The inconstant gut microbiota of Drosophila species revealed by 16S rRNA gene analysis

The gut microorganisms in some animals are reported to include a core microbiota of consistently associated bacteria that is ecologically distinctive and may have coevolved with the host. The core microbiota is promoted by positive interactions among bacteria, favoring shared persistence; its retention over evolutionary timescales is evident as congruence between host phylogeny and bacterial community composition. This study applied multiple analyses to investigate variation in the composition of gut microbiota in drosophilid flies. First, the prevalence of five previously described gut bacteria (Acetobacter and Lactobacillus species) in individual flies of 21 strains (10 Drosophila species) were determined. Most bacteria were not present in all individuals of most strains, and bacterial species pairs co-occurred in individual flies less frequently than predicted by chance, contrary to expectations of a core microbiota. A complementary pyrosequencing analysis of 16S rRNA gene amplicons from the gut microbiota of 11 Drosophila species identified 209 bacterial operational taxonomic units (OTUs), with near-saturating sampling of sequences, but none of the OTUs was common to all host species. Furthermore, in both of two independent sets of Drosophila species, the gut bacterial community composition was not congruent with host phylogeny. The final analysis identified no common OTUs across three wild and four laboratory samples of D. melanogaster. Our results yielded no consistent evidence for a core microbiota in Drosophila. We conclude that the taxonomic composition of gut microbiota varies widely within and among Drosophila populations and species. This is reminiscent of the patterns of bacterial composition in guts of some other animals, including humans.     

The Host as the Driver of the Microbiota in the Gut and External Environment of Drosophila melanogaster

Most associations between animals and their gut microbiota are dynamic, involving sustained transfer of food-associated microbial cells into the gut and shedding of microorganisms into the external environment with feces, but the interacting effects of host and microbial factors on the composition of the internal and external microbial communities are poorly understood. This study on laboratory cultures of the fruit fly Drosophila melanogaster reared in continuous contact with their food revealed time-dependent changes of the microbial communities in the food that were strongly influenced by the presence and abundance of Drosophila. When germfree Drosophila eggs were aseptically added to nonsterile food, the microbiota in the food and flies converged to a composition dramatically different from that in fly-free food, showing that Drosophila has microbiota-independent effects on the food microbiota. The microbiota in both the flies that developed from unmanipulated eggs (bearing microorganisms) and the associated food was dominated by the bacteria most abundant on the eggs, demonstrating effective vertical transmission via surface contamination of eggs. Food coinoculated with a four-species defined bacterial community of Acetobacter and Lactobacillus species revealed the progressive elimination of Lactobacillus from the food bearing few or no Drosophila, indicating the presence of antagonistic interactions between Acetobacter and Lactobacillus. Drosophila at high densities ameliorated the Acetobacter/Lactobacillus antagonism, enabling Lactobacillus to persist. This study with Drosophila demonstrates how animals can have major, coordinated effects on the composition of microbial communities in the gut and immediate environment.     

Interspecies Interactions Determine the Impact of the Gut Microbiota on Nutrient Allocation in Drosophila melanogaster

The animal gut is perpetually exposed to microorganisms, and this microbiota affects development, nutrient allocation, and immune homeostasis. A major challenge is to understand the contribution of individual microbial species and interactions among species in shaping these microbe-dependent traits. Using the Drosophila melanogaster gut microbiota, we tested whether microbe-dependent performance and nutritional traits of Drosophila are functionally modular, i.e., whether the impact of each microbial taxon on host traits is independent of the presence of other microbial taxa. Gnotobiotic flies were constructed with one or a set of five of the Acetobacter and Lactobacillus species which dominate the gut microbiota of conventional flies (Drosophila with untreated microbiota). Axenic (microbiota-free) flies exhibited prolonged development time and elevated glucose and triglyceride contents. The low glucose content of conventional flies was recapitulated in gnotobiotic Drosophila flies colonized with any of the 5 bacterial taxa tested. In contrast, the development rates and triglyceride levels in monocolonized flies varied depending on the taxon present: Acetobacter species supported the largest reductions, while most Lactobacillus species had no effect. Only flies with both Acetobacter and Lactobacillus had triglyceride contents restored to the level in conventional flies. This could be attributed to two processes: Lactobacillus-mediated promotion of Acetobacter abundance in the fly and a significant negative correlation between fly triglyceride content and Acetobacter abundance. We conclude that the microbial basis of host traits varies in both specificity and modularity; microbe-mediated reduction in glucose is relatively nonspecific and modular, while triglyceride content is influenced by interactions among microbes.     

