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Abstract Fixation of atmospheric N2 by free-living diazotrophs accounts for an important proportion of nitrogen naturally introduced to temperate grasslands. The effect of plants or fertilization on the general microbial community has been extensively studied, yet an understanding of the potential combinatorial effects on the community structure and activity of free-living diazotrophs is lacking. In this study we provide a multilevel assessment of the single and interactive effects of different long-term fertilization treatments, plant species and vicinity to roots on the free-living diazotroph community in relation to the general microbial community in grassland soils. We sequenced the dinitrogenase reductase (nifH) and the 16S rRNA genes of bulk soil and root-associated compartments (rhizosphere soil, rhizoplane and root) of two grass species (Arrhenatherum elatius and Anthoxanthum odoratum) and two herb species (Galium album and Plantago lanceolata) growing in Austrian grassland soils treated with different fertilizers (N, P, NPK) since 1960. Overall, fertilization has the strongest effect on the diazotroph and general microbial community structure, however with vicinity to the root, the plant effect increases. Despite the long-term fertilization, plants strongly influence the diazotroph communities emphasizing the complexity of soil microbial communities’ responses to changing nutrient conditions in temperate grasslands.

Abstract Background Fungi and ants belong to the most important organisms in terrestrial ecosystems on Earth. In nutrient-poor niches of tropical rainforests, they have developed steady ecological relationships as a successful survival strategy. In tropical ant-plant mutualisms worldwide, where resident ants provide the host plants with defense and nutrients in exchange for shelter and food, fungi are regularly found in the ant nesting space, inhabiting ant-made dark-colored piles (“patches”). Unlike the extensively investigated fungus-growing insects, where the fungi serve as the primary food source, the purpose of this ant-fungi association is less clear. To decipher the roles of fungi in these structures within ant nests, it is crucial to first understand the dynamics and drivers that influence fungal patch communities during ant colony development. Results In this study, we investigated how the ant colony age and the ant-plant species affect the fungal community in the patches. As model we selected one of the most common mutualisms in the Tropics of America, the Azteca-Cecropia complex. By amplicon sequencing of the internal transcribed spacer 2 (ITS2) region, we analyzed the patch fungal communities of 93 Azteca spp. colonies inhabiting Cecropia spp. trees. Our study demonstrates that the fungal diversity in patches increases as the ant colony grows and that a change in the prevalent fungal taxa occurs between initial and established patches. In addition, the ant species significantly influences the composition of the fungal community in established ant colonies, rather than the host plant species. Conclusions The fungal patch communities become more complex as the ant colony develops, due to an acquisition of fungi from the environment and a substrate diversification. Our results suggest a successional progression of the fungal communities in the patches during ant colony growth and place the ant colony as the main driver shaping such communities. The findings of this study demonstrate the unexpectedly complex nature of ant-plant mutualisms in tropical regions at a micro scale.

Abstract Background Microorganisms are responsible for nutrient removal and resource recovery in wastewater treatment plants (WWTPs), and their diversity is often studied by 16S rRNA gene amplicon sequencing. However, this approach underestimates the abundance and diversity of Patescibacteria due to the low coverage of commonly used PCR primers for this highly divergent bacterial phylum. Therefore, our current understanding of the global diversity, distribution, and ecological role of Patescibacteria in WWTPs is very incomplete. This is particularly relevant as Patescibacteria are considered to be associated with microbial host cells and can therefore influence the abundance and temporal variability of other microbial groups that are important for WWTP functioning. Results Here, we evaluated the in silico coverage of widely used 16S rRNA gene-targeted primer pairs and redesigned a primer pair targeting the V4 region of bacterial and archaeal 16S rRNA genes to expand its coverage for Patescibacteria. We then experimentally evaluated and compared the performance of the original and modified V4-targeted primers on 565 WWTP samples from the MiDAS global sample collection. Using the modified primer pair, the percentage of ASVs classified as Patescibacteria increased from 5.9 to 23.8%, and the number of detected patescibacterial genera increased from 560 to 1576, while the detected diversity of the remaining microbial community remained similar. Due to this significantly improved coverage of Patescibacteria, we identified 23 core genera of Patescibacteria in WWTPs and described the global distribution pattern of these unusual microbes in these systems. Finally, correlation network analysis revealed potential host organisms that might be associated with Patescibacteria in WWTPs. Interestingly, strong indications were found for an association between Patescibacteria of the Saccharimonadia and globally abundant polyphosphate-accumulating organisms of the genus Ca. Phosphoribacter. Conclusions Our study (i) provides an improved 16S rRNA gene V4 region-targeted amplicon primer pair inclusive of Patescibacteria with little impact on the detection of other taxa, (ii) reveals the diversity and distribution patterns of Patescibacteria in WWTPs on a global scale, and (iii) provides new insights into the ecological role and potential hosts of Patescibacteria in WWTPs . Video Abstract

