Search

Aim: To identify microorganisms unrecognized through culture in the lower airway of patients with chronic pulmonary disease through amplification and pyrosequencing of specific genes for microbial diversity assessment Method: To avoid culture-related bias, 1) DNA extraction from respiratory samples (sputum, bronchial aspirate (BAS), bronchoalveolar lavage (BAL) and bronchial mucosa); 2) 16S rRNA amplification and purification with modified primers which include a specific 8 nucleotides code per sample; 3) Pyrosequencing of amplified products by Titanium/GS-FLX System (454-Roche; 4) Obtention of >500 sequences per sample; and 6) Comparison of the results obtained with the diferent samples using bronchial mucosa as gold standard. Results: Sputum, BAS, BAL and bronchial mucosa samples from 6 patients were studied. Over 500 sequences of gen 16S were identified in 17 out of 24 samples. Total number of bacterial genera per patient were over 100. Analysis of coincidence obtained from bronchial mucosa showed low coincidence with BAS and sputum, and maximal coincidence with BAL, that showed a range of 55-74 bacterial genera. Streptococcus, Prevotella, Moraxella, Haemophilus, Acinetobacter, Fusobacterium and Neisseria were the most often identified genders, accounting for up to 60% of the overall. Conclusions: Pyrosequencing is applicable to samples of bronchial secretions and is able to demonstrate a microbial diversity over 600 microorganisms. Sputum and BAS samples were non-representative of bronchial mucosa, but BAL that mostrates over fifty bacterial genera, shows the closest results with bronchial mucosa. Sponsorship by Fundacio Parc Tauli.

When a bacterial genome is compared to the metagenome of an environment it inhabits, most genes recruit at high sequence identity. In free-living bacteria (for instance marine bacteria compared against the ocean metagenome) certain genomic regions are totally absent in recruitment plots, representing therefore genes unique to individual bacterial isolates. We show that these Metagenomic Islands (MIs) are also visible in bacteria living in human hosts when their genomes are compared to sequences from the human microbiome, despite the compartmentalized structure of human-related environments such as the gut. From an applied point of view, MIs of human pathogens (e.g. those identified in enterohaemorragic Escherichia coli against the gut metagenome or in pathogenic Neisseria meningitidis against the oral metagenome) include virulence genes that appear to be absent in related strains or species present in the microbiome of healthy individuals. We propose that this strategy (i.e. recruitment analysis of pathogenic bacteria against the metagenome of healthy subjects) can be used to detect pathogenicity regions in species where the genes involved in virulence are poorly characterized. Using this approach, we detect well-known pathogenicity islands and identify new potential virulence genes in several human pathogens.

Bacterial strains belonging to the same species vary considerably in gene content. Thus, the genetic repertoire of a given species (its "pan-genome") is much larger than the gene content of individual strains. These variations in DNA material, together with differences in genomic structure and nucleotide polymorphisms among strains, confer upon prokaryotic species a phenomenal adaptability. Although the approach of sequencing multiple strains from a single species remains the main and often easiest way to study the pan-genome, feasible alternatives include those related to DNA hybridization. In other cases, the use of metagenomic sequences is already applicable by data mining from the growing metagenomic databases. Eventually, the single-cell genome approach might be the ideal solution. The pan-genome concept has important consequences for the way we understand bacterial evolution, adaptation, and population structure, as well as for more applied issues such as vaccine design or the identification of virulence genes.

