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.     

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.     

Microbial Diversity in the Early In Vivo-Formed Dental Biofilm

Although the mature dental biofilm composition is well studied, there is very little information on the earliest phase of in vivo tooth colonization. Progress in dental biofilm collection methodologies and techniques of large-scale microbial identification have made new studies in this field of oral biology feasible. The aim of this study was to characterize the temporal changes and diversity of the cultivable and noncultivable microbes in the early dental biofilm. Samples of early dental biofilm were collected from 11 healthy subjects at 0, 2, 4, and 6 h after removal of plaque and pellicle from tooth surfaces. With the semiquantitative Human Oral Microbiome Identification Microarray (HOMIM) technique, which is based on 16S rRNA sequence hybridizations, plaque samples were analyzed with the currently available 407 HOMIM microbial probes. This led to the identification of at least 92 species, with streptococci being the most abundant bacteria across all time points in all subjects. High-frequency detection was also made with Haemophilus parainfluenzae, Gemella haemolysans, Slackia exigua, and Rothia species. Abundance changes over time were noted for Streptococcus anginosus and Streptococcus intermedius (P = 0.02), Streptococcus mitis bv. 2 (P = 0.0002), Streptococcus oralis (P = 0.0002), Streptococcus cluster I (P = 0.003), G. haemolysans (P = 0.0005), and Stenotrophomonas maltophilia (P = 0.02). Among the currently uncultivable microbiota, eight phylotypes were detected in the early stages of biofilm formation, one belonging to the candidate bacterial division TM7, which has attracted attention due to its potential association with periodontal disease.     

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.     

Exploring the oral microflora of preschool children

The oral cavity is one of the most important and complicated habitats in our body and supports diverse microbial communities. In this study, we aimed to determine the bacterial diversity and composition of various oral micro-niches. Samples were collected from supragingival plaque, saliva, and tongue coating from 10 preschool children (30 samples total). 16S rRNA gene pyrosequencing dataset generated 314,639 clean reads with an average of 10,488 ± 2,787 reads per sample. The phyla Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Fusobacteria were predominant, accounting for more than 90% of the total sequences. We found the highest α diversity, microbial richness, and evenness in plaque, compared with saliva and tongue coating. Plaque was also distinguished from saliva and tongue coating by phylogenetic distances (weighted UniFrac). Taxa with different relative abundances were further identified, confirming the existence of microbial differences across the three niches. Core microbiomes were defined of each niche; however, only a small proportion of operational taxonomic units (8.07%) were shared by the three niches. Coaggregation between Actinomyces spp. and Streptococcus spp. and other correlations among periodontal pathogens, such as Prevotella, Fusobacteria, Capnocytophaga, and Tannerella, were shown by a co-occurrence network. In summary, our study provides a framework of oral microbial communities in the population of preschool children as a baseline for further studies of oral diseases related to microbes.     

Terminal RFLP analysis to determine the oral microbiota with hyposalivation

Previous studies of oral microbiota by culture-dependent or targeted DNA approaches demonstrated that hyposalivation, a reduction in salivary secretions, might increase the amount of certain oral pathogens. However, the relationship between hyposalivation and the balance of oral microbiota, especially uncultivable bacteria, remains still unclear. The aim of this study was to elucidate the relationship between hyposalivation and oral microbiota by analyzing terminal restriction fragment length polymorphism (T-RFLP) of 16S rDNA. The 61 subjects were divided into two groups, hyposalivation group and normo-salivation group. The microbiota of tongue-coating samples was analyzed by T-RFLP. The amount of saliva, the number of Candida albicans, and also the dental status including plaque index, gingival index, bleeding on probing, probing pocket depth and decayed, missing, and filled teeth (DMFT) were assessed. Regarding the dental status, none of the evaluated factors were significantly different between the groups except the number of DMFT. According to the T-RFLP profiles, the patterns of microbiota in the tongue coating were classified into two groups, Clusters I and II. Cluster I is made up 76 % of subjects with hyposalivation, while Cluster II is made up 61 % of subjects with normo-salivation (p < 0.001). Compared with the microbiota found in Cluster II, that in Cluster I had higher proportions of T-RFs corresponding to genera Veillonella, Dialister, Prevotella, Fusobacterium, and Streptococcus. T-RFLP analysis showed a significant role of salivary volume in determining the composition of the microbial community, regardless of the cultivability of the bacteria.     