A comparative study of gut microbiota profiles of earthworms fed in three different substrates

The gut microbiome of earthworms has a complex interdependence with the host. When the soil minerals pass through earthworm’s gut, they may affect the gut microbiota. To gain insight into the response of gut microbiota to the passed minerals, we fed earthworm (Eisenia fetida) on nutrient-poor soil and ore powder, and used high throughput sequencing to characterize the earthworm intestinal microbial community to find evidence for a core bacterial community of the E. fetida. The results showed that earthworms’ gut maintained a core microbiome that appeared in all samples. These core microbiota may play a significant role in a species’ environmental interactions. The composition of intestinal microbiomes varied with substrates. The earthworm guts from two nutrient-poor substrates had similar microbial communities and they were different from nutrient-rich substrate. Proteobacteria and Bacteroidetes were more abundant in the gut of earthworms kept on a nutrient-poor substrate such as ore powder or mineral soil than in the gut of earthworms kept in organic-rich compost soil; some of these microorganisms may help earthworms survive in nutrient-poor substrates.     

Noninvasive Analysis of Microbiome Dynamics in the Fruit Fly Drosophila melanogaster

The diversity and structure of the intestinal microbial community has a strong influence on life history. To understand how hosts and microbes interact, model organisms with comparatively simple microbial communities, such as the fruit fly (Drosophila melanogaster), offer key advantages. However, studies of the Drosophila microbiome are limited to a single point in time, because flies are typically sacrificed for DNA extraction. In order to test whether noninvasive approaches, such as sampling of fly feces, could be a means to assess fly-associated communities over time on the same cohort of flies, we compared the microbial communities of fly feces, dissected fly intestines, and whole flies across three different Drosophila strains. Bacterial species identified in either whole flies or isolated intestines were reproducibly found in feces samples. Although the bacterial communities of feces and intestinal samples were not identical, they shared similarities and obviously the same origin. In contrast to material from whole flies and intestines, feces samples were not compromised by Wolbachia spp. infections, which are widespread in laboratory and wild strains. In a proof-of-principle experiment, we showed that simple nutritional interventions, such as a high-fat diet or short-term starvation, had drastic and long-lasting effects on the micobiome. Thus, the analysis of feces can supplement the toolbox for microbiome studies in Drosophila, unleashing the full potential of such studies in time course experiments where multiple samples from single populations are obtained during aging, development, or experimental manipulations.     

Host Species and Environmental Effects on Bacterial Communities Associated with Drosophila in the Laboratory and in the Natural Environment

The fruit fly Drosophila is a classic model organism to study adaptation as well as the relationship between genetic variation and phenotypes. Although associated bacterial communities might be important for many aspects of Drosophila biology, knowledge about their diversity, composition, and factors shaping them is limited. We used 454-based sequencing of a variable region of the bacterial 16S ribosomal RNA gene to characterize the bacterial communities associated with wild and laboratory Drosophila isolates. In order to specifically investigate effects of food source and host species on bacterial communities, we analyzed samples from wild Drosophila melanogaster and D. simulans collected from a variety of natural substrates, as well as from adults and larvae of nine laboratory-reared Drosophila species. We find no evidence for host species effects in lab-reared flies; instead, lab of origin and stochastic effects, which could influence studies of Drosophila phenotypes, are pronounced. In contrast, the natural Drosophila–associated microbiota appears to be predominantly shaped by food substrate with an additional but smaller effect of host species identity. We identify a core member of this natural microbiota that belongs to the genus Gluconobacter and is common to all wild-caught flies in this study, but absent from the laboratory. This makes it a strong candidate for being part of what could be a natural D. melanogaster and D. simulans core microbiome. Furthermore, we were able to identify candidate pathogens in natural fly isolates.     

