BackgroundAdvancements in Next Generation Sequencing (NGS) technologies regarding throughput, read length and accuracy had a major impact on microbiome research by significantly improving 16S rRNA amplicon sequencing. As rapid improvements in sequencing platforms and new data analysis pipelines are introduced, it is essential to evaluate their capabilities in specific applications. The aim of this study was to assess whether the same project-specific biological conclusions regarding microbiome composition could be reached using different sequencing platforms and bioinformatics pipelines.ResultsChicken cecum microbiome was analyzed by 16S rRNA amplicon sequencing using Illumina MiSeq, Ion Torrent PGM, and Roche 454 GS FLX Titanium platforms, with standard and modified protocols for library preparation. We labeled the bioinformatics pipelines included in our analysis QIIME1 and QIIME2 (de novo OTU picking [not to be confused with QIIME version 2 commonly referred to as QIIME2]), QIIME3 and QIIME4 (open reference OTU picking), UPARSE1 and UPARSE2 (each pair differs only in the use of chimera depletion methods), and DADA2 (for Illumina data only). GS FLX+ yielded the longest reads and highest quality scores, while MiSeq generated the largest number of reads after quality filtering. Declines in quality scores were observed starting at bases 150–199 for GS FLX+ and bases 90–99 for MiSeq. Scores were stable for PGM-generated data. Overall microbiome compositional profiles were comparable between platforms; however, average relative abundance of specific taxa varied depending on sequencing platform, library preparation method, and bioinformatics analysis. Specifically, QIIME with de novo OTU picking yielded the highest number of unique species and alpha diversity was reduced with UPARSE and DADA2 compared to QIIME.ConclusionsThe three platforms compared in this study were capable of discriminating samples by treatment, despite differences in diversity and abundance, leading to similar biological conclusions. Our results demonstrate that while there were differences in depth of coverage and phylogenetic diversity, all workflows revealed comparable treatment effects on microbial diversity. To increase reproducibility and reliability and to retain consistency between similar studies, it is important to consider the impact on data quality and relative abundance of taxa when selecting NGS platforms and analysis tools for microbiome studies.Electronic supplementary materialThe online version of this article (10.1186/s12866-017-1101-8) contains supplementary material, which is available to authorized users.

Background & AimsThe human gut microbiota is becoming increasingly recognized as a key factor in homeostasis and disease. The lack of physiologically relevant in vitro models to investigate host–microbe interactions is considered a substantial bottleneck for microbiota research. Organoids represent an attractive model system because they are derived from primary tissues and embody key properties of the native gut lumen; however, access to the organoid lumen for experimental perturbation is challenging. Here, we report the development and validation of a high-throughput organoid microinjection system for cargo delivery to the organoid lumen and high-content sampling.MethodsA microinjection platform was engineered using off-the-shelf and 3-dimensional printed components. Microinjection needles were modified for vertical trajectories and reproducible injection volumes. Computer vision (CVis) and microfabricated CellRaft Arrays (Cell Microsystems, Research Triangle Park, NC) were used to increase throughput and enable high-content sampling of mock bacterial communities. Modeling preformed using the COMSOL Multiphysics platform predicted a hypoxic luminal environment that was functionally validated by transplantation of fecal-derived microbial communities and monocultures of a nonsporulating anaerobe.ResultsCVis identified and logged locations of organoids suitable for injection. Reproducible loads of 0.2 nL could be microinjected into the organoid lumen at approximately 90 organoids/h. CVis analyzed and confirmed retention of injected cargos in approximately 500 organoids over 18 hours and showed the requirement to normalize for organoid growth for accurate assessment of barrier function. CVis analyzed growth dynamics of a mock community of green fluorescent protein– or Discosoma sp. red fluorescent protein-expressing bacteria, which grew within the organoid lumen even in the presence of antibiotics to control media contamination. Complex microbiota communities from fecal samples survived and grew in the colonoid lumen without appreciable changes in complexity.ConclusionsHigh-throughput microinjection into organoids represents a next-generation in vitro approach to investigate gastrointestinal luminal physiology and the gastrointestinal microbiota.

