Association of Colonization with Streptococcus mutans Genotypes from Mothers and Early Childhood Caries

Abstract

Purpose

This study evaluated Streptococcus mutans (Sm) genotypes (GT) between mother and child (M-C) in a high caries risk cohort to explore the association with early childhood caries (ECC).

Methods

Sixty-nine infants (~1 year-old) had periodic oral examination (dmfs) and collection of microbial samples from dental plaque, saliva and oral other surfaces. Their mothers had an examination and plaque collected. Sm isolates were genotyped using repetitive extragenic palindromic-PCR (rep-PCR). Statistical analyses were conducted for associations of Sm in M-C dyads with caries outcomes.

Results

Twenty-seven Sm genotypes (GT) from 3,414 isolates were identified. M-C were categorized as GT Match (N=40) or no-Match (N=29). When modeling the severity of ECC at 36-months (~4 years old), the estimated dmfs in the Match group was 2.61 times that in the no-Match group (P=.014).

Conclusions

Colonization of children with Sm GT that matched with mothers was shown to be highly associated with ECC. Although the data suggest vertical transmission of Sm in 40 of 69 children that shared GT with their mother, it is possible that other individuals transmitted the Sm. Nonetheless, these findings support the importance of the mother's oral microbial status as a contributing influence to their children's oral health.

INTRODUCTION

Early Childhood Caries (ECC); Caries that occurs prior to six years of age continues to be a national problem in the US.¹ As previously reported,² caries is not equally experienced by the population and although caries is on the decline in most population groups in the US, ECC prevalence continues to be high, especially in low socioeconomic minority groups.^{3, 4} As recently reported, in Alabama, caries prevalence is high among a community in one of the 19 Black Belt counties⁵ where the poverty level is widespread in these mostly African American rural counties.⁴

Dental caries is a multifactorial dietobacterial disease with complex socioeconomic, medical, genetic, and other indicators that affect risk for disease^{6, 7}. Nonetheless, the mutans streptococci (MS, i.e., Streptococcus mutans and Streptococcus sobrinus) have been implicated as the major etiologic agent involved in the initiation of human dental caries.^8-10 An important question is the source of initial MS colonization. To identify the source, genetic analysis is an accepted approach for strain identification due to enhanced reliability and reproducibility.¹¹ Prevailing concepts hypothesize that MS require non-shedding surfaces for effective colonization with the source of inoculation primarily from the mother via vertical transmission.^12-14 However, transmission fidelity of S. mutans (Sm) from mother to child varies from 38 percent to 70.6 percent with individuals carrying one or more genotypes.^{12, 15-17} Despite conflicting evidence that has challenged the generally accepted concept of vertical transmission as the mode of initial colonization with MS, the mother as a source is logical due to closest contact when initial colonization occurs^{12, 18}. Among the various genetic analyses that have been applied to screen species, repetitive extragenic palindromic PCR (rep-PCR) has been reported to effectively and consistently generate genotypic profiles of Sm isolates from genomic DNA, identifying genotypic differences of MS isolated from children and adults’ plaque.¹⁹

Maternal colonization with Sm has previously and recently²⁰ been associated with ECC in the offspring, while assessment of caries risk related to mother and child’s genotype further defines the association. The aim of this study was to identify colonization and genotypes (GT) with MS at specific times in children in relation with that of their mothers (mother-child dyads, M-C). This study explored the natural history of caries in children during time of early dental development and the initial characterization of MS in plaque through age four years.

METHODS

Participants

Originally, 96 unrelated African-American infants (average age 1.1 years) and their mothers were recruited to participate in this longitudinal study in a small community setting in Uniontown, Alabama beginning March 2008 and followed periodically for 36-months. The study was approved by the University of Alabama at Birmingham, (UAB) IRB. Parents gave informed consent and waiver of assent for participation of their children. Inclusion criteria were: Healthy infants and parental willingness to participate for the period of study. The infants/children/parent received anticipatory guidance, a professional fluoride treatment, customized oral hygiene guidance upon dental examination and oral sample collection at baseline and every six months (examinations every 12 months) for the three years. The mothers had dental examination and plaque sample collection at the baseline visit and at a two-year follow-up. Dental examinations were conducted by four calibrated dentists using artificial light source, two-by-two gauze, mirror and explorer with no dental radiographs, recording decayed (cavitated), missing, filled surface score (DMFS/dmfs), according to WHO criteria as previously reported.⁴ A total of 23 three to four year old children were examined to assess inter- and intra-examiner reliability.