Oral Microbiota and Risk for Esophageal Squamous Cell Carcinoma in a High-Risk Area of China

Poor oral health has been linked with an increased risk of esophageal squamous cell carcinoma (ESCC). We investigated whether alteration of oral microbiota is associated with ESCC risk. Fasting saliva samples were collected from 87 incident and histopathologicallly diagnosed ESCC cases, 63 subjects with dysplasia and 85 healthy controls. All subjects were also interviewed with a questionnaire. V3–V4 region of 16S rRNA was amplified and sequenced by 454-pyrosequencing platform. Carriage of each genus was compared by means of multivariate-adjusted odds ratios derived from logistic regression model. Relative abundance was compared using Metastats method. Beta diversity was estimated using Unifrac and weighted Unifrac distances. Principal coordinate analysis (PCoA) was applied to ordinate dissimilarity matrices. Multinomial logistic regression was used to compare the coordinates between different groups. ESCC subjects had an overall decreased microbial diversity compared to control and dysplasia subjects (P<0.001). Decreased carriage of genera Lautropia, Bulleidia, Catonella, Corynebacterium, Moryella, Peptococcus and Cardiobacterium were found in ESCC subjects compared to non-ESCC subjects. Multinomial logistic regression analyses on PCoA coordinates also revealed that ESCC subjects had significantly different levels for several coordinates compared to non-ESCC subjects. In conclusion, we observed a correlation between altered salivary bacterial microbiota and ESCC risk. The results of our study on the saliva microbiome are of particular interest as it reflects the shift in microbial communities. Further studies are warranted to verify this finding, and if being verified, to explore the underlying mechanisms.     

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.     

Extracellular Glycoside Hydrolase Activities in the Human Oral Cavity

Carbohydrate availability shifts when bacteria attach to a surface and form biofilm. When salivary planktonic bacteria form an oral biofilm, a variety of polysaccharides and glycoproteins are the primary carbon sources; however, simple sugar availabilities are limited due to low diffusion from saliva to biofilm. We hypothesized that bacterial glycoside hydrolase (GH) activities would be higher in a biofilm than in saliva in order to maintain metabolism in a low-sugar, high-glycoprotein environment. Salivary bacteria from 13 healthy individuals were used to grow in vitro biofilm using two separate media, one with sucrose and the other limiting carbon sources to a complex carbohydrate. All six GHs measured were higher in vitro when grown in the medium with complex carbohydrate as the sole carbon source. We then collected saliva and overnight dental plaque samples from the same individuals and measured ex vivo activities for the same six enzymes to determine how oral microbial utilization of glycoconjugates shifts between the planktonic phase in saliva and the biofilm phase in overnight dental plaque. Overall higher GH activities were observed in plaque samples, in agreement with in vitro observation. A similar pattern was observed in GH activity profiles between in vitro and ex vivo data. 16S rRNA gene analysis showed that plaque samples had a higher abundance of microorganisms with larger number of GH gene sequences. These results suggest differences in sugar catabolism between the oral bacteria located in the biofilm and those in saliva.     

Oral Microbiota Distinguishes Acute Lymphoblastic Leukemia Pediatric Hosts from Healthy Populations

In leukemia, oral manifestations indicate aberrations in oral microbiota. Microbiota structure is determined by both host and environmental factors. In human hosts, how health status shapes the composition of oral microbiota is largely unknown. Taking advantage of advances in high-throughput sequencing, we compared the composition of supragingival plaque microbiota of acute lymphoblastic leukemia (ALL) pediatric patients with healthy controls. The oral microbiota of leukemia patients had lower richness and less diversity compared to healthy controls. Microbial samples clustered into two major groups, one of ALL patients and another of healthy children, with different structure and composition. Abundance changes of certain taxa including the Phylum Firmicutes, the Class Bacilli, the Order Lactobacillales, the Family Aerococcaceae and Carnobacteriaceae, as well as the Genus Abiotrophia and Granulicatella were associated with leukemia status. ALL patients demonstrated a structural imbalance of the oral microbiota, characterized by reduced diversity and abundance alterations, possibly involved in systemic infections, indicating the importance of immune status in shaping the structure of oral microbiota.     

Supragingival Microbial Profiles of Permanent and Deciduous Teeth in Children with Mixed Dentition

Objectives

The present study was designed to investigate the microbial profiles of teeth in different locations in mixed-dentition-stage children, and to compare the microbiomes of permanent and deciduous teeth in the same healthy oral cavity.

