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Journal of Clinical Microbiology, September 2007, p. 3082-3085, Vol. 45, No. 9
0095-1137/07/$08.00+0     doi:10.1128/JCM.00771-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Similarity of Bacterial Populations in Saliva from African-American Mother-Child Dyads{triangledown}

Yihong Li,1* Amid I. Ismail,2 Yao Ge,1,{dagger} Marisol Tellez,2,{ddagger} and Woosung Sohn2

Department of Basic Science and Craniofacial Biology, New York University College of Dentistry, New York, New York 10010,1 Department of Cariology, Restorative Sciences, and Endodontics, University of Michigan School of Dentistry, Ann Arbor, Michigan 481092

Received 10 April 2007/ Returned for modification 20 May 2007/ Accepted 12 June 2007


   ABSTRACT
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 ABSTRACT
TEXT
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Using PCR-based denaturing gradient gel electrophoresis analyses of oral bacterial samples in 20 mother-child dyads, this study demonstrated a high degree of similarity of bacterial compositions between the mothers and their children; the two may share as much as 94% of their oral bacterial spectra, including cariogenic species.


   TEXT
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Early childhood caries (ECC) is a particularly destructive form of tooth decay that affects preschool children. The incidence of ECC ranges from 0.8% to 72%, depending on the caries diagnostic criteria and ethnic and socioeconomic backgrounds of the population of children examined (3, 11). A recent cohort study of African-American children from low-income families in Detroit, MI, reported the prevalence of ECC to be 44% among 5-year-olds (25). This is much higher than the U.S. national average (16 to 29%) (34). Although frequent consumption of fermentable carbohydrates, poor oral hygiene, and lack of access to dental care are risk factors associated with the occurrence of dental caries (9, 29, 30), a multilevel analysis of the Detroit study found that social, dietary, behavioral, and community risk factors could explain only a small fraction of the interindividual variability in the prevalence of the disease (33).

The Detroit study also found that over 80% of the children's primary caregivers experienced dental caries (6), and more than 50% of them had lost at least one tooth due to caries (28). Thus, there appears to be a strong correlation between the oral health status of the children's teeth and those of their mothers. Other studies have previously corroborated this conclusion (12, 25, 27).

Dental caries is an infectious disease caused by cariogenic bacteria in the oral cavity, mainly Streptococcus mutans (5, 20, 32). Substantial evidence indicates that early infection and high levels of S. mutans colonization in children are significantly associated with the prevalence and severity of ECC (12, 20, 22). Even though there is evidence that S. mutans plays an important etiological role in caries, more than 700 species of bacteria are known to colonize the oral cavity (1, 23, 24), so a mixture of bacterial species may be involved in caries development. We and others have found that children acquire S. mutans primarily from their mothers (4, 13, 15, 16, 21). However, no studies have investigated the correlation of complex oral microbiota between mother-child dyads. Therefore, the objective of this pilot study was to investigate if children acquire the entire cariogenic oral microflora of their mothers.

Twenty mother-child dyads were randomly selected from the Detroit Center study cohort (10). All 20 mothers experienced dental caries; 10 children (50%) had caries, and the other 10 children were caries free. The 20 children (9 boys and 11 girls) were 2.4 to 8.3 years old. Informed consent letters were obtained from all 20 mothers. The study proposal was approved by the Health Sciences IRB of the University of Michigan.

Stimulated whole-salivary samples were collected from all subjects as previously described (15). Immediately, after sample collection, 1.0 ml of the saliva was transferred into a prelabeled 2.0-ml sterile vial and centrifuged at 5,000 x g for 3 min. Then the supernatant was removed and the pellet was washed with 1 ml TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA). The sample vials were then sealed and placed in biological containers and transported on dry ice overnight to the microbiology laboratory at the New York University College of Dentistry and then stored at –80°C.

Total bacterial genomic DNA was isolated from each salivary sample as described previously (17, 18), and a standard concentration of 10 ng/µl was prepared for all PCR assays. A nested-PCR approach was performed with two sets of universal primers, 8F/1492R (14) and prbac1/prbac2 (26), to amplify the bacterial 16S rRNA genes. The final PCR products (~300 bp) were separated in 40% to 60% linear DNA denaturing gradient gels by using a DCode System (Bio-Rad, Hercules, CA) and stained with 0.5-µg/ml ethidium bromide solution as previously described (17-19). The denaturing gradient gel electrophoresis (DGGE) images were digitally captured (Alpha Innotech Co., San Leandro, CA) and analyzed using Fingerprinting II Informatix software (Bio-Rad) as presented elsewhere. A pairwise similarity index (Cs) was calculated for each set of mother-child or mother-nonchild paired samples. Analyses were performed using the SPSS 13.0 program (SPSS, Inc., Chicago, IL).