The species composition and metabolic potential of microbial and viral communities are predictable and stable for most ecosystems. This apparent stability contradicts theoretical models as well as the viral-microbial dynamics observed in simple ecosystems, both of which show Kill-the-Winner behavior causing cycling of the dominant taxa. Microbial and viral metagenomes were obtained from four human-controlled aquatic environments at various time points separated by one day to >1 year. These environments were maintained within narrow geochemical bounds and had characteristic species composition and metabolic potentials at all time points. However, underlying this stability were rapid changes at the fine-grained level of viral genotypes and microbial strains. These results suggest a model wherein functionally redundant microbial and viral taxa are cycling at the level of viral genotypes and virus-sensitive microbial strains. Microbial taxa, viral taxa, and metabolic function persist over time in stable ecosystems and both communities fluctuate in a Kill-the-Winner manner at the level of viral genotypes and microbial strains. The ISME Journal (2010) 4, 739-751; doi:10.1038/ismej.2010.1; published online 11 February 2010

Fusobacterium nucleatum is a Gram-negative anaerobic organism that plays a central role in the development of periodontal diseases. The progression of periodontitis is associated with a rise in pH of the gingival sulcus which promotes the growth and expression of virulence factors by periodontopathic bacteria. We have previously reported that the expression of specific cytoplasmic proteins is altered by a shift in growth pH. In the present study we have compared cell envelope protein expression of F. nucleatum during chemostat growth at pH 7.2 and 7.8. From a total of 176 proteins resolved from the cell envelope, 15 were found to have altered expression in response to an increase in growth pH and were identified by MS. Upregulated proteins included an outer membrane porin which has been identified as playing a role in virulence, a periplasmic chaperone which assists in the folding of outer membrane proteins, and a transporter thought to be involved with iron uptake. Proteins downregulated at pH 7.8 were consistent with our previous findings that the bacterium reduces its catabolism of energy-yielding substrates in favour of energy-storage pathways. Among the downregulated proteins, two transporters which are involved in the uptake of C4 dicarboxylates and phosphate were identified. A putative protease and an enzyme associated with the metabolism of glutamate were also identified. A high proportion of the cell envelope proteins suggested by these data to play a role in the organism's response to alkaline growth pH may have arisen by lateral gene transfer. This would support the hypothesis that genes that provide an ability to adapt to the changing conditions of the oral environment may be readily shared between oral bacteria.

Background Saturated brines are extreme environments of low diversity. Salinibacter ruber is the only bacterium that inhabits this environment in significant numbers. In order to establish the extent of genetic diversity in natural populations of this microbe, the genomic sequence of reference strain DSM 13855 was compared to metagenomic fragments recovered from climax saltern crystallizers and obtained with 454 sequencing technology. This kind of analysis reveals the presence of metagenomic islands, i.e. highly variable regions among the different lineages in the population. Results Three regions of the sequenced isolate were scarcely represented in the metagenome thus appearing to vary among co-occurring S. ruber cells. These metagenomic islands showed evidence of extensive genomic corruption with atypically low GC content, low coding density, high numbers of pseudogenes and short hypothetical proteins. A detailed analysis of island gene content showed that the genes in metagenomic island 1 code for cell surface polysaccharides. The strain-specific genes of metagenomic island 2 were found to be involved in biosynthesis of cell wall polysaccharide components. Finally, metagenomic island 3 was rich in DNA related enzymes. Conclusion The genomic organisation of S. ruber variable genomic regions showed a number of convergences with genomic islands of marine microbes studied, being largely involved in variable cell surface traits. This variation at the level of cell envelopes in an environment devoid of grazing pressure probably reflects a global strategy of bacteria to escape phage predation.

