Pyrosequencing Analysis of Oral Microbiota in Children with Severe Early Childhood Dental Caries

Severe early childhood caries are a prevalent public health problem among preschool children throughout the world. However, little is known about the microbiota found in association with severe early childhood caries. Our study aimed to explore the bacterial microbiota of dental plaques to study the etiology of severe early childhood caries through pyrosequencing analysis based on 16S rRNA gene V1–V3 hypervariable regions. Forty participants were enrolled in the study, and we obtained twenty samples of supragingival plaque from caries-free subjects and twenty samples from subjects with severe early childhood caries. A total of 175,918 reads met the quality control standards, and the bacteria found belonged to fourteen phyla and sixty-three genera. Our results show the overall structure and microbial composition of oral bacterial communities, and they suggest that these bacteria may present a core microbiome in the dental plaque microbiota. Three genera, Streptococcus, Granulicatella, and Actinomyces, were increased significantly in children with severe dental cavities. These data may facilitate improvements in the prevention and treatment of severe early childhood caries.     

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

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.     

Maturation of Oral Microbiota in Children with or without Dental Caries

Background

The aim of this longitudinal study was to evaluate the oral microbiota in children from age 3 months to 3 years, and to determine the association of the presence of caries at 3 years of age.

Methods and findings

Oral biofilms and saliva were sampled from children at 3 months (n = 207) and 3 years (n = 155) of age, and dental caries was scored at 3 years of age. Oral microbiota was assessed by culturing of total lactobacilli and mutans streptococci, PCR detection of Streptococcus mutans and Streptococcus sobrinus, 454 pyrosequencing and HOMIM (Human Oral Microbe Identification Microarray) microarray detection of more then 300 species/ phylotypes. Species richness and taxa diversity significantly increased from 3 months to 3 years. Three bacterial genera, present in all the 3-month-old infants, persisted at 3 years of age, whereas three other genera had disappeared by this age. A large number of new taxa were also observed in the 3-year-olds. The microbiota at 3 months of age, except for lactobacilli, was unrelated to caries development at a later age. In contrast, several taxa in the oral biofilms of the 3-year-olds were linked with the presence or absence of caries. The main species/phylotypes associated with caries in 3-year-olds belonged to the Actinobaculum, Atopobium, Aggregatibacter, and Streptococcus genera, whereas those influencing the absence of caries belonged to the Actinomyces, Bergeyella, Campylobacter, Granulicatella, Kingella, Leptotrichia, and Streptococcus genera.

Conclusions

Thus, during the first years of life, species richness and taxa diversity in the mouth increase significantly. Besides the more prevalent colonization of lactobacilli, the composition of the overall microbiota at 3 months of age was unrelated to caries development at a later age. Several taxa within the oral biofilms of the 3-year-olds could be linked to the presence or absence of caries.     

Pyrosequencing Analysis of the Salivary Microbiota of Healthy Chinese Children and Adults

Describing the biogeography of bacterial communities within the human body is critical for establishing healthy baselines from which to detect differences associated with diseases. Little is known, however, about the baseline of normal salivary microbiota from healthy Chinese children and adults. With parallel barcoded 454 pyrosequencing, the bacterial diversity and richness of saliva were thoroughly investigated from ten healthy Chinese children and adults. The overall taxonomic distribution of our metagenomic data demonstrated that the diversity of salivary microbiota from children was more complex than adults, while the composition and richness of salivary microbiota were similar in children and adults, especially for predominant bacteria. A large number of bacterial phylotypes were shared by healthy children and adults, indicating the existence of a core salivary microbiome. In children and adults, the vast majority of sequences in salivary microbiota belonged to Streptococcus, Prevotella, Neisseria, Haemophilus, Porphyromonas, Gemella, Rothia, Granulicatella, Fusobacterium, Actinomyces, Veillonella, and Aggregatibacter, which constituted the major components of normal salivary microbiota. With the exception of Actinomyces, the other seven non-predominant bacteria including Moraxella, Leptotrichia, Peptostreptococcus, Eubacterium, and members of Neisseriaceae, Flavobacteriaceae, and SR1 showed significant differences between children and adults (p?<?0.05). We first established the framework of normal salivary microbiota from healthy Chinese children and adults. Our data represent a critical step for determining the diversity of healthy microbiota in Chinese children and adults, and our data established a platform for additional large-scale studies focusing on the interactions between health and diseases in the future.     

