The gut microbiota is involved in metabolic and immune disorders associated with obesity and type 2 diabetes. We previously demonstrated that prebiotic treatment may significantly improve host health by modulating bacterial species related to the improvement of gut endocrine, barrier and immune functions. An analysis of the gut metagenome is needed to determine which bacterial functions and taxa are responsible for beneficial microbiota–host interactions upon nutritional intervention. We subjected mice to prebiotic (Pre) treatment under physiological (control diet: CT) and pathological conditions (high-fat diet: HFD) for 8 weeks and investigated the production of intestinal antimicrobial peptides and the gut microbiome. HFD feeding significantly decreased the expression of regenerating islet-derived 3-gamma (Reg3g) and phospholipase A2 group-II (PLA2g2) in the jejunum. Prebiotic treatment increased Reg3g expression (by ∼50-fold) and improved intestinal homeostasis as suggested by the increase in the expression of intectin, a key protein involved in intestinal epithelial cell turnover. Deep metagenomic sequencing analysis revealed that HFD and prebiotic treatment significantly affected the gut microbiome at different taxonomic levels. Functional analyses based on the occurrence of clusters of orthologous groups (COGs) of proteins also revealed distinct profiles for the HFD, Pre, HFD-Pre and CT groups. Finally, the gut microbiota modulations induced by prebiotics counteracted HFD-induced inflammation and related metabolic disorders. Thus, we identified novel putative taxa and metabolic functions that may contribute to the development of or protection against the metabolic alterations observed during HFD feeding and HFD-Pre feeding.

Caloric restriction (CR) stimulates development of functional beige fat and extends healthy lifespan. Here we show that compositional and functional changes in the gut microbiota contribute to a number of CR-induced metabolic improvements and promote fat browning. Mechanistically, these effects are linked to a lower expression of the key bacterial enzymes necessary for the lipid A biosynthesis, a critical lipopolysaccharide (LPS) building component. The decreased LPS dictates the tone of the innate immune response during CR, leading to increased eosinophil infiltration and anti-inflammatory macrophage polarization in fat of the CR animals. Genetic and pharmacological suppression of the LPS-TLR4 pathway or transplantation with Tlr4 bone-marrow-derived hematopoietic cells increases beige fat development and ameliorates diet-induced fatty liver, while Tlr4 or microbiota-depleted mice are resistant to further CR-stimulated metabolic alterations. These data reveal signals critical for our understanding of the microbiota-fat signaling axis during CR and provide potential new anti-obesity therapeutics.

Apical periodontitis is an inflammatory disease of the dental periradicular tissues triggered by bacteria colonizing necrotic root canals. Primary apical periodontitis results from the microbial colonization of necrotic pulp tissues. Secondary apical periodontitis results from a persistent infection of incorrectly treated root canals. The aim of this study was to characterize the microbiota present in primary and secondary intraradicular infections associated with apical periodontitis using 16S rRNA gene amplicon sequencing. Teeth exhibiting apical periodontitis with or without root canal treatment were extracted after informed consent. From each tooth, the intraradicular content as well as a dentin sample (control) were collected and subjected to DNA extraction. PCR amplicons of the V3–V4 region of the bacterial 16S rRNA gene were pooled and sequenced (2 × 300) on an Illumina MiSeq instrument. The bioinformatics analysis pipeline included quality filtering, merging of forward and reverse reads, clustering of reads into operational taxonomic units (OTUs), removal of putative contaminant OTUs and assigning taxonomy. The most prevalent and abundant OTU in both dentin and root canal samples was assigned to anaerobic bacterium Fusobacterium nucleatum. Multivariate analysis showed clustering of microbiota by sample type (dentin vs. intraradicular content) and, in root canals, by pathology (primary vs. secondary infection). The proportions of Enterococcus faecalis and F. nucleatum were, respectively, higher and lower when comparing secondary to primary infected root canals. Co-occurrence network analysis provided evidence of microbial interactions specific to the infection type. The identification of bacterial taxa differentially abundant in primary and secondary intraradicular infections may provide the basis for targeted therapeutic approaches aimed at reducing the incidence of apical periodontitis.

