 30 (56%) 0.44 c
Race:
 White 13 (32%) 16 (30%) 0.874 c
 Black 9 (28%) 20 (37%) 0.398 c
 Asian 12 (36%) 11 (32%) 0.568 c
 Other/mixed 1 ( 3%) 1 (2%) 0.608 c
Ethnicity: Hispanic 6 (17%) 13 (24%) 0.565 c
Clinical Characteristics
 Mean percent carious surfaces 0 32.4 ± 2.67
 Mean Plaque Index ± SEM 0.58 ± 0.08 4.79 ± 0.13 < 0.0001b
 Mean Gingival Index ± SEM 0.16 ± 0.07 1.54 ± 0.13 < 0.0001b
 Mean percent sites bleeding ± SEM 2.0 ± 1.2 52.0 ± 4.9 < 0.0001b
Survey Characteristics
 Parents with cavities 66 % 86% 0.027 c
 Smoker in household 44% 84% 0.026 c
 Number of juice drinks/wk ± SEM 6.5 ± 0.7 9.7 ± 0.9 0.011 b
 Drinks juice between meals 34% 65% 0.016 c
 Liquid cariogenic foods d ± SEM 2.31 ± 0.33 3.69 ± 0.31 0.004 b
  Juice and juice drinks, ice cream, sweetened yogurt, diet and regular soda
 Solid cariogenic foods d ± SEM 2.47 ± 0.31 4.88 ± 0.46 < 0.001b
  Bread, potato chips, crackers, jam/jelly, sweetened cereal, bananas, cookies
 Eating or drinking frequency/day 5.29 ± 0.24 6.16 ± 0.15 0.0016 b
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a

SEM = Standard Error of the Mean.

b

Student’s t test.

c

Chi-square test.

d

Cariogenicity coding (from Palmer et al., 2010).

Clinical measurements and plaque sampling were performed on children at baseline, and at 6 and at 12 mos after comprehensive restorative and extraction therapy under general anesthesia and caries-preventive measures for severe-ECC children (Hughes et al., 2011). Plaque samples were taken from the mesial interproximal sites of primary molars, placed in 100 µL TE buffer, and frozen at -70°C until microbial analysis (Palmer et al., 2010). For children with new caries lesions, the visit during which caries was detected (6 or 12 mos) was the last visit for that child.

Microbiological Methods

DNA from plaque samples was purified with the use of MasterPure kits (Epicenter, Madison, WI, USA) as previously described (Palmer et al., 2010).

Microarray analysis

Samples were analyzed by microarray to over 300 bacterial taxa by the Human Oral Microbe Identification Microarray (HOMIM) assay as previously described (Preza et al., 2009). The lower limit of detection for the HOMIM array is approximately 104 bacterial cells. Only probes that were reactive to at least one sample were evaluated.

Bacterial-specific PCR analysis

PCR was performed for the detection of S. mutans, S. sobrinus, and Bifidobacteriaceae (Palmer et al., 2010). Scardovia wiggsiae PCR was performed with species-specific primers designed with AlleleID® (PREMIER Biosoft International, Palo Alto, CA, USA) in 16S rRNA. The forward primer was (Scar448F) 5′- GTG GAC TTT ATG AAT AAG C -3′ and reverse primer (Scar619R) 5′- CTA CCG TTA AGC AGT AAG -3′. The samples were amplified by pre-heating (94°C, 2 min), denaturation (94°C, 20 sec), annealing (51°C, 20 sec), and elongation (72°C, 20 sec) (40 cycles), with a final extension (72°C, 5 min). Amplicons were verified on 1.5% low-melt agarose gels (NuSieve® GTG®, Lonza, Rockland, ME, USA). The specificity of the primers was evaluated by testing against Scardovia inopinata, Parascardovia denticolens, Alloscardovia omnicolens, Bifidobacterium dentium, Bifidobacterium subtile, and selected Actinomyces, Corynebacterium, Propionibacterium, Mobilun- cus, and Rothia species. Amplicons from samples from the S. wiggsiae primers were sequenced and compared against the Human Oral Microbiome Database (HOMD) (Dewhirst et al., 2010).

Statistical Analyses

Mean differences between caries-free and severe-ECC children in age, plaque, gingival, and bleeding indices and dietary characteristics were evaluated by t test. Data from microarray and PCR were analyzed as detected or not-detected by child. Differences in proportions between disease categories and demographic characteristics, probe reactions from the microarray, and PCR were tested by Chi-square. Severe-ECC children, divided into children with and without new lesions post-therapy, and baseline and follow-up microbial data were compared by McNemar Chi-square. A significance threshold of p ≤ 0.05 was used. In addition, adjustment for multiple comparisons was by the false-discovery rate (Benjamini and Hochberg, 1995). Statistical analyses were performed with SPSS® software.