Pyrosequencing of Plaque Microflora In Twin Children with Discordant Caries Phenotypes

Despite recent successes in the control of dental caries, the mechanism of caries development remains unclear. To investigate the causes of dental decay, especially in early childhood caries, the supragingival microflora composition of 20 twins with discordant caries phenotypes were analyzed using high-throughput pyrosequencing. In addition, the parents completed a lifestyle questionnaire. A total of 228,789 sequencing reads revealed 10 phyla, 84 genera, and 155 species of microflora, the relative abundances of these strains varied dramatically among the children, Comparative analysis between groups revealed that Veillonella, Corynebacterium and Actinomyces were presumed to be caries-related genera, Fusobacterium, Kingella and Leptotrichia were presumed to be healthy-related genus, yet this six genera were not statistically significant (P>0.05). Moreover, a cluster analysis revealed that the microbial composition of samples in the same group was often dissimilar but that the microbial composition observed in twins was usually similar. Although the genetic and environmental factors that strongly influence the microbial composition of dental caries remains unknown, we speculate that genetic factors primarily influence the individual''s susceptibility to dental caries and that environmental factors primarily regulate the microbial composition of the dental plaque and the progression to caries. By using improved twins models and increased sample sizes, our study can be extended to analyze the specific genetic and environmental factors that affect the development of caries.     

Comparison of Oral Microbial Profiles between Children with Severe Early Childhood Caries and Caries-Free Children Using the Human Oral Microbe Identification Microarray

Objective

Early childhood caries (ECC) has become a prevalent public health problem among Chinese preschool children. The bacterial microflora is considered to be an important factor in the formation and progress of dental caries. However, high-throughput and large-scale studies of the primary dentition are lacking. The present study aimed to compare oral microbial profiles between children with severe ECC (SECC) and caries-free children.

Methods

Both saliva and supragingival plaque samples were obtained from children with SECC (n = 20) and caries-free children (n = 20) aged 3 to 4 years. The samples were assayed using the Human Oral Microbe Identification Microarray (HOMIM).

Results

A total of 379 bacterial species were detected in both the saliva and supragingival plaque samples from all children. Thirteen (including Streptococcus) and two (Streptococcus and Actinomyces) bacterial species in supragingival plaque and saliva, respectively, showed significant differences in prevalence between the two groups. Of these, the frequency of Streptococcus mutans detection was significantly higher in both saliva (p = 0.026) and plaque (p = 0.006) samples from the SECC group than in those from the caries-free group.

Conclusions

The findings of our study revealed differences in the oral microbiota between the SECC and caries-free groups Several genera, including Streptococcus, Porphyromonas, and Actinomyces, are strongly associated with SECC and can be potential biomarkers of dental caries in the primary dentition.     

Exploring the Dynamic Core Microbiome of Plaque Microbiota during Head-and-Neck Radiotherapy Using Pyrosequencing

Radiotherapy is the primary treatment modality used for patients with head-and-neck cancers, but inevitably causes microorganism-related oral complications. This study aims to explore the dynamic core microbiome of oral microbiota in supragingival plaque during the course of head-and-neck radiotherapy. Eight subjects aged 26 to 70 were recruited. Dental plaque samples were collected (over seven sampling time points for each patient) before and during radiotherapy. The V1–V3 hypervariable regions of bacterial 16S rRNA genes were amplified, and the high-throughput pyrosequencing was performed. A total of 140 genera belonging to 13 phyla were found. Four phyla (Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria) and 11 genera (Streptococcus, Actinomyces, Veillonella, Capnocytophaga, Derxia, Neisseria, Rothia, Prevotella, Granulicatella, Luteococcus, and Gemella) were found in all subjects, supporting the concept of a core microbiome. Temporal variation of these major cores in relative abundance were observed, as well as a negative correlation between the number of OTUs and radiation dose. Moreover, an optimized conceptual framework was proposed for defining a dynamic core microbiome in extreme conditions such as radiotherapy. This study presents a theoretical foundation for exploring a core microbiome of communities from time series data, and may help predict community responses to perturbation as caused by exposure to ionizing radiation.     

