Oral Microbiome Profiles: 16S rRNA Pyrosequencing and Microarray Assay Comparison

The human oral microbiome is potentially related to diverse health conditions and high-throughput technology provides the possibility of surveying microbial community structure at high resolution. We compared two oral microbiome survey methods: broad-based microbiome identification by 16S rRNA gene sequencing and targeted characterization of microbes by custom DNA microarray.Oral wash samples were collected from 20 individuals at Memorial Sloan-Kettering Cancer Center. 16S rRNA gene survey was performed by 454 pyrosequencing of the V3–V5 region (450 bp). Targeted identification by DNA microarray was carried out with the Human Oral Microbe Identification Microarray (HOMIM). Correlations and relative abundance were compared at phylum and genus level, between 16S rRNA sequence read ratio and HOMIM hybridization intensity.; Correlation = 0.70–0.84).Microbiome community profiles assessed by 16S rRNA pyrosequencing and HOMIM were highly correlated at the phylum level and, when comparing the more commonly detected taxa, also at the genus level. Both methods are currently suitable for high-throughput epidemiologic investigations relating identified and more common oral microbial taxa to disease risk; yet, pyrosequencing may provide a broader spectrum of taxa identification, a distinct sequence-read record, and greater detection sensitivity

Correlation Network Analysis Applied to Complex Biofilm Communities

The complexity of the human microbiome makes it difficult to reveal organizational principles of the community and even more challenging to generate testable hypotheses. It has been suggested that in the gut microbiome species such as Bacteroides thetaiotaomicron are keystone in maintaining the stability and functional adaptability of the microbial community. In this study, we investigate the interspecies associations in a complex microbial biofilm applying systems biology principles. Using correlation network analysis we identified bacterial modules that represent important microbial associations within the oral community. We used dental plaque as a model community because of its high diversity and the well known species-species interactions that are common in the oral biofilm. We analyzed samples from healthy individuals as well as from patients with periodontitis, a polymicrobial disease. Using results obtained by checkerboard hybridization on cultivable bacteria we identified modules that correlated well with microbial complexes previously described. Furthermore, we extended our analysis using the Human Oral Microbe Identification Microarray (HOMIM), which includes a large number of bacterial species, among them uncultivated organisms present in the mouth. Two distinct microbial communities appeared in healthy individuals while there was one major type in disease. Bacterial modules in all communities did not overlap, indicating that bacteria were able to effectively re-associate with new partners depending on the environmental conditions. We then identified hubs that could act as keystone species in the bacterial modules. Based on those results we then cultured a not-yet-cultivated microorganism, Tannerella sp. OT286 (clone BU063). After two rounds of enrichment by a selected helper (Prevotella oris OT311) we obtained colonies of Tannerella sp. OT286 growing on blood agar plates. This system-level approach would open the possibility of manipulating microbial communities in a targeted fashion as well as associating certain bacterial modules to clinical traits (e.g.: obesity, Crohn's disease, periodontal disease, etc)

Use of 16S ribosomal RNA gene analyses to characterize the bacterial signature associated with poor oral health in West Virginia

<p>Abstract</p> <p>Background</p> <p>West Virginia has the worst oral health in the United States, but the reasons for this are unclear. This pilot study explored the etiology of this disparity using culture-independent analyses to identify bacterial species associated with oral disease.</p> <p>Methods</p> <p>Bacteria in subgingival plaque samples from twelve participants in two independent West Virginia dental-related studies were characterized using 16S rRNA gene sequencing and Human Oral Microbe Identification Microarray (HOMIM) analysis. Unifrac analysis was used to characterize phylogenetic differences between bacterial communities obtained from plaque of participants with low or high oral disease, which was further evaluated using clustering and Principal Coordinate Analysis.</p> <p>Results</p> <p>Statistically different bacterial signatures (<it>P </it>< 0.001) were identified in subgingival plaque of individuals with low or high oral disease in West Virginia based on 16S rRNA gene sequencing. Low disease contained a high frequency of <it>Veillonella </it>and <it>Streptococcus</it>, with a moderate number of <it>Capnocytophaga</it>. High disease exhibited substantially increased bacterial diversity and included a large proportion of Clostridiales cluster bacteria (<it>Selenomonas</it>, <it>Eubacterium, Dialister</it>). Phylogenetic trees constructed using 16S rRNA gene sequencing revealed that Clostridiales were repeated colonizers in plaque associated with high oral disease, providing evidence that the oral environment is somehow influencing the bacterial signature linked to disease.</p> <p>Conclusions</p> <p>Culture-independent analyses identified an atypical bacterial signature associated with high oral disease in West Virginians and provided evidence that the oral environment influenced this signature. Both findings provide insight into the etiology of the oral disparity in West Virginia.</p

Characterization of the Oral Fungal Microbiome (Mycobiome) in Healthy Individuals

