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J Dent Res. Apr 2010; 89(4): 378–383.
PMCID: PMC2880172
NIHMSID: NIHMS203892

Microbial Risk Markers for Childhood Caries in Pediatricians’ Offices

Abstract

Dental caries in pre-school children has significant public health and health disparity implications. To determine microbial risk markers for this infection, this study aimed to compare the microbiota of children with early childhood caries with that of caries-free children. Plaque samples from incisors, molars, and the tongue from 195 children attending pediatricians’ offices were assayed by 74 DNA probes and by PCR to Streptococcus mutans. Caries-associated factors included visible plaque, child age, race, and snacking habits. Species were detected more frequently from tooth than tongue samples. Lactobacillus gasseri (p < 0.01), Lactobacillus fermentum, Lactobacillus vaginalis, and S. mutans with Streptococcus sobrinus (all p < 0.05) were positively associated with caries. By multifactorial analysis, the probiotic Lactobacillus acidophilus was negatively associated with caries. Prevotella nigrescens was the only species (p < 0.05) significantly associated with caries by the ‘false discovery’ rate. Analysis of the data suggests that selected Lactobacillus species, in addition to mutans streptococci, are risk markers for early childhood caries.

Keywords: early childhood caries, S. mutans, Lactobacillus

Introduction

Despite a high dental caries prevalence (28%) in US pre-school children (Beltran-Aguilar et al., 2005), treatment is not universally available, particularly for disadvantaged children (Siegal et al., 2005). Thus, there is need for dental care through government and state programs (Edelstein, 2000; Lee et al., 2004), as well as for dental screenings, parental counseling, and fluoride application in pediatricians’ offices (Lewis et al., 2000). Since dental caries is a bacterial infection, testing for caries microbial biomarkers could facilitate identifying children at greatest risk for caries and most in need of care.

The primary pathogens associated with dental caries are Streptococcus mutans and Streptococcus sobrinus, the mutans streptococci. Other caries-associated species include non-mutans Streptococcus, Lactobacillus, Actinomyces, Bifidobacterium, and Veillonella species (Van Houte, 1993). Studies of early childhood caries (ECC) microbiota using cultural (Marchant et al., 2001) and molecular approaches (Corby et al., 2005; Aas et al., 2008) further expanded the range of species detected in caries. Several reports have also documented the detection of periodontal species in young children, including Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Parvimonas micra (Peptostreptococcus micros), Tannerella forsythia, and Treponema denticola (Kamma et al., 2000; Tanner et al., 2002b). Early colonization with periodontal pathogens probably reflects gingival inflammation, but could indicate risk of periodontitis in adulthood.

This study aimed primarily to determine microbial risk markers for early childhood caries that could be used in risk assessment for caries development. As a possible location for screening young children, microbial samples were obtained from pediatricians who served socio-economically disadvantaged children at increased risk for ECC. Positive associations between bacterial species with caries would suggest that they are caries biomarkers. These could be used in risk assessment to identify children most at risk for caries development and progression, and who would benefit from dental counseling and preventive protocols.

Materials & Methods

Study Population

Pre-school children were recruited in a cohort study from pediatric clinics (Boston Medical Center-Boston University, Floating Hospital-Tufts Medical Center, Boston, MA, USA). Inclusion criteria were that the child was from 1 to 6 yrs of age, had not taken antibiotics in the preceding 3 mos, and the parent or guardian was willing to consent to the child’s clinical examination and microbial sampling. The study design, protocol, and informed consent were examined and approved by the Institutional Review Boards of the institutions involved. Children consenting to microbial samplings were from a larger cohort of children participating in a study to evaluate the efficacy of pediatrician training to reduce rates of early childhood caries (Kressin et al., 2009).

Clinical Examination, Socio-demographic Information, and Bacterial Sampling

Children were examined by two trained and calibrated dental hygienists. The child’s socio-demographic characteristics, diet, and oral health practices were collected by survey (Table 1). Presence of teeth and their status as sound, pre-cavitated, cavitated, filled, or sealed were recorded for each tooth (Drury et al., 1999). Visible plaque was recorded on a 0-3 scale (no plaque, plaque covering a mean surface area of <1/3, 1/3 to <2/3, or >2/3 by tooth, respectively).