Temporal Stability of the Salivary Microbiota in Oral Health

Objectives

Saliva is a biological fluid suitable for biomarker analysis, and differences in the salivary microbiota in oral health and disease have been reported. For such comparative analyses, time of sampling is critical since the bacterial composition may vary throughout the day, i.e., diurnal variation. The purpose of this study is to compare the salivary microbiome over time to determine the optimal time for sampling.

Design

Stimulated saliva samples were collected from 5 orally healthy individuals in 4 h intervals for 24 h, and collection was repeated 7 days later (number of samples per person, n = 12, total number of samples, n = 60). Salivary microbiota was analyzed using the Human Oral Microbe Identification using Next Generation Sequencing (HOMINGS), and statistical analysis was performed using the Kruskal-Wallis test with Benjamini-Hochberg’s correction for multiple comparisons, cluster analysis, principal component analysis and correspondence analysis.

Results

From a total of 60 saliva samples, 477 probe targets were collectively identified with a mean number of probes per sample of 207 (range: 153–307). Little or no variation in microbial profiles within subjects was observed over time.

Conclusions

Although there was considerable variation between subjects, microbial profiles within subjects were stable throughout a 24 hour period and after 1 week. Since there is little or no evidence of diurnal variation of the salivary microbiome, time of sampling of saliva is not critical for perturbation or other microbial studies.     

Impact of Pelvic Radiotherapy on Gut Microbiota of Gynecological Cancer Patients Revealed by Massive Pyrosequencing

Although pelvic irradiation is effective for the treatment of various cancer types, many patients who receive radiotherapy experience serious complications. Gut microbial dysbiosis was hypothesized to be related to the occurrence of radiation-induced complications in cancer patients. Given the lack of clinical or experimental data on the impact of radiation on gut microbiota, a prospective observational study of gut microbiota was performed in gynecological cancer patients receiving pelvic radiotherapy. In the current study, the overall composition and alteration of gut microbiota in cancer patients receiving radiation were investigated by 454 pyrosequencing. Gut microbial composition showed significant differences (P < 0.001) between cancer patients and healthy individuals. The numbers of species-level taxa were severely reduced after radiotherapy (P < 0.045), and the abundance of each community largely changed. In particular, the phyla Firmicutes and Fusobacterium were significantly decreased by 10% and increased by 3% after radiation therapy, respectively. In addition, overall gut microbial composition was gradually remolded after the full treatment course of pelvic radiotherapy. In this set of cancer patients, dysbiosis of the gut microbiota was linked to health status, and the gut microbiota was influenced by pelvic radiotherapy. Although further studies are needed to elucidate the relationship between dysbiosis and complications induced by pelvic radiotherapy, the current study may offer insights into the treatment of cancer patients suffering from complications after radiation therapy.     

Robustness of Gut Microbiota of Healthy Adults in Response to Probiotic Intervention Revealed by High-Throughput Pyrosequencing

Probiotics are live microorganisms that potentially confer beneficial outcomes to host by modulating gut microbiota in the intestine. The aim of this study was to comprehensively investigate effects of probiotics on human intestinal microbiota using 454 pyrosequencing of bacterial 16S ribosomal RNA genes with an improved quantitative accuracy for evaluation of the bacterial composition. We obtained 158 faecal samples from 18 healthy adult Japanese who were subjected to intervention with 6 commercially available probiotics containing either Bifidobacterium or Lactobacillus strains. We then analysed and compared bacterial composition of the faecal samples collected before, during, and after probiotic intervention by Operational taxonomic units (OTUs) and UniFrac distances. The results showed no significant changes in the overall structure of gut microbiota in the samples with and without probiotic administration regardless of groups and types of the probiotics used. We noticed that 32 OTUs (2.7% of all analysed OTUs) assigned to the indigenous species showed a significant increase or decrease of ≥10-fold or a quantity difference in >150 reads on probiotic administration. Such OTUs were found to be individual specific and tend to be unevenly distributed in the subjects. These data, thus, suggest robustness of the gut microbiota composition in healthy adults on probiotic administration.     