Bile acid is a significant host factor shaping the gut microbiome of diet-induced obese mice

Background

Intestinal bacteria are known to regulate bile acid (BA) homeostasis via intestinal biotransformation of BAs and stimulation of the expression of fibroblast growth factor 19 through intestinal nuclear farnesoid X receptor (FXR). On the other hand, BAs directly regulate the gut microbiota with their strong antimicrobial activities. It remains unclear, however, how mammalian BAs cross-talk with gut microbiome and shape microbial composition in a dynamic and interactive way.

Results

We quantitatively profiled small molecule metabolites derived from host-microbial co-metabolism in mice, demonstrating that BAs were the most significant factor correlated with microbial alterations among all types of endogenous metabolites. A high-fat diet (HFD) intervention resulted in a rapid and significant increase in the intestinal BA pool within 12 h, followed by an alteration in microbial composition at 24 h, providing supporting evidence that BAs are major dietary factors regulating gut microbiota. Feeding mice with BAs along with a normal diet induced an obese phenotype and obesity-associated gut microbial composition, similar to HFD-fed mice. Inhibition of hepatic BA biosynthesis under HFD conditions attenuated the HFD-induced gut microbiome alterations. Both inhibition of BAs and direct suppression of microbiota improved obese phenotypes.

Conclusions

Our study highlights a liver–BA–gut microbiome metabolic axis that drives significant modifications of BA and microbiota compositions capable of triggering metabolic disorders, suggesting new therapeutic strategies targeting BA metabolism for metabolic diseases.

     

Effects of long-term antibiotic treatment on mice urinary aromatic amino acid profiles

The gut microbiota–host co-metabolites are good indicators for representing the cross-talk between host and gut microbiota in a bi-direct manner. There is increasing evidence that levels of aromatic amino acids (AAAs) are associated with the alteration of intestinal microbial community though the effects of long-term microbial disturbance remain unclear. Here we monitored the gut microbiota composition and host–microbiota co-metabolites AAA profiles of mice after gentamicin and ceftriaxone treatments for nearly 4 months since their weaning to reveal the relationship between host and microbiome in long- term microbial disturbances. The study was performed employing targeted LC-MS measurement of AAA-related metabolites and 16S RNA sequence of mice cecal contents. The results showed obvious decreased gut microbial diversity and decreased Firmicutes/Bacteroidetes ratio in the cecal contents after long-term antibiotics treatment. The accumulated AAA (tyrosine, phenylalanine and tryptophan) and re-distribution of their downstreaming metabolites that produced under the existence of intestinal flora were found in mice treated with antibiotics for 4 months. Our results suggested that the long-term antibiotic treatment significantly changed the composition of the gut microbiota and destroyed the homeostasis in the intestinal metabolism. And the urinary AAA could be an indicator for exploring interactions between host and gut microbiota.     

Spotting the differences: Probing host/microbiota interactions with a dedicated software tool for the analysis of faecal outputs in Drosophila

The intestinal physiology of Drosophila melanogaster can be monitored in an integrative, non-invasive manner by analysing graphical features of the excreta produced by flies fed on a dye-supplemented diet. This assay has been used by various labs to explore gut function and its regulation. To facilitate its use, we present here a free, stand-alone dedicated software tool for the analysis of fly excreta. The Ultimate Reader of Dung (T.U.R.D.) is designed to offer a flexible environment for a wide range of experimental designs, with special attention to automation and high-throughput processing. This software detects the distinctive changes in acid-base and water balance previously reported to occur in response to dietary challenges and mating. We have used T.U.R.D. to test the contribution of the bacterial environment of the flies to various intestinal parameters including the established diet- and mating-triggered responses. To this end, we have analysed the faecal patterns of flies reared in germ-free conditions, upon re-association with controlled microbiota and subjected to food-borne or systemic, non-lethal bacterial infections. We find that the tested faecal outputs are unchanged in all these conditions, suggesting that the impact of the bacterial environment on the intestinal features highlighted by faecal deposit analysis is minimal.     