Methods

Supragingival plaque samples of teeth in various locations—the first permanent molars, deciduous molars, deciduous canines and incisors and permanent incisors—were collected from 20 healthy mixed-dentition-stage children with 10–12 permanent teeth erupted. Plaque DNA was extracted, and the V3–V4 hypervariable region of the bacterial 16S rRNA gene was amplified and subjected to sequencing.

Results

On average, 18,051 high-quality sequences per sample were generated. Permanent tooth sites tended to host more diverse bacterial communities than those of deciduous tooth sites. A total of 12 phyla, 21 classes, 38 orders, 66 families, 74 genera were detected ultimately. Five predominant phyla (Proteobacteria, Firmicutes, Bacteroidetes, Fusobacteria and Actinobacteria) were highly variable among sites. Of 26 genera with a mean relative abundance of >0.1%, 16 showed significant differences in relative abundance among the groups. More than 20% of the total operational taxonomical units were detected only in permanent or deciduous teeth. The variation in the microbial community composition was due mainly to permanent teeth being enriched in Actinomyces and deciduous teeth in Treponema. The core microbiome of supragingival plaque in mixed dentition comprised 19 genera with complex correlationships.

Conclusion

Our results suggest differences in microbial diversity and composition between permanent and deciduous teeth sites in mixed dentition. Moreover, the core microbiome of these sites was determined. These findings enhance our understanding of the development of the native oral microbiota with age.     

Molecular Characterization of Subject-Specific Oral Microflora during Initial Colonization of Enamel

The initial microbial colonization of tooth surfaces is a repeatable and selective process, with certain bacterial species predominating in the nascent biofilm. Characterization of the initial microflora is the first step in understanding interactions among community members that shape ensuing biofilm development. Using molecular methods and a retrievable enamel chip model, we characterized the microbial diversity of early dental biofilms in three subjects. A total of 531 16S rRNA gene sequences were analyzed, and 97 distinct phylotypes were identified. Microbial community composition was shown to be statistically different among subjects. In all subjects, however, 4-h and 8-h communities were dominated by Streptococcus spp. belonging to the Streptococcus oralis/Streptococcus mitis group. Other frequently observed genera (comprising at least 5% of clone sequences in at least one of the six clone libraries) were Actinomyces, Gemella, Granulicatella, Neisseria, Prevotella, Rothia, and Veillonella. Fluorescence in situ hybridization (FISH) confirmed that the proportion of Streptococcus sp. sequences in the clone libraries coincided with the proportion of streptococcus probe-positive organisms on the chip. FISH also revealed that, in the undisturbed plaque, not only Streptococcus spp. but also the rarer Prevotella spp. were usually seen in small multigeneric clusters of cells. This study shows that the initial dental plaque community of each subject is unique in terms of diversity and composition. Repetitive and distinctive community composition within subjects suggests that the spatiotemporal interactions and ecological shifts that accompany biofilm maturation also occur in a subject-dependent manner.     

Microbiological Survey of the Human Gastric Ecosystem Using Culturing and Pyrosequencing Methods

Stomach mucosa biopsies and gastric juices samples of 12 healthy persons were analysed by culturing in selective- and non-selective-rich media. Microbial DNA from four mucosal samples was also amplified by nested PCR using universal bacterial primers, and the 16S rDNA amplicons pyrosequenced. The total number of cultivable microorganisms recovered from the samples ranged from 10² to 10⁴?cfu/g or ml. The isolates were identified at the species level by PCR amplification and sequencing of the 16S rDNA. Isolates belonged mainly to four genera; Propionibacterium, Lactobacillus, Streptococcus and Staphylococcus. A total of 15,622 high-quality 16S rDNA sequence reads were obtained by pyrosequencing from the four mucosal samples. Sequence analysis grouped the reads into 59 families and 69 genera, revealing wide bacterial diversity. Considerable differences in the composition of the gastric microbiota were observed among the subjects, although in all samples the most abundant operational taxonomic units belonged to Streptococcus, Propionibacterium and Lactobacillus. Comparison of the stomach microbiota with that present in other parts of the human gastrointestinal tract revealed distinctive microbial communities. This is the first study in which a combination of culture and culture-independent techniques has been used to explore the bacterial diversity of the human stomach.     