BackgroundHuman axillary odour is commonly attributed to the bacterial degradation of precursors in sweat secretions. To assess the role of bacterial communities in the formation of body odours, we used a culture-independent approach to study axillary skin microbiota and correlated these data with olfactory analysis.ResultsTwenty-four Caucasian male and female volunteers and four assessors showed that the underarms of non-antiperspirant (non-AP) users have significantly higher global sweat odour intensities and harboured on average about 50 times more bacteria than those of AP users. Global sweat odour and odour descriptors sulfury-cat urine and acid-spicy generally increased from the morning to the afternoon sessions. Among non-AP users, male underarm odours were judged higher in intensity with higher fatty and acid-spicy odours and higher bacterial loads. Although the content of odour precursors in underarm secretions varied widely among individuals, males had a higher acid: sulfur precursor ratio than females did. No direct correlations were found between measured precursor concentration and sweat odours. High-throughput sequencing targeting the 16S rRNA genes of underarm bacteria collected from 11 non-AP users (six females and five males) confirmed the strong dominance of the phyla Firmicutes and Actinobacteria, with 96% of sequences assigned to the genera Staphylococcus, Corynebacterium and Propionibacterium. The proportion of several bacterial taxa showed significant variation between males and females. The genera Anaerococcus and Peptoniphilus and the operational taxonomic units (OTUs) from Staphylococcus haemolyticus and the genus Corynebacterium were more represented in males than in females. The genera Corynebacterium and Propionibacterium were correlated and anti-correlated, respectively, with body odours. Within the genus Staphylococcus, different OTUs were either positively or negatively correlated with axillary odour. The relative abundance of five OTUs (three assigned to S. hominis and one each to Corynebacterium tuberculostearicum and Anaerococcus) were positively correlated with at least one underarm olfactory descriptor.ConclusionsPositive and negative correlations between bacterial taxa found at the phylum, genus and OTU levels suggest the existence of mutualism and competition among skin bacteria. Such interactions, and the types and quantities of underarm bacteria, affect the formation of body odours. These findings open the possibility of developing new solutions for odour control.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-014-0064-3) contains supplementary material, which is available to authorized users.

BackgroundIdentification of unexpected taxa in 16S rRNA surveys of low-density microbiota, diluted mock communities and cultures demonstrated that a variable fraction of sequence reads originated from exogenous DNA. The sources of these contaminants are reagents used in DNA extraction, PCR, and next-generation sequencing library preparation, and human (skin, oral and respiratory) microbiota from the investigators.ResultsFor in silico removal of reagent contaminants, a pipeline was used which combines the relative abundance of operational taxonomic units (OTUs) in V3–4 16S rRNA gene amplicon datasets with bacterial DNA quantification based on qPCR targeting of the V3 segment of the 16S rRNA gene. Serially diluted cultures of Escherichia coli and Staphylococcus aureus were used for 16S rDNA profiling, and DNA from each of these species was used as a qPCR standard. OTUs assigned to Escherichia or Staphylococcus were virtually unaffected by the decontamination procedure, whereas OTUs from Pseudomonas, which is a major reagent contaminant, were completely or nearly completely removed. The decontamination procedure also attenuated the trend of increase in OTU richness in serially diluted cultures.ConclusionsRemoval of contaminant sequences derived from reagents based on use of qPCR data may improve taxonomic representation in samples with low DNA concentration. Using the described pipeline, OTUs derived from cross-contamination of negative extraction controls were not recognized as contaminants and not removed from the sample dataset.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0689-4) contains supplementary material, which is available to authorized users.