The International Caries Detection and Assessment System (ICDAS) was used for the caries examination (10). Among the 20 mothers, the mean caries score (decayed, filled, or missing tooth surfaces due to caries) was 51.9 ± 27.5 (range, 14 to 107). Among the 10 children who were caries active, the mean caries score was 5.7 ± 7.4 (range, 1 to 27). Caries severity in the mothers was significantly correlated with that of the children (r = 0.71; P < 0.01).

DGGE analyses were performed based on the position and intensity of each detected band (Fig. 1). Normalized banding patterns were used to generate a similarity coefficient (Cs; range, 0 to 100%) and compared for each mother-child (Fig. 2A) and mother-nonchild (Fig. 2B) pair. The mean Cs for the mother-child pairs was 83.5%, ranging from 66.7% to 94.4%, whereas, the mean Cs for the mother-nonchild group was 69.3% (range from 58.9% to 74.2%). Table 1 also shows that the lowest and highest Cs for the mother-nonchild pairs were 44.0% and 86.2%, respectively. The difference in the mean Cs was statistically significant between the two groups (P < 0.001) (Fig. 3).


Figure 1
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  		FIG. 1. Negative DGGE images of the 20 mother-child pairs (lanes 1 to 20). All DGGE gels were normalized first and then analyzed based on the position and intensity of each band detected. The DGGE profile analysis clearly shows different banding patterns of bacterial populations in the saliva between mother-child pairs and mother-nonchild pairs. M, mother; C, child.

 

Figure 2
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  		FIG. 2. Comparison of the DGGE banding patterns between a mother-child pair (no. 12) (A) and mother-nonchild pairs (B). The Dice correlation coefficient for pairwise comparisons was generated by Fingerprinting II Informatix software (BioRed). (A) A Cs of 94.4% was calculated for mother-child pair 12, which indicates a high degree of similarity between the mother-child pair. (B) In contrast, the mean Cs was 69.3% when comparing a child with other female adults. M1, M4, M8, and M18 represent mothers 1, 4, 8, and 18; C12 represents child 12.

 

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  		TABLE 1. Comparison of Cs between mother-child and mother-nonchild dyads

 

Figure 3
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  		FIG. 3. The mean similarity value for the mother-child pairs was 83.5% ± 6.9% (range from 67% to 94%). In contrast, the mean similarity value for the mother-nonchild pairs was 69.3% ± 3.7% (range from 59% to 74%). The differences were highly statistically significant (nonparametric [NPar] Mann-Whitney test; P < 0.001).

 
In this study, we confirmed the association of caries severity in mother-child pairs. More importantly, our results revealed highly similar bacterial compositions between the mothers and their children. A mother-child pair may have up to 94.4% of their oral bacterial spectra in common. Recently, studies using novel culture-independent molecular methods suggested that, in addition to S. mutans, the diversity of cariogenic bacteria in saliva and dental plaque is much greater than previously believed (2, 7, 31).

Since over 50% of the microbiota in the oral cavity are uncultivable (1), conventional cultivation approaches are severely limited for comparative investigation of complex oral bacterial populations. Here, we use the PCR-DGGE assay because a DGGE-generated total bacterial profile consists of both cultivable and uncultivable microbiota, which enables us to survey the composition of oral microflora among different individuals. As we previously demonstrated, the numbers and types of bacteria most likely vary between caries-active and caries-free individuals (17, 18). Thus, an ecologically imbalanced oral biota may not only contribute to the clinical differences in caries activity of the mothers but also be transmitted to their children and consequently affect caries outcome of the children.

Based on the 20 mother-child dyads, this study found statistically significant correlations in the similarity in oral microbiota between children and their biological mothers. A comprehensive study with a large sample size has been planned to evaluate whether mothers transmit not only a single cariogenic bacterium but their entire cariogenic microbiota to their children, resulting in a high-caries experience in the children. Confirmation of this proposed hypothesis will have substantial impact on the development of new interventions—especially in prenatal care clinics for all caries-active mothers—aimed at reducing cariogenic bacterium transmission from mothers to their children and ultimately reducing dental caries in children.


   ACKNOWLEDGMENTS
 
This study was supported by Public Health Service grant U-54 DE14261 from the National Institute on Dental and Craniofacial Research, the Delta Dental Fund of Michigan, the University of Michigan's Office of Vice President for Research, and Dean's Faculty Research Fund of New York University College of Dentistry.

We thank the staff of the project for their diligence and commitment.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Basic Science and Craniofacial Biology, New York University College of Dentistry, 345 E. 24th St., New York, NY 10010. Phone: (212) 998-9607. Fax: (212) 995-4087. E-mail: yihong.li{at}nyu.edu Back

{triangledown} Published ahead of print on 18 July 2007. Back

{dagger} Present address: Peking University School of Stomatology, Beijing, People's Republic of China 100081. Back

{ddagger} Present address: Facultad de Odontologia Universidad El Bosque, Cra. 7B Bis no. 132-11, Colombia. Back


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Journal of Clinical Microbiology, September 2007, p. 3082-3085, Vol. 45, No. 9
0095-1137/07/$08.00+0     doi:10.1128/JCM.00771-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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