Pyrosequencing

We aimed to determine the bacterial diversity of different oral micro-niches and to assess whether saliva and plaque samples are representative of oral microbial composition. We took minute samples from each surface of the individual teeth and gingival crevices of two healthy volunteers (112 samples per donor), as well as samples from the tongue dorsum and non-stimulated and stimulated saliva. DNA was extracted from 67 selected samples of each donor, and the 16S rRNA gene was amplified by PCR and pyrosequenced to obtain, on average, over 2,700 reads per sample, which were taxonomically assigned to obtain a geographic map of bacterial diversity at each tooth and sulcus location. Analysis of the data shows considerable differences in bacterial composition between teeth at different intra-oral locations and between surfaces of the same tooth. The most pronounced differences were observed in incisors and canines, where genera like Streptococcus were found at 40% to 70% on the vestibular surfaces but were almost absent on the lingual sides. Saliva samples, especially non-stimulated saliva, were not representative of supra-and subgingival plaque in the two individuals tested. We suggest that more precise sampling is required for the proper determination of oral microbial composition and to relate that diversity to epidemiological, clinical, and etiological parameters.

One sentence summary: This study gives insight into the bacterial community composition related to iron-cycling in suboxic marine sediments based on 16S rRNA gene pyrosequencing. ABSTRACT To gain insight into the bacterial communities involved in iron-(Fe) cycling under marine conditions, we analysed sediments with Fe-contents (0.5–1.5 wt %) from the suboxic zone at a marine site in the Skagerrak (SK) and a brackish site in the Bothnian Bay (BB) using 16S rRNA gene pyrosequencing. Several bacterial families, including Desulfobulbaceae, Desulfuromonadaceae and Pelobacteraceae and genera, including Desulfobacter and Geobacter, known to reduce Fe were detected and showed highest abundance near the Fe(III)/Fe(II) redox boundary. Additional genera with microorganisms capable of coupling fermentation to Fe-reduction, including Clostridium and Bacillus, were observed. Also, the Fe-oxidizing families Mariprofundaceae and Gallionellaceae occurred at the SK and BB sites, respectively, supporting Fe-cycling. In contrast, the sulphate (SO 4 2−) reducing bacteria Desulfococcus and Desulfobacterium were more abundant at greater depths concurring with a decrease in Fe-reducing activity. The communities revealed by pyrosequencing, thus, match the redox stratification indicated by the geochemistry, with the known Fe-reducers coinciding with the zone of Fe-reduction. Not the intensely studied model organisms, such as Geobacter spp., but rather versatile microorganisms, including sulphate reducers and possibly unknown groups appear to be important for Fe-reduction in these marine suboxic sediments.

Olive ridley turtles, although widely distributed globally and in Indian coastal waters, have undergone declines in recent years due to anthropogenic factors, particularly fishery-related mortality. Assessment of genetic variability in existing populations is critical to the development of effective conservation strategies. Here we describe the development of six highly polymorphic microsatellite loci from a simple sequence repeat-enriched genomic DNA library of olive ridley turtle. Characterization of five of these loci using 83 individual olive ridley turtles revealed eight to 24 alleles per locus, high observed and expected heterozygosity values and broad cross-species amplifications. The sixth microsatellite was found to be monomorphic in the olive ridley samples but was polymorphic in two related marine turtle species. These microsatellites thus provide efficient genetic markers to understand the population structure, phylogeography and species relationships of olive ridley and other marine turtle species.

Objectives: Genetic variation in genes encoding for drug-metabolizing enzymes, drug targets and signaling pathways have proven to contribute significantly to differences in drug response. Pharmacogenetics is now expanding from clinical pharmacological research to its application in clinical practice. Genotyping of patients in a routine clinical setting requires robust and reliable genotyping methods. Materials & methods: A survey of pharmacogenetic association studies for quality control samples published from 2005 to 2007 in the two most prominent pharmacogenetic journals, and development of plasmid-derived external controls. Results: Surveying journals revealed that only a minority of papers report the use of quality controls, and no standard procedures are applied. We established 12 plasmid-derived external controls and applied these in pharmacogenetic testing. Conclusion: There still is a need for quality control materials, especially for application in pharmacogenetic testing. ...

In the present study we assessed the composition of Bacteria in four biotopes, namely sediment, seawater and two sponge species (Stylissa massa and Xestospongia testudinaria) at four different reef sites in a coral reef ecosystem in west Java, Indonesia. In addition to this, we used a predictive metagenomic approach to predict to what extent nitrogen metabolic pathways differ among bacterial communities in different biotopes. We observed marked differences in bacterial composition of the most abundant bacterial phyla, classes and orders among sponge species, water and sediment. Proteobacteria were by far the most abundant phylum in terms of both sequences and OTUs. Predicted counts for genes associated with the nitrogen metabolism suggest that several genes involved in the nitrogen cycle were enriched in sponge samples, including nosZ, nifD, nirK, NorB and nrfA genes Our data show that a combined barcoded pyrosequencing and predictive metagenomic- approach can provide novel insights...