Severe brain damage is common among premature infants, and the gut microbiota has been implicated in its pathology. Although the order of colonizing bacteria is well described, the mechanisms underlying aberrant assembly of the gut microbiota remain elusive. Here, we employed long-read nanopore sequencing to assess abundances of microbial species and their functional genomic potential in stool samples from a cohort of 30 extremely premature infants. We identify several key microbial traits significantly associated with severe brain damage, such as the genomic potential for nitrate respiration and iron scavenging. Members of the Enterobacteriaceae were prevalent across the cohort and displayed a versatile metabolic potential, including pathogenic and nonpathogenic traits. Predominance of Enterobacter hormaechei and Klebsiella pneumoniae were associated with an overall loss of genomic functional redundancy as well as poor neurophysiological outcome. These findings reveal microbial traits that may be involved in exacerbating brain injury in extremely premature infants and provide suitable targets for therapeutic interventions.

The gut microbiota has been implicated as a driver of irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). Recently we described, mucosal biofilms, signifying alterations in microbiota composition and bile acid (BA) metabolism in IBS and ulcerative colitis (UC). Luminal oxygen concentration is a key factor in the gastrointestinal (GI) ecosystem and might be increased in IBS and UC. Here we analyzed the role of archaea as a marker for hypoxia in mucosal biofilms and GI homeostasis. The effects of archaea on microbiome composition and metabolites were analyzed via amplicon sequencing and untargeted metabolomics in 154 stool samples of IBS-, UC-patients and controls. Mucosal biofilms were collected in a subset of patients and examined for their bacterial, fungal and archaeal composition. Absence of archaea, specifically Methanobrevibacter, correlated with disrupted GI homeostasis including decreased microbial diversity, overgrowth of facultative anaerobes and conjugated secondary BA. IBS-D/-M was associated with absence of archaea. Presence of Methanobrevibacter correlated with Oscillospiraceae and epithelial short chain fatty acid metabolism and decreased levels of Ruminococcus gnavus. Absence of fecal Methanobrevibacter may indicate a less hypoxic GI environment, reduced fatty acid oxidation, overgrowth of facultative anaerobes and disrupted BA deconjugation. Archaea and Ruminococcus gnavus could distinguish distinct subtypes of mucosal biofilms. Further research on the connection between archaea, mucosal biofilms and small intestinal bacterial overgrowth should be performed.

Bryophytes and biological soil crusts (biocrusts) are the two major biological soil cover types of maritime Antarctica and play a crucial role for key ecosystem functions in the barely vegetated and little developed soils. Besides their profound impacts on nutrient cycling, they also provide habitats and activity hotspots for unique soil microbial communities. Yet, the effects of biological soil cover on the physical and chemical soil environment and belowground microbial communities have not been comprehensively studied in this fragile ecosystem.We here address the research question how biocrusts and mosses shape the quantity and structure of the soil organic matter pool, and the activity and composition of subjacent microbial communities. Towards this end, we sampled soils under two common, but physiologically distinct moss species, Polytrichastrum alpinum and Sanionia unicinata, and adjacent biological soil crusts at two sites on Deception Island, South Shetland Islands.We found that biocrusts and mosses differentially influenced central soil properties and subjacent soil microbial communities. All major SOM compound groups (carbohydrates, aromatics and phenols, lipids, N-containing polymers) as well as microbial biomass were more abundant in soil under biocrusts. However, microbial mass-specific growth rates were higher in soil under mosses. Our results showed moss-species-specific effects in addition to effects of soil cover type, as P. alpinum affected the activity and structure of soil microbial communities and the composition of soil organic matter stronger than S. unicinata.Our study highlights the interconnectedness between soil cover and soil biogeochemistry, which is crucial for deepening our understanding of belowground functioning in Antarctic soils. This linkage is of particular importance in the context of ongoing rapid climate change on the Antarctic Peninsula, as future shifts in the distribution and abundance of soil cover may substantially impact multiple soil processes in this vulnerable ecosystem.

