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RESEARCH REPORT |
1 The Forsyth Institute, 140 Fenway, Boston, MA 02115;
2 University of Washington, Department of Dental Public Health Sciences, Box 357475, and Department of Periodontics, Box 357480, Seattle, WA 98195-7475;
3 King Saud University, PO Box 85032, Riyadh 11691, Saudi Arabia; and
4 Department of Public Health Services, Box 409 CK, Saipan, Mariana Islands 96950;
*corresponding author, atanner{at}forsyth.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: Streptococcus mutans • Porphyromonas gingivalis • Actinomyces • Prevotella • pre-school children • dental caries • tongue samples
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Much of our understanding of the oral microbiota and the age at which colonization occurs comes from studies that used cultural methods. Colonization by Streptococcus mutans, the principal species associated with caries, was found to occur during a window of infectivity between 19 and 31 months of age (Caufield et al., 1993). The principal periodontal pathogens Porphyromonas gingivalis, Bacteroides forsythus, and Actinobacillus actinomycetemcomitans were not detected by culture in children under 2.5 years of age (Frisken et al., 1990; Kononen, 2000), although other Gram-negative anaerobes were detected. Using PCR-based methods, however, investigators have detected P. gingivalis and A. actinomycetemcomitans in the saliva of infants and young children (McClellan et al., 1996; Lamell et al., 2000). What is not clear from these studies is the proportion of children in the general population that may be colonized by dental pathogens, and at what age. S. mutans in the presence of a high-sucrose diet was associated with the development of caries in young children (Fujiwara et al., 1991; Grindefjord et al., 1996). Whether early colonization by periodontal pathogens is a risk factor for periodontitis is not known, although detection in children would suggest that periodontitis is an endogenous infection.
The purpose of the present study was to determine colonization frequencies of oral species in a population-based sample of young children. Population-based studies have been limited because of difficulties in accessing populations and technical difficulties in processing large numbers of samples. We studied a randomly selected population and used the checkerboard DNA probe hybridization method (Socransky et al., 1994) that allowed for simultaneous detection of multiple species in the large number of samples generated in epidemiological studies. This study determined the prevalence of 38 oral species in children from 6 to 36 months of age, examined the association and relative frequency of species detection in tooth and tongue samples, and evaluated species associations with dental caries.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Clinical Measurements and Sample Sites
The age and gender of study children were recorded, and the numbers of teeth with lesions were measured as white-spot lesions (white, chalky areas of enamel in the gingival third of the crown, hypoplasia (in the incisal two-thirds of the crown), or enamel cavitation (coronal dentin exposed) (Milgrom et al., 2000).
In children with caries, a sample was taken from either a caries lesion or a white spot. In caries-free children, a sample was taken from the cervical supragingival region of the upper anterior right incisor. Teeth were sampled with sterile toothpicks with tapered ends, and a tongue sample was obtained by means of a flat-ended toothpick scraped over the tongue dorsum (Milgrom et al., 2000).
Microbial Analysis
Samples were placed in 0.1 mL of TE buffer (10 mM Tris, 1 mM EDTA, pH 7.6), and 0.1 mL of 0.5 M NaOH was added. The microbiota of samples was characterized by means of the checkerboard hybridization method (Socransky et al., 1994). Up to 30 samples were simultaneously hybridized with DNA probes to 38 oral species. Whole chromosomal DNA probes were prepared with DNA extracted from reference strains of oral species (Table 1
). Digoxigenin-labeled, whole chromosomal DNA probes were prepared by random primer labeling with the GeniusTM 2 kit (Boehringer Mannheim, Indianapolis, IN, USA). Each hybridization run had quantitation standards consisting of a mixture of each probe species at concentrations equivalent to 105 and 106 cells per sample. DNA probe reactions were detected with anti-digoxigenin alkaline phosphatase conjugate, with CDP-StarTM (Boehringer Mannheim) as substrate. Chemiluminescent signals were detected by exposure to Hyperfilm ECL (Amersham Corp., Arlington Heights, IL, USA). Signal intensities of each probe for each sample were expressed by reference to the quantitation standards and were scored visually on a scale of 0 to 3 relative to controls, where 0 = no reaction detected, 1 = a reaction approximating > 104 to < 105, 2 = a reaction approximating 105 to < 106, and 3 = a reaction approximating > 106 bacterial cells per sample.