Archaea and bacteria are important drivers for nutrient transformations in soils and catalyse
the production and consumption of important greenhouse gases. In this study, we investigate
changes in archaeal and bacterial communities of four Czech grassland soils affected
by outdoor cattle husbandry. Two show short-term (3 years; STI) and long-term impact
(17 years; LTI), one is regenerating from cattle impact (REG) and a control is unaffected by
cattle (CON). Cattle manure (CMN), the source of allochthonous microbes, was collected
from the same area. We used pyrosequencing of 16S rRNA genes to assess the composition
of archaeal and bacterial communities in each soil type and CMN. Both short- and longterm
cattle impact negatively altered archaeal and bacterial diversity, leading to increase of
homogenization of microbial communities in overwintering soils over time. Moreover, strong
shifts in the prokaryotic communities were observed in response to cattle overwintering,
with the greatest impact on archaea. Oligotrophic and acidophilic microorganisms (e.g.
Thaumarchaeota, Acidobacteria, and α-Proteobacteria) dominated in CON and expressed
strong negative response to increased pH, total C and N. Whereas copiotrophic and alkalophilic
microbes (e.g. methanogenic Euryarchaeota, Firmicutes, Chloroflexi, Actinobacteria,
and Bacteroidetes) were common in LTI showing opposite trends. Crenarchaeota were
also found in LTI, though their trophic interactions remain cryptic. Firmicutes, Bacteroidetes,
Methanobacteriaceae, and Methanomicrobiaceae indicated the introduction and establishment
of faecal microbes into the impacted soils, while Chloroflexi and Methanosarcinaceae
suggested increased abundance of soil-borne microbes under altered environmental conditions.
The observed changes in prokaryotic community composition may have driven corresponding
changes in soil functioning.

The jungle crow (Corvus macrorhynchos) belongs to the order Passeriformes of bird species and is important for avian ecological and evolutionary genetics studies. However, there is limited information on the transcriptome data of this species. In the present study, we report the characterization of the lung transcriptome of the jungle crow using GS FLX Titanium XLR70. Altogether, 1 510 303 high-quality sequence reads with 581 198 230 bases was de novo assembled into 22 169 isotigs (isotig represents an individual transcript) and 784 009 singletons. Using these isotigs and 581 681 length-filtered (greater than 300 bp) singletons, 20 010 unique protein-coding genes were identified by BLASTx comparison against a nonredundant (nr) protein sequence database. Comparative analysis revealed that 46 604 (70.29%) and 51 642 (72.48%) of the assembled transcripts have significant similarity to zebra finch and chicken RefSeq proteins, respectively. As determined by GO annotation and KEGG pathway...

We present the almost complete (16,007 bp) mitochondrial genome of a Colossendeis megalonyx specimen from the Southern Ocean and discuss gene order and tRNA structure in a comparative phylogenetic context. Our data suggest a basal position of the colossendeid lineage ...

Background and AimIn irritable bowel syndrome (IBS), the gut microbiota may be altered. Probiotic bacteria appear to be therapeutically effective. We characterized the mucosa-associated microbiota, and determined the clinical and microbiological effects of orally administered probiotic bacteria, in patients with IBS.In irritable bowel syndrome (IBS), the gut microbiota may be altered. Probiotic bacteria appear to be therapeutically effective. We characterized the mucosa-associated microbiota, and determined the clinical and microbiological effects of orally administered probiotic bacteria, in patients with IBS.MethodsMucosal microbiota from rectal biopsies of IBS patients and controls were assessed on the V1 and V2 variable regions of the 16S ribosomal RNA gene amplified using 454 pyrosequencing. Clinical symptoms and changes in mucosal microbiota were assessed in IBS patients before and after 4 weeks of treatment with probiotic mix VSL#3.Mucosal microbiota from rectal biopsies of IBS patients and controls were assessed on the V1 and V2 variable regions of the 16S ribosomal RNA gene amplified using 454 pyrosequencing. Clinical symptoms and changes in mucosal microbiota were assessed in IBS patients before and after 4 weeks of treatment with probiotic mix VSL#3.ResultsTen IBS subjects (eight female; mean age 46 years) were included. At week 4 of probiotic therapy, six patients showed symptom improvement on global symptom assessment compared with baseline (P = 0.031). Before therapy, intestinal microbiota of IBS subjects differed significantly from that of healthy controls, with less diversity and evenness than controls (n = 9; P < 0.05), increased abundance of Bacteroidetes (P = 0.014) and Synegitestes (P = 0.017), and reduced abundance of Actinobacteria (P = 0.004). The classes Flavobacteria (P = 0.028) and Epsilonproteobacteria (P = 0.017) were less enriched in IBS. Abundance differences were largely consistent from the phylum to genus level. Probiotic treatment in IBS patients was associated with a significant reduction of the genus Bacteroides (all taxonomy levels; P < 0.05) to levels similar to that of controls.Ten IBS subjects (eight female; mean age 46 years) were included. At week 4 of probiotic therapy, six patients showed symptom improvement on global symptom assessment compared with baseline (P = 0.031). Before therapy, intestinal microbiota of IBS subjects differed significantly from that of healthy controls, with less diversity and evenness than controls (n = 9; P < 0.05), increased abundance of Bacteroidetes (P = 0.014) and Synegitestes (P = 0.017), and reduced abundance of Actinobacteria (P = 0.004). The classes Flavobacteria (P = 0.028) and Epsilonproteobacteria (P = 0.017) were less enriched in IBS. Abundance differences were largely consistent from the phylum to genus level. Probiotic treatment in IBS patients was associated with a significant reduction of the genus Bacteroides (all taxonomy levels; P < 0.05) to levels similar to that of controls.ConclusionIn this pilot study, global and deep molecular analysis demonstrates an altered mucosal microbiota composition in IBS. Probiotic leads to detectable changes in the microbiota. These effects of probiotic bacteria may contribute to their therapeutic benefit.In this pilot study, global and deep molecular analysis demonstrates an altered mucosal microbiota composition in IBS. Probiotic leads to detectable changes in the microbiota. These effects of probiotic bacteria may contribute to their therapeutic benefit.

