Metagenomic study of the oral microbiota by Illumina high throughput sequencing

Genomic Research Laboratory, Geneva University Hospitals, Rue Gabrielle-Perret-Gentil 4, CH-1211 Geneva 14, Switzerland.
Journal of microbiological methods (Impact Factor: 2.03). 09/2009; 79(3):266-71. DOI: 10.1016/j.mimet.2009.09.012
Source: PubMed


To date, metagenomic studies have relied on the utilization and analysis of reads obtained using 454 pyrosequencing to replace conventional Sanger sequencing. After extensively scanning the 16S ribosomal RNA (rRNA) gene, we identified the V5 hypervariable region as a short region providing reliable identification of bacterial sequences available in public databases such as the Human Oral Microbiome Database. We amplified samples from the oral cavity of three healthy individuals using primers covering an approximately 82-base segment of the V5 loop, and sequenced using the Illumina technology in a single orientation. We identified 135 genera or higher taxonomic ranks from the resulting 1,373,824 sequences. While the abundances of the most common phyla (Firmicutes, Proteobacteria, Actinobacteria, Fusobacteria and TM7) are largely comparable to previous studies, Bacteroidetes were less present. Potential sources for this difference include classification bias in this region of the 16S rRNA gene, human sample variation, sample preparation and primer bias. Using an Illumina sequencing approach, we achieved a much greater depth of coverage than previous oral microbiota studies, allowing us to identify several taxa not yet discovered in these types of samples, and to assess that at least 30,000 additional reads would be required to identify only one additional phylotype. The evolution of high-throughput sequencing technologies, and their subsequent improvements in read length enable the utilization of different platforms for studying communities of complex flora. Access to large amounts of data is already leading to a better representation of sample diversity at a reasonable cost.