Abstract Prebiotics are defined as non-digestible dietary components that promote the growth of beneficial gut microorganisms. In many cases, however, this capability is not systematically evaluated. Here, we develop a methodology for determining prebiotic-responsive bacteria using the popular dietary supplement inulin. We first identify microbes with a capacity to bind inulin using mesoporous silica nanoparticles functionalized with inulin. 16S rRNA gene amplicon sequencing of sorted cells revealed that the ability to bind inulin was widespread in the microbiota. We further evaluate which taxa are metabolically stimulated by inulin and find that diverse taxa from the phyla Firmicutes and Actinobacteria respond to inulin, and several isolates of these taxa can degrade inulin. Incubation with another prebiotic, xylooligosaccharides (XOS), in contrast, shows a more robust bifidogenic effect. Interestingly, the Coriobacteriia Eggerthella lenta and Gordonibacter urolithinfaciens are indirectly stimulated by the inulin degradation process, expanding our knowledge of inulin-responsive bacteria.

Abstract Climate change increases the frequency and intensity of drought events, affecting soil functions including carbon sequestration and nutrient cycling, which are driven by growing microorganisms. Yet we know little about microbial responses to drought due to methodological limitations. Here, we estimate microbial growth rates in montane grassland soils exposed to ambient conditions, drought, and potential future climate conditions (i.e., soils exposed to 6 years of elevated temperatures and elevated CO2 levels). For this purpose, we combined 18O-water vapor equilibration with quantitative stable isotope probing (termed ‘vapor-qSIP’) to measure taxon-specific microbial growth in dry soils. In our experiments, drought caused >90% of bacterial and archaeal taxa to stop dividing and reduced the growth rates of persisting ones. Under drought, growing taxa accounted for only 4% of the total community as compared to 35% in the controls. Drought-tolerant communities were dominated by specialized members of the Actinobacteriota, particularly the genus Streptomyces. Six years of pre-exposure to future climate conditions (3 °C warming and + 300 ppm atmospheric CO2) alleviated drought effects on microbial growth, through more drought-tolerant taxa across major phyla, accounting for 9% of the total community. Our results provide insights into the response of active microbes to drought today and in a future climate, and highlight the importance of studying drought in combination with future climate conditions to capture interactive effects and improve predictions of future soil-climate feedbacks.

Abstract Taurine-respiring gut bacteria produce H2S with ambivalent impact on host health. We report the isolation and ecophysiological characterization of a taurine-respiring mouse gut bacterium. Taurinivorans muris strain LT0009 represents a new widespread species that differs from the human gut sulfidogen Bilophila wadsworthia in its sulfur metabolism pathways and host distribution. T. muris specializes in taurine respiration in vivo, seemingly unaffected by mouse diet and genotype, but is dependent on other bacteria for release of taurine from bile acids. Colonization of T. muris in gnotobiotic mice increased deconjugation of taurine-conjugated bile acids and transcriptional activity of a sulfur metabolism gene-encoding prophage in other commensals, and slightly decreased the abundance of Salmonella enterica, which showed reduced expression of galactonate catabolism genes. Re-analysis of metagenome data from a previous study further suggested that T. muris can contribute to protection against pathogens by the commensal mouse gut microbiota. Together, we show the realized physiological niche of a key murine gut sulfidogen and its interactions with selected gut microbiota members.

Abstract The role of the gut microbiome in developing Inflammatory Bowel Disease (IBD) in humans and dogs has received attention in recent years. Evidence suggests that IBD is associated with alterations in gut microbial composition, but further research is needed in veterinary medicine. The impact of IBD treatment on the gut microbiome needs to be better understood, especially in a breed-specific form of IBD in Yorkshire Terriers known as Yorkshire Terrier Enteropathy (YTE). This study aimed to investigate the difference in gut microbiome composition between YTE dogs during disease and remission and healthy Yorkshire Terriers. Our results showed a significant increase in specific taxa such as Clostridium sensu stricto 1, Escherichia-Shigella, and Streptococcus, and a decrease in Bacteroides, Prevotella, Alloprevotella, and Phascolarctobacterium in YTE dogs compared to healthy controls. No significant difference was found between the microbiome of dogs in remission and those with active disease, suggesting that the gut microbiome is affected beyond clinical recovery.

