Changes In Alpha Diversity Research Articles

Roux-en-Y gastric bypass and sleeve gastrectomy induce substantial and persistent changes in microbial communities and metabolic pathways

ABSTRACT Bariatric surgery induces significant microbial and metabolomic changes, however, links between microbial and metabolic pathways have not been fully elucidated. The objective of this study was to conduct a comprehensive investigation of the microbial, metabolomic, and inflammatory changes that occur following Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG). A prospective clinical trial was conducted with participants undergoing RYGB, SG, and non-operative controls (CTRL). Clinical parameters, blood samples, and fecal samples were collected pre-intervention and at 3 and 9 months. A multi-omics approach was used to perform integrated microbial-metabolomic analysis to identify functional pathways in which weight loss and metabolic changes occur after surgery. RYGB led to profound microbial changes over time that included reductions in alpha-diversity, increased Proteobacteria and Verrucomicrobiota, decreased Firmicutes, and numerous changes at the genera level. These changes were associated with a reduction in inflammation and significant weight loss. A reduction in Romboutsia genera correlated strongly with weight loss and integrated microbial-metabolomic analysis revealed the importance of Romboutsia. Its obliteration correlated with improved weight loss and insulin resistance, possibly through decreases in glycerophospholipids. In contrast, SG was associated with no changes in alpha-diversity, and only a small number of changes in microbial genera. A cluster of Firmicutes genera including Butyriciccocus, Eubacterium ventriosum, and Monoglobus was decreased, which correlated with decreased weight, insulin resistance, and systemic inflammation. This work represents comprehensive analyses of microbial-metabolomic changes that occur following bariatric surgery and identifies several pathways that are associated with beneficial metabolic effects of surgery.

Biannual Administrations of Azithromycin and the Gastrointestinal Microbiome of Malawian Children: A Nested Cohort Study Within a Randomized Controlled Trial.

Community-level mass treatment with azithromycin has been associated with a mortality benefit in children. However, antibiotic exposures result in disruption of the gut microbiota and repeated exposures may reduce recovery of the gut flora. We conducted a nested cohort study within the framework of a randomized controlled trial to examine associations between mass drug administration (MDA) with azithromycin and the gut microbiota of rural Malawian children aged between 1 and 59 months. Fecal samples were collected from the children at baseline and 6 months after two or four biannual rounds of azithromycin treatment. DNA was extracted from fecal samples and V4-16S rRNA sequencing used to characterize the gut microbiota. Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria were the dominant phyla while Faecalibacterium and Bifidobacterium were the most prevalent genera. There were no associations between azithromycin treatment and changes in alpha diversity, however, four biannual rounds of treatment were associated with increased abundance of Prevotella. The lack of significant changes in gut microbiota after four biannual treatments supports the use of mass azithromycin treatment to reduce mortality in children living in low- and middle-income settings.

Dynamic succession patterns and interactions of phyllospheric microorganisms during NOx exposure

The phyllosphere plays a role in alleviating air pollution, potentially leveraging the native microorganisms for further enhancement. It remains unclear how phyllospheric microorganisms respond to nitrogen oxide (NOx) pollution and participate in abatement. Here, we exposed Schefflera octophylla to NOx to reveal microbial succession patterns and interactions in the phyllosphere. During exposure, phyllospheric ammonium (NH4+-N) significantly increased, with different alpha diversity changes between bacteria and fungi. Community successions enclosed core taxa with relatively excellent tolerance, represented by bacterial genera (Norcardiodes, Aeromicrobium) and fungal genera (Talaromyces, Acremonium). The exposure eliminated specific pathogens (e.g., Zymoseptoria) and benefitted plant growth-promoting populations (e.g., Talaromyces, Exiguobacterium), which might favor plant disease control, improve plant health and thus buffer NOx pollution. Cooccurrence networks revealed more negative correlations among bacteria and closer linkages among fungi during exposure. Our results also showed a functional shift from the predominance of pathotrophs to saprotrophs. Our study identified microbial successions and interactions during NOx pollution and thus enlightened prospective taxa and potential roles of phyllospheric microorganisms in NOx remediation.

Dynamic Upper and Lower Airway Microbiotas in Paediatric Bronchiectasis Exacerbations: A Pilot Study.