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Data Analysis
Clinical characteristics of children under and over 18 mos of age were compared by the Mann-Whitney U test. Comparisons were made between the frequency of species detection from tooth and from tongue samples between the two age groups by Chi-square test. Comparisons were made of the frequency of species detection in paired tooth and tongue samples within each age group by McNemar's Chi-square test. The significance of odds ratios between species detection in tooth and tongue samples was compared by Chi-square test.
The levels (probe score) of species detected were compared in tooth samples from children with and those without dental caries by Wilcoxon rank-sum tests. The Mantel-Haenszel Chi-square test of trend was used for comparison of the detection of S. mutans from tooth and tongue samples between caries-free children and children with either 1-2 or 3 or more teeth with dental caries.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). The numbers of teeth erupted and teeth with white-spot lesions or enamel cavitation were significantly higher in the older children (Table 2). The most frequently detected species in 6- to 18-month-old children included S. mutans, S. sobrinus, Streptococcus oralis, Streptococcus mitis, and Lactobacillus acidophilus (Table 1
). While periodontal pathogens were detected, the detection frequency was generally lower than for other species assayed. In children aged 6-18 mos, the detection frequencies of periodontal pathogens from tongue samples were: A. actinomycetemcomitans 30%, Prevotella intermedia 29%, P. gingivalis 23%, B. forsythus 11%, and Treponema denticola 36%.
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Detection frequencies in tooth samples were similar (within ± 5%) for 18 species, increased in older children by > 5% in 25 species, and decreased by > 5% in only 1 species, Actinomyces gerencseriae (Table 1, Fig. 1). When tongue samples were compared, the prevalence of only 4 species increased by > 5% in the older children (none significantly), 24 showed similar frequencies (within ± 5%) in both age groups, and 16 species decreased > 5% in the older age group. Only S. sobrinus decreased significantly in the 19- to 36-month-old children.
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), whereas S. mutans (p < 0.01) was the principal species associated with carious teeth in the 19- to 36-month-old children. However, these differences were not significant after Bonferroni adjustment for multiple comparisons. No other species evaluated (Table 1) were detected at higher levels in carious teeth. In both age groups, S. mutans detection was highest in tooth and tongue samples of children with more than two (> 2) carious teeth (Fig. 2). This association was significant in the 19- to 36-month-old children.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Differences in populations studied and in microbiological assays may account for species detection rates. In Alabama, a window of infectivity for S. mutans, detected by culture, was 25% children colonized by 19 mos and 75% children colonized by 31 mos of age (Caufield et al., 1993), with a caries rate of 35% in the S. mutans-positive children. In our study, 28% of the 6- to 18-month-old children had caries and a 55% S. mutans detection rate from tooth samples, which rose to 72% caries and 75% S. mutans detection in the 19- to 36-month-old children (Table 1, Fig. 1). A detection rate of S. mutans of over 80% associated with a high caries rate was reported in 1- to 2.5-year-old Brazilian children (Mattos-Graner et al., 1998). S. mutans was also detected in predentate children, despite a low caries rate (Mohan et al., 1998). Together, these reports suggest that children may be colonized by S. mutans before the "window of infectivity" opens (Caufield et al., 1993).
The high caries rate and high detection frequency of S. mutans and periodontal pathogens could indicate that our child population had increased amounts and maturity of plaque. Microbial complexes have been described in subgingival plaque (Socransky et al., 1998), with a sequence of dependence for species detection that resembled plaque development, leading to a climax community of the periodontal pathogens P. gingivalis and B. forsythus. In the current study, a higher proportion of the children were colonized by Streptococcus, Actinomyces, and Capnocytophaga species than by a complex of P. intermedia, Prevotella nigrescens, Prevotella melaninogenica, Campylobacter species, and Fusobacterium nucleatum, with even lower detection frequencies of P. gingivalis and B. forsythus. The colonization pattern observed was consistent with the subgingival microbial complexes reported in adults (Socransky et al., 1998).