Intestinal Microbiota in Early Life and Its Implications on Childhood Health

Trillions of microbes reside in the human body and participate in multiple physiological and pathophysiological processes that affect host health throughout the life cycle. The microbiome is hallmarked by distinctive compositional and functional features across different life periods.Accumulating evidence has shown that microbes residing in the human body may play fundamental roles in infant development and the maturation of the immune system. Gut microbes are thought to be essential for the facilitation of infantile and childhood development and immunity by assisting in breaking down food substances to liberate nutrients, protecting against pathogens, stimulating or modulating the immune system, and exerting control over the hypothalamic–pituitary–adrenal axis.This review aims to summarize the current understanding of the colonization and development of the gut microbiota in early life, highlighting the recent findings regarding the role of intestinal microbes in pediatric diseases. Furthermore, we also discuss the microbiota-mediated therapeutics that can reconfigure bacterial communities to treat dysbiosis.     

Community Analysis of Chronic Wound Bacteria Using 16S rRNA Gene-Based Pyrosequencing: Impact of Diabetes and Antibiotics on Chronic Wound Microbiota

Background

Bacterial colonization is hypothesized to play a pathogenic role in the non-healing state of chronic wounds. We characterized wound bacteria from a cohort of chronic wound patients using a 16S rRNA gene-based pyrosequencing approach and assessed the impact of diabetes and antibiotics on chronic wound microbiota.

Methodology/Principal Findings

We prospectively enrolled 24 patients at a referral wound center in Baltimore, MD; sampled patients'' wounds by curette; cultured samples under aerobic and anaerobic conditions; and pyrosequenced the 16S rRNA V3 hypervariable region. The 16S rRNA gene-based analyses revealed an average of 10 different bacterial families in wounds—approximately 4 times more than estimated by culture-based analyses. Fastidious anaerobic bacteria belonging to the Clostridiales family XI were among the most prevalent bacteria identified exclusively by 16S rRNA gene-based analyses. Community-scale analyses showed that wound microbiota from antibiotic treated patients were significantly different from untreated patients (p = 0.007) and were characterized by increased Pseudomonadaceae abundance. These analyses also revealed that antibiotic use was associated with decreased Streptococcaceae among diabetics and that Streptococcaceae was more abundant among diabetics as compared to non-diabetics.

Conclusions/Significance

The 16S rRNA gene-based analyses revealed complex bacterial communities including anaerobic bacteria that may play causative roles in the non-healing state of some chronic wounds. Our data suggest that antimicrobial therapy alters community structure—reducing some bacteria while selecting for others.     

Spatial Variation of the Gut Microbiota in Broiler Chickens as Affected by Dietary Available Phosphorus and Assessed by T-RFLP Analysis and 454 Pyrosequencing

Molecular fingerprinting and sequencing based techniques have been widely used to characterize microbial communities. Terminal restriction fragment length polymorphism (T-RFLP) and 454-pyrosequencing were used to determine the microorganisms present in the different sections of the chicken gastrointestinal tract (GIT) (crop, jejunum, ileum and caeca). Broilers fed with diets differing in phosphorous (P) and calcium (Ca) as well as in phytase levels were used to study the microbiota of the upper and lower part of the GIT. A database with terminal restriction fragments (T-RF) of the most important organism present in the different gastrointestinal sections was constructed. The analysis revealed a distinct microbial assemblage on each section. Regardless of the diet, crop, jejunum and ileum were mainly colonized by Lactobacillaceae, and caeca were the most diverse site. The correlation between Lactobacillus crispatus and L. reuteri was positive in the crop, but negative in the jejunum. In crop samples, higher P and Ca levels led to a shift in the abundance of L. reuteri and L. crispatus to L. salivarius and L. taiwanensis whereas in the ileum supplementation of phytase favored L. salivarius and L. taiwanensis but resulted in decreased abundance of L. crispatus. Both methods were correlating significantly, being T-RFLP a reliable fingerprinting method to rapidly analyze large numbers of samples in a cost-effective and rapid manner. Results are easy to interpret with no need of deep bioinformatics knowledge and can be integrated with taxonomic information.     

