Gut Microbiota and SCFAs Play Key Roles in QingFei Yin Recipe Anti-Streptococcal Pneumonia Effects

Emerging evidence has revealed the presence in animals of a bidirectional regulatory “lung-gut axis” that provides resistance to respiratory infections. Clues to the existence of this system stem from observations that respiratory infections are often accompanied by gastrointestinal symptoms, whereby intestinal microbiota appear to play pivotal roles in combating pathogenic infections. Importantly, short-chain fatty acids (SCFAs) produced by the gut microbiota appear to serve as the biological link between host immune defenses and gut flora. Streptococcus pneumoniae (S.pn), the main cause of lower respiratory tract infections, is involved in more than 1.189 million deaths per year. QingFei Yin (QFY) is known for its excellent therapeutic efficacy in combating bacterial lung infections. In this study, effects of S.pn infection on gut homeostasis were assessed using 16S RNA-based microbiota community profiling analysis. In addition, potential mechanisms underlying QFY recipe beneficial therapeutic effects against bacterial pneumonia were explored using S.pn-infected gut microbiota-depleted mice. Results of data analysis indicated that QFY treatment alleviated lung infection-associated pathogenic processes, while also promoting repair of disordered gut flora and counteracting S.pn infection-associated decreases in levels of SCFAs, particularly of acetate and butyrate. Mechanistically, QFY treatment suppressed inflammatory lung injury through inhibition of the host NF-κB-NLRP3 pathway. These results inspired us to identify precise QFY targets and mechanisms underlying QFY anti-inflammatory effects. In addition, we conducted an in-depth evaluation of QFY as a potential treatment for bacterial pneumonia.

The accuracy of absolute differential abundance analysis from relative count data

Concerns have been raised about the use of relative abundance data derived from next generation sequencing as a proxy for absolute abundances. In the differential abundance setting compositional effects are hypothesized to contribute to increased rates of spurious differences (false positives). However in practice, partial reconstruction of total abundance can be imputed through renormalization of observed per-sample abundance. Given the renormalized data differential abundance need not be called on relative counts themselves but on estimates of absolute counts. We use simulated data to explore the consistency of differential abundance calls made on these adjusted relative abundances and find that while overall rates of false positive calls are low substantial error is possible. Conditions consistent with microbial community profiling are the most at risk of error induced by compositional effects. Increasing complexity of composition (i.e. increasing feature number) is generally protective against this effect. In real data sets drawn from 16S metabarcoding, expression array, bulk RNA-seq, and single-cell RNA-seq experiments, results are similar: though median accuracy is high, microbial community profiling and single-cell transcriptomic data sets can have poor outcomes. However, we show that problematic data sets can often be identified by summary characteristics of their relative abundances alone, giving researchers a means of anticipating problems and adjusting analysis strategies where appropriate.

A wipe-based stool collection and preservation kit for microbiome community profiling

While a range of methods for stool collection exist, many require complicated, self-directed protocols and stool transfer. In this study, we introduce and validate a novel, wipe-based approach to fecal sample collection and stabilization for metagenomics analysis. A total of 72 samples were collected across four different preservation types: freezing at -20°C, room temperature storage, a commercial DNA preservation kit, and DESS (dimethyl sulfoxide, ethylenediaminetetraacetic acid, sodium chloride) solution. These samples were sequenced and analyzed for taxonomic abundance metrics, metabolic pathway classification, and diversity analysis. Overall, the DESS wipe results validated the use of a wipe-based capture method to collect stool samples for microbiome analysis, showing an R2 of 0.96 for species across all kingdoms, as well as exhibiting a maintenance of Shannon diversity (3.1-3.3) and species richness (151-159) compared to frozen samples. Moreover, DESS showed comparable performance to the commercially available preservation kit (R2 of 0.98), and samples consistently clustered by subject across each method. Future studies will be needed to further explore sample processing options and their applications in non-healthy subjects, particularly patients with irritable bowel syndrome, inflammatory bowel disease, and colorectal cancer, but these data suggest the DESS wipe method can be used for stable, room temperature collection and transport of human stool specimens.