Culture-independent high-throughput sequencing-based methods are widely used to study bacterial communities. Although these approaches are superior to traditional culture-based methods, they introduce bias at the experimental and bioinformatics levels. We assessed the diversity of the human salivary microbiome by pyrosequencing of the 16S rDNA V1–3 amplicons using metagenomic DNA extracted by two different protocols: a simple proteinase K digestion without a subsequent DNA clean-up step, and a bead-beating mechanical lysis protocol followed by column DNA purification. A high degree of congruence was found between the two extraction methods, most notably in regard to the microbial community composition. The results showed that for a given bioinformatics pipeline, all the taxa with an average proportion >0.12% in samples processed using one extraction method were also detected in samples extracted using the other method. The same taxa tended to be abundant and frequent for both extraction methods. The relative abundance of sequence reads assigned to the phyla Actinobacteria, Spirochaetes, TM7, Synergistetes, and Tenericutes was significantly higher in the mechanically-treated samples than in the enzymatically-treated samples, whereas the phylum Firmicutes showed the opposite pattern. No significant differences in diversity indices were found between the extraction methods, although the mechanical lysis method revealed higher operational taxonomic unit richness. Differences between the extraction procedures outweighed the variations due to the bioinformatics analysis pipelines used.

Purpose To compare bacteria recovered by standard cultures and metataxonomics, particularly with regard to ventilator-associated pneumonia (VAP) pathogens, and to determine if the presence of particular bacteria or microbiota in tracheal and oropharyngeal secretions during the course of intubation was associated with the development of VAP. Methods In this case–control study, oropharyngeal secretions and endotracheal aspirate were collected daily in mechanically ventilated patients. Culture and metataxonomics (16S rRNA gene-based taxonomic profiling of bacterial communities) were performed on serial upper respiratory samples from patients with late-onset definite VAP and their respective controls. Results Metataxonomic analyses showed that a low relative abundance of Bacilli at the time of intubation in the oropharyngeal secretions was strongly associated with the subsequent development of VAP. On the day of VAP, the quantity of human and bacterial DNA in both tracheal and oropharyngeal secretions was significantly higher in patients with VAP than in matched controls with similar ventilation times. Molecular techniques identified the pathogen(s) of VAP found by culture, but also many more bacteria, classically difficult to culture, such as Mycoplasma spp. and anaerobes. Conclusions Molecular analyses of respiratory specimens identified markers associated with the development of VAP, as well as important differences in the taxa abundance between VAP and controls. Further prospective trials are needed to test the predictive value of these markers, as well as the relevance of uncultured bacteria in the pathogenesis of VAP. Electronic supplementary material The online version of this article (10.1007/s00134-019-05660-8) contains supplementary material, which is available to authorized users.

Recent treatment guidelines for uncomplicated urinary tract infections (UTIs) discourage fluoroquinolone prescription because of collateral damage to commensal microbiota, but the ecologic impact of alternative agents has not been evaluated by culture-free techniques. We prospectively collected faecal samples at three time points from ambulatory patients with UTIs treated with ciprofloxacin or nitrofurantoin, patients not requiring antibiotics and household contacts of ciprofloxacin-treated patients. We described changes in gut microbiota using a culture-independent approach based on pyrosequencing of the V3-V4 region of the bacterial 16S rRNA gene. All groups were similar at baseline. Ciprofloxacin had a significant global impact on the gut microbiota whereas nitrofurantoin did not. The end of ciprofloxacin treatment correlated with a reduced proportion of Bifidobacterium (Actinobacteria), Alistipes (Bacteroidetes) and four genera from the phylum Firmicutes (Faecalibacterium, Oscillospira, Ruminococcus and Dialister) and an increased relative abundance of Bacteroides (Bacteroidetes) and the Firmicutes genera Blautia, Eubacterium and Roseburia. Substantial recovery had occurred 4 weeks later. Nitrofurantoin treatment correlated with a reduced relative proportion of the genus Clostridium and an increased proportion of the genus Faecalibacterium. This study supports use of nitrofurantoin over fluoroquinolones for treatment of uncomplicated UTIs to minimize perturbation of intestinal microbiota.