sccomp: Robust differential composition and variability analysis for single-cell data.

Cellular omics such as single-cell genomics, proteomics, and microbiomics allow the characterization of tissue and microbial community composition, which can be compared between conditions to identify biological drivers. This strategy has been critical to revealing markers of disease progression, such as cancer and pathogen infection. A dedicated statistical method for differential variability analysis is lacking for cellular omics data, and existing methods for differential composition analysis do not model some compositional data properties, suggesting there is room to improve model performance. Here, we introduce sccomp, a method for differential composition and variability analyses that jointly models data count distribution, compositionality, group-specific variability, and proportion mean-variability association, being aware of outliers. sccomp provides a comprehensive analysis framework that offers realistic data simulation and cross-study knowledge transfer. Here, we demonstrate that mean-variability association is ubiquitous across technologies, highlighting the inadequacy of the very popular Dirichlet-multinomial distribution. We show that sccomp accurately fits experimental data, significantly improving performance over state-of-the-art algorithms. Using sccomp, we identified differential constraints and composition in the microenvironment of primary breast cancer.

Women with type 1 diabetes exhibit a progressive increase in gut Saccharomyces cerevisiae in pregnancy associated with evidence of gut inflammation.

AIMS: Studies of the gut microbiome have focused on its bacterial composition. We aimed to characterize the gut fungal microbiome (mycobiome) across pregnancy in women with and without type 1 diabetes. METHODS: Faecal samples (n = 162) were collected from 70 pregnant women (45 with and 25 without type 1 diabetes) across all trimesters. Fungi were analysed by internal transcribed spacer 1 amplicon sequencing. Markers of intestinal inflammation (faecal calprotectin) and intestinal epithelial integrity (serum intestinal fatty acid binding protein; I-FABP), and serum antibodies to Saccharomyces cerevisiae (ASCA) were measured. RESULTS: Women with type 1 diabetes had decreased fungal alpha diversity by the third trimester, associated with an increased abundance of Saccharomyces cerevisiae that was inversely related to the abundance of the anti-inflammatory butyrate-producing bacterium Faecalibacterium prausnitzii. Women with type 1 diabetes had higher concentrations of calprotectin, I-FABP and ASCA. CONCLUSIONS: Women with type 1 diabetes exhibit a shift in the gut mycobiome across pregnancy associated with evidence of gut inflammation and impaired intestinal barrier function. The relevance of these findings to the higher rate of pregnancy complications in type 1 diabetes warrants further study.

Type 1 diabetes in pregnancy is associated with distinct changes in the composition and function of the gut microbiome.

BACKGROUND: The gut microbiome changes in response to a range of environmental conditions, life events and disease states. Pregnancy is a natural life event that involves major physiological adaptation yet studies of the microbiome in pregnancy are limited and their findings inconsistent. Pregnancy with type 1 diabetes (T1D) is associated with increased maternal and fetal risks but the gut microbiome in this context has not been characterized. By whole metagenome sequencing (WMS), we defined the taxonomic composition and function of the gut bacterial microbiome across 70 pregnancies, 36 in women with T1D. RESULTS: Women with and without T1D exhibited compositional and functional changes in the gut microbiome across pregnancy. Profiles in women with T1D were distinct, with an increase in bacteria that produce lipopolysaccharides and a decrease in those that produce short-chain fatty acids, especially in the third trimester. In addition, women with T1D had elevated concentrations of fecal calprotectin, a marker of intestinal inflammation, and serum intestinal fatty acid-binding protein (I-FABP), a marker of intestinal epithelial damage. CONCLUSIONS: Women with T1D exhibit a shift towards a more pro-inflammatory gut microbiome during pregnancy, associated with evidence of intestinal inflammation. These changes could contribute to the increased risk of pregnancy complications in women with T1D and are potentially modifiable by dietary means. Video abstract.