Background & Aims: Alcohol-related liver disease (ALD) is a global healthcare challenge with limited treatment options. 24-Norursodeoxycholic acid (NorUDCA) is a synthetic bile acid with anti-inflammatory properties in experimental and human cholestatic liver diseases. In the present study, we explored the efficacy of norUDCA in experimental ALD. Methods: NorUDCA was tested in a preventive and therapeutic setting in an experimental ALD model (Lieber–DeCarli diet enriched with ethanol). Liver disease was phenotypically evaluated using histology and biochemical methods, and anti-inflammatory properties and peroxisome proliferator-activated receptor gamma activation by norUDCA were evaluated in cellular model systems. Results: NorUDCA administration ameliorated ethanol-induced liver injury, reduced hepatocyte death, and reduced the expression of hepatic pro-inflammatory cytokines including tumour necrosis factor (Tnf), Il-1β, Il-6, and Il-10. NorUDCA shifted hepatic macrophages towards an anti-inflammatory M2 phenotype. Further, norUDCA administration altered the composition of the intestinal microbiota, specifically increasing the abundance of Roseburia, Enterobacteriaceae, and Clostridum spp. In a therapeutic model, norUDCA also ameliorated ethanol-induced liver injury. Moreover, norUDCA suppressed lipopolysaccharide-induced IL-6 expression in human peripheral blood mononuclear cells and evoked peroxisome proliferator-activated receptor gamma activation. Conclusions: NorUDCA ameliorated experimental ALD, protected against hepatic inflammation, and affected gut microbial commensalism. NorUDCA could serve as a novel therapeutic agent in the future management of patients with ALD. Impact and implications: Alcohol-related liver disease is a global healthcare concern with limited treatment options. 24-Norursodeoxycholic acid (NorUDCA) is a modified bile acid, which was proven to be effective in human cholestatic liver diseases. In the present study, we found a protective effect of norUDCA in experimental alcoholic liver disease. For patients with ALD, norUDCA could be a potential new treatment option.

Host phylogeny and the environment play vital roles in shaping animal microbiomes. However, the effects of these variables on the diversity and richness of the gut microbiome in different bioclimatic zones remain underexplored. In this study, we investigated the effects of host phylogeny and bioclimatic zone on the diversity and composition of the gut microbiota of two heterospecific rodent species, the spiny mouse Acomys cahirinus and the house mouse Mus musculus, in three bioclimatic zones of the African Great Rift Valley (GRV). We confirmed host phylogeny using the D-loop sequencing method and analyzed the influence of host phylogeny and bioclimatic zone parameters on the rodent gut microbiome using high-throughput amplicon sequencing of 16S rRNA gene fragments. Phylogenetic analysis supported the morphological identification of the rodents and revealed a marked genetic difference between the two heterospecific species. We found that bioclimatic zone had a significant effect on the gut microbiota composition while host phylogeny did not. Microbial alpha diversity of heterospecific hosts was highest in the Mediterranean forest bioclimatic zone, followed by the Irano–Turanian shrubland, and was lowest in the Sudanian savanna tropical zone. The beta diversity of the two rodent species showed significant differences across the Mediterranean, Irano–Turanian, and Sudanian regions. The phyla Firmicutes and Bacteroidetes were highly abundant, and Deferribacterota, Cyanobacteria and Proteobacteria were also prominent. Amplicon sequence variants (ASVs) were identified that were unique to the Sudanian bioclimatic zone. The core microbiota families recovered in this study were consistent among heterospecific hosts. However, diversity decreased in conspecific host populations found at lower altitudes in Sudanian bioclimatic zone. The composition of the gut microbiota is linked to the adaptation of the host to its environment, and this study underscores the importance of incorporating climatic factors such as elevation and ambient temperature, in empirical microbiome research and is the first to describe the rodent gut microbiome from the GRV.

Chronic rhinosinusitis (CRS) is a chronic inflammatory disease phenotypically classified by the absence (CRSsNP) or presence of nasal polyps (CRSwNP). The latter may also be associated with asthma and hypersensitivity towards non-steroidal anti-inflammatory drugs (NSAID) as a triad termed NSAID-exacerbated respiratory disease (N-ERD). The role of the microbiome in these different disease entities with regard to the underlying inflammatory process and disease burden is yet not fully understood. To address this question, we measured clinical parameters and collected nasal samples (nasal mucosal fluids, microbiome swabs from middle meatus and anterior naris) of patients suffering from CRSsNP (n=20), CRSwNP (n=20) or N-ERD (n=20) as well as from patients without CRS (=disease controls, n=20). Importantly, all subjects refrained from taking local or systemic corticosteroids or immunosuppressants for at least two weeks prior to sampling. The nasal microbiome was analyzed using 16S rRNA gene amplicon sequencing, and levels of 33 inflammatory cytokines were determined in nasal mucosal fluids using the MSD platform. Patients suffering from N-ERD and CRSwNP showed significantly worse smell perception and significantly higher levels of type 2 associated cytokines IL-5, IL-9, Eotaxin and CCL17. Across all 4 patient groups, Corynebacteria and Staphylococci showed the highest relative abundances. Although no significant difference in alpha and beta diversity was observed between the control and the CRS groups, pairwise testing revealed a higher relative abundance of Staphylococci in the middle meatus in N-ERD patients as compared to CRSwNP (p

