Characterizing Diversity of Lactobacilli Associated with Severe Early Childhood Caries: A Study Protocol.
Characterizing Diversity of Lactobacilli Associated with Severe Early Childhood Caries: A Study Protocol.
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DOI：
10.4236/aim.2015.51002
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发表时间：
2015-01-01
期刊：
影响因子：
--
通讯作者：
Caufield PW
中科院分区：
文献类型：
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作者：
Li Y;Argimón S;Schön CN;Saraithong P;Caufield PW
数十年来，乳酸杆菌一直与龋齿相关。然而，对这组细菌在疾病病因学方面的了解仅限于定量阐明。如今，尽管口腔乳杆菌的分类学很复杂，但明确鉴定它们的种类是可能的。在这里，我们描述了一种涉及培养和遗传方法的组合方法，以确定和表征患有严重儿童早期龋齿（S-ECC）的儿童口腔中乳酸菌种群的多样性和丰度。 80 名在纽约市贝尔维尤医院儿童牙科诊所寻求牙科护理的 3 至 6 岁儿童（40 名 S-ECC 和 40 名无龋齿）被邀请参加这项研究。有关社会人口统计信息和口腔健康行为的临床数据是从主要护理人员处获得的。这些数据包括详细的牙科检查、儿童病史和问卷调查。从每个儿童收集未刺激唾液和龈上菌斑的组合样本，并在选择性培养基上培养，以定量测量乳酸杆菌水平。乳杆菌物种筛选的程序包括随机选择每块板 50 个菌落，从每个菌落中提取 DNA，并通过任意引物聚合酶链式反应 (AP-PCR) 进行基因分型。将选择每种独特的乳杆菌 AP-PCR 基因型，通过 16S rRNA 基因测序分析进行分类学评估。将通过将 16S rRNA 序列与核糖体数据库和人类口腔微生物组数据库进行比较来鉴定乳酸菌物种。同时，同一套临床样本将通过定制的流程在属和种水平上独立进行基因组DNA分离、使用乳杆菌属特异性引物进行16S rRNA扩增、测序和分类学鉴定。将在患有或不患有 S-ECC 的儿童之间比较这些乳杆菌物种的分布和系统发育差异。本研究的主要目标之一是建立口腔中乳酸杆菌的鉴定和表征的研究方案。未来的龋齿风险评估可包括乳杆菌计数（定量）以及与 S-ECC 相关的乳杆菌物种（定性）特定致龋基因特征的存在/不存在。
Lactobacilli have been consistently associated with dental caries for decades; however, knowledge of this group of bacteria in the etiology of the disease is limited to quantitative elucidation. Nowadays, explicit identification of oral Lactobacillus species is possible, despite their taxonomic complexity. Here we describe a combined approach involving both cultivation and genetic methods to ascertain and characterize the diversity and abundance of the Lactobacillus population in the oral cavities of children with severe early childhood caries (S-ECC). Eighty 3- to 6-year-old children (40 S-ECC and 40 caries free) who were seeking dental care at the Pediatric Dental Clinic of Bellevue Hospital in New York City were invited to participate in this study. Clinical data on socio-demographic information and oral health behavior were obtained from the primary caregiver. The data included a detailed dental examination, children’s medical history, and a questionnaire survey. Combined non-stimulated saliva and supra-gingival plaque samples were collected from each child and cultivated on selective media for quantitative measures of lactobacilli levels. The procedure for Lactobacillus species screening will include the random selection of 50 colonies per plate, extraction of DNA from each colony, and genotyping by arbitrarily primed polymerase chain reaction (AP-PCR). Each unique Lactobacillus AP-PCR genotype will be selected for taxonomic assessment by 16S rRNA gene sequencing analysis. Lactobacillus species will be identified by comparing the 16S rRNA sequences with the Ribosomal Database and the Human Oral Microbiome Database. Meanwhile, the same set of clinical samples will be independently subjected to genomic DNA isolation, 16S rRNA amplification with Lactobacillus genus-specific primers, sequencing, and taxonomic identification, both at genus and species levels with a customized pipeline. The distribution and phylogenetic differences of these Lactobacillus species will be compared between children with or without S-ECC. One of the main objectives of this study is to establish a study protocol for the identification and characterization of lactobacilli in the oral cavity. Future caries risk assessments can include lactobacilli counts (quantitative) and the presence/absence of specific cariogenic genetic signatures of a Lactobacillus species (qualitative) associated with S-ECC.