Sample collections & Bacterial cultures for S. mutans

At each visit, infants/children had pooled and/or individual plaque samples (i.e., primary molars that had erupted after baseline) collected from all tooth surfaces using sterile toothpicks. Samples were transferred to one mL of sterile transport fluid²¹ and transported on ice to the laboratory at UAB (100 miles) for processing within 24 hours. Additionally, oral samples were collected from each child for MS count analysis using sterile tongue scraping (i.e., tongue sample, TS) or cotton swab (i.e, saliva and biofilm sample, SBS) placing in the mouth and rubbing along cheeks, tongue and teeth until saturated with saliva, then placed into 4.5 mL transport fluid. Samples were sonicated (Vibra Cell, Sonics & Materials Inc., Newtown, CT, USA) and dispersed onto MS selective media (mitis salivarius agar, Difco/Beckton Dickinson, Sparks, MD, USA) supplemented with sucrose and Bacitracin²² using a spiral inoculator (Spiral System™ Inc, Cincinnati, OH, USA). Agar plates were anaerobically incubated at 37 degrees Celsius for 48 hours. Resulting colonies were counted using a stereomicroscope for total MS counts quantitation of MS colony-forming units per ml transport fluid (CFU/ml). MS isolates were distinguished by colony morphology on bacterial culture plates. When multiple MS colonies resulted from a sample, up to 10 representative colonies identified as MS were pure streaked and individually inoculated into Todd Hewitt Broth (THB, Beckton Dickinson, Sparks, Maryland, USA). Cell pellets were saved at −20 degrees Celsius until processing for extraction of genomic DNA (see below) and confirmed to be Sm or S. sobrinus (Ss) by SYBR Green PCR using primers published by Yoshida²³ as previously reported²⁴ prior to being genotyped by rep-PCR.

Rep-PCR analysis

THB inocula were centrifuged and cell pellets processed for isolation of genomic DNA as previously described.²⁴ The DNA was quantified using a Nanodrop 1000 (Thermo Scientific, Wilmington, DE) yielding at least 25 ng/microliter of DNA. Rep-PCR reactions were carried out with DiversiLab streptococcus kit (bioMerieux, Durham, NC, USA) and ABI9700 thermocycler (Applied Biosystems, Foster City, CA, USA). DNA amplicons were fractionated with DNA Chip kit (bioMerieux) using microfluidics LabChip device inserted on a model B 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA)^{25, 26}. Resulting dendrograms and virtual gel images indicating strain level groupings of the Sm were generated by Diversilab software (Pearson Correlation coefficient method and unweighted-pair group methodology with arithmetic mean).²⁴ The analyzed individual reports were saved at the DiversiLab website to be utilized for the Sm library and comparisons. Data for GT of each isolate from each sample from a given individual for each period of analysis were stored in a database (FileMaker Pro, version 11, FileMaker Inc., Santa Clara, CA) for generation of reports to analyze in context of commonality and caries activity.

Statistical analysis

Independent variables included Sm in mother at baseline and in children at age two and one-half years. Caries outcomes were defined as both binary (yes/no) and count (dmfs score) in children at an average age of four years, where logistic regression and negative binomial modeling were used, respectively, to analyze the data. All analyses were adjusted for number of child’s and mother’s bacterial isolates. Also, interaction terms were assessed between number of bacterial isolates and other independent variables in different models. For analysis, 69 M-C were organized as to availability of MS isolates for comparison of GT in common. Comparisons of caries history and plaque biology in the Match and no-Match groups were analyzed by Mann-Whitney U test, SAS version 9.3 (SAS Institute Inc., Cary, NC, USA). Statistical significance was set at the P<0.05 level.