IntroductionNon-cystic fibrosis bronchiectasis is a respiratory health condition with many possible aetiologies, some of which are potentially reversible in childhood with early diagnosis and appropriate treatment. It is important to understand factors which contribute to progression or potential resolution of bronchiectasis. It is evident that respiratory exacerbations are a key feature of bronchiectasis disease progression. In this pilot study we document how the microbiota of the upper and lower airways presents during the course of an exacerbation and treatment.MethodsWe recruited children (aged 1-15) undergoing antibiotic treatment for bronchiectasis exacerbations at Starship Children’s Hospital and outpatient clinics. Sputum and nasal swabs were taken before and after antibiotic treatment. Sample DNA was extracted, then bacterial 16S rRNA genes amplified and sequenced via Illumina MiSeq.ResultsThirty patients were recruited into this study with 81 samples contributing to the final dataset, including 8 patients with complete sets of upper and lower airway samples at both (before and after antibiotics) timepoints. Changes in alpha-diversity over the course of an exacerbation and treatment were non-significant. However, sample composition did alter over the course of an exacerbation, with most notably a reduction in the relative abundance of amplicon sequence variants assigned to Haemophilus.Discussion Haemophilus has been associated with more severe symptoms in respiratory infections and a reduction in its relative abundance may represent a positive shift in a patient’s microbiota. Current treatments for bronchiectasis may preserve bacterial diversity while altering microbiota composition.

Dietary shifts and social interactions drive temporal fluctuations of the gut microbiome from wild redfronted lemurs

Animals living in highly seasonal environments adapt their diets accordingly to changes in food availability. The gut microbiome as an active participant in the metabolization of the host’s diet should adapt and change with temporal diet fluctuations, but dietary shifts can be short-term and, hence, difficult to detect in cross-sectional studies. Therefore, we performed a longitudinal study combining repeated sampling of fecal samples with observations of feeding behavior in wild redfronted lemurs. We amplified taxonomical marker genes for assessing the bacteria, archaea, protozoa, helminths, and fungi, as well as the active bacterial community inhabiting their gut. We found that the most abundant protozoans were Trichostomatia and Trichomonadida, and the most abundant helminths were Chromadorea. We detected known members of the gut mycobiome from humans but in low abundances. The archaeal community is composed only of members of Methanomethylophilaceae. The predominant phyla in the entire bacterial community were Bacteroidota and Firmicutes while the most abundant genera harbor so far unknown bacteria. Temporal fluctuations at the entire community level were driven by consumption of fruits and flowers, and affiliative interactions. Changes in alpha diversity correlated only with the consumption of flowers and leaves. The composition of the entire and active bacterial community was not significantly different, but the most abundant taxa differed. Our study revealed that monthly changes in the bacterial community composition were linked to fruit and flower consumption and affiliative interactions. Thus, portraying the importance of longitudinal studies for understanding the adaptations and alterations of the gut microbiome to temporal fluctuations.

Gut microbiota modulation by prednisolone in a rat kindling model of pentylenetetrazol (PTZ)-induced seizure

The gut microbiota is a complex community composed by several microorganisms that interact in the maintenance of homeostasis and contribute to physiological processes, including brain function. The relationship of the taxonomic composition of the gut microbiota with neurological diseases such as autism, Parkinson's, Alzheimer's, anxiety, and depression is widely recognized. The immune system is an important intermediary between the gut microbiota and the central nervous system, being one of the communication routes of the gut-brain axis. Although the complexity of the relationship between inflammation and epilepsy has not yet been elucidated, inflammatory processes are similar in many ways to the consequences of dysbiosis and contribute to disease progression. This study aimed to analyze the taxonomic composition of the gut microbiota of rats treated with prednisolone in a kindling model of epilepsy. Male Wistar rats (90 days, n = 24) divided into four experimental groups: sodium chloride solution 0.9 g%, diazepam 2 mg/kg, prednisolone 1 mg/kg, and prednisolone 5 mg/kg administered intraperitoneally (i.p.) for 14 days. The kindling model was induced by pentylenetetrazole (PTZ) 25 mg/kg i.p. on alternate days. The taxonomic profile was established by applying metagenomic DNA sequencing. There was no change in alpha diversity, and the composition of the gut microbiota between prednisolone and diazepam was similar. The significant increase in Verrucomicrobia, Saccharibacteria, and Actinobacteria may be related to the protective activity against seizures and inflammatory processes that cause some cases of epilepsy. Further studies are needed to investigate the functional influence that these species have on epilepsy and the inflammatory processes that trigger it.