Abstract Background Dietary fiber is an integral part of a healthy diet, but questions remain about the mechanisms that underlie effects and the causal contributions of the gut microbiota. Here, we performed a 6-week exploratory trial in adults with excess weight (BMI: 25–35 kg/m2) to compare the effects of a high-dose (females: 25 g/day; males: 35 g/day) supplement of fermentable corn bran arabinoxylan (AX; n = 15) with that of microbiota-non-accessible microcrystalline cellulose (MCC; n = 16). Obesity-related surrogate endpoints and biomarkers of host-microbiome interactions implicated in the pathophysiology of obesity (trimethylamine N-oxide, gut hormones, cytokines, and measures of intestinal barrier integrity) were assessed. We then determined whether clinical outcomes could be predicted by fecal microbiota features or mechanistic biomarkers. Results AX enhanced satiety after a meal and decreased homeostatic model assessment of insulin resistance (HOMA-IR), while MCC reduced tumor necrosis factor-α and fecal calprotectin. Machine learning models determined that effects on satiety could be predicted by fecal bacterial taxa that utilized AX, as identified by bioorthogonal non-canonical amino acid tagging. Reductions in HOMA-IR and calprotectin were associated with shifts in fecal bile acids, but correlations were negative, suggesting that the benefits of fiber may not be mediated by their effects on bile acid pools. Biomarkers of host-microbiome interactions often linked to bacterial metabolites derived from fiber fermentation (short-chain fatty acids) were not affected by AX supplementation when compared to non-accessible MCC. Conclusion This study demonstrates the efficacy of purified dietary fibers when used as supplements and suggests that satietogenic effects of AX may be linked to bacterial taxa that ferment the fiber or utilize breakdown products. Other effects are likely microbiome independent. The findings provide a basis for fiber-type specific therapeutic applications and their personalization. Trial registration Clinicaltrials.gov, NCT02322112 , registered on July 3, 2015. Video Abstract

IntroductionGlycosaminoglycans (GAGs) present in the mucosal layer can be used as nutrients by certain intestinal bacteria, particularly members of the Bacteroides. GAG abundances are altered in some diseases such as inflammatory bowel diseases, which may affect microbial composition and activity, and it is therefore important to understand GAG utilization by members of the gut microbiota.MethodsWe used growth assays, transcriptomics, and comparative genomics to evaluate chondroitin sulfate (CS) and hyaluronan (HA) degradation ability by multiple gut Bacteroides species.Results and discussionWe found that not all Bacteroides species able to degrade CS could also degrade HA, despite having lyases which act on both compounds. We propose that in the model organism Bacteroides thetaiotaomicron, the lyase BT_3328 in combination with surface binding proteins BT_3329 and BT_3330 and potentially BT_4411 are involved in HA breakdown. Furthermore, degradation of both compounds provides public goods for other Bacteroides, including non-degraders, suggesting that cooperative degradation as well as cross-feeding may be widespread in the mucosal glycan utilization clade.

ABSTRACT The initial contact between humans and their colonizing gut microbiota after birth is thought to have expansive and long-lasting consequences for physiology and health. Premature infants are at high risk of suffering from lifelong impairments, due in part to aberrant development of gut microbiota that can contribute to early-life infections and inflammation. Despite their importance to health, the ecological assembly and succession processes governing gut microbiome composition in premature infants remained incompletely understood. Here, we quantified these ecological processes in a spatiotemporally resolved 16S rRNA gene amplicon sequencing data set of 60 extremely premature neonates using an established mathematical framework. We found that gut colonization during the first months of life is predominantly stochastic, whereby interindividual diversification of microbiota is driven by ecological drift. Dispersal limitations are initially small but have increasing influence at later stages of succession. Furthermore, we find similar trends in a cohort of 32 healthy term-born infants. These results suggest that the uniqueness of individual gut microbiota of extremely premature infants is largely due to stochastic assembly. IMPORTANCE Our knowledge concerning the initial gut microbiome assembly in human neonates is limited, and scientific progression in this interdisciplinary field is hindered due to the individuality in composition of gut microbiota. Our study addresses the ecological processes that result in the observed individuality of microbes in the gastrointestinal tract between extremely premature and term-born infants. We find that initial assembly is mainly driven by neutral ecological processes. Interestingly, while this progression is predominantly random, limitations to the dispersal of microbiota between infants become increasingly important with age and are concomitant features of gut microbiome stability. This indicates that while we cannot predict gut microbiota assembly due to its random nature, we can expect the establishment of certain ecological features that are highly relevant for neonatal health.