RESULTS

Of 96 infants/children recruited to this study at baseline (average age 1.1 years), subject attrition was approximately five percent per year (mostly because of moving out of the community) resulting in 69 M-C that continued to participate through the 36-month follow-up. By the 36-month follow-up visit, 68 children had at least one Sm isolate available (average 40, range 1-111) for rep-PCR genotyping. Most children had Sm isolated from plaque but no Ss was detected from any samples collected from the children in this cohort. Sixty-six of the mothers had Sm identified for genotyping from plaque samples. The other three mothers did not have Sm recovered for genotyping. A total of 3,412 Sm isolates were confirmed to be Sm and analyzed by rep-PCR (2,702 were from children and 710 from mothers) for comparison of GT. The median number of GT observed in both mother and children was two (range one to four and one to seven, respectively). Twenty seven different GT were identified among the isolates tested (children and mothers each had 23). As previously reported,¹⁹ most children harbored a GT that predominated (i.e., at least seven isolates, equal to or greater than 50 percent of isolates with same GT) over a period of 36-months (i.e., 62 of 69, 90 percent). Many of the children shared common GT with other children (two to 27 children found to harbor a same GT). Among the 69 M-C, 40 children harbored a GT in common with the mother while 29 did not have any matching GT. For the children, the numbers of different GT observed in the GT Match group was not significantly different from the non-Match M-C group (P=.33 Mann-Whitney U, Table 1).

Table 1.

Comparison of S. mutans and caries history between two groups of mother-child dyads (M-C).

Variable	M-C GT Match* (N=40) Median (mean±SD)	No GT Match M-C (N=29) Median (mean±SD)	P-value**
Child’s age at 36 month follow-up visit (years)	4.0 (4.0±0.4)	3.8 (3.9±0.3)	0.22
Number of isolates analyzed in children	45.5 (45.4±32.4)	25 (31.6±28.3)	0.09
Number of GT of S. mutans in children	2.0 (2.8±1.7)	2.0 (2.2±1.0)	0.33
Number of isolates analyzed in mothers	10.0 (12.2±7.4)	7.0 (8.6±5.6)	0.06
Number of GT of S. mutans in mothers	2.0 (2.0±0.8)	2.0 (1.9±1.0)	0.65
Sm (CFU/ml) in children at 2½ years§	3.9×104 (5.4×105±1.1×106)	6.6×102 (1.4×105±4.0×105)	0.03
Child’s dmfs score at 36 month follow-up	6.5 (17.1±22.8)	1.00 (6.4±13.0)	0.012
Mother’s Sm (CFU/ml) at baseline in pooled plaque samples§	7.3×102 (3.1×104±6.4×104)	2.0×102 (7.3×104±1.9×105)	0.47
Mother’s DMFS score at baseline	16.5 (22.3±21.04)	13 (17.7±16.0)	0.45

^*M-C with S. mutans Match Genotype (GT) Match (or No Match) Group identified by Repetitive Extragenic Palindromic Polymerase Chain Reaction (rep-PCR)

^§Saliva and biofilm sample obtained by rubbing teeth, tongue, and mucosa with a sterile cotton swab. CFU, colony forming units of S. mutans identified by morphology on Sm select media plates²⁰

^**P value obtained by Mann-Whitney U test. P values that result in significant differences are identified in bold.

Sm counts from SBS of children at 18 month follow-up visit (average age two and one-half years), were found to be significantly different between M-C with matching GT and those that were in the No-Match M-C group (P<.03 Mann-Whitney U test, Table 1). Plaque Sm plate counts for mothers at baseline was lower in the No-Match M-C group, however was not significantly different (P=.47).

The median dmfs for the children at 36-month follow-up dental examination and baseline DMFS for the mothers are presented in Table 1. Intra-examiner weighted kappa for 17 children aged three to four years was 0.91, and inter-examiner weighted kappa for 23 children aged three to four years was 0.93 when comparing the results of the total dmfs count at the person level. Caries prevalence by the 36-month dental examination (average age of the children four years) was found to be 68 percent (74 percent, 54 percent for GT Match and No-Match M-C groups, respectively). There was an association with M-C match and caries history with the children experiencing significantly higher caries (P=.012, Table 1), however the differences for the mother’s caries DMFS at baseline was not significant. The multivariable results of this study showed that the odds of having ECC in children at age 36 months in the GT Match group were 2.98 time that in children in the No-Match group (the results approached significance, P=.063 after adjustment for the number of Sm isolates variable, Table 2). Further, children in the GT Match group had statistically significantly higher dmfs counts at age 36 months than those who were in the No-Match group (Incidence rate ratio: IRR=2.61 after adjustment, P=.023). Interaction terms were not statistically significant at α<0.05.