Actinomyces viscosus T14V and Streptococcus sanguis 34 coaggregate by a mechanism which is not inhibited by 1 M NaCl, is dextran independent, requires calcium, is pH dependent with an optimum at pH 8.0 to 8.5, and appears to require the interaction of a protein or glycoprotein on A. viscosus with a carbohydrate on S. sanguis. The coaggregation is inhibited more than 80% by 0.01 M lactose, 0.02 M /l-methyl-D-galactoside, or 0.05 M D-galactose; inhibition of coaggregation was less than 10% in 0.1 M a-methyl-D-galactoside, melibiose, maltose, cellobiose, sucrose, and a number of monosaccharides. At very high concentrations of enzyme, protease from S. griseus destroyed the reactive site on A. viscosus but not on S. sanguis. Both were totally resistant to dextranase. Periodate (0.01 M; pH 4) inactivated both bacteria. The ability of S. sanguis to coaggregate with A. viscosus was not destroyed by phenol-water extraction at 65°C for 15 min. When the bacteria were cultured under specified conditions, the coaggregation was highly reproducible. Under the same conditions, T14AV, the avirulent mutant of A. viscosus T14V, did not coaggregate with S. sanguis 34. Electron microscopic studies of coaggregates, labeled immunochemically with antibody to A. viscosus, indicated that fibrils on A. viscosus may be involved in the coaggregation.

Understanding the importance of the gut microbiome on modulation of host health has become a subject of great interest for researchers across disciplines. As an intrinsically multidisciplinary field, microbiome research has been able to reap the benefits of technological advancements in systems and synthetic biology, biomaterials engineering, and traditional microbiology. Gut microbiome research has been revolutionized by high-throughput sequencing technology, permitting compositional and functional analyses that were previously an unrealistic undertaking. Emerging technologies including engineered organoids derived from human stem cells, high-throughput culturing, and microfluidics assays allowing for the introduction of novel approaches will improve the efficiency and quality of microbiome research. Here, we will discuss emerging technologies and their potential impact on gut microbiome studies.

Phage-encoded Shiga toxin (Stx) acts as a bacterial defence against the eukaryotic predator Tetrahymena. To function as an effective bacterial anti-predator defence, Stx must kill a broad spectrum of predators. Consistent with that assertion, we show here that bacterially encoded Stx efficiently kills the bacteriovore Acanthamoeba castellanii in co-culture. We also show that, in addition to Stx, the phage-encoded exotoxin, diphtheria toxin (Dtx) expressed by Corynebacterium diphtheriae also can function as part of an anti-predator strategy; it kills Acanthamoeba in co-culture. Interestingly, only exotoxins produced by bacteria internalized by the Acanthamoeba predator are cytolethal; the presence of purified Dtx or Stx in culture medium has no effect on predator viability. This finding is consistent with our results indicating that intoxication of Acanthamoeba by these exotoxins does not require a receptor. Thus bacteria, in the disguise of a food source, function as a 'Trojan Horse', carrying genes encoding an exotoxin into target organisms. This 'Trojan Horse' mechanism of exotoxin delivery into predator cells allows intoxication of predators that lack a cell surface receptor for the particular toxin, allowing bacteria-bearing exotoxins to kill a broader spectrum of predators, increasing the fitness of the otherwise 'defenceless' prey bacteria.

Using a screening protocol we have identified 68 genes that are required for female development in the filamentous fungus Neurospora crassa. We find that we can divide these genes into five general groups: 1) Genes encoding components of the PACC signal transduction pathway, 2) Other signal transduction pathway genes, including genes from the three N. crassa MAP kinase pathways, 3) Transcriptional factor genes, 4) Autophagy genes, and 5) Other miscellaneous genes. Complementation and RIP studies verified that these genes are needed for the formation of the female mating structure, the protoperithecium, and for the maturation of a fertilized protoperithecium into a perithecium. Perithecia grafting experiments demonstrate that the autophagy genes and the cell-to-cell fusion genes (the MAK-1 and MAK-2 pathway genes) are needed for the mobilization and movement of nutrients from an established vegetative hyphal network into the developing protoperithecium. Deletion mutants for the PACC pathway genes palA, palB, palC, palF, palH, and pacC were found to be defective in two aspects of female development. First, they were unable to initiate female development on synthetic crossing medium. However, they could form protoperithecia when grown on cellophane, on corn meal agar, or in response to the presence of nearby perithecia. Second, fertilized perithecia from PACC pathway mutants were unable to produce asci and complete female development. Protein localization experiments with a GFP-tagged PALA construct showed that PALA was localized in a peripheral punctate pattern, consistent with a signaling center associated with the ESCRT complex. The N. crassa PACC signal transduction pathway appears to be similar to the PacC/Rim101 pathway previously characterized in Aspergillus nidulans and Saccharomyces cerevisiae. In N. crassa the pathway plays a key role in regulating female development.