Background The Mycobacterium leprae genome has less than 50% coding capacity and 1,133 pseudogenes. Preliminary evidence suggests that some pseudogenes are expressed. Therefore, defining pseudogene transcriptional and translational potentials of this genome should increase our understanding of their impact on M. leprae physiology. Results Gene expression analysis identified transcripts from 49% of all M. leprae genes including 57% of all ORFs and 43% of all pseudogenes in the genome. Transcribed pseudogenes were randomly distributed throughout the chromosome. Factors resulting in pseudogene transcription included: 1) co-orientation of transcribed pseudogenes with transcribed ORFs within or exclusive of operon-like structures; 2) the paucity of intrinsic stem-loop transcriptional terminators between transcribed ORFs and downstream pseudogenes; and 3) predicted pseudogene promoters. Mechanisms for translational "silencing" of pseudogene transcripts included the lack of both translational start codons and strong Shine-Dalgarno (SD) sequences. Transcribed pseudogenes also contained multiple "in-frame" stop codons and high Ka/Ks ratios, compared to that of homologs in M. tuberculosis and ORFs in M. leprae. A pseudogene transcript containing an active promoter, strong SD site, a start codon, but containing two in frame stop codons yielded a protein product when expressed in E. coli. Conclusion Approximately half of M. leprae's transcriptome consists of inactive gene products consuming energy and resources without potential benefit to M. leprae. Presently it is unclear what additional detrimental affect(s) this large number of inactive mRNAs has on the functional capability of this organism. Translation of these pseudogenes may play an important role in overall energy consumption and resultant pathophysiological characteristics of M. leprae. However, this study also demonstrated that multiple translational "silencing" mechanisms are present, reducing additional energy and resource expenditure required for protein production from the vast majority of these transcripts.

Not all isolates of a species contain the same set of genes. In this Opinion article, Rodriguez-Valera and colleagues propose the constant-diversity model to account for these differences. In this model, predation by phages promotes bacterial diversity and allows more efficient use of the nutrients in the environment. The remarkable differences that have been detected by metagenomics in the genomes of strains of the same bacterial species are difficult to reconcile with the widely accepted paradigm that periodic selection within bacterial populations will regularly purge genomic diversity by clonal replacement. We have found that many of the genes that differ between strains affect regions that are potential phage recognition targets. We therefore propose the constant-diversity dynamics model, in which the diversity of prokaryotic populations is preserved by phage predation. We provide supporting evidence for this model from metagenomics, mathematical analysis and computer simulations. Periodic selection and phage predation dynamics are not mutually exclusive; we compare their predictions to shed light on the ecological circumstances under which each type of dynamics could predominate.

Alteromonas macleodii is a common marine heterotrophic gamma-proteobacterium. Isolates from this microbe cluster by molecular analysis into two major genotypic groups or ecotypes, one found in temperate latitudes in the upper water column and another that is for the most part found in the deep water column of the Mediterranean. Here, we describe the genome of one strain of the 'deep ecotype' (AltDE) isolated from 1000 m in the Eastern Mediterranean and compare this genome with that of the type strain ATCC 27126, a representative of the global 'surface' ecotype. The genomes are substantially different with DNA sequence similarity values that are borderline for microbes belonging to the same species, and a large differential gene content, mainly found in islands larger than 20 kbp, that also recruit poorly to the Global Ocean Sampling project (GOS). These genomic differences indicate that AltDE is probably better suited to microaerophilic conditions and for the degradation of recalcitrant compounds such as urea. These, together with other features, and the distribution of this genotypic group, indicate that this microbe colonizes relatively large particles that sink rapidly to meso and bathypelagic depths. The genome of ATCC 27126 on the other hand has more potential for regulation (two component systems) and degrades more sugars and amino acids, which is consistent with a more transient particle attachment, as would be expected for lineages specialized in colonizing smaller particulate organic matter with much slower sinking rates. The genomic data are also consistent with a picture of incipient speciation driven by niche specialization.

The halophilic archaeon Haloarcula marismortui contains three ribosomal RNA operons, designated rrnA, rrnB, and rrnC. Operons A and C are virtually identical, whereas operon B presents a high divergence in nucleotide sequence, having up to 135 nucleotide polymorphisms among the three 16S, 23S, and 5S ribosomal RNA genes. Quantitative PCR and structural analyses have been performed to elucidate whether the presence of this intragenomic heterogeneity could be an adaptation to the variable environmental conditions in the natural habitat of H. marismortui. Variation in salt concentration did not affect expression but variation in incubation temperature did produce significant changes, with operon B displaying an expression level four times higher than the other two together at 50 degrees C and three times lower at 15 degrees C. We show that the putative promoter region of operon B is also different. In addition, the predicted secondary structure of these genes indicated that they have distinct stabilities at different temperatures and a mutant strain lacking operon B grew slower at high temperatures. This study supports the idea that divergent rRNA genes can be adaptive, with different variants being functional under different environmental conditions (e.g., temperature). The same phenomenon could take place in other halophiles or thermophiles with intragenomic rDNA heterogeneity, where the use of 16S rDNA as a phylogenetic marker and indicator of biodiversity should be used with caution.