Table 2.

Results of statistical models with different caries outcome definitions in children

Model		Statistical test	Magnitude of associations (95% CI)	P-value
Outcome (age 4)	Covariate
dmfs>0 (yes)	Mother-Child Pair with S. mutans GT Match*	Logistic regression	OR=2.98 (0.987-7.936)	0.063
dmfs count	Mother-Child Pair with S. mutans GT Match**	Negative binomial modeling	IRR§=2.61 (NA)	0.014
dmfs>0 (yes)	S. mutans in children at age 18 months (≥500 CFU/ml) †	Logistic regression	OR=5.604 (1.86-16.86)	0.002
dmfs count	S. mutans in children at age 18 months (≥500 CFU/ml) †	Negative binomial modeling	IRR§=2.83 (NA)	0.014

^*Multivariable results are adjustment for the number of bacterial isolates in children (p=0.011) and number of bacterial isolates in mothers (p=0.728).

^**Multivariable results are adjustment for the number of bacterial isolates in children (p<0.001) and number of bacterial isolates in mothers (p=0.347).

^†Bivariate results, CFU, colony forming units of S. mutans identified by morphology on Sm select media plates²⁰

^§Incidence Rate Ratio (IRR) obtained from the exponentiation of the parameter estimates.

The bivariate results showed that among children with Sm count ≥ 500 CFU/ml at age two and one-half years, the odds of having dental caries at age 36 months were 5.60 that of children with Sm count<500 CFU/ml (P =.002, Table 2). Also, children with Sm count equal to or greater than 500 CFU/ml at age two and one-half years had significantly higher dmfs counts at age 36 months than those with Sm count<500 CFU/ml (IRR=2.83, P=.014).

DISCUSSION

This study provides prospective data for Sm and dental caries activity for children and their mothers spanning early childhood monitoring the development of ECC. Although multiple time-points of data were collected, the results presented herein focus on relevant specific time points (i.e., Sm counts 18 months prior to caries assessment at approximately four years of age). These findings improve the basic understanding of the natural history and epidemiology of caries experience in a minority high-risk group. Further, we examined the colonization of infants/children determining the prevalence, and commonality of Sm GT and their relationship to caries pathogenesis. The data was analyzed in conjunction with analysis of samples collected from the children’s mothers to investigate the association between mother’s MS counts, MS GT and the children’s ECC experience. In this regard, we evaluated the commonality of MS GT among children and their mothers by collecting samples before, during, and after the emergence of primary molar teeth. ECC was found to be highly associated with Sm in children, as well as mother’s matching GT (Table 1). Although Sheiham suggests that dental caries is a dietary disease,²⁷ evidence for the infectious nature, specifically Sm should not be discounted. We are investigating and have reported dietary habits as related to caries in this population and have found some relationship,²⁸ moreover, the multifactorial nature of caries is self-evident.

In addition to the data obtained in this study, participants were provided preventive education and care. However, despite the preventive approach to their care, a high prevalence of dental disease was observed (65 percent)⁴. The parents of this cohort were encouraged to seek a dental home, and when oral disease was observed, assistance was provided to obtain (i.e., refer for) dental treatment. The involvement in our study appeared to improve access to care for this community and possibly resulted in less severe disease outcomes (i.e., most treatment was restorative, not extractions, data not shown).

One powerful aspect of this study was the analyses of Sm GT as an epidemiological tool to track the commonality, and potentially, transmission. Presentation of the temporal GT findings are beyond the scope of this manuscript, for most children with isolates from multiple visits, the predominant genotype was observed throughout all periods, while other genotypes were rare and/or added as children aged (i.e., from age 1-4). The GT data presented herein is compatible with the concept of vertical transmission (i.e., mother to child), with 40 of 68 children (58 percent) that had Sm isolates/GT found to be shared between M-C. However, due to the extensive commonality of GT in this cohort (27 GT in 3,412 isolates), it is not possible to confirm by this method alone. Although evidence for maternal transmission of specific GTs was possible in more than half of the children with at least one GT in common, there was a large proportion (42 percent) that we observed no evidence of sharing any GT. Those with no sharing of GT also were found to have a non-significantly lower number of isolates available for genotyping. Whether the etiology of caries in children is related to a specific infection from the mother’s cannot be determined by these findings. However, mothers with some matching GTs to their children was associated with ECC. Therefore, the oral health of the mother’s, whether related to specific GT, a higher level of colonization of Sm, or maternal bacterial detection as a surrogate of active disease, is clearly associated to caries in their children. Future studies are ongoing that will use multi-locus sequence typing (MLST) as an additional tool to provide clearer association of GT of Sm to potential sources of transmission to the children.²⁹