Partial least-squares analysis (OPLS procedure) with SIMCA P+ (version 12.0, Umetrics AB, Umeå, Sweden) was used for multivariate analysis of microarray, PCR, dietary, clinical, and demographic information (Table) as previously described (Kanasi et al., 2010b; Lif Holgerson et al., 2011). PLS is a multivariate linear regression model method suitable for the detection of correlations between matrices of descriptor and response variables in datasets where the descriptor variables co-vary and/or the numbers exceed the numbers of observations. Variable auto-scaling to unit variance and cross-validation (Q2) was done by the software.

Results

There were no differences in child age, gender, race, or ethnicity between the 53 severe-ECC and 32 caries-free children (Table). Severe-ECC children had higher plaque and gingival indices and bleeding scores than caries-free children. Putative cariogenic foods and eating and drinking frequencies were more frequent in severe-ECC than caries-free children (Table).

Taxa detected more frequently by microarray at baseline in severe-ECC than caries-free children included Slackia exigua (p = 0.002), Streptococcus parasanguinis I and II (p = 0.013), Prevotella melaninogenica (p = 0.013), and Prevotella Cluster IV (p = 0.018) (Fig. 1A, Appendix Table). Species detected more frequently in caries-free children included Cardiobacterium hominis (p = 0.003), Leptotrichia hofstadii (p = 0.006), and Corynebacterium matruchotii (p = 0.017). Species detected more frequently at baseline in severe-ECC children who subsequently had new caries lesions than in ECC children without new lesions included Prevotella Cluster IV (p = 0.001), Prevotella nigrescens (p = 0.01), and several Capnocytophaga taxa. None of the differences in microarray data was significant after adjustment for multiple comparisons.

Figure 1.

Figure 1.

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(A) Microbiota of severe-ECC and caries-free children by microarray. Percentage of children with reactivity to probes in HOMIM microarray. Taxa were selected as showing some difference between disease categories, with taxa detected more frequently in severe ECC to the left and in caries-free to the right. For data from all probes, see Appendix Table. Taxa showing associations with severe ECC included Slackia exigua, S. parasanguinis I & II, Prevotella species, and Neisseria flavescens. S. mutans was detected in 25% severe-ECC children in the microarray. (B) Microbiota of severe-ECC and caries-free children by taxon-specific PCR. Taxa showing greatest association with severe ECC were Bifidobacteriaceae, S. wiggsiae, and S. mutans in combination with these taxa. S. mutans was detected in 56% severe-ECC children by species-specific PCR.

The PCR primers for S. wiggsiae detected S. wiggsiae but did not react with species in Bifidobacteriaceae or the Phylum/Class Actinobacteria. All amplicons from PCR of plaque samples for S. wiggsiae showed homology to S. wiggsiae sequences in HOMD. By specific PCR, S. mutans, S. sobrinus, Bifidobacteriaceae, and Scardovia wiggsiae individually, and the combinations S. mutans with S. sobrinus (p < 0.02), S. mutans with Bifidobacteriaceae (p < 0.0001), and S. mutans with Scardovia wiggsiae (p < 0.005) were associated with severe ECC (Fig. 1B).

Children with subsequent lesions (n = 17) compared with none (n = 36) had no differences in baseline gingival index, bleeding, number of carious surfaces, restorations placed, or teeth extracted (data not shown). For 16 out of 17 children, the new lesions on follow-up were on surfaces without a previous restoration.

By microarray, severe-ECC children without new lesions (Fig. 2A) showed post-therapy reductions in S. mutans (not detected in follow-up), Streptococcus mitis biovar 2 (p < 0.035), Campylobacter gracilis (p < 0.02), Campylobacter concisus/rectus (p = 0.004), Capnocytophaga species Human Oral Taxon from HOMD (HOT) 335 (p = 0.004), and Selenomonas taxa. Species that were detected more frequently in these children post-therapy were Propionibacterium propionicum (p < 0.002), Lautropia mirabilis (p < 0.026), and 3 Capnocytophaga species. Children without new lesions also had significantly lower plaque index (p < 0.001), gingival index (p < 0.0001), and bleeding gingival sites (p < 0.0001) in follow-up compared with pre-treatment. By PCR, only S. mutans was detected less frequently in follow-up (p = 0.035).

Figure 2.

Figure 2.

