Oral, Vaginal, and Stool Microbial Signatures in Patients With Endometriosis as Potential Diagnostic Non-Invasive Biomarkers: A Prospective Cohort Study.

OBJECTIVE: To identify a microbial signature for endometriosis for use as a diagnostic non-invasive biomarker. DESIGN: Prospective cohort pilot study. SETTING: Nepean Hospital and UNSW Microbiome Research Centre, Australia. POPULATION: Sixty-four age- and sex-matched subjects (n = 19 healthy control (HC); n = 24 non-endometriosis (N-ENDO) and n = 21 confirmed endometriosis (ENDO)). All study participants, besides healthy controls, underwent laparoscopic surgical assessment for endometriosis, and histology was performed on excised lesions. METHODS: Oral, stool and, vaginal samples were self-collected at a single time point for healthy controls, and preoperatively for patients undergoing laparoscopy. Samples underwent 16S rRNA amplicon sequencing, followed by bioinformatics analysis. MAIN OUTCOME MEASURES: Compositional differences between cohorts as identified by diversity analyses, and differentially abundant microbial taxa, as identified by LEfSE analysis. RESULTS: The composition of the oral (adjusted p = 0.003), and stool (adjusted p = 0.042) microbiota is different between the three cohorts. Differentially abundant taxa are present within each cohort as identified by LEfSE analysis. Particularly, Fusobacterium was enriched in the oral samples of patients with moderate/severe endometriosis. CONCLUSIONS: Taxonomic and compositional differences were found between the microbiota in the mouth, gut and, vagina of patients with and without endometriosis and healthy controls. Fusobacterium was enriched in patients with moderate/severe endometriosis. Fusobacterium is noted as a key pathogen in periodontal disease, a common comorbidity in endometriosis. These findings suggest a role for the oral, stool and, vaginal microbiome in endometriosis, and present potential for microbial-based treatments and the design of a diagnostic swab.

Alignment-free inference of hierarchical and reticulate phylogenomic relationships.

We are amidst an ongoing flood of sequence data arising from the application of high-throughput technologies, and a concomitant fundamental revision in our understanding of how genomes evolve individually and within the biosphere. Workflows for phylogenomic inference must accommodate data that are not only much larger than before, but often more error prone and perhaps misassembled, or not assembled in the first place. Moreover, genomes of microbes, viruses and plasmids evolve not only by tree-like descent with modification but also by incorporating stretches of exogenous DNA. Thus, next-generation phylogenomics must address computational scalability while rethinking the nature of orthogroups, the alignment of multiple sequences and the inference and comparison of trees. New phylogenomic workflows have begun to take shape based on so-called alignment-free (AF) approaches. Here, we review the conceptual foundations of AF phylogenetics for the hierarchical (vertical) and reticulate (lateral) components of genome evolution, focusing on methods based on k-mers. We reflect on what seems to be successful, and on where further development is needed.

Assessing DNA for fish identifications from reference collections: the good, bad and ugly shed light on formalin fixation and sequencing approaches.

Natural history collections are repositories of biodiversity and are potentially used by molecular ecologists for comparative taxonomic, phylogenetic, biogeographic and forensic purposes. Specimens in fish collections are preserved using a combination of methods with many fixed in formalin and then preserved in ethanol for long-term storage. Formalin fixation damages DNA, thereby limiting genetic analyses. In this study, the authors compared the DNA barcoding and identification success for frozen and formalin-fixed tissues obtained from specimens in the CSIRO Australian National Fish Collection. They studied 230 samples from fishes (consisting of >160 fish species). An optimized formalin-fixed, paraffin-embedded DNA extraction method resulted in usable DNA from degraded tissues. Four mini barcoding assays of the mitochondrial DNA (mtDNA) were characterized with Sanger and Illumina amplicon sequencing. In the good quality DNA (without exposure to formalin), up to 88% of the specimens were correctly matched at the species level using the cytochrome oxidase subunit 1 (COI) mini barcodes, whereas up to 58% of the specimens exposed to formalin for less than 8 weeks were correctly identified to species. In contrast, 16S primers provided higher amplification success with formalin-exposed tissues, although the COI gene was more successful for identification. Importantly, the authors found that DNA of a certain size and quality can be amplified and sequenced despite exposure to formalin, and Illumina sequencing provided them with greater power of resolution for taxa identification even when there was little DNA present. Overall, within parameter constraints, this study highlights the possibilities of recovering DNA barcodes for identification from formalin-fixed fish specimens, and the authors provide guidelines for when successful identification could be expected.

