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the detoxifying enzyme lactoylglutathione lyase in Streptococcus mutans aciduricity. J Bacteriol 2007;189:7586–7592. 9. Zhou Y, Lin HC, Lo EC, Wong MC.
Australian Dental Journal

The official journal of the Australian Dental Association

Australian Dental Journal 2013; 58: 507–513 doi: 10.1111/adj.12113

Factors associated with colonization of Streptococcus mutans in 8- to 32-month-old children: a cohort study Y Zhou,*†§ JY Yang,‡§ QH Zhi,*† Y Tao,*† RM Qiu,*† HC Lin*† *Department of Preventive Dentistry, Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, China. †Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, China. ‡Department of Stomatology, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.

ABSTRACT Background: The factors involved in Streptococcus mutans colonization in young children are not clear. The aim of this study was to determine the factors associated with S. mutans colonization in 8- to 32-month-old children. Methods: A group of 225 caries-free 8-month-old children was recruited for the study. They were examined every six months until they were 32 months old to investigate their environmental factors, host factors and bacterial transmission factors. At baseline and during each examination, their teeth were checked for the presence of dental plaque and developmental defects of enamel, and S. mutans plaque status was assessed using a real-time PCR test. Results: Eight children (3.6%) showed S. mutans colonization by the age of 8 months. The percentages of colonization were 6.0% at 14 months, 16.2% at 20 months, 26.7% at 26 months, and 33.5% at 32 months. The results showed that females (p = 0.006), children with enamel hypoplasia (p = 0.024), children with low birth weights (p = 0.005), those who consume more sweets (p < 0.001), and those with a higher proportion of visible plaque (p = 0.020 and p = 0.041) were more likely to be colonized by S. mutans. Conclusions: Streptococcus mutans colonization in young children was associated with gender, tooth enamel hypoplasia, low birth weight, frequent consumption of sweets and poor oral hygiene. Keywords: Cohort study, colonization, Streptococcus mutans. Abbreviations and acronyms: DDE = developmental defects of enamel; ECC = early childhood caries; GEE = generalized estimating equation; MS = mutans streptococci; PCR = polymerase chain reaction; TYCSB = tryptone-yeast-cysteine-sucrose-bacitracin; VPI = Visible Plaque Index. (Accepted for publication 26 March 2013.)

INTRODUCTION Early childhood caries (ECC) is a term that describes the presence of one or more decayed (non-cavitated or cavitated lesions), missing (due to caries), or filled tooth surfaces in any primary tooth in a child under the age of 6.1 Despite significant advances in dentistry, ECC remains a serious problem in China.2 ECC develops at an early age and progresses quickly. Mutans streptococci (MS) have been implicated as the principal oral bacteria responsible for the initiation and development of ECC.3 A strong relationship has been established between MS colonization and caries experience in the primary dentition.4 Recently, Laitala5 reported that avoiding early MS colonization may lead to favourable long-term effects on caries experience and subsequent need for restorative treatment. The younger the children when MS §Y Zhou and JY Yang contributed equally to this work. © 2013 Australian Dental Association

colonization occurs, the higher the risk of acquiring ECC in the future.6 However, the factors involved in MS colonization in young children are not clear. Law reviewed the factors that affect MS colonization in young children and divided them into bacterial factors, host factors and environmental factors.7 Bacterial factors include biofilm, bacterial virulence and bacterial transmission. Most studies have focused on biofilm and bacterial virulence from a microscopic perspective.8 Bacterial transmission results from increased numbers of MS in mothers/close contacts as well as increased frequency of contact with MS carriers. Host and environmental factors that affect MS colonization have not been fully elucidated. Such host factors include heredity, surfaces for microbial adherence, diet, oral hygiene, saliva and immunological factors. Environmental factors include demographic characteristics and socio-economic status of children. Complex interactions among these factors determine the timing of MS colonization in children.7 507

Y Zhou et al. MS consists mainly of the Streptococcus mutans and Streptococcus sobrinus species. Streptococcus mutans has been identified as an important bacterial species in caries initiation, while S. sobrinus plays a role in enhancing caries progression and development. Both cross-sectional9 and longitudinal studies10 have provided evidence that high levels of S. mutans contribute to ECC. The role of S. mutans in the aetiology of dental caries in children is important and of great interest to researchers. However, due to the paucity of longitudinal studies in young children, relatively little is known regarding the factors associated with S. mutans colonization in young children. The present study investigated factors affecting S. mutans colonization in 8-month-old children through a two-year cohort study. METHODS Study sample Subjects were participants in an earlier study living in Xinhua Town, Huadu District, Guangzhou City, in southern China. The study design was longitudinal, observational and community-based, which has been previously described in detail.11 Briefly, we recruited 225 children from an initial sample of 267 healthy 8-month-old children who visited the hospital for routine vaccinations or health examinations. Inclusion criteria were that the parents of the children had lived in the district for more than two years and the children were physically healthy at birth. Children with a systemic illness at birth were excluded from the study. Data were recorded from the same infants at 8, 14, 20, 26 and 32 months. The study duration was two years. Ethical approval for this study was obtained from an ethics committee at Sun Yat-sen University. Study model The theoretical model for this study was converted from a table in a previous study.7 The model assumes that environmental, host and bacterial transmission factors contribute to the colonization of S. mutans in children. Environmental factors included the socioeconomic status of children. Parental socio-economic status determined the socio-economic background of children and was reflected by parental occupation, income and educational attainment. Other studies have reported that the mother plays a more influential role than the father in the colonization of S. mutans in children.12 Therefore, in this study we were interested in the maternal role with respect to S. mutans colonization. Host factors included gender, developmental characteristics at and around birth, feeding habits and oral hygiene. The child’s developmental characteristics at and around birth included the child’s weight at birth, 508