Gut microbial alterations in neonatal jaundice pre- and post-treatment

Neonatal jaundice is a common disease that affects up to 60% of newborns. Herein, we performed a comparative analysis of the gut microbiome in neonatal jaundice and non-neonatal jaundice infants (NJIs) and identified gut microbial alterations in neonatal jaundice pre- and post-treatment. We prospectively collected 232 fecal samples from 51 infants at five time points (0, 1, 3, 6, and 12 months). Finally, 114 samples from 6 NJIs and 19 non-NJI completed MiSeq sequencing and analysis. We characterized the gut microbiome and identified microbial differences and gene functions. Meconium microbial diversity from NJI was decreased compared with that from non-NJI. The genus Gemella was decreased in NJI versus non-NJI. Eleven predicted microbial functions, including fructose 1,6-bisphosphatase III and pyruvate carboxylase subunit B, decreased, while three functions, including acetyl-CoA acyltransferase, increased in NJI. After treatments, the microbial community presented significant alteration-based β diversity. The phyla Firmicutes and Actinobacteria were increased, while Proteobacteria and Fusobacteria were decreased. Microbial alterations were also analyzed between 6 recovered NJI and 19 non-NJI. The gut microbiota was unique in the meconium microbiome from NJI, implying that early gut microbiome intervention could be promising for the management of neonatal jaundice. Alterations of gut microbiota from NJI can be of great value to bolster evidence-based prevention against ‘bacterial dysbiosis’.     

Correlation of Gut Microbiome Between ASD Children and Mothers and Potential Biomarkers for Risk Assessment

Variation of maternal gut microbiota may increase the risk of autism spectrum disorders(ASDs) in offspring. Animal studies have indicated that maternal gut microbiota is related to neurodevelopmental abnormalities in mouse offspring, while it is unclear whether there is a correlation between gut microbiota of ASD children and their mothers. We examined the relationships between gut microbiome profiles of ASD children and those of their mothers, and evaluated the clinical discriminatory power of discovered bacterial biomarkers. Gut microbiome was profiled and evaluated by 16S ribosomal RNA gene sequencing in stool samples of 59 mother–child pairs of ASD children and 30 matched mother–child pairs of healthy children. Significant differences were observed in the gut microbiome composition between ASD and healthy children in our Chinese cohort. Several unique bacterial biomarkers, such as Alcaligenaceae and Acinetobacter, were identified. Mothers of ASD children had more Proteobacteria, Alphaproteobacteria, Moraxellaceae, and Acinetobacter than mothers of healthy children. There was a clear correlation between gut microbiome profiles of children and their mothers; however, children with ASD still had unique bacterial biomarkers, such as Alcaligenaceae, Enterobacteriaceae, and Clostridium. Candidate biomarkers discovered in this study had remarkable discriminatory power. The identified patterns of mother–child gut microbiome profiles may be important for assessing risks during the early stage and planning of personalized treatment and prevention of ASD via microbiota modulation.     

Strong responses of Drosophila melanogaster microbiota to developmental temperature

Physiological responses to changes in environmental conditions such as temperature may partly arise from the resident microbial community that integrates a wide range of bio-physiological aspects of the host. In the present study, we assessed the effect of developmental temperature on the thermal tolerance and microbial community of Drosophila melanogaster. We also developed a bacterial transplantation protocol in order to examine the possibility of reshaping the host bacterial composition and assessed its influence on the thermotolerance phenotype. We found that the temperature during development affected thermal tolerance and the microbial composition of male D. melanogaster. Flies that developed at low temperature (13°C) were the most cold resistant and showed the highest abundance of Wolbachia, while flies that developed at high temperature (31°C) were the most heat tolerant and had the highest abundance of Acetobacter. In addition, feeding newly eclosed flies with bacterial suspensions from intestines of flies developed at low temperatures changed the heat tolerance of recipient flies. However, we were not able to link this directly to a change in the host bacterial composition.     