Table 1.
Demographic and Clinical Characteristics of Study Population

Plaque samples were taken with sterile toothpicks (Milgrom et al., 2000) from: (i) the gingival third of the labial coronal surfaces of incisors, (ii) the buccal and interproximal surfaces of molars, and (iii) across the midline of the tongue. In children with caries (pre-cavitated and cavitated), plaque was sampled from lesions. Samples were collected in 100 μL of 50 mM Tris-EDTA buffer (pH 7.6) and kept frozen at -70°C until microbial analysis.

Microbial Analyses

Plaque samples were analyzed by DNA probes to 74 species (Table 2) in a checkerboard assay as previously described (Tanner et al., 2002a; Socransky et al., 2004). Samples were denaturated, neutralized, and fixed onto a nylon membrane, the last lanes of which were used for quantitation, containing all probe species mixed as a DNA standard at 105 and 106 bacterial cell equivalents.

Table 2.
Microbiota of Incisor, Molar, and Tongue Samples of Pre-school Children

For S. mutans PCR detection, samples were treated with proteinase K (Dewhirst et al., 2000). The assay was run with S. mutans-specific reverse primer 5′-ACT CCA GAC TTT CCT GAC CG-3′ and forward universal primer 5′-GAG TTT GAT YMT GGC TCA G-3′ (Paster et al., 1998). The amplification products were visualized with 1% agarose gels. If an amplicon was not detected, a universal PCR (primers: forward as above, reverse 5′-AAG GAG GTG WTC CAR CC-3′) was run to verify the presence of sample DNA.

Data Analyses

Decayed (d), comprising pre-cavitated and cavitated lesions, and filled (f), not including sealants, primary teeth (t) for each child were summarized by a decayed-filled tooth score (dft). Plaque was dichotomized to ‘presence’ and ‘absence’, since half (48%) of the observations were ‘no plaque’ and half (52%) were ‘plaque detected’. Species detection was at ≥ 105 cells with the use of DNA probes (equivalent to the lower DNA internal standard), or a positive PCR reaction. Tooth and tongue samples were matched by child. We used Student’s t test to compare differences in means, chi-square test for proportions between socio-demographics, plaque, species detection, and caries, and McNemar’s test for species detection at paired intra-oral sites. A p-value ≤ 0.05 was considered significant. Results were also adjusted for multiple comparisons by the false-discovery rate (α = 0.05) (Benjamini and Hochberg, 1995). We used regression analysis to evaluate child caries extent with age and plaque and their interactions, and model sensitivity analysis was performed by overdispersed Poisson regression. Statistical analyses were performed with SPSS® software.

Multivariate analysis was performed with Partial Least Squares (PLS) modeling (SIMCA P, Umetrics, Umeå, Sweden) (Hellberg et al., 1986). PLS is a linear model that detects correlations between matrices of each independent (x) variable and outcome (y) variables to generate model R2- (estimated model explanation) and Q2- (estimated model prediction) values, and variable Importance in Projection (VIP) values, which, if >1.0, indicates influential and, if VIP ≥ 1.5, highly influential x-variables. Separate and combined microbial and survey data were modeled on caries (dft) by logarithmic transformation [log10(dft) after input of 0.01 for 0] and dichotomization (0 for dft = 0 and 1 for dft > 0) of dft values. SIMCA P auto-centering and scaling to unit variance were applied to all variables, and leave-one-out cross-validation was used. Generally, R2- and Q2- values should not differ by more than 0.2.

Results

Demographic and Clinical Characteristics

The population sample was comprised of 195, predominantly Black (77%), children (Table 1). For most children, parental education was ≤ 12 yrs (57%), and annual household income was < $20,000 (46%). Caries was detected in 18% of the children, mainly affecting Asian (42%) and Black (19%) children (p < 0.05) (Table 1). Survey variables associated with caries were: previous child dental visits, crackers, potato chips, and cereal consumption (all p < 0.05), and visible plaque (p < 0.001) (Table 1). In the children without visible plaque, caries (6%) was unrelated to age. In the children with plaque (52% of children), caries (28%) was related to age (p = 0.02). Caries extent (dft) increased with plaque presence and higher age (p < 0.05, data not shown). Child age (p = 0.0041) and detectable plaque (p < 0.0001) were also significant after sensitivity analysis by overdispersed Poisson regression.

