title: Characterizing Diversity of Lactobacilli Associated with Severe Early Childhood Caries: A Study Protocol.
abstract: 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 oralLactobacillusspecies 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 theLactobacilluspopulation 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 forLactobacillusspecies 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 uniqueLactobacillusAP-PCR genotype will be selected for taxonomic assessment by 16S rRNA gene sequencing analysis.Lactobacillusspecies 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 withLactobacillusgenus-specific primers, sequencing, and taxonomic identification, both at genus and species levels with a customized pipeline. The distribution and phylogenetic differences of theseLactobacillusspecies 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 aLactobacillusspecies (qualitative) associated with S-ECC.
authors.affiliation: Department of Basic Science and Craniofacial Biology, College of Dentistry, New York University, New York, USA.
authors.forename: Yihong
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Cariology and Comprehensive Care, College of Dentistry, New York University, New York, USA.
authors.forename: Silvia
authors.initials: S
authors.lastname: Argimón
authors.affiliation: Department of Cariology and Comprehensive Care, College of Dentistry, New York University, New York, USA.
authors.forename: Catherine N
authors.initials: CN
authors.lastname: Schön
authors.affiliation: Department of Basic Science and Craniofacial Biology, College of Dentistry, New York University, New York, USA.
authors.forename: Prakaimuk
authors.initials: P
authors.lastname: Saraithong
authors.affiliation: Department of Cariology and Comprehensive Care, College of Dentistry, New York University, New York, USA.
authors.forename: Page W
authors.initials: PW
authors.lastname: Caufield
keywords: 16S rRNA; AP-PCR; Bacterial Diversity; Dental Plaque; Early Childhood Caries; Lactobacilli; Saliva