While the species reported and sampling procedures varied, there were similar patterns of species detection in other studies of young children. The early colonizers S. mitis, S. oralis, S. salivarius, S. sanguis, and S. gordonii have been identified in young infants and predentate children (Tappuni and Challacombe, 1993; Pearce et al., 1995; Könönen, 2000). Actinomyces species, particularly A. odontolyticus and A. naeslundii, were detected in young children (Cole et al., 1998; Könönen, 2000; Sarkonen et al., 2000). The latter study also detected A. georgiae, the most frequently detected Actinomyces in the current study. Veillonella and Capnocytophaga species, and representatives of the Prevotella/Fusobacterium complex that supported colonization of P. gingivalis and B. forsythus (Socransky et al., 1998), have been cultured from young children (Frisken et al., 1990; Könönen et al., 1999). For example, P. intermedia and P. melaninogenica were cultured from infants 1 month after birth (Frisken et al., 1990). P. melaninogenica, P. nigrescens, other non-pigmented Prevotella species, Selenomonas, M. micros, and F. nucleatum were cultured from children younger than 1 yr (Könönen, 2000). These culture-based studies differ from the present study in that P. gingivalis, B. forsythus, T. denticola, and A. actinomycetemcomitans were not detected in young children (Frisken et al., 1990; Könönen, 2000).
The increased detection of fastidious periodontal pathogens may have resulted from the use of DNA hybridization rather than culture. P. gingivalis was detected less frequently by culture than by either immunofluorescence or DNA probe assay (Tanner et al., 1998). P. gingivalis and A. actinomycetemcomitans were detected in young children by PCR assay (McClellan et al., 1996; Lamell et al., 2000) and a slot-immunoblotting assay (Morinushi et al., 2000). In the current study, the detection frequency for most probe species was similar in younger and older children, suggesting that these species colonize in the first 18 months of life.
More species were detected on the tongues of young children than has previously been reported, with a high association between species detection in tooth and tongue samples. Numerous studies demonstrate that anaerobic bacteria can successfully colonize young children before tooth eruption (Frisken et al., 1990). Higher species-detection frequencies from tongue-compared with tooth-associated samples have been reported for A. actinomycetemcomitans, P. gingivalis, and P. intermedia by means of immunofluorescence (Timmerman et al., 1998), for P. intermedia and P. nigrescens by means of AP-PCR (Matto et al., 1996), and for A. actinomycetemcomitans by means of selective culture (Muller et al., 1996). This suggests that the dorsum of the tongue houses organized biofilms to which anaerobic bacteria may locate and thrive to seed anaerobic locations around teeth, consistent with the concept that mucosal surfaces serve as the initial colonization site (Gibbons, 1989) and reservoir (Frisken et al., 1990; Asikainen et al., 1991; McClellan et al., 1996) for oral sites. The increased detection frequency observed from tongue samples from the 6- to 18-month-old children supports such a pathway of transmission and colonization.
The current study detected associations (P < 0.05) between several species—including S. mutans, S. sobrinus, A. israelii, and Lactobacillus uli—and dental caries. Incipient caries in 4- to 9-year-old children was associated with S. mutans, Lactobacillus, and Actinomyces (Boyar and Bowden, 1985). Another study of school-aged children associated S. sobrinus, S. oralis, A. israelii, and a Lactobacillus species with caries (Babaahmady et al., 1997). In some studies, S. sobrinus was not detected in association with caries (Matee et al., 1992), whereas in other studies, S. sobrinus detection indicated a higher risk of smooth-surface than did S. mutans (Hirose et al., 1993). Children with caries probably had significant plaque, yielding larger samples, increasing the possibility of species cross-reactivity of streptococci and actinomyces. The association of S. mutans detection from both tooth and tongue samples with caries in the 18- to 36-month-old children suggests validity for tongue samples as a means of assaying for S. mutans as a risk indicator for caries.
In conclusion, this study indicated that a wide range of species, including S. mutans and putative periodontal pathogens, can be detected in children under 3 years old. Higher species detection frequencies in the younger children from tongue compared with tooth samples suggest that the tongue serves as a reservoir for tooth-associated species. If detection of periodontal pathogens in childhood indicates permanent colonization, these putative pathogens may belong to the commensal microbiota and represent an endogenous infection.
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ACKNOWLEDGMENTS |
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Received October 26, 2000; Last revision November 6, 2001; Accepted November 14, 2001
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| IADR Journals | Advances in Dental Research ® |
| Journal of Dental Research ® | Critical Reviews (1990-2004) |
105 to < 106, 3 equivalent to 
0.0025 (Wilcoxon ranked-sum test), a level accounting for the multiple species comparisons.