The oral microbiome–organisms residing in the oral cavity and their collective genome–are critical components of health and disease. The fungal component of the oral microbiota has not been characterized. In this study, we used a novel multitag pyrosequencing approach to characterize fungi present in the oral cavity of 20 healthy individuals, using the pan-fungal internal transcribed spacer (ITS) primers. Our results revealed the “basal” oral mycobiome profile of the enrolled individuals, and showed that across all the samples studied, the oral cavity contained 74 culturable and 11 non-culturable fungal genera. Among these genera, 39 were present in only one person, 16 genera were present in two participants, and 5 genera were present in three people, while 15 genera (including non-culturable organisms) were present in ≥4 (20%) participants. Candida species were the most frequent (isolated from 75% of participants), followed by Cladosporium (65%), Aureobasidium, Saccharomycetales (50% for both), Aspergillus (35%), Fusarium (30%), and Cryptococcus (20%). Four of these predominant genera are known to be pathogenic in humans. The low-abundance genera may represent environmental fungi present in the oral cavity and could simply be spores inhaled from the air or material ingested with food. Among the culturable genera, 61 were represented by one species each, while 13 genera comprised between 2 and 6 different species; the total number of species identified were 101. The number of species in the oral cavity of each individual ranged between 9 and 23. Principal component (PCO) analysis of the obtained data set followed by sample clustering and UniFrac analysis revealed that White males and Asian males clustered differently from each other, whereas both Asian and White females clustered together. This is the first study that identified the “basal mycobiome” of healthy individuals, and provides the basis for a detailed characterization of the oral mycobiome in health and disease

Bacterial Diversity in Oral Samples of Children in Niger with Acute Noma, Acute Necrotizing Gingivitis, and Healthy Controls

Noma is a devastating gangrenous disease that leads to severe facial disfigurement, but its cause remains unknown. It is associated with high morbidity and mortality and affects almost exclusively young children living in remote areas of developing countries, particularly in Africa. Several factors have been linked to the disease, including malnutrition, immune dysfunction, lack of oral hygiene, and lesions of the mucosal gingival barrier, particularly the presence of acute necrotizing gingivitis, and a potentially non-identified bacterial factor acting as a trigger for the disease. This study assessed the total bacterial diversity present in 69 oral samples of 55 children in Niger with or without acute noma or acute necrotizing gingivitis using culture-independent molecular methods. Analysis of bacterial composition and frequency showed that diseased and healthy site bacterial communities are composed of similar bacteria, but differ in the prevalence of a limited group of phylotypes. We failed to identify a causative infectious agent for noma or acute necrotizing gingivitis as the most plausible pathogens for both conditions were present also in sizeable numbers in healthy subjects. Most likely, the disease is initiated by a synergistic combination of several bacterial species, and not a single agent

Exploring the oral microbiota of children at various developmental stages of their dentition in the relation to their oral health

<p>Abstract</p> <p>Background</p> <p>An understanding of the relation of commensal microbiota to health is essential in preventing disease. Here we studied the oral microbial composition of children (N = 74, aged 3 - 18 years) in natural transition from their deciduous to a permanent dentition and related the microbial profiles to their oral health status. The microbial composition of saliva was assessed by barcoded pyrosequencing of the V5-V6 hypervariable regions of the 16 S rRNA, as well as by using phylogenetic microarrays.</p> <p>Results</p> <p>Pyrosequencing reads (126174 reads, 1045 unique sequences) represented 8 phyla and 113 higher taxa in saliva samples. Four phyla - Firmicutes, Bacteriodetes, Proteobacteria and Actinobacteria - predominated in all groups. The deciduous dentition harboured a higher proportion of Proteobacteria (Gammaproteobacteria, Moraxellaceae) than Bacteroidetes, while in all other groups Bacteroidetes were at least as abundant as Proteobacteria. Bacteroidetes (mainly genus <it>Prevotella</it>), Veillonellaceae family, Spirochaetes and candidate division TM7 increased with increasing age, reflecting maturation of the microbiome driven by biological changes with age.</p> <p>Microarray analysis enabled further analysis of the individual salivary microbiota. Of 350 microarray probes, 156 gave a positive signal with, on average, 77 (range 48-93) probes per individual sample.</p> <p>A caries-free oral status significantly associated with the higher signal of the probes targeting <it>Porphyromonas catoniae </it>and <it>Neisseria flavescens</it>.</p> <p>Conclusions</p> <p>The potential role of <it>P. catoniae </it>and <it>N. flavescens </it>as oral health markers should be assessed in large-scale clinical studies. The combination of both, open-ended and targeted molecular approaches provides us with information that will increase our understanding of the interplay between the human host and its microbiome.</p

Prevalence of systemic immunoreactivity to Aggregatibacter actinomycetemcomitans leukotoxin in relation to the incidence of myocardial infarction