Rivers are the “tip of the iceberg”, with the underlying groundwater being the unseen freshwater majority. Microbial community composition and the dynamics of shallow groundwater ecosystems are thus crucial, due to their potential impact on ecosystem processes and functioning. In early summer and late autumn, samples of river water from 14 stations and groundwater from 45 wells were analyzed along a 300 km transect of the Mur River valley, from the Austrian alps to the flats at the Slovenian border. The active and total prokaryotic communities were characterized using high-throughput gene amplicon sequencing. Key physico-chemical parameters and stress indicators were recorded. The dataset was used to challenge ecological concepts and assembly processes in shallow aquifers. The groundwater microbiome is analyzed regarding its composition, change with land use, and difference to the river. Community composition and species turnover differed significantly. At high altitudes, dispersal limitation was the main driver of groundwater community assembly, whereas in the lowland, homogeneous selection explained the larger share. Land use was a key determinant of the groundwater microbiome composition. The alpine region was more diverse and richer in prokaryotic taxa, with some early diverging archaeal lineages being highly abundant. This dataset shows a longitudinal change in prokaryotic communities that is dependent on regional differences affected by geomorphology and land use.

Abstract Alterations in the human microbiome have been linked to several malignant diseases. Here, we investigated the oral microbiome of 79 patients with relapsed/refractory multiple myeloma (MM) treated with ixazomib‐thalidomide‐dexamethasone. Increased alpha diversity (Shannon index) at the phylum level was associated with longer progression‐free survival (PFS) (10.2 vs 8.5 months, P = .04), particularly in patients with very long (>75% quartile) PFS . Additionally, alpha diversity was lower in patients with progressive disease (P

Emerging evidence points to a major role of salivary flow and viscoelastic properties in taste perception and mouthfeel. It has been proposed that sweet-tasting compounds influence salivary characteristics. However, whether perceived differences in the sensory properties of structurally diverse sweet-tasting compounds contribute to salivary flow and saliva viscoelasticity as part of mouthfeel and overall sweet taste perception remains to be clarified. In this study, we hypothesized that the sensory diversity of sweeteners would differentially change salivary characteristics in response to oral sweet taste stimulation. Therefore, we investigated salivary flow and saliva viscoelasticity from 21 healthy test subjects after orosensory stimulation with sucrose, rebaudioside M (RebM), sucralose, and neohesperidin dihydrochalcone (NHDC) in a crossover design and considered the basal level of selected influencing factors, including the basal oral microbiome. All test compounds enhanced the salivary flow rate by up to 1.51 ± 0.12 g/min for RebM compared to 1.10 ± 0.09 g/min for water within the 1st min after stimulation. The increase in flow rate was moderately correlated with the individually perceived sweet taste (r = 0.3, p < 0.01) but did not differ between the test compounds. The complex viscosity of saliva was not affected by the test compounds, but the analysis of covariance showed that it was associated (p < 0.05) with mucin 5B (Muc5B) concentration. The oral microbiome was of typical composition and diversity but was strongly individual-dependent (permutational analysis of variance (PERMANOVA): R2 = 0.76, p < 0.001) and was not associated with changes in salivary characteristics. In conclusion, this study indicates an impact of individual sweet taste impressions on the flow rate without measurable changes in the complex viscosity of saliva, which may contribute to the overall taste perception and mouthfeel of sweet-tasting compounds.