Microbiology

DNA probe data from matched tooth and tongue samples (Table 2 and Appendix Table) showed a consistently higher species detection frequency from teeth compared with the tongue. Species were detected at similar frequencies from incisor and molar samples (p = 0.09-1.00). The most frequently detected species (> 60% of children) included Streptococcus and Actinomyces species, Rothia dentocariosa, Filifactor alocis, and Veillonella parvula. Caries-associated species (p ≤ 0.05) from molar plaque samples (Fig. 1 and Appendix Fig) included: S. mutans,S. mutans with S. sobrinus (p = 0.03), Streptococcus intermedius, Lactobacillus vaginalis, Lactobacillus fermentum, Lactobacillus gasseri (p < 0.01), Actinomyces odontolyticus, Actinomyces israelii, and V. parvula (p < 0.01). Subgingival species detected more frequently from caries-affected children (p ≤ 0.05) included Eubacterium brachy, P. micra, F. alocis, P. gingivalis (p < 0.01), Prevotella nigrescens, Porphyromonas endodontalis, A. actinomycetemcomitans, and Leptotrichia buccalis (p < 0.05). P. nigrescens remained significant (p < 0.05) after multiple-comparisons adjustment.

Figure 1.
Microbiota of caries-affected and caries-free children. Bacterial species withz > 5% difference in detection between caries-affected and caries-free children are in the same order as in Table 2. Most bacterial species assayed were detected more ...

S. mutans was detected by PCR in 41% molar, 32% incisor, and 14% tongue plaque samples from 169 children with valid samples from all sites (data not shown). Detection frequencies of S. mutans from caries-affected and caries-free children, respectively, were: molar, 69% and 35%; incisor, 63% and 25% (both p < 0.001); and tongue, 22% and 12% (p = 0.167). S. mutans was associated with children with plaque compared with plaque-free children in molar (51% and 31%) (p < 0.01), incisor (42% and 21%) (p < 0.01), and tongue (20% and 6%) (p <0.05) samples, respectively.

Initial multivariate analyses (PLS) identified age and number of teeth at risk as influencing caries detection. Modeling, performed in age groups (< 2 yrs, ≥ 2 to < 3 yrs, and ≥ 3 yrs of age) with dft as the dependent variable, indicated that bacterial detection and the presence of visible plaque yielded statistically significant models for the three age groups (R2 = 0.78, Q2 = 0.21; R2 = 0.54, Q2 = 0.15; and R2 = 0.53, Q2 = 0.14, respectively). There were no significant models for caries with the survey data alone, and combining microbial and survey data improved the model values only marginally.

When data from all children were analyzed, plaque and S. mutans detection by PCR were highly influential variables for higher dft scores (VIP > 1.5) (data not shown). L. vaginalis, A. odontolyticus, A. israelii, P. nigrescens, Prevotella loescheii, P. gingivalis, A. actinomycetemcomitans, and V. parvula were consistently influential in children with higher dft (VIP > 1.0). In contrast, Lactobacillus acidophilus, Lactobacillus reuteri, Eubacterium saburreum, Eubacterium nodatum, and Campylobacter gracilis were consistently influential in children with lower dft (VIP > 1.0).

When data were restricted to children with visible plaque, S. mutans by PCR was highly associated with caries (model R2 = 0.48, Q2 = 0.23) (VIP>1.5, Fig. 2). A. israelii, Eubacterium timidum, A. actinomycetemcomitans, and V. parvula were influential with caries extent (higher dft) (VIP > 1.0-1.3), whereas L. gasseri, B. dentium, A. odontolyticus, and P. nigrescens were borderline influential (VIP = 0.9). In children with plaque, the significant negative influence of L. acidophilus, E. saburreum, and E. nodatum on caries was confirmed (VIP > 1.5), and several additional species exhibited highly influential negative associations with caries (VIP = 1.0-1.4, Fig. 2). Caries-associated species explained 50% of caries extent.

Figure 2.
Scatter plot of Partial Least Squares (PLS) weights w*c for bacterial data modeled on caries prevalence (dft) in children with visible plaque (n = 102). For a given PLS model, one vector of X-weights w*a and one vector of Y-weights ca are obtained for ...

Log-transformed and dichotomized dft gave similar results in all PLS models.

Discussion

Microbiology sampling was successfully integrated into pediatric practices serving low-income populations as a means of seeking microbial biomarkers for ECC. This study expanded microbial risk markers for ECC beyond mutans streptococci to include selected Lactobacillus species. Non-microbial caries risk factors included visible plaque, child age, race, and snacking habits. Detection of subgingival/periodontal species in children was confirmed. While the PLS statistical models showed low predictive power, R2-Q2 > 0.2, and suggested cautious interpretation of findings, overall the data are generally consistent with previous reports, as discussed below.