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(A) Microbiota pre-treatment and at follow-up of severe-ECC children without new lesions after 12 mos monitoring. Taxa were selected as showing some difference between pre-treatment and follow-up, with taxa detected more frequently pre-treatment to the left and in follow-up to the right. Taxa detected less frequently 12 mos after treatment included S. mutans, C. concisus/C. rectus, C. gracilis, S. mitis biovar 2, and unnamed Capnocytophaga and Selenomonas taxa. Species detected more frequently in follow-up included P. propionicum, L. mirabilis, and C. sputigena. (B) Microbiota pre-treatment and at follow-up of severe-ECC children with new lesions during 12 mos of monitoring. Taxa are the same and in the same order as in Fig. 2A. While there is variation in taxa detection between pre-treatment and follow-up, none of the differences was significantly different. Comparison of Figs. 2A and 2B also illustrates baseline differences between the microbiota of children with and without new lesions in follow-up, including Prevotella Cluster IV (p = 0.001), Prevotella nigrescens (p = 0.01), and several Capnocytophaga taxa (Appendix Table).

There were no significant differences in baseline and follow-up microbiota of severe-ECC children with new caries lesions (Fig. 2B), although there were reductions at follow-up in plaque index (p < 0.02), gingival index (p < 0.0001), and bleeding gingival sites (p < 0.04).

The multivariate model of baseline microbial, clinical, and food cariogenicity data yielded a strong model with 2 significant components (R2= 0.646, Q2= 0.462). This model clustered the children into caries-free children (n = 32) and two subgroups [C1 (n = 34) and C2 (n = 19)] of severe-ECC children (Fig. 3). The factors associated with being caries-free were consistent with the univariate observations (Fig. 3, Appendix Figure).

Figure 3.

Figure 3.

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PLS modeling plots. Inset: PLS score plot based on demographics, diet, and microbiota of severe-ECC and caries-free children. Children clustered into three groups, caries-free (Inline graphic) and two ECC subgroups, C1 and C2 (Inline graphic). The PLS model used caries at baseline as the dependent variable, and the microarray, PCR, dietary, clinical, and demographic information as the independent matrix. Main Plot: PLS loading scatter plot illustrating important variables for clustering. Cluster groups are as in the score plot (inset). Taxa with VIP-values > 1.0 are indicated as follows: HOMIM-detected taxa located in the lower left characterize caries-free children (Inline graphic), whereas taxa located in the top and to the right characterize severe-ECC children [Inline graphic and Inline graphic (for species detected by PCR)]. Circles (Inline graphic) represent other variables used in the analysis but not influential for clustering (VIP-values ≤ 1.0). Important taxa differentiating caries-free and severe-ECC children are generally consistent with those that differed in the univariate analyses.

Restricting the multivariate model to severe-ECC children (model R2= 0.784, Q2= 0.622) confirmed separation into two caries subgroups (C1 and C2). Scores for plaque, gingivitis and bleeding; PCR detection of S. mutans, S. wiggsiae, S. mutans combined with S. wiggsiae, S. mutans combined with S. sobrinus; and microarray detection of Slackia exigua did not differ significantly between the two caries subgroups, though values tended to be higher in group C2. Children in subgroup C1, however, were specifically characterized by higher intake of solid cariogenic foods, being White and presence of Haemophilus parainfluenzae (Fig. 3, Appendix Fig.). Children in caries subgroup C2 were specifically characterized by higher detection frequencies of multiple bacterial species, including Prevotella by microarray, and PCR detection of S. sobrinus and Bifidobacteraceae alone or in combination with S. mutans (Fig. 3, Appendix Fig.). The differences between cluster groups were also significant by univariate analyses (data not shown).

Clinically, although not significant, children in subgroup C1 had more carious surfaces (44%) than those in subgroup C2 (32%) before treatment (p = 0.07), and on follow-up, more children in subgroup C2 (47%) experienced new caries lesions than those in subgroup C1 (15%) (p = 0.06).

Discussion

In this study, we confirmed differences in the microbiota of caries-free children and S-ECC children, and observed differences in the microbiota of children with and without caries progression after treatment. In the children without new lesions, there was reduction in several species in addition to S. mutans. Using modeling based on the microbiota, high cariogenic diet, and clinical features, we observed two groups of severe-ECC children, suggesting diversity within severe ECC.