Acrobeloides maximus consistently comprised a set of different microorganisms compared to the microbiome of Caenorhabditis elegans or the metagenome of the soil sample to which nematodes were exposed. Using 16S rRNA gene amplification, the bacterial genera Ochrobactrum, Pedobacter, and Chitinophaga were identified at significant levels within the A. maximus communities only and were found to be consistently present during each of the three sampling periods over six months. We were able to isolate and grow colonies of Ochrobactrum and Pedobacter from the microbiome of the nematode on Lysogeny Broth-Miller (LB) and Yeast Extract (YE) agar plates. We were unable to isolate the associate from the genus Chitinophaga. The results obtained suggest a possible specific and persistent association between A. maximus and the two bacterial isolates we were able to culture.

With fluorescence in situ hybridization (FISH) with probes specific for Ochrobactrum and Pedobacter, we were able to stain these bacterial cells and show that they reside within the intestinal tract of A. maximus. It is possible that these gut bacteria may be mutualistic by providing A. maximus with essential metabolites, by aiding the nematode in the digestion of other bacteria, or by providing it with protection against pathogens.

Using 16S rRNA amplification and sequencing, three bacterial strains belonging to the genera Ochrobactrum, Pedobacter, and Chitinophaga were detected at significant levels within cultured Acrobeloides maximus populations exposed to soil for 24 hours before extraction and re-inoculation on agar. All three strains were found to persist in re-inoculated A. maximus cultures over a period of six months. For each strain, sequences from our A. maximus associated clones along with relevant 16S rRNA sequences from congeners or related genera available in GenBank were subjected to comprehensive phylogenetic analyses. Sequences were aligned using the multiple sequence alignment algorithms implemented in T-Coffee, MAFFT, Kalign, and WebPRANK, and phylogenetic trees were constructed using Bayesian and maximum-likelihood methods. Similarities and differences among the different alignment and tree-building methods suggest tree building methods with T-Coffee performed more poorly while maximum-likelihood methods with MAFFT and Kalign produced more mutually consistent topologies that correspond better to recognized genus boundaries. Inferences are made about the known species that are phylogenetically closest to each of our three strains. Our Ochrobactrum strains appear to be most closely related to Ochrobactrum tritici, while our Chitinophaga strains are most similar to a bacterium previously identified as “Flexibacter cf. sancti.” Our Pedobacter strains seem to be a previously undescribed sphingobacterium within the genus.

A good definition of commensal microflora and an understanding of its relation to health are essential in preventing and combating disease. We hypothesized that the species richness of human oral microflora is underestimated. Saliva and supragingival plaque were sampled from 71 and 98 healthy adults, respectively. Amplicons from the V6 hypervariable region of the small-subunit ribosomal RNA gene were generated by PCR, pooled into saliva and plaque pools, and sequenced by means of the Genome Sequencer 20 system at 454 Life Sciences. Data were evaluated by taxonomic and rarefaction analyses. The 197,600 sequences generated yielded about 29,000 unique sequences, representing 22 taxonomic phyla. Grouping the sequences in operational taxonomic units (6%) yielded 3621 and 6888 species-level phylotypes in saliva and plaque, respectively. This work gives a radically new insight into the diversity of human oral microflora, which, with an estimated number of 19,000 phylotypes, is considerably...