S. sobrinus detection with S. mutans showed high associations with ECC, which is consistent with studies correlating this species combination with caries development (Seki et al., 2006) and higher caries prevalence than S. mutans alone (Okada et al., 2005). Among the non-mutans streptococci, S. intermedius was associated with ECC, as previously reported in a different population of young children (Tanner et al., 2002b).

Caries-associated Lactobacillus species in the current report included L. gasseri, previously detected in childhood and adult caries (Munson et al., 2004; Corby et al., 2005), L. fermentum, detected in childhood caries (Marchant et al., 2001; Aas et al., 2008), and L. vaginalis, reported in caries-active young women’s saliva (Caufield et al., 2007). Most of these Lactobacillus species were also detected by molecular methods from adult carious dentin (Chhour et al., 2005). In contrast, the Lactobacillus species L. rhamnosus, L. acidophilus, L. plantarum, and L. reuteri, which are used in probiotic infant enteric and/or oral anti-caries therapy (Savino et al., 2007; Twetman and Stecksén-Blicks, 2008), showed negative associations with caries.A. israelii and A. odontolyticus were detected more frequently in caries, as previously reported (Marchant et al., 2001; Tanner et al., 2002b).

Subgingival periodontal species were also detected in these young children, particularly from the molar plaque samples that included gingival bacteria. Subgingival species detected included A. actinomycetemcomitans, P. micra, P. gingivalis, T. forsythia, and T. denticola, as has previously been reported in young children (Könönen et al., 1992; Tanner et al., 2002b), at detection frequencies consistent with cultural assay (Kamma et al., 2000). Other subgingival species associated with adult periodontitis (Kumaret al., 2005), but infrequently reported in pre-school children, included E. brachy, Eubacterium saphenum, F. alocis, and Selenomonas flueggei. Colonization of these species probably reflected gingivitis (not consistently measured in this study), but may pose risk for future periodontitis.

Increased species detection in caries may be a reflection of larger plaque samples. Nonetheless, comparable caries-associated species were detected in all children compared with children with visible plaque. In other studies, visible plaque was strongly associated with caries (Wennhall et al., 2002; Mohebbi et al., 2006), and was an indicator for future caries development in young children (Alaluusua and Malmivirta, 1994).

For microbial screening, plaque sampling should be easy and rapid. Similar species detection rates between incisors and molars suggest that either tooth site could be sampled, in contrast to tongue samples, which exhibited lower detection frequencies of DNA probe species. Lower species detection from the tongue than from the teeth suggests that screening for caries pathogens in tongue samples is less sensitive than screening samples from teeth, and may result in false-negative data.

Caries prevalence in this study was similar to US national levels (Beltran-Aguilar et al., 2005). Increased caries experience in Asian and Black compared with White children has also been reported, with Asians having the highest caries prevalence (30%) and untreated caries (49%) (Shiboski et al., 2003). There was more caries (dft) in the older children, reflecting the longer time teeth were exposed to caries-risk factors. Other caries-associated risk factors were consistent with previous findings. Frequent snacking was associated with caries. High-starch foods like potato chips and crackers stay in the mouth longer and aid in increased acid production, contributing to caries development (Kashket et al., 1996). The association of ECC and having a dentist might seem counterintuitive. Families with children with cavities and possibly pain, however, are more likely to seek treatment than are dentally healthy children.

In conclusion, children in disadvantaged populations were sampled to evaluate microbial risk biomarkers for caries, indicating the feasibility of accessing these children while attending routine and emergency visits in pediatricians’ offices. Using univariate and multivariate approaches, we associated selected Lactobacillus species, in addition to mutans streptococci, with early childhood caries. Future study will indicate if these species are risk indicators for future caries, and if they can be used to select children most in need of preventive and treatment regimens.

Supplementary Material

Supplemental Data:

Acknowledgments

We thank Harpreet Singh and Cindy Cadoret for subject recruitment, clinical measurements, and sampling.

Footnotes

A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental.

This investigation was supported by USPHS grants U54 DE-014264, DE-015847, DE-007151-18, DE-007327-07, and K24 DE000419 from the National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA, and by a Medical Faculty PhD grant, Umeå University, Sweden.

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