Effects of Heat Stress on Gut Microbiome in Rats

Gut microbiome, as the largest and most important micro-ecosystem, plays a critical role in health. The purpose of this study was to evaluate whether heat stress modulates the composition and diversity of the gut microbiome in rats. The heat stress model was prepared in rats with the heating temperature maintained at 35–38°C. Cecum contents were collected after heat stress for 3 h and days 1, 3 and 7. Total DNA was extracted for 16 S rRNA sequencing and analysis of intestinal microbiome composition and diversity. The study showed that the composition of the intestinal microbiome of heat stress group was changed. And the heat stress modulated key phylotypes of gut microbiota at the level of phylum and genus. In particular, the genus of Lactobacillus and Bacteroides were significantly reduced, whereas the Oscillospira and Clostridium were increased by heat stress. Meanwhile, the rats under the heat stress encountered the change in carbohydrate metabolism, amino acid metabolism, and membrane transport to defense against stress. Taken together, the composition and structure of gut microbiome were affected by heat stress and some key phylotypes were also significantly altered. We conclude that the heat stress could impact multiple biological functions, via altering the gut microbiome.     

Lactobacillus plantarum IFPL935 impacts colonic metabolism in a simulator of the human gut microbiota during feeding with red wine polyphenols

The colonic microbiota plays an important role in the bioavailibility of dietary polyphenols. This work has evaluated the impact on the gut microbiota of long-term feeding with both a red wine polyphenolic extract and the flavan-3-ol metabolizer strain Lactobacillus plantarum IFPL935. The study was conducted in the dynamic Simulator of the Human Intestinal Microbial Ecosystem (SHIME). The feeding of the gut microbiota model with red wine polyphenols caused an initial decrease in the counts of total bacteria in the ascending colon (AC), with Bacteroides, Clostridium coccoides/Eubacterium rectale and Bifidobacterium being the most affected bacterial groups. The bacterial counts recovered to initial numbers faster than the overall microbial fermentation and proteolysis, which seemed to be longer affected by polyphenols. Addition of L. plantarum IFPL935 helped to promptly recover total counts, Lactobacillus and Enterobacteriaceae and led to an increase in lactic acid formation in the AC vessel at the start of the polyphenol treatment as well as butyric acid in the transverse (TC) and descending (DC) vessels after 5 days. Moreover, L. plantarum IFPL935 favoured the conversion in the DC vessel of monomeric flavan-3-ols and their intermediate metabolites into phenylpropionic acids and in particular 3-(3′-hydroxyphenyl)propionic acid. The results open the possibilities of using L. plantarum IFPL935 as a food ingredient for helping individuals showing a low polyphenol-fermenting metabotype to increase their colonic microbial capacities of metabolizing dietary polyphenols.     

Distinct composition and metabolic functions of human gut microbiota are associated with cachexia in lung cancer patients

Cachexia is associated with decreased survival in cancer patients and has a prevalence of up to 80%. The etiology of cachexia is poorly understood, and limited treatment options exist. Here, we investigated the role of the human gut microbiome in cachexia by integrating shotgun metagenomics and plasma metabolomics of 31 lung cancer patients. The cachexia group showed significant differences in the gut microbial composition, functional pathways of the metagenome, and the related plasma metabolites compared to non-cachectic patients. Branched-chain amino acids (BCAAs), methylhistamine, and vitamins were significantly depleted in the plasma of cachexia patients, which was also reflected in the depletion of relevant gut microbiota functional pathways. The enrichment of BCAAs and 3-oxocholic acid in non-cachectic patients were positively correlated with gut microbial species Prevotella copri and Lactobacillus gasseri, respectively. Furthermore, the gut microbiota capacity for lipopolysaccharides biosynthesis was significantly enriched in cachectic patients. The involvement of the gut microbiome in cachexia was further observed in a high-performance machine learning model using solely gut microbial features. Our study demonstrates the links between cachectic host metabolism and specific gut microbial species and functions in a clinical setting, suggesting that the gut microbiota could have an influence on cachexia with possible therapeutic applications.Subject terms: Microbiome, Metagenomics, Next-generation sequencing, Metabolomics     