Hierarchical modelling of immunoglobulin coated bacteria in dogs with chronic enteropathy shows reduction in coating with disease remission but marked inter-individual and treatment-response variability.

Chronic enteropathies are a common problem in dogs, but many aspects of the pathogenesis remain unknown, making the therapeutic approach challenging in some cases. Environmental factors are intimately related to the development and perpetuation of gastrointestinal disease and the gut microbiome has been identified as a contributing factor. Previous studies have identified dysbiosis and reduced bacterial diversity in the gastrointestinal microbiota of dogs with chronic enteropathies. In this case-controlled study, we use flow cytometry and 16S rRNA sequencing to characterise bacteria highly coated with IgA or IgG in faecal samples from dogs with chronic enteropathy and evaluated their correlation with disease and resolution of the clinical signs. IgA and IgG-coated faecal bacterial counts were significantly higher during active disease compared to healthy dogs and decreased with the resolution of the clinical signs. Characterisation of taxa-specific coating of the intestinal microbiota with IgA and IgG showed marked variation between dogs and disease states, and different patterns of immunoglobulin enrichment were observed in dogs with chronic enteropathy, particularly for Erysipelotrichaceae, Clostridicaceae, Enterobacteriaceae, Prevotellaceae and Bacteroidaceae, families. Although, members of these bacterial groups have been associated with strong immunogenic properties and could potentially constitute important biomarkers of disease, their significance and role need to be further investigated.

Field evaluation of the gut microbiome composition of pre-school and school-aged children in Tha Song Yang, Thailand, following oral MDA for STH infections.

Soil-transmitted helminths, such as roundworms (Ascaris lumbricoides), whipworms (Trichuris trichiura) and hookworms (Necator americanus and Ancylostoma spp.), are gastrointestinal parasites that occur predominantly in low- to middle-income countries worldwide and disproportionally impact children. Depending on the STH species, health status of the host and infection intensity, direct impacts of these parasites include malnutrition, anaemia, diarrhoea and physical and cognitive stunting. The indirect consequences of these infections are less well understood. Specifically, gastrointestinal infections may exert acute or chronic impacts on the natural gut microfauna, leading to increased risk of post-infectious gastrointestinal disorders, and reduced gut and overall health through immunomodulating mechanisms. To date a small number of preliminary studies have assessed the impact of helminths on the gut microbiome, but these studies are conflicting. Here, we assessed STH burden in 273 pre-school and school-aged children in Tha Song Yang district, Tak province, Thailand receiving annual oral mebendazole treatment. Ascaris lumbricoides (107/273) and Trichuris trichiura (100/273) were the most prevalent species and often occurred as co-infections (66/273). Ancylostoma ceylanicum was detected in a small number of children as well (n = 3). All of these infections were of low intensity (<4,999 or 999 eggs per gram for Ascaris and Trichuris respectively). Using this information, we characterised the baseline gut microbiome profile and investigated acute STH-induced alterations, comparing infected with uninfected children at the time of sampling. We found no difference between these groups in bacterial alpha-diversity, but did observe differences in beta-diversity and specific differentially abundant OTUs, including increased Akkermansia muciniphila and Bacteroides coprophilus, and reduced Bifidobacterium adolescentis, each of which have been previously implicated in STH-associated changes in the gut microfauna.

Expanding the taxonomic range in the fecal metagenome.

BACKGROUND: Except for bacteria, the taxonomic diversity of the human fecal metagenome has not been widely studied, despite the potential importance of viruses and eukaryotes. Widely used bioinformatic tools contain limited numbers of non-bacterial species in their databases compared to available genomic sequences and their methodologies do not favour classification of rare sequences which may represent only a small fraction of their parent genome. In seeking to optimise identification of non-bacterial species, we evaluated five widely-used metagenome classifier programs (BURST, Kraken2, Centrifuge, MetaPhlAn2 and CCMetagen) for their ability to correctly assign and count simulations of bacterial, viral and eukaryotic DNA sequence reads, including the effect of taxonomic order of analysis of bacteria, viruses and eukaryotes and the effect of sequencing depth. RESULTS: We found that the precision of metagenome classifiers varied significantly between programs and between taxonomic groups. When classifying viruses and eukaryotes, ordering the analysis such that bacteria were classified first significantly improved classification precision. Increasing sequencing depth decreased classification precision and did not improve recall of rare species. CONCLUSIONS: Choice of metagenome classifier program can have a marked effect on results with respect to precision of species assignment in different taxonomic groups. The order of taxonomic classification can markedly improve precision. Increasing sequencing depth can decrease classification precision and yields diminishing returns in probability of species detection.

