TITLE:
Characterizing Diversity of Lactobacilli Associated with Severe Early Childhood Caries: A Study Protocol
AUTHORS:
Yihong Li, Silvia Argimón, Catherine N. Schön, Prakaimuk Saraithong, Page W. Caufield
KEYWORDS:
Lactobacilli, Early Childhood Caries, Bacterial Diversity, 16S rRNA, AP-PCR, Saliva, Dental Plaque
JOURNAL NAME:
Advances in Microbiology,
Vol.5 No.1,
January
12,
2015
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 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 sociodemographic
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 supragingival
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, ex- traction 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 cario- genic genetic signatures of a Lactobacillus
species (qualitative) associated with S-ECC.