A new finding was the association of Slackia exigua with severe ECC, suggesting that the species may be a risk factor for this infection. S. exigua is a fastidious anaerobic Gram-positive rod (Wade et al., 1999) in Actinobacteria (Dewhirst et al., 2010) and has been associated with periodontitis (Abiko et al., 2010). Prevotella species were also associated with severe ECC. Prevotella species were detected in ECC (Corby et al., 2005), and cluster IV species P. melaninogenica and Prevotella nigrescens were cultured from the current population (Tanner et al., 2011). Prevotella species were not strongly aciduric (Tanner et al., 2011) or acidogenic, suggesting that their presence reflects the ecological niche of deep dentinal caries (Nadkarni et al., 2004; Chhour et al., 2005), rather than contributing to caries progression.

We developed a PCR assay for S. wiggsiae because of its association with severe ECC by culture (Tanner et al., 2011) and by 16S rRNA gene probes as CX101 (Becker et al, 2002), but this species was detected relatively infrequently in the HOMIM assay. In contrast to our culture data, S. wiggsiae by PCR showed a stronger association with severe ECC than S. mutans, suggesting that the relative importance of these species needs further study. Combinations of S. mutans and S. sobrinus were associated with S-ECC, consistent with improved risk assessment over these species individually for ECC (Okada et al., 2005; Choi et al., 2009). The associations of combinations of bifidobacteria and S. wiggsiae with S. mutans suggest that additional species combinations could be valuable in risk assessment for ECC. None of the species combinations tested, however, was associated with detection of new lesions post-therapy, including S. mutans with S. sobrinus, suggesting that risk assessment for ECC in the general population is different from predicting new lesions in children who already have advanced disease.

Species at baseline associated with children without new lesions post-therapy included Cardiobacterium hominis and S. mitis biovar 2. C. hominis was also associated with control sites for root caries (Preza et al., 2009), and S. mitis was associated with caries-free children by culture as compared with severe-ECC children (Marchant et al., 2001; Tanner et al., 2011), consistent with the current study findings. Several unnamed Capnocytophaga species were detected, but their taxonomy needs clarification before their role in disease can be assessed.

We examined pre-treatment and follow-up microbiotas and observed that several species, including S. mutans, changed in detection in the children without new lesions, suggesting a shift in the plaque microbial complex. There were no significant changes in children with new caries, suggesting minimal change in the microbiota, and that persistence of the pre-treatment microbiota was responsible for caries progression. These observations are in accord with the role of the composition of dental plaque as a bacterial community in health and caries (Marsh, 2006; Takahashi and Nyvad, 2011). Studies focusing on S. mutans observed reduction in this species post-treatment in children without new caries lesions (Simratvir et al., 2010), as observed in the current report and by selective isolation of S. mutans (Hughes et al., 2011).

Multivariate modeling by baseline characteristics grouped children by caries-free or severe-ECC status, with characteristics that were similar to those in univariate analysis. Modeling also revealed diversity within severe ECC. Both severe-ECC groups had poor oral hygiene and a similar level of S. mutans infection, but one group ate more cariogenic foods while the other was characterized by a multispecies complex including Bifidobacteriaceae and S. sobrinus. It seems possible that one group of children had disease associated with an increased infection by cariogenic bacteria compared with the second group. Diversity within ECC is consistent with the observed differences within ECC, based on higher counts of cariogenic bacteria in subsets of children (Beighton et al., 2004), and warrants further investigation.

A strength of this study was the use of the HOMIM microarray that facilitated detection of multiple taxa and revealed ecological changes that occurred after therapy that would not have been possible by assaying only for mutans streptococci and lactobacilli. The major study limitation was in loss to follow-up of children with severe ECC, which limited the post-treatment monitoring time to 12 mos, and reduced statistical power for examining pre- and post-treatment microbiotas in children in the new-lesion group. While a longer follow-up time could have identified additional children with new lesions, in children with 2 years’ follow-up, most of the new lesions were recurrent caries (Jamieson and Vargas, 2007). In the current study, children had new lesions on unrestored surfaces, suggesting that we captured a population with aggressive caries. A technical limitation was reduced detection for specific species with the universal primers needed for the microarray assay. We addressed this by adding specific PCR to selected taxa.

This study aimed to understand the influence of the microbiota on treatment relapses after therapy for severe ECC. Our findings suggest that there are differences in the microbiota before treatment, with increased infection in children with new lesions post-treatment, and differences in treatment response. Further, we propose that severe ECC is a complex infection, with the possibility that the microbiota and diet may have different degrees of influence in different children.

Footnotes

This investigation was supported by USPHS grants DE-015847 and DE-007327 from the NIDCR of NIH, the Colgate-Palmolive Company, and the Henning and Johan Throne-Holst’s Foundation. We acknowledge Vanja Klepac-Ceraj for exploratory data analyses.

The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

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