Use of Pyrosequencing to Characterize the Microbiota in the Ileum of Goats Fed with Increasing Proportion of Dietary Grain

This study evaluated the effects of an increasing proportion of dietary grain on changes in bacterial populations in the goat ileum. Nine ruminally fistulated, castrated male goats were assigned to three diets in a completely randomized design. Goats were fed three different dietary treatments containing different proportions of corn grain (0, 25, and 50 %). The pH of the ileal contents and rumen fluid (P = 0.015) linearly decreased (P < 0.001), and the acetate, propionate, butyrate, and total volatile fatty acid in ileal contents increased (P < 0.05) with increases in dietary corn, and similar results were also observed in rumen fluid. The barcoded DNA pyrosequencing method was used to reveal 8 phyla, 70 genera, and 1,693 16S operational taxonomic units (OTUs). At the genus level, the proportions of Acetitomaculum, Enterococcus, Atopobium, unclassified Coriobacteriaceae, and unclassified Planctomycetaceae were linearly decreased (P < 0.05) with increases in corn grain. At the species level, high grain feeding linearly decreased the percentage of OTU8686 (unclassified Bacteria) (P = 0.004). To the best of our knowledge, this is the first study using barcoded DNA pyrosequencing method to survey the ileal microbiome of goats and the results suggest that increasing levels of dietary corn change the composition of the ileal bacterial community. These findings provide previously unknown information about the ileal microbiota of goats and a new understanding of the ileal microbial ecology, which may be useful in modulating the gut microbiome.     

Analysis of read length limiting factors in Pyrosequencing chemistry

Pyrosequencing is a bioluminometric DNA sequencing technique that measures the release of pyrophosphate during DNA synthesis. The amount of pyrophosphate is proportionally converted into visible light by a cascade of enzymatic reactions. Pyrosequencing has heretofore been used for generating short sequence reads (1-100 nucleotides) because certain factors limit the system's ability to perform longer reads accurately. In this study, we have characterized the main read length limiting factors in both three-enzyme and four-enzyme Pyrosequencing systems. A new simulation model was developed to simulate the read length of both systems based on the inhibitory factors in the chemical equations governing each enzymatic cascade. Our results indicate that nonsynchronized extension limits the obtained read length, albeit to a different extent for each system. In the four-enzyme system, nonsynchronized extension due mainly to a decrease in apyrase's efficiency in degrading excess nucleotides proves to be the main limiting factor of read length. Replacing apyrase with a washing step for removal of excess nucleotide proves to be essential in improving the read length of Pyrosequencing. The main limiting factor of the three-enzyme system is shown to be loss of DNA fragments during the washing step. If this loss is minimized to 0.1% per washing cycle, the read length of Pyrosequencing would be well beyond 300 bases.     

Investigation of Supragingival Plaque Microbiota in Different Caries Status of Chinese Preschool Children by Denaturing Gradient Gel Electrophoresis

This study aimed to detect differences in the richness of total supragingival plaque microbiota as well as the species composition of oral streptococci involved in the different stages of dental caries. Forty-five plaque samples were collected from caries-moderate (CM, 4 ≤ dmfs ≤ 6), caries-susceptible (CS, dmfs ≥ 10), and age-matched caries-free children separately. Total DNA was isolated directly from each sample, and polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) analyses using universal and primers specific for oral streptococci were carried out. Using 16S rDNA PCR-DGGE, 34 different species of bacteria were identified in a culture-independent manner and classified into 11 genera according to phylogenetic analysis. Among them, Mitis group streptococci and Campylobacter, which were present in health status, no longer appeared in caries-susceptible samples. In addition, Capnocytophaga, Burkholderia, and Prevotella were found significantly less frequently in the CS group samples (P < 0.05), while there were no significant differences among the prevalence of Neisseria, Leptotrichia, Haemophilus, Mutans group streptococci, Corynebacterium, and Actinomyces in the three groups. Further DGGE analysis of rnpB gene amplicons obtained with oral streptococci species-specific primers showed that a total of 23 species of oral streptococci were identified. Streptococcus sanguinis, Streptococcus mitis, and Streptococcus oralis showed a significantly higher prevalence in healthy children (P < 0.05), while that of Streptococcus mutans and Streptococcus sobrinus did not vary among the three groups. Overall, these results suggest that supragingival plaque microbiota as a whole undergoes a more complicated shift in the caries process.