Diversidad de arañas (Arachnida, Araneae) sinantrópicas de la ciudad de Morelia, Michoacán, México, ¿qué tanto influye el tiempo de construcción de las viviendas en la comunidad de arañas?

Spiders have been used to evaluate changes in systems by anthropization effect, some species showing sensitivity to gradual and drastic changes such as urbanization, and other species have been documented as tolerant to this effect. The goal of this work was to describe the change in the spider community in relation to the age of buildings. at the west of Morelia, Michoacán, Mexico. Three categories of construction time and the neighboring vegetation as the pre-urbanization environment were used for comparison. Using direct capture, pit-fall traps and beating nets, 3,619 spiders were collected, and 3,219 (315 males, 630 females and 2,274 immatures) were used for the analysis after removal juveniles that was not possible to identify. A total of 28 families, 93 genera, 47 species and 55 morphospecies were identified. This represents the greatest richness and abundance recorded in urban environments of the country. It describes changes in alpha diversity from colonization in recent constructions to those built 17 years ago. I t was observed that abundance, richness, and diversity decrease with the age of a building, but with high equitability in every age of construction. The age of a building is an important factor for the process of succession in urban environments.

Bifidobacterium breve CCFM1025 attenuates major depression disorder via regulating gut microbiome and tryptophan metabolism: A randomized clinical trial

ObjectivePsychobiotics, as a novel class of probiotics mainly acting on the gut-brain axis, have shown promising prospects in treating psychiatric disorders. Bifidobacterium breve CCFM1025 was validated to have an antidepressant-like effect in mice. This study aims to assess its psychotropic potential in managing major depression disorder (MDD) and unravel the underlying mechanisms. MethodsClinical Trial Registration: https://www.chictr.org.cn/index.aspx (identifier: NO. ChiCTR2100046321). Patients (n = 45) diagnosed with MDD were randomly assigned to the Placebo (n = 25) and CCFM1025 (n = 20) groups. The freeze-dried CCFM1025 in a dose of viable bacteria of 1010 CFU was given to MDD patients daily for four weeks, while the placebo group was given maltodextrin. Changes from baseline in psychometric and gastrointestinal symptoms were evaluated using Hamilton Depression Rating scale-24 Items (HDRS-24), Montgomery-Asberg Depression Rating Scale (MADRS), Brief Psychiatric Rating Scale (BPRS), and Gastrointestinal Symptom Rating Scale (GSRS). Serum measures were also determined, i.e., cortisol, TNF-α, and IL-β. Serotonin turnover in the circulation, gut microbiome composition, and tryptophan metabolites were further investigated for clarifying the probiotics’ mechanisms of action. ResultsCCFM1025 showed a better antidepressant-like effect than placebo, based on the HDRS-24 (placebo: M = 6.44, SD = 5.44; CCFM1025: M = 10.40, SD = 6.85; t(43) = 2.163, P = 0.036, d = 0.640) and MADRS (placebo: M = 4.92, SD = 7.15; CCFM1025: M = 9.60, SD = 7.37; t(43) = 2.152, P = 0.037, d = 0.645) evaluation. The factor analysis of BPRS and GSRS suggested that patients’ emotional and gastrointestinal problems may be affected by the serotonergic system. Specifically, CCFM1025 could significantly and to a larger extend reduce the serum serotonin turnover compared with the placebo (placebo: M = -0.01, SD = 0.41; CCFM1025: M = 0.27, SD = 0.40; t(43) = 2.267, P = 0.029, d = 0.681). It may be due to changes in gut microbiome and gut tryptophan metabolism under the probiotic treatment, such as changes in alpha diversity, tryptophan, and indoles derivatives. ConclusionB. breve CCFM1025 is a promising candidate psychobiotic strain that attenuates depression and associated gastrointestinal disorders. The mechanisms may be relevant to the changes in the gut microbiome and tryptophan metabolism. These findings support the future clinical applications of psychobiotics in the treatment of psychiatric disorders.