Current human activities undoubtedly impact natural ecosystems. However, the influence of Homo sapiens on living organisms must have also occurred in the past. Certain genomic characteristics of prokaryotes can be used to study the impact of ancient human activities on microorganisms. By analyzing DNA sequence similarity features of transposable elements, dramatic genomic changes have been identified in bacteria that are associated with large and stable human communities, agriculture and animal domestication: three features unequivocally linked to the Neolithic revolution. It is hypothesized that bacteria specialized in human-associated niches underwent an intense transformation after the social and demographic changes that took place with the first Neolithic settlements. These genomic changes are absent in related species that are not specialized in humans.

Background The phylogenetic position and evolutionary relationships of Fusobacteria remain uncertain. Especially intriguing is their relatedness to low G+C Gram positive bacteria (Firmicutes) by ribosomal molecular phylogenies, but their possession of a typical gram negative outer membrane. Taking advantage of the recent completion of the Fusobacterium nucleatum genome sequence we have examined the evolutionary relationships of Fusobacterium genes by phylogenetic analysis and comparative genomics tools. Results The data indicate that Fusobacterium has a core genome of a very different nature to other bacterial lineages, and branches out at the base of Firmicutes. However, depending on the method used, 35–56% of Fusobacterium genes appear to have a xenologous origin from bacteroidetes, proteobacteria, spirochaetes and the Firmicutes themselves. A high number of hypothetical ORFs with unusual codon usage and short lengths were found and hypothesized to be remnants of transferred genes that were discarded. Some proteins and operons are also hypothesized to be of mixed ancestry. A large portion of the Gram-negative cell wall-related genes seems to have been transferred from proteobacteria. Conclusions Many instances of similarity to other inhabitants of the dental plaque that have been sequenced were found. This suggests that the close physical contact found in this environment might facilitate horizontal gene transfer, supporting the idea of niche-specific gene pools. We hypothesize that at a point in time, probably associated to the rise of mammals, a strong selective pressure might have existed for a cell with a Clostridia-like metabolic apparatus but with the adhesive and immune camouflage features of Proteobacteria.

Many insects eat their cast cuticle (exuviae) after moulting. The functional significance of this behaviour has not been addressed experimentally. I tested the hypothesis that exuviae eating constitutes a meal, so the animal recycles its nitrogen content. Nitrogenous compounds (protein and chitin) are major components of the cuticle in Periplaneta americana, accounting for as much as 87% of the total weight. It was found that insects almost invariably ate their exuviae during their larval life. The frequency of the behaviour decreased in newly emerged adults and varied between the sexes, males eating their exuviae less frequently than females. This may be due to the extra nitrogen endowment which females need for reproduction. Aposymbiotic animals, which lack the supply of essential amino acids from endosymbiotic bacteria, always ate their exuviae regardless of sex. When animals were reared on different diets throughout their larval life protein level in the diet correlated with exuviae eating. Animals reared on a low protein diet showed the highest levels of exuviae eating; animals reared on a high protein diet showed the highest levels of exuviae rejection. Analysis of the frass produced after exuviae meals showed that over 58% of the nitrogen present in the exuviae was recycled. This demonstrated that cockroaches digested nitrogenous compounds contained in the cuticle. The possibility that the exuviae meal has other functions is discussed, although the evidence supports a nutritional role.