Altered Mucus Glycosylation in Core 1 O-Glycan-Deficient Mice Affects Microbiota Composition and Intestinal Architecture

A functional mucus layer is a key requirement for gastrointestinal health as it serves as a barrier against bacterial invasion and subsequent inflammation. Recent findings suggest that mucus composition may pose an important selection pressure on the gut microbiota and that altered mucus thickness or properties such as glycosylation lead to intestinal inflammation dependent on bacteria. Here we used TM-IEC C1galt ^-/- mice, which carry an inducible deficiency of core 1-derived O-glycans in intestinal epithelial cells, to investigate the effects of mucus glycosylation on susceptibility to intestinal inflammation, gut microbial ecology and host physiology. We found that TM-IEC C1galt ^-/- mice did not develop spontaneous colitis, but they were more susceptible to dextran sodium sulphate-induced colitis. Furthermore, loss of core 1-derived O-glycans induced inverse shifts in the abundance of the phyla Bacteroidetes and Firmicutes. We also found that mucus glycosylation impacts intestinal architecture as TM-IEC C1galt^-/- mice had an elongated gastrointestinal tract with deeper ileal crypts, a small increase in the number of proliferative epithelial cells and thicker circular muscle layers in both the ileum and colon. Alterations in the length of the gastrointestinal tract were partly dependent on the microbiota. Thus, the mucus layer plays a role in the regulation of gut microbiota composition, balancing intestinal inflammation, and affects gut architecture.     

Gut microbiota composition of Japanese macaques associates with extent of human encroachment

In recent decades, human–wildlife interaction and associated anthropogenic food provisioning has been increasing and becoming more severe due to fast population growth and urban development. Noting the role of the gut microbiome in host physiology like nutrition and health, it is thus essential to understand how human–wildlife interactions and availability of anthropogenic food in habitats can affect an animal's gut microbiome. This study, therefore, set out to examine the gut microbiota of Japanese macaques (Macaca fuscata) with varying accessibility to anthropogenic food and the possibility of using gut microbiota as indicator for macaques’ reliance on anthropogenic food. Using 16S ribosomal RNA gene sequencing, we described the microbial composition of Japanese macaques experiencing different types of human disturbance and anthropogenic food availability—captive, provisioned, crop‐raiding, and wild. In terms of alpha diversity, our results showed that observed richness of gut microbiota did not differ significantly between disturbance types but among collection sites, whereas Shannon diversity index differed by both disturbance types and sites. In terms of beta diversity, captive populations harbored the most distinctive gut microbial composition, and had the greatest difference compared with wild populations. Whereas for provisioned and crop‐raiding groups, the macaques exhibited intermediate microbiota between wild and captive. We identified several potential bacterial taxa at different taxonomic ranks whose abundance potentially could help in assessing macaques’ accessibility to anthropogenic food. This study revealed the flexibility of the gut microbiome of Japanese macaques and provided possible indices based on the gut microbiome profile in assessing macaques’ accessibility to/reliance on anthropogenic foods.     

Decipher enzymes from human microbiota for drug discovery and development

The human microbiota plays an important role in human health and contributes to the metabolism of therapeutic drugs affecting their potency. However, the current knowledge on human gut bacterial metabolism is limited and lacks an understanding of the underlying mechanisms of observed drug biotransformations. Despite the complexity of the gut microbial community, genomic and metagenomic sequencing provides insights into the diversity of chemical reactions that can be carried out by the microbiota and poses new challenges to functionally annotate thousands of bacterial enzymes. Here, we outline methods to systematically address the structural and functional space of the human microbiome, highlighting a combination of in silico and in vitro approaches. Systematic knowledge about microbial enzymes could eventually be applied for personalized therapy, the development of prodrugs and modulators of unwanted bacterial activity, and the further discovery of new antibiotics.