Distinct response patterns of bacterial communities in Ag- and ZnO-rGO nanocomposite-amended silt loam soils

The widespread application of metal-based nanoparticle (MNPs)/reduced graphene oxide (rGO) composites inevitably leads to their release into soils. However, we lack a detailed understanding of the bacterial community response to MNPs-rGO exposure in farmland soils. Here, we conducted a soil microcosm experiment to analyze the potential impact of MNPs-rGO on bacterial communities in two field soils via high-throughput sequencing. The change in alpha diversity of bacterial communities was more susceptible to Ag-rGO and ZnO-rGO treatments than CuO-rGO. In both soils, MNPs-rGO significantly changed the bacterial community structure even at a low dose (1 mg kg−1). The bacterial community structure was most strongly affected by Ag-rGO at 30 days, but the greatest changes occurred in ZnO-rGO at 60 days. The differences in soil properties could shape bacterial communities to MNPs-rGO exposure. Distance-based redundancy analysis and functional annotation of prokaryotic taxa showed that some bacterial species associated with nitrogen cycling were greatly influenced by Ag-rGO and ZnO-rGO exposure. In sum, Ag-rGO and ZnO-rGO may potentially affect bacterial communities and nitrogen turnover under long-term realistic field exposure. These findings present a perspective on the response of bacterial communities to MNPs-rGO and provide a fundamental basis for estimating the ecological behavior of MNPs-rGO.

S156 Microbiome Alteration via Fecal Microbiota Transplantation (FMT) Is Effective for Immune Checkpoint Inhibitor (ICI) Induced-Colitis (IMC) Refractory to Immunosuppressive Therapy

Introduction: Immune checkpoint inhibitors (ICI) target advanced malignancies with high efficacy, but also predispose to immune related adverse events such as immune-mediated colitis (IMC). While evidence-based management is lacking, medical therapy is limited to immunosuppressant therapy which may compromise cancer outcome. The composition of gut commensal bacteria is associated with response to ICI therapy and IMC severity, but optimal strategies to target the microbiome have yet to be identified. Fecal microbiota transplantation (FMT) represents a feasible way to restore microbial diversity and composition in patients for clinical benefit. Methods: We present a case series of 12 patients with IMC who received FMT following unsuccessful treatment to immunosuppression with a detailed stool microbiome analysis. Of note, donor stool had successfully been employed previously in treatment of recurrent C.difficile diarrhea. Results: All patients had advanced cancer with grade 3-4 diarrhea that failed immunosuppression. 3 patients received FMT twice. 83% had symptom improvement at a median duration of 14 days (IQR 9-16). Seven (58.3%) patients had complete clinical response, while five (41.7%) had either partial or no response. 42% of patients achieved both endoscopic and histologic remission at the last follow up. 4 (33%) resumed cancer therapy post FMT. 4 (33%) patients required immunosuppression for GI irAE after failing FMT. There were no FMT related complications at 7 and 30 days respectively. At the end of the study period, 92% achieved clinical remission. 3 patients in the cohort succumbed to underlying malignancy. The clinical characteristics are summarized in Table. 16S rRNA sequencing of patient stools before and following FMT revealed that complete response to FMT was significantly associated with greater gut dysbiosis before FMT, higher alpha diversity and an increased abundance in bacterial families Coriobacteriaceae and Bifidobacteriaceae from the phylum Actinobacteria post FMT. (Figure 1). Conclusion: FMT for refractory IMC represents a novel approach wherein the gut microbial diversity and composition may be targeted to confer therapeutic benefit for IMC. The data from this study gives greater insight into the unique microbial signatures that may play a role in FMT response to IMC. We speculate that routine use of FMT may have value in enhancing effectiveness of immunotherapy in some cancers while preventing gastrointestinal drug side effects.Figure 1.: A)Paired analysis depicting changes in alpha diversity B) Coriobacteriaceae and Bifidobacteriaceae comparing pre-FMT and post-FMT. C) beta diversity of donors, Complete responders (CR) and Partial/Non-responders (PR/NR) at baseline (pre-FMT). Significance was assessed by comparing donor to CR (P= 0.012) and donor to PR/NR (P=0.4).Table 1.: Demographic, Oncologic, IMC and FMT-related characteristics of patients.