Background Prebiotic galacto-oligosaccharides (GOS) have an extensively demonstrated beneficial impact on intestinal health. In this study, we determined the impact of GOS diets on hallmarks of gut aging: microbiome dysbiosis, inflammation, and intestinal barrier defects (“leaky gut”). We also evaluated if short-term GOS feeding influenced how the aging gut responded to antibiotic challenges in a mouse model of Clostridioides difficile infection. Finally, we assessed if colonic organoids could reproduce the GOS responder—non-responder phenotypes observed in vivo. Results Old animals had a distinct microbiome characterized by increased ratios of non-saccharolytic versus saccharolytic bacteria and, correspondingly, a lower abundance of β-galactosidases compared to young animals. GOS reduced the overall diversity, increased the abundance of specific saccharolytic bacteria (species of Bacteroides and Lactobacillus), increased the abundance of β-galactosidases in young and old animals, and increased the non-saccharolytic organisms; however, a robust, homogeneous bifidogenic effect was not observed. GOS reduced age-associated increased intestinal permeability and increased MUC2 expression and mucus thickness in old mice. Clyndamicin reduced the abundance Bifidobacterium while increasing Akkermansia, Clostridium, Coprococcus, Bacillus, Bacteroides, and Ruminococcus in old mice. The antibiotics were more impactful than GOS on modulating serum markers of inflammation. Higher serum levels of IL-17 and IL-6 were observed in control and GOS diets in the antibiotic groups, and within those groups, levels of IL-6 were higher in the GOS groups, regardless of age, and higher in the old compared to young animals in the control diet groups. RTqPCR revealed significantly increased gene expression of TNFα in distal colon tissue of old mice, which was decreased by the GOS diet. Colon transcriptomics analysis of mice fed GOS showed increased expression of genes involved in small-molecule metabolic processes and specifically the respirasome in old animals, which could indicate an increased oxidative metabolism and energetic efficiency. In young mice, GOS induced the expression of binding-related genes. The galectin gene Lgals1, a β-galactosyl-binding lectin that bridges molecules by their sugar moieties and is an important modulator of the immune response, and the PI3K-Akt and ECM-receptor interaction pathways were also induced in young mice. Stools from mice exhibiting variable bifidogenic response to GOS injected into colon organoids in the presence of prebiotics reproduced the response and non-response phenotypes observed in vivo suggesting that the composition and functionality of the microbiota are the main contributors to the phenotype. Conclusions Dietary GOS modulated homeostasis of the aging gut by promoting changes in microbiome composition and host gene expression, which was translated into decreased intestinal permeability and increased mucus production. Age was a determining factor on how prebiotics impacted the microbiome and expression of intestinal epithelial cells, especially apparent from the induction of galectin-1 in young but not old mice.

Large-scale microbiome studies have established that most of the diversity contained in the gastrointestinal tract is represented at the strain level; however, exhaustive genomic and physiological characterization of human isolates is still lacking. With increased use of probiotics as interventions for gastrointestinal disorders, genomic and functional characterization of novel microorganisms becomes essential. In this study, we explored the impact of strain-level genomic variability on bacterial physiology of two novel human Lactobacillus rhamnosus strains (AMC143 and AMC010) of probiotic potential in relation to stress resistance. The strains showed differences with known probiotic strains (L. rhamnosus GG, Lc705, and HN001) at the genomic level, including nucleotide polymorphisms, mutations in non-coding regulatory regions, and rearrangements of genomic architecture. Transcriptomics analysis revealed that gene expression profiles differed between strains when exposed to simulated gastrointestinal stresses, suggesting the presence of unique regulatory systems in each strain. In vitro physiological assays to test resistance to conditions mimicking the gut environment (acid, alkali, and bile stress) showed that growth of L. rhamnosus AMC143 was inhibited upon exposure to alkaline pH, while AMC010 and control strain LGG were unaffected. AMC143 also showed a significant survival advantage compared to the other strains upon bile exposure. Reverse transcription qPCR targeting the bile salt hydrolase gene (bsh) revealed that AMC143 expressed bsh poorly (a consequence of a deletion in the bsh promoter and truncation of bsh gene in AMC143), while AMC010 had significantly higher expression levels than AMC143 or LGG. Insertional inactivation of the bsh gene in AMC010 suggested that bsh could be detrimental to bacterial survival during bile stress. Together, these findings show that coupling of classical microbiology with functional genomics methods for the characterization of bacterial strains is critical for the development of novel probiotics, as variability between strains can dramatically alter bacterial physiology and functionality.