Analysis of Oral Microbiota in Children with Dental Caries by PCR-DGGE and Barcoded Pyrosequencing

Abstract

Oral microbiota plays a vital role in maintaining the homeostasis of oral cavity. Dental caries are among the most common oral diseases in children and pathogenic bacteria contribute to the development of the disease. However, the overall structure of bacterial communities in the oral cavity from children with dental caries has not been explored deeply heretofore. We used high-throughput barcoded pyrosequencing and PCR-denaturing gradient gel electrophoresis (DGGE) to examine bacterial diversity of oral microbiota in saliva and supragingival plaques from 60 children aged 3 to 6 years old with and without dental caries from China. The multiplex barcoded pyrosequencing was performed in a single run, with multiple samples tagged uniquely by multiplex identifiers. As PCR-DGGE analysis is a conventional molecular ecological approach, this analysis was also performed on the same samples and the results of both approaches were compared. A total of 186,787 high-quality sequences were obtained for evaluating bacterial diversity and 41,905 unique sequences represented all phylotypes. We found that the oral microbiota in children was far more diverse than previous studies reported, and more than 200 genera belonging to ten phyla were found in the oral cavity. The phylotypes in saliva and supragingival plaques were significantly different and could be divided into two distinct clusters (p < 0.05). The bacterial diversity in oral microbiome analyzed by PCR-DGGE and barcoded pyrosequencing was employed to cross validate the data sets. The genera of Streptococcus, Veillonella, Actinomyces, Granulicatella, Leptotrichia, and Thiomonas in plaques were significantly associated with dental caries (p < 0.05). The results showed that there was no one specific pathogen but rather pathogenic populations in plaque that significantly correlated with dental caries. The enormous diversity of oral microbiota allowed for a better understanding of oral microecosystem, and these pathogenic populations in plaque provide new insights into the etiology of dental caries and suggest new targets for interventions of the disease.

Association of oral streptococci community dynamics with severe early childhood caries as assessed by PCR-denaturing gradient gel electrophoresis targeting the rnpB gene

“…These findings imply that the onset of dental caries has an association with a lower diversity of oral streptococci, which is consistent with a cross-section study of preschool children in the various stages of dental caries (Jiang et al, 2011). According to these findings and the previous studies, we infer that the decline in streptococcal diversity in S-ECC plaques is possible because of shifts in the balance of the resident plaque microbiota (Jiang et al, 2011;Li et al, 2007;Ling et al, 2010). Bacterial species present in the dental biofilm communities have cooperative or competitive interactions with other members (Guo et al, 2014).…”

“…Such data could reflect the general microbial composition in dental plaque of the exposed tooth surface, compared with sitespecific plaque sampling, for which data reflect only the situation of specific colonized sites (Tanzer et al, 2001). Previously, we and others demonstrated that PCR-DGGE is a valuable tool for intuitively observing the dynamics of microbial communities composing the dental plaque of children (Jiang et al, 2011;Li et al, 2007;Ling et al, 2010;Tao et al, 2013). In the present study, we sequenced the rnpB gene combined with DGGE to evaluate the diversity and dynamics of oral streptococci associated with S-ECC.…”

“…Previous studies by our group (Tao et al, 2013) and others (Jiang et al, 2011;Li et al, 2007;Ling et al, 2010) demonstrated the superior utility of PCR-denaturing gel gradient electrophoresis (PCR-DGGE) relative to other methods in evaluating the oral microbiota composition. With this approach, microbial profiles of plaques or saliva from various individuals can be compared, and shifts in the ecological balance of the biofilms can be evaluated.…”

Leptotrichia species in human infections II

“…polymorphum , Veillonella spp., Actinomyces, K. oralis , C. matruchotii , C. gracilis , C. curvus , S. gordonii , S. mutans , S. mitis bv. 2, S. cristatus , S. anginosus , cloning, 16S rRNA gene sequencing 84M 73 L. wadei , Leptotrichia spp.Immunocompetent, pneumonia, hypoxemia, sore throat, fever, dyspnea, cough, leukocytosis, RNote: 1st case of pneumoniaBALF, mouth-gargled water Staphylococcus spp., Acidaminococcus spp., Veillonella spp., V. parvula , V. atypica , V. dispar , Lactobacillus spp., Enterococcus spp., E. faecalis , E. casseliflavus , P. nanceiensis , culture, cloning, 16S rRNA gene sequencing[24]85M/F 3–6 Leptotrichia Caries-free male, male with caries, caries-free females, female with cariesSaliva, plaque Capnocytophaga , Peptostreptococcus, Corynebacterium , Rothia, Veillonella , Prevotella, Granulicatella , Streptococcus, Actinomyces , Thiomonas, Kingella , Campylobacter, Fusobacterium , Erysipelothrix, Atopobium , Oribacterium, Haemophilus , Neisseria , DGGE, HTS[25]86M/F 3–5 Leptotrichia spp.Moderate cariesPlaque Capnocytophaga , Corynebacterium, Campylobacter , Haemophilus, mitis group streptococci, mutans group streptococci, Neisseria, Burkholderia , Actinomyces, Prevotella , DGGE, cloning, 16S rRNA gene sequencing[26]M/F 3–5 Leptotrichia spp.Caries-susceptiblePlaque Capnocytophaga, Corynebacterium, Actinomyces , Burkholderia, mutans group streptococci, Neisseria, Haemophilus , Prevotella , DGGE, cloning, 16S rRNA gene sequencing M/F 3–5 Leptotrichia spp.Caries-freePlaque Mitis group streptococci, mutans group streptococci, Neisseria, Prevotella , Campylobacter, Burkholderia , Capnocytophaga, Corynebacterium , Actinomyces, Haemophilus , DGGE, cloning, 16S rRNA gene sequencing 89M/F 25–39 Leptotrichia spp.Unhealthy, gingivitisPlaque, saliva S. sanguinis , Veillonella, Prevotella , Neisseria, Fusobacterium , Rothia , TM7, H. parainfluenzae , Granulicatella, L. mirabilis , Selenomonas, Actinomyces , HTS, PCA[27]F 21–23 ± 18 Leptotrichia spp.…”

“…They were also recovered from oral plaque of guinea pigs [21] and feces of piglets [54], dental plaque from healthy individuals, plaque and saliva from patients with various types of caries, gingivitis, chronic periodontitis, and peri-implantitis [23,25–27,34,35,37,38,44,45,47,49,52,59,60,62,66–69,91,94], decayed tooth surfaces and discordant caries from intact enamel surfaces [53]. Leptotrichia species were also isolated from bronchoalveolar lavage fluid, and patients with leukocytosis, hypoxemia, and dyspnea [24].…”