The aquaculture industry is one of the fastest-growing sectors in animal food production. However, farming of carnivorous fish strongly relies on the use of wild fish-based meals, a practice that is environmentally and economically unsustainable. Insect-based diets constitute a strong candidate for fishmeal substitution, due to their high nutritional value and low environmental footprint. Nevertheless, data on the impact of insect meal (IM) on the gut microbiome of farmed fish are so far inconclusive, and very scarce in what concerns modulation of microbial-mediated functions. Here we use high-throughput 16S rRNA gene amplicon sequencing and quantitative PCR to evaluate the impact of different IMs on the composition and chitinolytic potential of the European sea bass gut digesta- and mucosa-associated communities. Our results show that insect-based diets of distinct origins differently impact the gut microbiota of the European sea bass (Dicentrarchus labrax). We detected clear modulatory effects of IM on the gut microbiota, which were more pronounced in the digesta, where communities differed considerably among the diets tested. Major community shifts were associated with the use of black soldier fly larvae (Hermetia illucens, HM) and pupal exuviae (HEM) feeds and were characterized by an increase in the relative abundance of the Firmicutes families Bacillaceae, Enterococcaceae, and Lachnospiraceae and the Actinobacteria family Actinomycetaceae, which all include taxa considered beneficial for fish health. Modulation of the digesta community by HEM was characterized by a sharp increase in Paenibacillus and a decrease of several Gammaproteobacteria and Bacteroidota members. In turn, a mealworm larvae-based diet (Tenebrio molitor, TM) had only a modest impact on microbiota composition. Further, using quantitative PCR, we demonstrate that shifts induced by HEM were accompanied by an increase in copy number of chitinase ChiA-encoding genes, predominantly originating from Paenibacillus species with effective chitinolytic activity. Our study reveals an HEM-driven increase in chitin-degrading taxa and associated chitinolytic activity, uncovering potential benefits of adopting exuviae-supplemented diets, a waste product of insect rearing, as a functional ingredient.

Understanding microbial community dynamics in the alpine cryosphere is an important step toward assessing climate change impacts on these fragile ecosystems and meltwater-fed environments downstream. In this study, we analyzed microbial community composition, variation in community alpha and beta diversity, and the number of prokaryotic cells and virus-like particles (VLP) in seasonal snowpack from two consecutive years at three high altitude mountain summits along a longitudinal transect across the European Alps. Numbers of prokaryotic cells and VLP both ranged around 104 and 105 per mL of snow meltwater on average, with variation generally within one order of magnitude between sites and years. VLP-to-prokaryotic cell ratios spanned two orders of magnitude, with median values close to 1, and little variation between sites and years in the majority of cases. Estimates of microbial community alpha diversity inferred from Hill numbers revealed low contributions of common and abundant microbial taxa to the total taxon richness, and thus low community evenness. Similar to prokaryotic cell and VLP numbers, differences in alpha diversity between years and sites were generally relatively modest. In contrast, community composition displayed strong variation between sites and especially between years. Analyses of taxonomic and phylogenetic community composition showed that differences between sites within years were mainly characterized by changes in abundances of microbial taxa from similar phylogenetic clades, whereas shifts between years were due to significant phylogenetic turnover. Our findings on the spatiotemporal dynamics and magnitude of variation of microbial abundances, community diversity, and composition in surface snow may help define baseline levels to assess future impacts of climate change on the alpine cryosphere.

The gut mucosal environment is key in host health; protecting against pathogens and providing a niche for beneficial bacteria, thereby facilitating a mutualistic balance between host and microbiome. Lack of dietary fiber results in erosion of the mucosal layer, suggested to be a result of increased mucus-degrading gut bacteria. This study aimed to use quantitative analyses to investigate the diet-induced imbalance of mucosal homeostasis. Seven days of fiber-deficiency affected intestinal anatomy and physiology, seen by reduced intestinal length and loss of the colonic crypt-structure. Moreover, the mucus layer was diminished, muc2 expression decreased, and impaired mucus secretion was detected by stable isotope probing. Quantitative microbiome profiling of the gut microbiota showed a diet-induced reduction in bacterial load and decreased diversity across the intestinal tract, including taxa with fiber-degrading and butyrate-producing capabilities. Most importantly, there was little change in the absolute abundance of known mucus-degrading bacteria, although, due to the general loss of taxa, relative abundance would erroneously indicate an increase in mucus degraders. These findings underscore the importance of using quantitative methods in microbiome research, suggesting erosion of the mucus layer during fiber deprivation is due to diminished mucus production rather than overgrowth of mucus degraders.

