Al-Hudhairy, Salahddin Abdulqader Othman
(2016)
Genotypic identification of oral bacteria using 16s rRNA gene in children with and without early childhood caries in Kelantan.
Masters thesis, Universiti Sains Malaysia.
Abstract
Oral microorganisms are considered as one of the primary aetiological factors
in Early Childhood Caries (ECC) but they have not been completely identified. The
present study aimed to identify oral bacterial genotypes in 12 caries-free children
(CF) and 36 children with ECC aged 71 month and below by using 16S ribosomal
RNA gene sequence. In ECC children, plaque and dentine samples were collected
from intact enamel surfaces, over cavitated lesion and from dentine lesion surface.
For CF children, plaque samples were collected from sound tooth surfaces at baseline
and after one year follow up. The genomic DNA was extracted from all samples,
subjected to 16S rRNA PCR amplification and the end products were cloned into
pCR®2.1-TOPO® Vector. Five randomly selected positive clones collected from
each surface were sent for sequencing. Identification of the bacterial clones was
performed using BLAST search against GeneBank database. A total of 660 clones
were collected from enrolled children. From ECC group, 540 clones were obtained
from three different surfaces while, in CF group, 120 clones were collected from
sound tooth surfaces at baseline and after one year. Several comparisons were
performed between those identified oral bacteria using Pearson Chi-square test or
Fisher’s exact test between different categorical variables, while McNemar test was
used to compare among the individual categories. A total of 39 oral bacterial generawere identified from the ECC group. At genus level, Streptococcus sp. was the most
predominant bacteria among ECC group. Fusobacterium sp. is significantly higher in
the intact enamel while Lactobacillus sp. is significantly higher in the dentine surface
(p < 0.05). At species level, Fusobacterium nucleatum subsp. polymorphum was
detected in the intact surface (33.3%) while Streptococcus mutans was detected over
the carious lesions and dentine (33.3% and 52.7% respectively). A total 18 oral
bacteria genera were identified from CF group at baseline and after 1 year follow up,
but there were no significant differences between groups. At species level,
Fusobacterium nucleatum subsp. polymorphum is found highest in the CF group.
After follow up, Corynebacterium matruchotii is highest in those who remained
caries free, while Porphyromonas catoniae is highest in those who developed caries.
In conclusion, Streptococcus sp. is strongly associated with caries progression in
children with ECC especially for Streptococcus mutans. Lactobacillus sp. is
restricted to deep carious lesions. Fusobacterium sp., Leptotrichia sp., and
Corynebacterium sp. may play a role in sustaining the healthy environment.
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