Change in microbiota profile after vaginal estriol cream in postmenopausal women with stress incontinence.

Introduction: Vaginal estrogen is a treatment for genitourinary symptoms of menopause (GSM), which comprises vaginal atrophy and urinary dysfunction, including incontinence. Previous studies show that estrogen therapy promotes lactobacilli abundance and is associated with reduced GSM symptoms, including reduction of stress incontinence. However, detailed longitudinal studies that characterize how the microbiome changes in response to estrogen are scarce. We aimed to compare the vaginal microbiota of postmenopausal women, before and 12 weeks after vaginal estrogen cream. Methods: A total of 44 paired samples from 22 postmenopausal women with vaginal atrophy and stress incontinence were collected pre-vaginal estrogens and were compared to 12 weeks post-vaginal estrogen. Microbiota was characterized by 16S rRNA amplicon sequencing and biodiversity was investigated by comparing the alpha- and beta-diversity and potential markers were identified using differential abundance analysis. Results: Vaginal estrogen treatment was associated with a reduction in vaginal pH and corresponded with a significant reduction in alpha diversity of the microbiota. Healthy vaginal community state type was associated with lower mean pH 4.89 (SD = 0.6), in contrast to dysbiotic state which had a higher mean pH 6.4 (SD = 0.74). Women with lactobacilli dominant community pre-treatment, showed stable microbiota and minimal change in their pH. Women with lactobacilli deficient microbiome pre-treatment improved markedly (p = 0.004) with decrease in pH -1.31 and change to heathier community state types. Conclusion: In postmenopausal women with stress incontinence, vaginal estrogen promotes Lactobacillus and Bifidobacterium growth and lowers vaginal pH. Maximum response is seen in those with a dysbiotic vaginal microbiota pre-treatment.

Relationship between Diet Quality and Maternal Stool Microbiota in the MUMS Australian Pregnancy Cohort.

Dietary intake during pregnancy may influence the antenatal microbiome, which is proposed to impact maternal and infant health during the pregnancy and beyond. The aim of this sub-study was to examine associations between dietary intake and microbiota diversity during pregnancy using whole metagenomic sequencing and examine associations in low-risk versus high-risk pregnancies, as well as complicated versus uncomplicated pregnancies. Pregnancy data were analysed from women participating in the MUMS cohort study in Sydney, Australia (women followed from trimester 1 of pregnancy to 1-year postpartum), who had dietary intake data at either trimester 1 or 3, assessed using the Australian Eating Survey, and a matched stool sample (n = 86). Correlations of microbial alpha diversity with dietary intake data were determined using the repeated-measures correlation, rmcorr, in R. In the combined cohort, no associations were found between diet quality or diet composition and microbial alpha diversity or beta diversity. However, trends in our analysis suggested that dietary intake of specific macro- and micronutrients may influence microbial diversity differently, depending on particular pregnancy conditions. Our findings suggest that dietary intake during pregnancy may have a variable influence on the maternal microbiota, unique to the individual maternal pregnancy phenotype. More research is needed to disentangle these associations.

The MothersBabies Study, an Australian Prospective Cohort Study Analyzing the Microbiome in the Preconception and Perinatal Period to Determine Risk of Adverse Pregnancy, Postpartum, and Child-Related Health Outcomes: Study Protocol.