Measuring temporal change in alpha diversity: A framework integrating taxonomic, phylogenetic and functional diversity and the iNEXT.3D standardization

Abstract Biodiversity is a multifaceted concept covering different levels of organization from genes to ecosystems. Biodiversity has at least three dimensions: (a) Taxonomic diversity (TD): a measure that is sensitive to the number and abundances of species. (b) Phylogenetic diversity (PD): a measure that incorporates not only species abundances but also species evolutionary histories. (c) Functional diversity (FD): a measure that considers not only species abundances but also species' traits. We integrate the three dimensions of diversity under a unified framework of Hill numbers and their generalizations. Our TD quantifies the effective number of equally abundant species, PD quantifies the effective total branch length, mean‐PD (PD divided by tree depth) quantifies the effective number of equally divergent lineages, and FD quantifies the effective number of equally distinct virtual functional groups (or functional ‘species’). Thus, TD, mean‐PD and FD are all in the same units of species/lineage equivalents and can be meaningfully compared. Like species richness, empirical TD, PD and FD based on sampling data depend on sampling effort and sample completeness. For TD (Hill numbers), the iNEXT (interpolation and extrapolation) standardization was developed for standardizing sample size or sample completeness (as measured by sample coverage, the fraction of individuals that belong to the observed species) to make objective comparisons across studies. This paper extends the iNEXT method to the iNEXT.3D standardization to encompass all three dimensions of diversity via sample size‐ and sample coverage‐based rarefaction and extrapolation under the unified framework. The asymptotic diversity estimates (i.e. sample size tends to infinity and sample coverage tends to unity) are also derived. In addition to individual‐based abundance data, the proposed iNEXT.3D standardization is adapted to deal with incidence‐based occurrence data. We apply the integrative framework and the proposed iNEXT.3D standardization to measure temporal alpha‐diversity changes for estuarine fish assemblage data spanning four decades. The influence of environmental drivers on diversity change are also assessed. Our analysis informs a mechanistic interpretation of biodiversity change in the three dimensions of diversity. The accompanying freeware, iNEXT.3D, developed during this project, facilitates all computation and graphics.

The Impact of Acne Treatment on Skin Bacterial Microbiota: A Systematic Review.

BackgroundMicrobial strains such as Cutibacterium acnes have been examined as contributors to the pathogenesis of acne. Given the prevalence of the disease among adolescents and adults, the overutilization of antimicrobial agents may breed resistance and alter commensal microflora.ObjectivesTo characterize the impact of acne treatment on the diversity and relative abundance of the cutaneous microbial community, particularly of the bacterial floraMethodsAn electronic search was conducted of Embase, MEDLINE, and the Cochrane Central Register of Controlled Trials (CENTRAL) on June 5, 2020. Interventional and observational studies examining patients receiving acne treatment with culture-independent, community-level analysis of the cutaneous microbiome were included.ResultsNine studies with 170 treated acne patients were included. Five studies reported a significant change in alpha diversity following treatment, 3 of which examining systemic antibiotics reported significant increases in diversity. Two of 3 studies examining effects of benzoyl peroxide reported a decrease in diversity. However, trends in diversity were heterogeneous among studies.ConclusionsWhile individual variability in microbiome composition, and study-level heterogeneity in study sampling techniques may limit quantitative synthesis, our results support findings that acne treatment, including those not considered to have antimicrobial properties, alters the composition of the cutaneous microbiome.PROSPERO registration: CRD42020190629

Comparative Analysis of 16S rRNA Gene and Metagenome Sequencing in Pediatric Gut Microbiomes

The colonization of the human gut microbiome begins at birth, and over time, these microbial communities become increasingly complex. Most of what we currently know about the human microbiome, especially in early stages of development, was described using culture-independent sequencing methods that allow us to identify the taxonomic composition of microbial communities using genomic techniques, such as amplicon or shotgun metagenomic sequencing. Each method has distinct tradeoffs, but there has not been a direct comparison of the utility of these methods in stool samples from very young children, which have different features than those of adults. We compared the effects of profiling the human infant gut microbiome with 16S rRNA amplicon vs. shotgun metagenomic sequencing techniques in 338 fecal samples; younger than 15, 15–30, and older than 30 months of age. We demonstrate that observed changes in alpha-diversity and beta-diversity with age occur to similar extents using both profiling methods. We also show that 16S rRNA profiling identified a larger number of genera and we find several genera that are missed or underrepresented by each profiling method. We present the link between alpha diversity and shotgun metagenomic sequencing depth for children of different ages. These findings provide a guide for selecting an appropriate method and sequencing depth for the three studied age groups.