Effective low-cost preservation of human stools in field-based studies for helminth and microbiota analysis.

Molecular studies of gastrointestinal infections or microbiotas require either rapid sample processing or effective interim preservation. This is difficult in remote settings in low-income countries, where the majority of the global infectious disease burden exists. Processing or freezing of samples immediately upon collection is often not feasible and the cost of commercial preservatives is prohibitive. We compared fresh freezing (the 'gold standard' method), with low-cost chemical preservation in (i) a salt-based buffer consisting of DMSO, EDTA and NaCl (DESS) or (ii) 2.5% potassium dichromate (PD), for soil-transmitted helminth detection and microbiota characterisation in pre-school and school-aged children from north-western Thailand. Fresh frozen samples were frozen at -20°C on collection and maintained at -80°C within ~3 days of collection until molecular analysis, with international shipping on dry ice. In contrast, chemically preserved samples were collected and stored at ~4°C, transported on wet ice and only stored at -20°C on arrival in Australia ~8 weeks after collection, with international shipping on wet ice. DESS and PD provided better sensitivity for STH diagnosis, estimating higher infection rates (>80% for Ascaris lumbricoides and >60% for Trichuris trichiura; versus 56% and 15% for these parasites in fresh frozen samples) and egg abundance (inferred as gene copy number estimates). All methods performed similarly for microbiota preservation, showing no significant differences in alpha-diversity based on overall richness or inverted Simpson's Index. All three methods performed similarly for RNA and protein preservation in a small subset of samples. Overall, DESS provided the best performance, with the added benefit of being non-toxic, compared with PD, hence making it particularly applicable for studies in remote and resource-poor settings.

Associations between diet, the gut microbiome and short chain fatty acids in youth with islet autoimmunity and type 1 diabetes.

AIM: We aimed to characterize associations between diet and the gut microbiome and short chain fatty acid (SCFA) products in youth with islet autoimmunity or type 1 diabetes (IA/T1D) in comparison with controls. RESEARCH DESIGN AND METHODS: Eighty participants (25 diagnosed with T1D, 17 with confirmed IA, 38 sibling or unrelated controls) from the Australian T1D Gut Study cohort were studied (median [IQR] age 11.7 [8.9, 14.0] years, 43% female). A Food Frequency Questionnaire characterized daily macronutrient intake over the preceding 6 months. Plasma and fecal SCFA were measured by gas chromatography; gut microbiome composition and diversity by 16S rRNA gene sequencing. RESULTS: A 10 g increase in daily carbohydrate intake associated with higher plasma acetate in IA/T1D (adjusted estimate +5.2 (95% CI 1.1, 9.2) µmol/L p = 0.01) and controls (adjusted estimate +4.1 [95% CI 1.7, 8.5] µmol/L p = 0.04). A 5 g increase in total fat intake associated with lower plasma acetate in IA/T1D and controls. A 5% increase in noncore (junk) food intake associated with reduced richness (adjusted estimate -4.09 [95%CI -7.83, -0.35] p = .03) and evenness (-1.25 [95% CI -2.00, -0.49] p < 0.01) of the gut microbiome in IA/T1D. Fiber intake associated with community structure of the microbiome in IA/T1D. CONCLUSIONS: Modest increments in carbohydrate and fat intake associated with plasma acetate in all youth. Increased junk food intake associated with reduced diversity of the gut microbiome in IA/T1D alone. These associations with the gut microbiome in IA/T1D support future efforts to promote SCFA by using dietary interventions.