We are interested in quantifying the contribution of gene acquisition, loss, expansion and rearrangements to the evolution of microbial genomes. Here, we discuss factors influencing microbial genome divergence based on pair-wise genome comparisons of closely related strains and species with different lifestyles. A particular focus is on intracellular pathogens and symbionts of the genera Rickettsia, Bartonella and Buchnera. Extensive gene loss and restricted access to phage and plasmid pools may provide an explanation for why single host pathogens are normally less successful than multihost pathogens. We note that species-specific genes tend to be shorter than orthologous genes, suggesting that a fraction of these may represent fossil-orfs, as also supported by multiple sequence alignments among species. The results of our genome comparisons are placed in the context of phylogenomic analyses of alpha and gamma proteobacteria. We highlight artefacts caused by different rates and patterns of mutations, suggesting that atypical phylogenetic placements can not a priori be taken as evidence for horizontal gene transfer events. The flexibility in genome structure among free-living microbes contrasts with the extreme stability observed for the small genomes of aphid endosymbionts, in which no rearrangements or inflow of genetic material have occurred during the past 50 millions years (1). Taken together, the results suggest that genomic stability correlate with the content of repeated sequences and mobile genetic elements, and thereby indirectly with bacterial lifestyles.

Many species of bacterial endosymbionts are acquired by animal hosts before birth, through direct transmission from mothers to eggs or embryos. This vertical transmission imposes a reduction in numbers or "bottleneck," and the size of this bottleneck affects the population structure and evolution of symbiotic lineages. We have estimated the size of the transmission bottleneck in Buchnera, the bacterial symbiont of aphids, using basic light and electron microscopy techniques. By serial-sectioning whole aphid abdomens, their eggs, and embryos, we determined the following parameters: (i) The average size of a Buchnera cell is 2.9 mum in diameter. (ii) The total number of Buchnera in an Acyrthosiphon pisum embryo was around 36,700, whereas a first instar nymph contained more than 119,000. (iii) The number of symbionts per bacteriocyte was around 800 in an embryo and 3200 in a first instar nymph. (iv) The total number of Buchnera transmitted to each sexual egg ranged from 850 in Nasonovia to 1800 in A. pisum to more than 8000 in Uroleucon ambrosiae. (v) The total number of secondary endosymbionts in A. pisum was 12,170 for an embryo and 18,360 for a first instar nymph. Secondary symbionts were arranged both extracellularly and in clusters of 2000-8000 bacteria inside bacteriocytes. These numbers are consistent with the few previous estimates of symbiont population sizes based on counts of gene copies.

We compared nutrient selection in a laboratory culture of the American cockroach (Periplaneta americana: Insecta, Blattodea) and a feral population which was founded by escapees from the culture approximately 14 generations prior to the study. In a first experiment, adult male and female cockroaches were provided with two nutritionally complementary synthetic foods, and thus allowed to select the protein and carbohydrate components of their diet independently. There were no differences in the amounts of carbohydrate eaten by the two populations. However, feral males ingested more protein than cultured males. In a second experiment, the construction of nitrogen budgets showed that the additional nitrogen ingested by feral males was allocated preferentially to accessory sex glands, rather than somatic tissue or excretion via the faeces. This suggests a possible role for sexual selection in the dietary difference between the strains. By contrast with males, there was no statistically significant difference in the amount of protein eaten by females of the two populations. However, feral females were found to have a higher density of bacterial endosymbionts than cultured females. Since these symbionts are involved in the synthesis of essential amino acids, this might account for greater reproductive output observed in a previous study in the feral compared with the culture females.

In birds and mammals, most of the female germ cells are destroyed before fertilization in a process known as atresia, reducing the population of cells to a small fraction of that present in early fetal life. We suggest that this death of germ cells can be interpreted as a developmental solution to the accumulation of mutations (Muller's ratchet) in mitochondria, an idea that is supported by comparative analysis. Atresia in effect therefore removes oocytes carrying mutant mitochondria1.