Understanding the effects of intramuscular injection and feed withdrawal on Salmonella Typhimurium shedding and gut microbiota in pullets

BackgroundGut microbiota plays a key role in health, immunity, digestion, and production in layers. Factors such as environment, diet, diseases, stress, and flock management significantly affect gut microbiota; however, it is not known how potential stressors such as intramuscular injections or feed withdrawal alter the composition of gut microbiota that result in increased the shedding level of foodborne pathogens. In the current study, the effects of intramuscular corticosterone injection and feed withdrawal were evaluated to understand their role in Salmonella Typhimurium shedding and changes in the composition of gut microbiota in layers.ResultsSalmonella shedding was observed for 8 weeks post-infection. There was a significant increase in Salmonella Typhimurium count after intramuscular injection and feed withdrawal. The Salmonella infected and the negative control groups showed significant differences in the abundance of different genera in gut microbiota at week 1 and up to week 7 post infection. The infected group showed a significant reduction in alpha diversity of gut microbiota. Firmicutes reduced significantly (P < 0.05) after intramuscular injection, while the feed withdrawal groups did not cause any significant changes in Proteobacteria-Firmicutes ratio. Furthermore, intramuscular injection resulted in a significant change in alpha diversity of gut microbiota.ConclusionsExposure of chicks to relatively low dose of Salmonella Typhimurium can lead to persistent shedding in pullets. The Salmonella Typhimurium infection disrupted the gut microbiota composition immediately after infection. The potential stress of intramuscular injection and feed withdrawal significantly increased the Salmonella Typhimurium count in faeces. The intramuscular injection also resulted in a significant alteration of the Proteobacteria-Firmicutes ratio, which could increase the risk of dysbiosis.

Effects of Inulin Consumption on the Gut Microbiome During Fasting: A Pilot Study in Healthy Volunteers

ObjectivesCaloric restriction has been associated with beneficial effects on metabolic diseases such as type 2 diabetes, cardiovascular diseases, and cancer but the mechanisms are diverse and not fully understood. Interactions between the human host and its gut microbiota might play a crucial role in this context, but these have not yet been analysed in detail. Therefore, we conducted a pilot intervention study to investigate how the microbial community in the human gut adapts to caloric restriction. Additionally, we investigated whether the compliance improved if the participants during fasting consumed prebiotics, which are supposed to reduce sensations of hunger while not providing calories through digestion in the small intestine. MethodsWe recruited six healthy volunteers (age: 20–50 years, BMI: 20–25 kg/m2) for a pilot intervention study using a randomized crossover design. The study consisted of two sequential fasting periods of which one included additional prebiotics. Participants were fasting for three consecutive days consuming a total of 300 kcal daily provided by vegetable juices, framed by two days with a total daily calorie intake of 800 kcal, respectively. During one of the fasting periods, participants consumed additionally 24 g of inulin daily. Stool samples were collected for the analysis of the microbial composition by 16S rRNA sequencing. ResultsWe observed no clear change in alpha diversity during the period of caloric reduction. However, quantitative changes in the composition of the microbiome was visible in mainly all participants. Additional intake of the prebiotic inulin did not influence compliance for the fasting intervention. However, we observed a clear reduction in alpha diversity in the gut microbiome after inulin ingestion during the fasting period. ConclusionsCaloric restriction appears to have no significant influence on the gut microbiome itself but inulin consumption during fasting seems to selectively promote the growth of some bacterial families leading to an overall decrease of alpha diversity in the gut microbiome. Further studies with a larger sample size are warranted to verify our observations. Funding SourcesInstitutional budget, no external funding.

Prospective manipulation of the gut microbiome with Microbial Ecosystem Therapeutic 4 (MET4) in locoregionally advanced oropharyngeal squamous cell carcinoma (LA-OPSCC) undergoing primary chemoradiation (ROMA2).