Gut microbiome dysbiosis and increased intestinal permeability in children with islet autoimmunity and type 1 diabetes: A prospective cohort study.

AIMS/HYPOTHESIS: To investigate the longitudinal relationship between the gut microbiome, circulating short chain fatty acids (SCFAs) and intestinal permeability in children with islet autoimmunity or type 1 diabetes and controls. METHODS: We analyzed the gut bacterial microbiome, plasma SCFAs, small intestinal permeability and dietary intake in 47 children with islet autoimmunity or recent-onset type 1 diabetes and in 41 unrelated or sibling controls over a median (range) of 13 (2-34) months follow-up. RESULTS: Children with multiple islet autoantibodies (≥2 IA) or type 1 diabetes had gut microbiome dysbiosis. Anti-inflammatory Prevotella and Butyricimonas genera were less abundant and these changes were not explained by differences in diet. Small intestinal permeability measured by blood lactulose:rhamnose ratio was higher in type 1 diabetes. Children with ≥2 IA who progressed to type 1 diabetes (progressors), compared to those who did not progress, had higher intestinal permeability (mean [SE] difference +5.14 [2.0], 95% confidence interval [CI] 1.21, 9.07, P = .006), lower within-sample (alpha) microbial diversity (31.3 [11.2], 95% CI 9.3, 53.3, P = .005), and lower abundance of SCFA-producing bacteria. Alpha diversity (observed richness) correlated with plasma acetate levels in all groups combined (regression coefficient [SE] 0.57 [0.21], 95% CI 0.15, 0.99 P = .008). CONCLUSIONS/INTERPRETATION: Children with ≥2 IA who progress to diabetes, like those with recent-onset diabetes, have gut microbiome dysbiosis associated with increased intestinal permeability. Interventions that expand gut microbial diversity, in particular SCFA-producing bacteria, may have a role to decrease progression to diabetes in children at-risk.

Influence of fecal collection conditions and 16S rRNA gene sequencing at two centers on human gut microbiota analysis.

To optimise fecal sampling for reproducible analysis of the gut microbiome, we compared different methods of sample collection and sequencing of 16S rRNA genes at two centers. Samples collected from six individuals on three consecutive days were placed in commercial collection tubes (OMNIgeneGut OMR-200) or in sterile screw-top tubes in a home fridge or home freezer for 6-24 h, before transfer and storage at -80 °C. Replicate samples were shipped to centers in Australia and the USA for DNA extraction and sequencing by their respective PCR protocols, and analysed with the same bioinformatic pipeline. Variation in gut microbiome was dominated by differences between individuals. Minor differences in the abundance of taxa were found between collection-processing methods and day of collection, and between the two centers. We conclude that collection with storage and transport at 4 °C within 24 h is adequate for 16S rRNA analysis of the gut microbiome. Other factors including differences in PCR and sequencing methods account for relatively minor variation compared to differences between individuals.

Functional biogeography and host specificity of bacterial communities associated with the Marine Green Alga Ulva spp.

Bacterial communities play an essential role for the function of marine macroalgae. Recent work has shown that bacterial communities associated with individual macroalgae possess on a local scale a functional core that is likely derived from diverse members of functional guilds. It is not known whether such functional cores also exist across large spatial scales or between closely related host species. To address this, we studied here the bacterial communities on three species of the green macroalgal genus Ulva from different geographic locations. While the taxonomic composition was too variable to describe a community core, we identified genes that were enriched across all Ulva samples as compared to the communities of the surrounding seawater. Of these core functions, 70% were consistently found and independent of the Ulva species and biogeography, while the remaining functions (~30%) are possibly involved in local or host-specific adaptations. For each host individual, the core functions are provided by bacteria with distinct phylogenetic origin and these bacteria could constitute a global guild of Ulva-associated bacteria. Together, our results demonstrate the presence of a stable core set of functional genes in the bacterial communities associated with closely related host species and across large biogeographies.