The authors of this article wish it to be known that full contact details for Dr Pasic were accidentally omitted from the final version of their article. The correct contact information for Dr Pasic is as follows:University of Ljubljana, Biotechnical Faculty, Department of Biology, Vecna pot 111, 1000 Ljubljana, Slovenia, SLOVENIA (lejla.pasic@bf.uni-lj.si).The authors apologise for this oversight.

Background The phylogenetic position and evolutionary relationships of Fusobacteria remain uncertain. Especially intriguing is their relatedness to low G+C Gram positive bacteria (Firmicutes) by ribosomal molecular phylogenies, but their possession of a typical gram negative outer membrane. Taking advantage of the recent completion of the Fusobacterium nucleatum genome sequence we have examined the evolutionary relationships of Fusobacterium genes by phylogenetic analysis and comparative genomics tools.

Background: The wealth of genomic data in bacteria is helping microbiologists understand the factors involved in gene innovation. Among these, the expansion and reduction of gene families appears to have a fundamental role in this, but the factors influencing gene family size are unclear. Results: The relative content of paralogous genes in bacterial genomes increases with genome size, largely due to the expansion of gene family size in large genomes. Bacteria undergoing genome reduction display a parallel process of redundancy elimination, by which gene families are reduced to one or a few members. Gene family size is also influenced by sequence divergence and physiological function. Large gene families show wider sequence divergence, suggesting they are probably older, and certain functions (such as metabolite transport mechanisms) are overrepresented in large families. The size of a given gene family is remarkably similar in strains of the same species and in closely related species, suggesting that homologous gene families are vertically transmitted and depend little on horizontal gene transfer (HGT). Conclusions: The remarkable preservation of copy numbers in widely different ecotypes indicates a functional role for the different copies rather than simply a back-up role. When different genera are compared, the increase in phylogenetic distance and/or ecological specialization disrupts this preservation, albeit in a gradual manner and maintaining an overall similarity, which also supports this view. HGT can have an important role, however, in nonhomologous gene families, as exemplified by a comparison between saprophytic and enterohemorrhagic strains of Escherichia coli.

Very small genomes have evolved repeatedly in eubacterial lineages that have adopted obligate associations with eukaryotic hosts. Complete genome sequences have revealed that small genomes retain very different gene sets, raising the question of how final genome content is determined. To examine the process of genome reduction, the tiny genome of the endosymbiont Buchnera aphidicola was compared to the larger ancestral genome, reconstructed on the basis of the phylogenetic distribution of gene orthologs among fully sequenced relatives of Escherichia coli and Buchnera. The reconstructed ancestral genome contained 2,425 open reading frames (ORFs). The Buchnera genome, containing 564 ORFs, consists of 153 fragments of 1-34 genes that are syntenic with reconstructed ancestral regions. On the basis of this reconstruction, 503 genes were eliminated within syntenic fragments, and 1,403 genes were lost from the gaps between syntenic fragments, probably in connection with genome rearrangements. Lost regions are sometimes large, and often span functionally unrelated genes. In addition, individual genes and regulatory regions have been lost or eroded. For the categories of DNA repair genes and rRNA genes, most lost loci fall in regions between syntenic fragments. This history of gene loss is reflected in the sequences of intergenic spacers at positions where genes were once present. The most plausible interpretation of this reconstruction is that Buchnera lost many genes through the fixation of large deletions soon after the acquisition of an obligate endosymbiotic lifestyle. An implication is that final genome composition may be partly the chance outcome of initial deletions and that neighboring genes influence the likelihood of loss of particular genes and pathways.