A limitation of the study presented is that the numbers of isolates of Sm from each individual over the six sample collections (children) and from the mothers (two collection periods) varied. However, this is also a strength, since multiple sampling increased the numbers of isolates available for genotyping and generally, the variability is most likely due to the level of colonization at the time of collection. Our objective was to test seven to ten isolates per sample (65 of 69 children) based on the likelihood of identifying up to three, equally distributed GTs.³⁰ Despite the large variability among the number of isolates analyzed in the cohort, an important finding is that maternal colonization with Sm was clearly associated with higher caries experience of the child and mother. The mother’s in the study identified themselves as the “primary caregiver” for the children, however, stay at home mothers versus daycare or other family member daytime environments was not considered in the analyses. The scope of this study did not include other family members, but they were involved in this study and will be important for future analyses.

One obvious question is whether there is an association of any specific GT with higher dental caries experience. However, due to the large number of genotypes (i.e., 23 GTs requiring adjustment for multiple comparisons) and relatively small sample size (low statistical power), we were unable to assess for a relationship between GT and ECC. Our group and others previously reported that, although we observed no single GT is associated with caries, multiple GT within an individual was associated with caries.^{19, 31-35}

The only MS detected in this study was Sm. Although we have demonstrated that S. sobrinus can be identified with the selective culture media we used,³⁶ Ss has rarely observed from the entire 13,000 isolates we have analyzed thus far (less than one percent, data not shown). This finding is interesting since other studies have shown Ss to be associated with high-caries in some populations,^{37, 38} however, in this high-caries risk population we failed to observe Ss. Follow-up studies in our laboratory using PCR for Sm and Ss specific primers confirm the findings with the media (unpublished data). The data and analyses we report supports the importance of the oral microbial status, including colonization with MS, of mothers to be influential in the child’s oral health and caries-risk assessment. Further, Sm colonization at age two and one-half years was found to be a risk factor for caries incidence at age four. A limitation of interpretation is the generalizability of these findings to other population groups. This population is somewhat unique, which was a benefit in the design of the study (i.e., African American cohort, low socioeconomic, with stable residence in the community) but may limit conclusions related to other socioeconomic, geographic, ethnic groups. Previous studies involved treatment of mothers aimed at prevention of MS transmission to their children was encouraging,^{39, 40} but definitive RCT are lacking.

Generally, these findings support the concept that the mother's oral flora may be reflection of risk for caries in their offspring. It is possible that the close contact with mothers that are colonized with S. mutans results in early colonization and increased caries risk. Although dental examination of the mothers, or maternal S. mutans counts were not significantly associated with ECC, matching GT was found to be associated. It is unclear whether the GT is key to the caries susceptibility based on these findings. The results gained by this approach provide fundamental information for risk assessment and the development of preventive strategies toward eliminating caries. There is a need for additional research, to assess the virulence of specific genotypes (or combinations) of S. mutans in ECC and interventional studies to assess the feasibility of improving oral health, specifically interfering with colonization with high levels of virulent Sm. High-caries risk populations such as the cohort of this study could provide the statistical power to demonstrate efficacy.

CONCLUSIONS

1. Most children were colonized with S. mutans by age two and one-half years, which was associated with early childhood caries at age four.

2. None of the children had S. sobrinus isolated from any of the samples collected in this high caries risk population of children and their mothers.

3. Generally, most children had a predominant S. mutans genotype, however we were unable to assess the relationship of so many genotypes to early childhood caries due to small sample size.

4. Fidelity of S. mutans genotype between mother and child was found to be 58 percent with 40 of 69 mother-child sharing genotypes, however much commonality of genotype among others was also observed and therefore makes it difficult to determine mode of transmission.

5. At age four years, caries in children was significantly higher in the group where mother-child S. mutans isolates were found to match even after adjustment for number of bacteria isolated.