gestational age, mode of delivery and developmental defects of enamel (DDE). Low birth weight was defined as the birth weight of a liveborn infant of less than 2500 g regardless of gestational age. Feeding habits included breastfeeding and bottle feeding practices, sleeping with a pacifier, and the frequency of eating staple foods and sweets. Oral hygiene included toothbrushing habits and visible plaque on teeth. Bacterial transmission factors involved presence of MS in maternal dental plaque, sharing tableware with children and bacterial virulence. All of the above factors are thought to affect S. mutans colonization in children and play different roles in the colonization process over time. Instruments and measures The data were collected using structured questionnaires, clinical oral examinations, and laboratory tests of samples obtained from the participants and their mothers. Completion of the questionnaires and the clinical examinations took place in the same examination room at the hospital. The questionnaire had been verified for question clarity in a previous study.9 The questionnaire was used to collect the related demographic, socio-economic, developmental and behavioural information, and was completed by the parents. Visual examination of the teeth was performed by a trained examiner (YZ) who recorded the level of plaque accumulation and DDE. The Visible Plaque Index (VPI) was used to assess oral hygiene.13 DDE type was recorded separately using criteria recommended by the Federation Dentaire Internationale (FDI) for general epidemiological surveys.14 As enamel opacities and enamel hypoplasia were the two major types of DDE, this variable was converted into enamel opacities only, hypoplasia and no presence of DDE. From the sample, 10% of subjects were re-examined to determine intra-examiner reliability. Plaque samples were collected from the surfaces of anterior teeth or alveolar ridges (for predentate infants) using sterile cotton tips at baseline and at each followup. Real-time polymerase chain reaction (PCR) was employed to identify the presence of S. mutans in the plaque. After centrifugation, the bacterial chromosomal deoxyribonucleic acid was extracted by precipitation, as has been described previously.15 Real-time PCR assays for S. mutans were performed using the TaqMan system and a modified version of the method described by Oho.16 In brief, an S. mutans-specific primer pair (forward, GATGATAGCAATGCAGCCAATC; reverse TACGAACTTTGCCGTTATGTCA) and a probe (5′-FAM-AACTTCCCAATGTAAAAGAAATTGATGG TATAMRA-3′) targeting the glucosyltransferase B gene was used. The amplification was performed using an ABI Prism 7900 (Applied Biosystems; Foster City, CA, USA). © 2013 Australian Dental Association

Factors associated with colonization of S. mutans Plaque samples from the mothers were collected at baseline by swabbing all surfaces of the anterior teeth with sterile cotton tips. The swabs were cut off and immediately placed in a 2-ml vial containing sterile thioglycolate transport solution and stored on ice before being transferred to the laboratory within four hours. Ten microlitre aliquots of the diluted sample were plated onto MS-selective tryptoneyeast-cysteine-sucrose-bacitracin (TYCSB) agar.17 After 48 hours of incubation at 37 °C in an anaerobic atmosphere of 85% N2, 10% H2, and 5% CO2, the bacterial colony on each plate was examined macroscopically for an estimation of MS in the mothers. Fourteen mothers refused to undergo oral examination; 211 (93.8%) mothers underwent oral examination. MS colonization in adults was reported to be stable;18 our baseline results indicated the presence of MS in the maternal dental plaques. As the purpose of this study was to explore the existence of MS in the dental plaque of the mothers, we did not distinguish each species of the MS genus.

RESULTS General characteristics of the sample The response rates for the six-month follow-up were sequentially: 215 (95.6%); 191 (84.9%); 172 (76.4%); and 155 (68.9%). The declining response rates were mainly due to participants who had moved out of the study area or could not be contacted. The data from children with S. mutans colonization detected by PCR during the 8–32 month period are shown in Table 1. The number of S. mutans-positive children was 52 (33.5%) at 32 months. Some children who were S. mutans-positive at each examination were S. mutans-negative afterwards. Kappa values for intra-examiner reliability of the visible plaque and DDE assessments were 0.91 and 0.92, respectively. No significant differences were found in the demographic and socio-economic profiles, including gender (p = 0.224), family monthly income (p = 0.568), mother’s schooling (p = 0.698), and mother’s occupation (p = 0.144), between the lost-to-follow-up children and those undergoing complete follow-up.

Statistic analysis The generalized estimating equation (GEE) was used to assess the relationship between potential risk factors and S. mutans colonization over the two-year study period. This statistical approach takes advantage of all data contributed by each subject, as well as enabling repeated measurements over time for both risk factors and outcome variables.19 GEE adjusts for the correlated nature of the error associated with repeated measurements over time.19 The outcome was the detection of S. mutans in the dental plaque of children. Bivariate analyses were used to assess differences between those children with and without S. mutans colonization for each variable. Variables with a p-value