Bacteria Cultivated From Sponges and Bacteria Not Yet Cultivated From Sponges—A Review

The application of high-throughput microbial community profiling as well as “omics” approaches unveiled high diversity and host-specificity of bacteria associated with marine sponges, which are renowned for their wide range of bioactive natural products. However, exploration and exploitation of bioactive compounds from sponge-associated bacteria have been limited because the majority of the bacteria remains recalcitrant to cultivation. In this review, we (i) discuss recent/novel cultivation techniques that have been used to isolate sponge-associated bacteria, (ii) provide an overview of bacteria isolated from sponges until 2017 and the associated culture conditions and identify the bacteria not yet cultured from sponges, and (iii) outline promising cultivation strategies for cultivating the uncultivated majority of bacteria from sponges in the future. Despite intensive cultivation attempts, the diversity of bacteria obtained through cultivation remains much lower than that seen through cultivation-independent methods, which is particularly noticeable for those taxa that were previously marked as “sponge-specific” and “sponge-enriched.” This poses an urgent need for more efficient cultivation methods. Refining cultivation media and conditions based on information obtained from metagenomic datasets and cultivation under simulated natural conditions are the most promising strategies to isolate the most wanted sponge-associated bacteria.

Arabidopsis assemble distinct root-associated microbiomes through the synthesis of an array of defense metabolites

Plant associated microbiomes are known to confer fitness advantages to the host. Understanding how plant factors including biochemical traits influence host associated microbiome assembly could facilitate the development of microbiome-mediated solutions for sustainable plant production. Here, we examined microbial community structures of a set of well-characterized Arabidopsis thaliana mutants disrupted in metabolic pathways for the production of glucosinolates, flavonoids, or a number of defense signalling molecules. A. thaliana lines were grown in a natural soil and maintained under greenhouse conditions for 4 weeks before collection of roots for bacterial and fungal community profiling. We found distinct relative abundances and diversities of bacterial and fungal communities assembled in the individual A. thaliana mutants compared to their parental lines. Bacterial and fungal genera were mostly enriched than depleted in secondary metabolite and defense signaling mutants, except for flavonoid mutations on fungi communities. Bacterial genera Azospirillum and Flavobacterium were significantly enriched in most of the glucosinolate, flavonoid and signalling mutants while the fungal taxa Sporobolomyces and Emericellopsis were enriched in several glucosinolates and signalling mutants. Whilst the present study revealed marked differences in microbiomes of Arabidopsis mutants and their parental lines, it is suggestive that unknown enzymatic and pleiotropic activities of the mutated genes could contribute to the identified host-associated microbiomes. Notwithstanding, this study revealed interesting gene-microbiota links, and thus represents valuable resource data for selecting candidate A. thaliana mutants for analyzing the links between host genetics and the associated microbiome.

Metagenomic Analysis of Lichen Associated Bacterial Community Profiling In Roccella Montagnei

A lichen is a composite organism formed of algae or cyanobacteria that live in a mutually advantageous symbiotic relationship with the filaments (hyphae) of fungus. Three lichen samples were obtained from diverse sites at Kuppanasamy temple, Pollachi, a terrestrial habitat located in Coimbatore, Nithiravilai, Nagercoil, and Ramarpatham, Vedaranyam, both coastal habitats located in Kanyakumari and Nagapattinam districts of Tamil Nadu, for this study. Amplification and sequencing of 16S rRNA V3 - V4 regions were used for metagenomic study. Aside from the NGS data, distinct types of lichen microbiome profiles were clearly shown. The bacterial diversity in the lichen genera of Roccella montagnei growing in coastal and terrestrial environments was further investigated using common and unique operational taxonomic units (OTUs) and the QIIME pipeline (1.9.1). By using similarity clustering, the heat map analysis depicts the abundance information of chosen OTUs as well as the similarity and difference between OTUs and lichen samples. Using multiple methods, the Alpha and Beta diversity analysis revealed that there were differences in all of the samples. However, UPGMA tree inference of comparable bacterial community in coastal habitat lichen samples compared to terrestrial habitat validates their evolutionary lineage. As a result, the bacterial population associated with corticolous lichen is dependent on geographic locations, growth substrate, and climatic circumstances of similar lichen genera produced in different habitats and tree substrates.