ABSTRACT High-affinity terminal oxidases (TOs) are believed to permit microbial respiration at low oxygen (O2) levels. Genes encoding such oxidases are widespread, and their existence in microbial genomes is taken as an indicator for microaerobic respiration. We combined respiratory kinetics determined via highly sensitive optical trace O2 sensors, genomics, and transcriptomics to test the hypothesis that high-affinity TOs are a prerequisite to respire micro- and nanooxic concentrations of O2 in environmentally relevant model soil organisms: acidobacteria. Members of the Acidobacteria harbor branched respiratory chains terminating in low-affinity (caa3-type cytochrome c oxidases) as well as high-affinity (cbb3-type cytochrome c oxidases and/or bd-type quinol oxidases) TOs, potentially enabling them to cope with varying O2 concentrations. The measured apparent Km (Km(app)) values for O2 of selected strains ranged from 37 to 288 nmol O2 liter−1, comparable to values previously assigned to low-affinity TOs. Surprisingly, we could not detect the expression of the conventional high-affinity TO (cbb3 type) at micro- and nanomolar O2 concentrations but detected the expression of low-affinity TOs. To the best of our knowledge, this is the first observation of microaerobic respiration imparted by low-affinity TOs at O2 concentrations as low as 1 nM. This challenges the standing hypothesis that a microaerobic lifestyle is exclusively imparted by the presence of high-affinity TOs. As low-affinity TOs are more efficient at generating ATP than high-affinity TOs, their utilization could provide a great benefit, even at low-nanomolar O2 levels. Our findings highlight energy conservation strategies that could promote the success of Acidobacteria in soil but might also be important for as-yet-unrevealed microorganisms. IMPORTANCE Low-oxygen habitats are widely distributed on Earth, ranging from the human intestine to soils. Microorganisms are assumed to have the capacity to respire low O2 concentrations via high-affinity terminal oxidases. By utilizing strains of a ubiquitous and abundant group of soil bacteria, the Acidobacteria, and combining respiration kinetics, genomics, and transcriptomics, we provide evidence that these microorganisms use the energetically more efficient low-affinity terminal oxidases to respire low-nanomolar O2 concentrations. This questions the standing hypothesis that the ability to respire traces of O2 stems solely from the activity of high-affinity terminal oxidases. We propose that this energetically efficient strategy extends into other, so-far-unrevealed microbial clades. Our findings also demonstrate that physiological predictions regarding the utilization of different O2 concentrations based solely on the presence or absence of terminal oxidases in bacterial genomes can be misleading.

Many phycological applications require the growth and maintenance of pure algae cultures. In some research areas, such as biochemistry and physiology, axenic growth is essential to avoid misinterpretations caused by contaminants. Nonetheless, axenicity—defined as the state of only a single strain being present, free of any other organism—needs to be verified. We compare the available methods to assess axenicity. We first purified unialgal Limnospira fusiformis cultures with an established series of axenicity treatments, and by including two additional treatment steps. The presumable axenic cultures were then tested for their axenic state by applying conventional tests on LB (lysogeny broth) agar-plates, 16S rRNA gene amplicon sequencing, flow-cytometry and epifluorescence microscopy. Only the plate tests indicated axenic conditions. We found a linear relationship between total cell counts of contaminants achieved by flow cytometry and epifluorescence microscopy, with flow cytometry counts being consistently higher. In addition, 16S rRNA gene amplicon sequencing demonstrated its superiority by not only being an efficient tool for axenicity testing, but also for identification of persistent contaminants. Although classic plate tests are still commonly used to verify axenicity, we found the LB-agar-plate technique to be inappropriate. Cultivation-independent methods are highly recommended to test for axenic conditions. A combination of flow-cytometry and 16S rRNA gene amplicon sequencing complement each other and will yield the most reliable result.

In microbiome research, phylogenetic and functional marker gene amplicon sequencing is the most commonly-used community profiling approach. Consequently, a plethora of protocols for the preparation and multiplexing of samples for amplicon sequencing have been developed. Here, we present two economical high-throughput gene amplification and sequencing workflows that are implemented as standard operating procedures at the Joint Microbiome Facility of the Medical University of Vienna and the University of Vienna. These workflows are based on a previously-published two-step PCR approach, but have been updated to either increase the accuracy of results, or alternatively to achieve orders of magnitude higher numbers of samples to be multiplexed in a single sequencing run. The high-accuracy workflow relies on unique dual sample barcoding. It allows the same level of sample multiplexing as the previously-published two-step PCR approach, but effectively eliminates residual read missasignments between samples (crosstalk) which are inherent to single barcoding approaches. The high-multiplexing workflow is based on combinatorial dual sample barcoding, which theoretically allows for multiplexing up to 299,756 amplicon libraries of the same target gene in a single massively-parallelized amplicon sequencing run. Both workflows presented here are highly economical, easy to implement, and can, without significant modifications or cost, be applied to any target gene of interest.

ABSTRACT Microbial diversity in the environment is mainly concealed within the rare biosphere (all species with