Exploring the Sources of Bacterial Spoilers in Beefsteaks by Culture-Independent High-Throughput Sequencing

Microbial growth on meat to unacceptable levels contributes significantly to change meat structure, color and flavor and to cause meat spoilage. The types of microorganisms initially present in meat depend on several factors and multiple sources of contamination can be identified. The aims of this study were to evaluate the microbial diversity in beefsteaks before and after aerobic storage at 4°C and to investigate the sources of microbial contamination by examining the microbiota of carcasses wherefrom the steaks originated and of the processing environment where the beef was handled. Carcass, environmental (processing plant) and meat samples were analyzed by culture-independent high-throughput sequencing of 16S rRNA gene amplicons. The microbiota of carcass swabs was very complex, including more than 600 operational taxonomic units (OTUs) belonging to 15 different phyla. A significant association was found between beef microbiota and specific beef cuts (P<0.01) indicating that different cuts of the same carcass can influence the microbial contamination of beef. Despite the initially high complexity of the carcass microbiota, the steaks after aerobic storage at 4°C showed a dramatic decrease in microbial complexity. Pseudomonas sp. and Brochothrix thermosphacta were the main contaminants, and Acinetobacter, Psychrobacter and Enterobacteriaceae were also found. Comparing the relative abundance of OTUs in the different samples it was shown that abundant OTUs in beefsteaks after storage occurred in the corresponding carcass. However, the abundance of these same OTUs clearly increased in environmental samples taken in the processing plant suggesting that spoilage-associated microbial species originate from carcasses, they are carried to the processing environment where the meat is handled and there they become a resident microbiota. Such microbiota is then further spread on meat when it is handled and it represents the starting microbial association wherefrom the most efficiently growing microbial species take over during storage and can cause spoilage.     

High Abundance of Escherichia During the Establishment of Fecal Microbiota in Brazilian Children

The sequence of bacterial events that occurs during the colonization of the gastrointestinal tract may affect the future health of the host. A clear understanding of the colonization process of the human neonatal gut in developing countries is lacking because the few available studies were mostly performed using culture techniques. Using molecular approaches, this study analyzed the fecal microbiota of children of low socioeconomic status in São Paulo, Brazil, during their first year of life. We collected fecal samples of healthy children at 3, 6, and 12 months of life. Total DNA was extracted directly from feces, and the bacteria-specific primers 27F-1492R were used to construct 16S rRNA libraries. Clones were randomly selected and partially sequenced. The main phylogenetic groups identified at 3 months were Streptococcus, unidentified bacteria, and Escherichia. At 6 months, Escherichia remained predominant, while the unidentified bacterial population increased significantly. At 12 months, a more complex composition of fecal microbiota was observed, represented by unidentified bacteria and microorganisms found at low rates at earlier ages. The genus Escherichia remained the most abundant microorganism (34 % relative abundance and 75 % prevalence). Principal component analysis (PCA) revealed changes in the composition of the microbiota at 6 months and an increase of diversity at 12 months of life. Bifidobacterium was identified by quantitative PCR (qPCR) and showed a high incidence in the microbiota at 3 months. The present results corroborate the global observation of inter-individual variability with an early establishment of microbial complexity at the end of the first year of life and highlight the presence of the Escherichia as abundant in microbiota composition of this group of children.     

The oral microbiome of pregnant women facilitates gestational diabetes discrimination

The oral microbiota plays an important role in the development of various diseases,whereas its association with gestational diabetes mellitus (GDM) remains largely unclear.The aim of this study is to identify biomarkers from the oral microbiota of GDM patients by analyzing the microbiome of the saliva and dental plaque samples of 111 pregnant women.We find that the microbiota of both types of oral samples in GDM patients exhibits differences and significantly varies from that of patients with periodontitis or dental caries.Using bacterial biomarkers from the oral microbiota,GDM classification models based on support vector machine and random forest algorithms are constructed.The area under curve (AUC) value of the classification model constructed by combination of Lautropia and Neisseria in dental plaque and Streptococcus in saliva reaches 0.83,and the value achieves a maximum value of 0.89 by adding clinical features.These findings suggest that certain bacteria in either saliva or dental plaque can effectively distinguish women with GDM from healthy pregnant women,which provides evidence of oral microbiome as an informative source for developing noninvasive biomarkers of GDM.     