Plant neighborhood shapes diversity and reduces interspecific variation of the phyllosphere microbiome

Microbial communities associated with plant leaf surfaces (i.e. the phyllosphere) are increasingly recognized for their role in plant health. While accumulating evidence suggests a role for host filtering of its microbiota, far less is known about how community composition is shaped by dispersal, including from neighboring plants. We experimentally manipulated the local plant neighborhood within which tomato, pepper, or bean plants were grown in a three-month field trial. Focal plants were grown in the presence of con- or hetero-specific neighbors (or no neighbors) in a fully factorial combination. At 30-day intervals, focal plants were harvested and replaced with a new age- and species-matched cohort while allowing neighborhood plants to continue growing. 16S community profiling revealed that the strength of host filtering effects (i.e. interspecific differences in composition) decreased over time. In contrast, the strength of neighborhood effects increased over time, suggesting dispersal from neighboring plants becomes more important as neighboring plant biomass increases. We next implemented a cross-inoculation study in the greenhouse using inoculum generated from the field plants to directly test host filtering of microbiomes while controlling for directionality and source of dispersal. This experiment further demonstrated that focal host species, the host from which the microbiome came, and in one case the donor hosts' neighbors, contribute to variation in phyllosphere bacterial composition. Overall, our results suggest that local dispersal is a key factor in phyllosphere assembly, and that demographic factors such as nearby neighbor identity and biomass or age are important determinants of phyllosphere microbiome diversity.

Cooperation between host immunity and the gut bacteria is essential for helminth-evoked suppression of colitis

Background Studies on the inhibition of inflammation by infection with helminth parasites have, until recently, overlooked a key determinant of health: the gut microbiota. Infection with helminths evokes changes in the composition of their host’s microbiota: one outcome of which is an altered metabolome (e.g., levels of short-chain fatty acids (SCFAs)) in the gut lumen. The functional implications of helminth-evoked changes in the enteric microbiome (composition and metabolites) are poorly understood and are explored with respect to controlling enteric inflammation. Methods Antibiotic-treated wild-type, germ-free (GF) and free fatty-acid receptor-2 (ffar2) deficient mice were infected with the tapeworm Hymenolepis diminuta, then challenged with DNBS-colitis and disease severity and gut expression of the il-10 receptor-α and SCFA receptors/transporters assessed 3 days later. Gut bacteria composition was assessed by 16 s rRNA sequencing and SCFAs were measured. Other studies assessed the ability of feces or a bacteria-free fecal filtrate from H. diminuta-infected mice to inhibit colitis. Results Protection against disease by infection with H. diminuta was abrogated by antibiotic treatment and was not observed in GF-mice. Bacterial community profiling revealed an increase in variants belonging to the families Lachnospiraceae and Clostridium cluster XIVa in mice 8 days post-infection with H. diminuta, and the transfer of feces from these mice suppressed DNBS-colitis in GF-mice. Mice treated with a bacteria-free filtrate of feces from H. diminuta-infected mice were protected from DNBS-colitis. Metabolomic analysis revealed increased acetate and butyrate (both or which can reduce colitis) in feces from H. diminuta-infected mice, but not from antibiotic-treated H. diminuta-infected mice. H. diminuta-induced protection against DNBS-colitis was not observed in ffar2−/− mice. Immunologically, anti-il-10 antibodies inhibited the anti-colitic effect of H. diminuta-infection. Analyses of epithelial cell lines, colonoids, and colon segments uncovered reciprocity between butyrate and il-10 in the induction of the il-10-receptor and butyrate transporters. Conclusion Having defined a feed-forward signaling loop between il-10 and butyrate following infection with H. diminuta, this study identifies the gut microbiome as a critical component of the anti-colitic effect of this helminth therapy. We suggest that any intention-to-treat with helminth therapy should be based on the characterization of the patient’s immunological and microbiological response to the helminth.