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Available from: Vladimir Lazarevic, Mar 17, 2015
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    • "Oral streptococci are pioneer colonizers of the oral cavity and are abundant in the dental plaque, a tooth-associated biofilm (Lazarevic et al., 2009). Streptococcus mutans is an important constituent of the dental plaque biofilm (Kolenbrander et al., 2006). "
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    ABSTRACT: Bacteria are considered “social” organisms able to communicate with one another using small hormone-like molecules (pheromones) in a process called quorum-sensing (QS). These signaling molecules increase in concentration as a function of bacterial cell density. For most human pathogens, QS is critical for virulence and biofilm formation, and the opportunity to interfere with bacterial QS could provide a sophisticated means for manipulating the composition of pathogenic biofilms, and possibly eradicating the infection. Streptococcus mutans is a well-characterized resident of the dental plaque biofilm, and is the major pathogen of dental caries (cavities). In S. mutans, its CSP QS signaling peptide does not act as a classical QS signal by accumulating passively in proportion to cell density. In fact, particular stresses such as those encountered in the oral cavity, induce the production of the CSP pheromone, suggesting that the pheromone most probably functions as a stress-inducible alarmone by triggering the signaling to the bacterial population to initiate an adaptive response that results in different phenotypic outcomes. This mini-review discusses two different CSP-induced phenotypes, bacterial “suicide” and dormancy, and the underlying mechanisms by which S. mutans utilizes the same QS signaling peptide to regulate two opposite phenotypes.
    Full-text · Article · Oct 2015 · Frontiers in Microbiology
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    • "H, G, and P indicate the subject with healthy periodontium, gingivitis, and periodontitis, respectively. Lazarevic et al. 2009; Zaura et al. 2009). Thus, Proteobacteria seems to be a predominant phylum in the subgingival PP samples , but Firmicutes in other oral regions except subgingiva. "
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    ABSTRACT: Subgingival microorganisms are potentially associated with periodontal diseases. However, changes in the subgingival microbiota during the progress of periodontal diseases are poorly understood. In this study, we analyzed bacterial communities in the subgingival paper point samples from 32 Korean individuals with no sign of disease, gingivitis, or periodontitis using 454 FLX Titanium pyrosequencing. A total of 256,113 reads representing 26 phyla, 433 genera, and 1,016 species were detected. Bacteroidetes, Fusobacteria, Synergistetes, and Spirochaetes were the abundant phyla in periodontitis subjects, whereas Firmicutes and Proteobacteria were identified as the dominant phyla in the gingivitis and healthy subjects, respectively. Although high levels of Porphyromonas, Fusobacterium, Fretibacterium, Rothia, Filifactor, and Treponema genera were observed in the periodontitis subjects, Streptococcus, Capnocytophaga, Leptotrichia, and Haemophilus genera were found at high frequency in the gingivitis subjects. Species including Porphyromonas gingivalis, Fusobacterium nucleatum, and Fretibacterium fastidiosum were significantly increased in periodontitis subjects. On the other hand, Streptococcus pseudopneumoniae, Haemophilus parainfluenzae, and Leptotrichia hongkongensis were preferentially observed in the gingivitis subjects. Intriguingly, the halophile Halomonas hamiltonii was revealed as a predominant species in the healthy subjects. Based on Fast UniFrac analysis, distinctive bacterial clusters were classified for the healthy, gingivitis, and periodontitis state. The current findings might be useful for understanding the pathogenesis, diagnosis, and treatment of periodontal diseases. © International & American Associations for Dental Research 2015.
    Full-text · Article · Mar 2015 · Journal of Dental Research
    • "DNA was extracted using the PowerSoil DNA Extraction kit (MoBio, Carlsbad, CA, USA). The V5 hypervariable region of the 16S rRNA gene was amplified and sequenced via Illumina HiSeq2000 analyzer (Illumina Inc., San Diego, CA, USA) (forward primer: 5 0 -GGMT TAGATACCC-3 0 , reverse primer: 5 0 -CCGYCA ATTYMTTTRAGTTT-3 0 (Lazarevic et al., 2009) (Eureka Genomics, Hercules, CA, USA). Paired-end sequences (2 Â 100 bp) were aligned to form 120 bp "
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    ABSTRACT: A variety of microbially mediated metabolic pathways impact biogeochemical cycling in terrestrial subsurface environments. However, the role that viruses have in influencing microbial mortality and microbial community structure is poorly understood. Here we investigated the production of viruses and change in microbial community structure within shallow alluvial aquifer sediment slurries amended with (13)C-labeled acetate and nitrate. Biostimulation resulted in production of viruses concurrent with acetate oxidation, (13)CO2 production and nitrate reduction. Interestingly, change in viral abundance was positively correlated to acetate consumption (r(2)=0.6252, P<0.05) and (13)CO2 production (r(2)=0.6572, P<0.05); whereas change in cell abundance was not correlated to acetate consumption or (13)CO2 production. Viral-mediated cell lysis has implications for microbial community structure. Betaproteobacteria predominated microbial community composition (62% of paired-end reads) upon inoculation but decreased in relative abundance and was negatively correlated to changes in viral abundance (r(2)=0.5036, P<0.05). As members of the Betaproteobacteria decreased, Gammaproteobacteria, specifically Pseudomonas spp., increased in relative abundance (82% of paired-end reads) and was positively correlated with the change in viral abundance (r(2)=0.5368, P<0.05). A nitrate-reducing bacterium, Pseudomonas sp. strain Alda10, was isolated from these sediments and produced viral-like particles with a filamentous morphology that did not result in cell lysis. Together, these results indicate that viruses are linked to carbon biogeochemistry and community structure in terrestrial subsurface sediments. The subsequent cell lysis has the potential to alter available carbon pools in subsurface environments, additionally controlling microbial community structure from the bottom-up.The ISME Journal advance online publication, 27 March 2014; doi:10.1038/ismej.2014.38.
    No preview · Article · Mar 2014 · The ISME Journal
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