Phenotypic and Genotypic Selection of Microbiota Surviving under Dental Restorations

The effects of sealing infected carious dentine below dental restorations on the phenotypic and genotypic diversity of the surviving microbiota was investigated. It was hypothesized that the microbiota would be subject to nutrient limitation or nutrient simplification, as it would no longer have access to dietary components or salivary secretion for growth. The available nutrients would be limited primarily to serum proteins passing from the pulp through the patent dentinal tubules to the infected dentine. Ten lesions were treated, and infected dentine was sealed below dental restorations for approximately 5 months. Duplicate standardized samples of infected dentine were taken at baseline and after the removal of the restorations. The baseline microbiota were composed primarily of Lactobacillus spp., Streptococcus mutans, Streptococcus parasanguinis, Actinomyces israelii, and Actinomyces gerencseriae. None of these taxa were isolated among the microbiota of the dentine samples taken after 5 months, which consisted of only Actinomyces naeslundii, Streptococcus oralis, Streptococcus intermedius, and Streptococcus mitis. The microbiota of the final sample exhibited a significantly (P < 0.001) increased ability to produce glycosidic enzymes (sialidase, β-N-acetylglucosaminidase, and β-galactosidase), which liberate sugars from glycoproteins. The genotypic diversity of S. oralis and A. naeslundii was significantly (P = 0.002 and P = 0.001, respectively) reduced in the final samples. There was significantly (P < 0.001) greater genotypic diversity within these taxa between the pairs of dentine samples taken at baseline than was found in the 5-month samples, indicating that the dentine was more homogenous than it was at baseline. We propose that during the interval between placement of the restorations and their removal, the available nutrient, primarily serum proteins, or the relative simplicity and homogeneity of the nutrient supply significantly affected the surviving microbiota. The surviving microbiota was less complex, based on compositional, phenotypic, and genotypic analyses, than that isolated from carious lesions which were also exposed to salivary secretions and pH perturbations.     

The Repertoire of Archaea Cultivated from Severe Periodontitis

In previous studies, the abundance and diversity of methanogenic archaea in the dental microbiota have been analysed by the detection of specific DNA sequences by PCR-based investigations and metagenomic studies. Few data issued regarding methanogens actually living in dental plaque. We collected dental plaque specimens in 15 control individuals and 65 periodontitis patients. Dental plaque specimens were cultured in an anoxic liquid medium for methanogens in the presence of negative control tubes. Dental plaque methanogens were cultured from 1/15 (6.67%) control and 36/65 (55.38%) periodontitis patient samples (p<0.001). The cultures yielded Methanobrevibacter oralis in one control and thirty-one patients, Methanobrevibacter smithii in two patients and a potential new species named Methanobrevibacter sp. strain N13 in three patients with severe periodontitis. Our observations of living methanogens, strengthen previous observations made on DNA-based studies regarding the role of methanogens, in periodontitis.     

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.     

Impact of Fishmeal Replacement in Diets for Gilthead Sea Bream (Sparus aurata) on the Gastrointestinal Microbiota Determined by Pyrosequencing the 16S rRNA Gene

Recent studies have demonstrated the impact of diet on microbiota composition, but the essential need for the optimization of production rates and costs forces farms and aquaculture production to carry out continuous dietary tests. In order to understand the effect of total fishmeal replacement by vegetable-based feed in the sea bream (Sparus aurata), the microbial composition of the stomach, foregut, midgut and hindgut was analysed using high-throughput 16S rDNA sequencing, also considering parameters of growth, survival and nutrient utilisation indices.A total of 91,539 16S rRNA filtered-sequences were analysed, with an average number of 3661.56 taxonomically assigned, high-quality sequences per sample. The dominant phyla throughout the whole gastrointestinal tract were Actinobacteria, Protebacteria and Firmicutes. A lower diversity in the stomach in comparison to the other intestinal sections was observed. The microbial composition of the Recirculating Aquaculture System was totally different to that of the sea bream gastrointestinal tract. Total fishmeal replacement had an important impact on microbial profiles but not on diversity. Streptococcus (p-value: 0.043) and Photobacterium (p-value: 0.025) were highly represented in fish fed with fishmeal and vegetable-meal diets, respectively. In the stomach samples with the vegetable diet, reads of chloroplasts and mitochondria from vegetable dietary ingredients were rather abundant. Principal Coordinate Analysis showed a clear differentiation between diets in the microbiota present in the gut, supporting the presence of specific bacterial consortia associated with the diet.Although differences in growth and nutritive parameters were not observed, a negative effect of the vegetable diet on the survival rate was determined. Further studies are required to shed more light on the relationship between the immune system and sea bream gastrointestinal tract microbiota and should consider the modulation of the microbiota to improve the survival rate and nutritive efficacy when using plant-based diets.