The microbiome has emerged as a key determinant of human health and reproduction, with recent evidence suggesting a dysbiotic microbiome is implicated in adverse perinatal health outcomes. The existing research has been limited by the sample collection and timing, cohort design, sample design, and lack of data on the preconception microbiome. This prospective, longitudinal cohort study will recruit 2000 Australian women, in order to fully explore the role of the microbiome in the development of adverse perinatal outcomes. Participants are enrolled for a maximum of 7 years, from 1 year preconception, through to 5 years postpartum. Assessment occurs every three months until pregnancy occurs, then during Trimester 1 (5 + 0-12 + 6 weeks gestation), Trimester 2 (20 + 0-24 + 6 weeks gestation), Trimester 3 (32 + 0-36 + 6 weeks gestation), and postpartum at 1 week, 2 months, 6 months, and then annually from 1 to 5 years. At each assessment, maternal participants self-collect oral, skin, vaginal, urine, and stool samples. Oral, skin, urine, and stool samples will be collected from children. Blood samples will be obtained from maternal participants who can access a study collection center. The measurements taken will include anthropometric, blood pressure, heart rate, and serum hormonal and metabolic parameters. Validated self-report questionnaires will be administered to assess diet, physical activity, mental health, and child developmental milestones. Medications, medical, surgical, obstetric history, the impact of COVID-19, living environments, and pregnancy and child health outcomes will be recorded. Multiomic bioinformatic and statistical analyses will assess the association between participants who developed high-risk and low-risk pregnancies, adverse postnatal conditions, and/or childhood disease, and their microbiome for the different sample types.

Effects of sequential inoculation of Lachancea thermotolerans and Oenococcus oeni on chemical composition of spent coffee grounds hydrolysates.

Spent coffee grounds (SCG) disposal is an environmental problem. These residues from coffee brewing and instant coffee production have potential to produce novel alcoholic beverages. SCG valorization through sequential alcoholic and malolactic fermentation was investigated using a yeast, Lachancea thermotolerans Concerto and a lactic acid bacterium (LAB), Oenococcus oeni Lalvin 31 in this study. Our results showed that sequential inoculation prevented early death of yeast confronted when simultaneous inoculation was adopted, allowing for growth and persistence of both yeast and LAB till the end of fermentation. Adequate ethanol production (4.91 ± 0.13 %, v/v) with low residual sugar content was also attained. In addition, relatively lower levels of acetic, lactic, and succinic acids were produced by sequential inoculation than that of simultaneous inoculation. Furthermore, SCG hydrolysates fermented via sequential inoculation had the widest variety of volatiles (e.g. esters and ketones). Overall, our results indicated that sequential inoculation of L. thermotolerans and O. oeni in SCG hydrolysates might be a way to develop novel beverages with pleasant flavor profiles.

The Role of the Microbiome in the Metabolic Health of People with Schizophrenia and Related Psychoses: Cross-Sectional and Pre-Post Lifestyle Intervention Analyses.

The microbiome has been implicated in the development of metabolic conditions which occur at high rates in people with schizophrenia and related psychoses. This exploratory proof-of-concept study aimed to: (i) characterize the gut microbiota in antipsychotic naïve or quasi-naïve people with first-episode psychosis, and people with established schizophrenia receiving clozapine therapy; (ii) test for microbiome changes following a lifestyle intervention which included diet and exercise education and physical activity. Participants were recruited from the Eastern Suburbs Mental Health Service, Sydney, Australia. Anthropometric, lifestyle and gut microbiota data were collected at baseline and following a 12-week lifestyle intervention. Stool samples underwent 16S rRNA sequencing to analyse microbiota diversity and composition. Seventeen people with established schizophrenia and five people with first-episode psychosis were recruited and matched with 22 age-sex, BMI and ethnicity matched controls from a concurrent study for baseline comparisons. There was no difference in α-diversity between groups at baseline, but microbial composition differed by 21 taxa between the established schizophrenia group and controls. In people with established illness pre-post comparison of α-diversity showed significant increases after the 12-week lifestyle intervention. This pilot study adds to the current literature that detail compositional differences in the gut microbiota of people with schizophrenia compared to those without mental illness and suggests that lifestyle interventions may increase gut microbial diversity in patients with established illness. These results show that microbiome studies are feasible in patients with established schizophrenia and larger studies are warranted to validate microbial signatures and understand the relevance of lifestyle change in the development of metabolic conditions in this population.