The size of a given gene family is remarkably similar in strains of the same species and in closely related species, suggesting that homologous gene families are vertically transmitted and depend little on horizontal gene transfer., Background The wealth of genomic data in bacteria is helping microbiologists understand the factors involved in gene innovation. Among these, the expansion and reduction of gene families appears to have a fundamental role in this, but the factors influencing gene family size are unclear. Results The relative content of paralogous genes in bacterial genomes increases with genome size, largely due to the expansion of gene family size in large genomes. Bacteria undergoing genome reduction display a parallel process of redundancy elimination, by which gene families are reduced to one or a few members. Gene family size is also influenced by sequence divergence and physiological function. Large gene families show wider sequence divergence, suggesting they are probably older, and certain functions (such as metabolite transport mechanisms) are overrepresented in large families. The size of a given gene family is remarkably similar in strains of the same species and in closely related species, suggesting that homologous gene families are vertically transmitted and depend little on horizontal gene transfer (HGT). Conclusions The remarkable preservation of copy numbers in widely different ecotypes indicates a functional role for the different copies rather than simply a back-up role. When different genera are compared, the increase in phylogenetic distance and/or ecological specialization disrupts this preservation, albeit in a gradual manner and maintaining an overall similarity, which also supports this view. HGT can have an important role, however, in nonhomologous gene families, as exemplified by a comparison between saprophytic and enterohemorrhagic strains of Escherichia coli.

Sub-acute mastitis (SAM) is a prevalent disease among lactating women, being one of the main reasons for early weaning. Although the etiology and diagnosis of acute mastitis (AM) is well established, little is known about the underlying mechanisms causing SAM. We collected human milk samples from healthy and SAM-suffering mothers, during the course of mastitis and after symptoms disappeared. Total (DNA-based) and active (RNA-based) microbiota were analysed by 16S rRNA gene sequencing and qPCR. Furthermore, mammary epithelial cell lines were exposed to milk pellets, and levels of the pro-inflammatory interleukin IL8 were measured. Bacterial load was significantly higher in the mastitis samples and decreased after clinical symptoms disappeared. Bacterial diversity was lower in SAM milk samples, and differences in bacterial composition and activity were also found. Contrary to AM, the same bacterial species were found in samples from healthy and SAM mothers, although at different proportions, indicating a dysbiotic ecological shift. Finally, mammary epithelial cell exposure to SAM milk pellets showed an over-production of IL8. Our work therefore supports that SAM has a bacterial origin, with increased bacterial loads, reduced diversity and altered composition, which partly recovered after treatment, suggesting a polymicrobial and variable etiology., This work was funded by the ERC Starting Grant 639226-MAMI, and the RTI2018-102032-B-I00 Grant from the Spanish Ministry of Science, Technology and Universities

Background Micro-organisms inhabiting teeth surfaces grow on biofilms where a specific and complex succession of bacteria has been described by co-aggregation tests and DNA-based studies. Although the composition of oral biofilms is well established, the active portion of the bacterial community and the patterns of gene expression in vivo have not been studied. Results Using RNA-sequencing technologies, we present the first metatranscriptomic study of human dental plaque, performed by two different approaches: (1) A short-reads, high-coverage approach by Illumina sequencing to characterize the gene activity repertoire of the microbial community during biofilm development; (2) A long-reads, lower-coverage approach by pyrosequencing to determine the taxonomic identity of the active microbiome before and after a meal ingestion. The high-coverage approach allowed us to analyze over 398 million reads, revealing that microbial communities are individual-specific and no bacterial species was detected as key player at any time during biofilm formation. We could identify some gene expression patterns characteristic for early and mature oral biofilms. The transcriptomic profile of several adhesion genes was confirmed through qPCR by measuring expression of fimbriae-associated genes. In addition to the specific set of gene functions overexpressed in early and mature oral biofilms, as detected through the short-reads dataset, the long-reads approach detected specific changes when comparing the metatranscriptome of the same individual before and after a meal, which can narrow down the list of organisms responsible for acid production and therefore potentially involved in dental caries. Conclusions The bacteria changing activity during biofilm formation and after meal ingestion were person-specific. Interestingly, some individuals showed extreme homeostasis with virtually no changes in the active bacterial population after food ingestion, suggesting the presence of a microbial community which could be associated to dental health.