Microbial community function in the bleaching disease of the marine macroalgae Delisea pulchra.

Disease is increasingly viewed as a major factor impacting the health of both natural and cultured populations of marine organisms, including macroalgae. The red macroalga Delisea pulchra suffers from a bleaching disease resulting from host stress and infection by opportunistic bacterial pathogens. However, how pathogens cause the disease and how the entire macro algal-associated community is involved in the process is unclear. Here, we perform a metagenomic analysis of microbial communities associated with diseased and healthy D. pulchra across multiple bleaching events. Analysis of reconstructed 16S rRNA gene sequences showed that bacteria belonging to the families Rhodobacteraceae, Saprospiraceae and Flavobacteriaceae, including bacteria previously implicated in algal bleaching, to be enriched in diseased D. pulchra. Genes with predicted functions related to chemotaxis, motility, oxidative stress response, vitamin biosynthesis and nutrient acquisition were also prevalent in microbiomes of bleached algae, which may have a role in pathogenicity. Reconstruction of genomes that were abundant on bleached samples revealed that no single organism contains all bleaching-enriched functional genes. This observation indicates that potential virulence traits are distributed across multiple bacteria and that the disease in D. pulchra may result from a consortium of opportunistic pathogens, analogous to dysbiotic or polymicrobial diseases.

Partitioning of functional and taxonomic diversity in surface-associated microbial communities.

Surfaces, including those submerged in the marine environment, are subjected to constant interactions and colonisation by surrounding microorganisms. The principles that determine the assembly of those epibiotic communities are however poorly understood. In this study, we employed a hierarchical design to assess the functionality and diversity of microbial communities on different types of host surfaces (e.g. macroalgae, seagrasses). We found that taxonomic diversity was unique to each type of host, but that the majority of functions (> 95%) could be found in any given surface community, suggesting a high degree of functional redundancy. However, some community functions were enriched on certain surfaces and were related to host-specific properties (e.g. the degradation of specific polysaccharides). Together these observations support a model, whereby communities on surfaces are assembled from guilds of microorganisms with a functionality that is partitioned into general properties for a surface-associated life-style, but also specific features that mediate host-specificity.

Effects of Temperature Stress and Aquarium Conditions on the Red Macroalga Delisea pulchra and its Associated Microbial Community.

In recent years, there has been an increase in the rate and severity of diseases affecting habitat-forming marine organisms, such as corals, sponges, and macroalgae. Delisea pulchra is a temperate red macroalga that suffers from a bleaching disease that is more frequent during summer, when seawater temperatures are elevated and the alga's chemical defense is weakened. A bacterial cause for the disease is implied by previous studies showing that some isolated strains can cause bleaching in vitro and that host-associated microbial communities are distinct between diseased and healthy individuals. However, nothing is known about the successional events in the microbial community that occur during the development of the disease. To study this aspect in the future, we aimed here to develop an experimental setup to study the bleaching disease in a controllable aquarium environment. Application of a temperature stress (up to 27°C) did not cause a clear and consistent pattern of bleaching, suggesting that temperature alone might not be the only or main factor to cause the disease. The results also showed that the aquarium conditions alone are sufficient to produce bleaching symptoms. Microbial community analysis based on 16S rRNA gene fingerprinting and sequencing showed significant changes after 15 days in the aquarium, indicating that the native microbial associates of D. pulchra are not stably maintained. Microbial taxa that were enriched in the aquarium-held D. pulchra thalli, however, did not match on a taxonomic level those that have been found to be enriched in natural bleaching events. Together our observations indicate that environmental factors, other than the ones investigated here, might drive the bleaching disease in D. pulchra and that the aquarium conditions have substantial impact on the alga-associated microbiome.