Counts: an outstanding challenge for log-ratio analysis of compositional data in the molecular biosciences.

Thanks to sequencing technology, modern molecular bioscience datasets are often compositions of counts, e.g. counts of amplicons, mRNAs, etc. While there is growing appreciation that compositional data need special analysis and interpretation, less well understood is the discrete nature of these count compositions (or, as we call them, lattice compositions) and the impact this has on statistical analysis, particularly log-ratio analysis (LRA) of pairwise association. While LRA methods are scale-invariant, count compositional data are not; consequently, the conclusions we draw from LRA of lattice compositions depend on the scale of counts involved. We know that additive variation affects the relative abundance of small counts more than large counts; here we show that additive (quantization) variation comes from the discrete nature of count data itself, as well as (biological) variation in the system under study and (technical) variation from measurement and analysis processes. Variation due to quantization is inevitable, but its impact on conclusions depends on the underlying scale and distribution of counts. We illustrate the different distributions of real molecular bioscience data from different experimental settings to show why it is vital to understand the distributional characteristics of count data before applying and drawing conclusions from compositional data analysis methods.

MetaDEGalaxy: Galaxy workflow for differential abundance analysis of 16s metagenomic data.

Metagenomic sequencing is an increasingly common tool in environmental and biomedical sciences yet analysis workflows remain immature relative to other field such as DNASeq and RNASeq analysis pipelines.  While software for detailing the composition of microbial communities using 16S rRNA marker genes is constantly improving, increasingly researchers are interested in identifying changes exhibited within microbial communities under differing environmental conditions. In order to gain maximum value from metagenomic sequence data we must improve the existing analysis environment by providing accessible and scalable computational workflows able to generate reproducible results. Here we describe a complete end-to-end open-source metagenomics workflow running within Galaxy for 16S differential abundance analysis. The workflow accepts 454 or Illumina sequence data (either overlapping or non-overlapping paired end reads) and outputs lists of the operational taxonomic unit (OTUs) exhibiting the greatest change under differing conditions. A range of analysis steps and graphing options are available giving users a high-level of control over their data and analyses. Additionally, users are able to input complex sample-specific metadata information which can be incorporated into differential analysis and used for grouping / colouring within graphs.  Detailed tutorials containing sample data and existing workflows are available for three different input types: overlapping and non-overlapping read pairs as well as for pre-generated Biological Observation Matrix (BIOM) files. Using the Galaxy platform we developed MetaDEGalaxy, a complete metagenomics differential abundance analysis workflow. MetaDEGalaxy is designed for bench scientists working with 16S data who are interested in comparative metagenomics.  MetaDEGalaxy builds on momentum within the wider Galaxy metagenomics community with the hope that more tools will be added as existing methods mature.

MixMC: A Multivariate Statistical Framework to Gain Insight into Microbial Communities.

Culture independent techniques, such as shotgun metagenomics and 16S rRNA amplicon sequencing have dramatically changed the way we can examine microbial communities. Recently, changes in microbial community structure and dynamics have been associated with a growing list of human diseases. The identification and comparison of bacteria driving those changes requires the development of sound statistical tools, especially if microbial biomarkers are to be used in a clinical setting. We present mixMC, a novel multivariate data analysis framework for metagenomic biomarker discovery. mixMC accounts for the compositional nature of 16S data and enables detection of subtle differences when high inter-subject variability is present due to microbial sampling performed repeatedly on the same subjects, but in multiple habitats. Through data dimension reduction the multivariate methods provide insightful graphical visualisations to characterise each type of environment in a detailed manner. We applied mixMC to 16S microbiome studies focusing on multiple body sites in healthy individuals, compared our results with existing statistical tools and illustrated added value of using multivariate methodologies to fully characterise and compare microbial communities.