<p>Abstract</p> <p>Background</p> <p>Chronic infections and associated inflammatory markers are suggested risk factors for cardiovascular disease (CVD). The proinflammatory cytokine, interleukin (IL)-1β, is suggested to play a role in the regulation of local inflammatory responses in both CVD and periodontitis. The leukotoxin from the periodontal pathogen <it>Aggregatibacter actinomycetemcomitans </it>has recently been shown to cause abundant secretion of IL-1β from macrophages. The aim of the present study was to compare the prevalence of systemic immunoreactivity to <it>A. actinomycetemcomitans </it>leukotoxin in myocardial infarction (MI) cases (n = 532) and matched controls (n = 1,000) in a population-based case and referents study in northern Sweden.</p> <p>Methods</p> <p>Capacity to neutralize <it>A. actinomycetemcomitans </it>leukotoxin was analyzed in a bioassay with leukocytes, purified leukotoxin, and plasma. Plasma samples that inhibited lactate-dehydrogenase release from leukotoxin-lysed cells by ≥50% were classified as positive.</p> <p>Results</p> <p>Neutralizing capacity against <it>A. actinomycetemcomitans </it>leukotoxin was detected in 53.3% of the plasma samples. The ability to neutralize leukotoxin was correlated to increasing age in men (n = 1,082) but not in women (n = 450). There was no correlation between presence of systemic leukotoxin-neutralization capacity and the incidence of MI, except for women (n = 146). Women with a low neutralizing capacity had a significantly higher incidence of MI than those who had a high neutralizing capacity.</p> <p>Conclusion</p> <p>Systemic immunoreactivity against <it>A. actinomycetemcomitans </it>leukotoxin was found at a high prevalence in the analyzed population of adults from northern Sweden. The results from the present study do not support the hypothesis that systemic leukotoxin-neutralizing capacity can decrease the risk for MI.</p

Anaerobic Carbon Monoxide Dehydrogenase Diversity in the Homoacetogenic Hindgut Microbial Communities of Lower Termites and the Wood Roach

Anaerobic carbon monoxide dehydrogenase (CODH) is a key enzyme in the Wood-Ljungdahl (acetyl-CoA) pathway for acetogenesis performed by homoacetogenic bacteria. Acetate generated by gut bacteria via the acetyl-CoA pathway provides considerable nutrition to wood-feeding dictyopteran insects making CODH important to the obligate mutualism occurring between termites and their hindgut microbiota. To investigate CODH diversity in insect gut communities, we developed the first degenerate primers designed to amplify cooS genes, which encode the catalytic (β) subunit of anaerobic CODH enzyme complexes. These primers target over 68 million combinations of potential forward and reverse cooS primer-binding sequences. We used the primers to identify cooS genes in bacterial isolates from the hindgut of a phylogenetically lower termite and to sample cooS diversity present in a variety of insect hindgut microbial communities including those of three phylogenetically-lower termites, Zootermopsis nevadensis, Reticulitermes hesperus, and Incisitermes minor, a wood-feeding cockroach, Cryptocercus punctulatus, and an omnivorous cockroach, Periplaneta americana. In total, we sequenced and analyzed 151 different cooS genes. These genes encode proteins that group within one of three highly divergent CODH phylogenetic clades. Each insect gut community contained CODH variants from all three of these clades. The patterns of CODH diversity in these communities likely reflect differences in enzyme or physiological function, and suggest that a diversity of microbial species participate in homoacetogenesis in these communities

Pyrosequencing as a tool for better understanding of human microbiomes

Next-generation sequencing technologies have revolutionized the analysis of microbial communities in diverse environments, including the human body. This article reviews several aspects of one of these technologies, the pyrosequencing technique, including its principles, applications, and significant contribution to the study of the human microbiome, with especial emphasis on the oral microbiome. The results brought about by pyrosequencing studies have significantly contributed to refining and augmenting the knowledge of the community membership and structure in and on the human body in healthy and diseased conditions. Because most oral infectious diseases are currently regarded as biofilm-related polymicrobial infections, high-throughput sequencing technologies have the potential to disclose specific patterns related to health or disease. Further advances in technology hold the perspective to have important implications in terms of accurate diagnosis and more effective preventive and therapeutic measures for common oral diseases

The Canine Oral Microbiome

Determining the bacterial composition of the canine oral microbiome is of interest for two primary reasons. First, while the human oral microbiome has been well studied using molecular techniques, the oral microbiomes of other mammals have not been studied in equal depth using culture independent methods. This study allows a comparison of the number of bacterial taxa, based on 16S rRNA-gene sequence comparison, shared between humans and dogs, two divergent mammalian species. Second, canine oral bacteria are of interest to veterinary and human medical communities for understanding their roles in health and infectious diseases. The bacteria involved are mostly unnamed and not linked by 16S rRNA-gene sequence identity to a taxonomic scheme. This manuscript describes the analysis of 5,958 16S rRNA-gene sequences from 65 clone libraries. Full length 16S rRNA reference sequences have been obtained for 353 canine bacterial taxa, which were placed in 14 bacterial phyla, 23 classes, 37 orders, 66 families, and 148 genera. Eighty percent of the taxa are currently unnamed. The bacterial taxa identified in dogs are markedly different from those of humans with only 16.4% of oral taxa are shared between dogs and humans based on a 98.5% 16S rRNA sequence similarity cutoff. This indicates that there is a large divergence in the bacteria comprising the oral microbiomes of divergent mammalian species. The historic practice of identifying animal associated bacteria based on phenotypic similarities to human bacteria is generally invalid. This report describes the diversity of the canine oral microbiome and provides a provisional 16S rRNA based taxonomic scheme for naming and identifying unnamed canine bacterial taxa