6059 Background: Therapeutic manipulation of the gut microbiome in cancer patients (pts) is an area of active investigation. MET4 (NuBiyota) is an oral alternative to fecal transplant consisting of a mixture of human gut bacteria associated with immunotherapy (IO) response. We previously reported variation in IO-responsive taxa across stages in human papilloma virus related (HPV+) LA-OPSCC pts treated with chemoradiotherapy (CRT) (Oliva et al., ASCO 2020). ROMA-2 is the first interventional study evaluating the safety, feasibility and ecological effect of MET4, in combination with definitive CRT in HPV+ LA-OPSCC (NCT03838601). Methods: This is an investigator-initiated study of pts with HPV+ LA-OPSCC treated with standard of care CRT. MET4 is administered daily until week 4 of CRT or unacceptable toxicity. Stool samples are collected at baseline, week 4, week 8-10, and 2-months post CRT. Bacterial V4 16S rDNA was extracted from stool and sequenced. Microbiome analyses were conducted in R using DADA2, phyloseq and DESeq2. Results: As of February 11 2021, 25 pts have been enrolled. A total of 50 stool samples from the first 14 pts were collected (98% adherence) and analyzed. Baseline cohort characteristics: median age = 62.5 (range, 48-69); Stage I/II/III = 5/1/8; use of antibiotics = 1pt. 3 pts did not complete the 3-week course of MET4 treatment due to non-compliance (n = 1), withdrawal of consent (n = 1) and grade 2 diarrhea (n = 1). Other reported MET4-related adverse events (all grade 1) included bloating (n = 2), flatulence (n = 1) and belching (n = 1). No longitudinal changes in alpha-diversity were seen from baseline through follow up. Administration of MET4 resulted in a transient trend towards increased cumulative MET4 taxa relative abundance (RA) by week 4. Stage III patients demonstrated the lowest MET4 taxa RA at baseline, and the greatest increase in MET4 taxa RA from baseline to week 4. By week 4 the following taxa in all pts were increased compared to baseline: Eubacterium hallii (21.71 Log2Fold change[L2FC], padj &lt; 0.001) and Parabacteroides johnsonii (23.67 L2FC, padj &lt; 0.001). An increase in the following taxa was observed by weeks 8-10 compared to baseline: Akkermansia muciniphilla (3.75 L2FC, padj = 0.027), Bacteroides fragilis (6.73 L2FC, padj = 0.010), Alistipes onderdonkii (3.30 L2FC, padj = 0.049) and Parabacteroides distasonis (24.43 L2FC, padj &lt; 0.001). Conclusions: Manipulation of the gut microbiota in these pts was feasible and safe. MET4-induced ecological changes are heterogenous and vary by taxa. MET4 taxa implicated in IO-response were increased by week 4 and week 8-10. This increase was higher in pts with stage III disease. These data suggest that specific subgroups may benefit from combination IO therapy and may guide pt selection for further interventional clinical trial design. Clinical trial information: NCT03838601.

Combined hormonal contraceptives are associated with minor changes in composition and diversity in gut microbiota of healthy women

Recent human and animal studies have found associations between gut microbiota composition and serum levels of sex hormones, indicating that they could be an important factor in shaping the microbiota. However, little is known about the effect of regular hormonal fluctuations over the menstrual cycle or CHC-related changes of hormone levels on gut microbiota structure, diversity and dynamics. The aim of this study was to investigate the effect of CHCs on human gut microbiota composition. The effect of CHC pill intake on gut microbiota composition was studied in a group of 7 healthy pre-menopausal women using the CHC pill, compared to the control group of 9 age-matched healthy women that have not used hormonal contraceptives in the six months prior the start of the study. By analyzing the gut microbiota composition in both groups during one menstrual cycle, we found that CHC usage is associated with a minor decrease in gut microbiota diversity and differences in the abundance of several bacterial taxa. These results call for further investigation of the mechanisms underlying hormonal and hormonal contraceptive-related changes of the gut microbiota and the potential implications of these changes for women's health. This article is protected by copyright. All rights reserved.

Impact of Forest Management on the Temporal Dynamics of Herbaceous Plant Diversity in the Carpathian Beech Forests over 40 Years.

Simple SummaryVegetation composition and plant diversity of mixed deciduous forests in Europe is strongly linked with the dynamics of the forest stand and/or the regimes of forest management. In this work, we showed the influence of temporal interactions among the changes in the management system—the dominant development stage—the intensity of forest treatments, and herbaceous plant diversity. We argued that different arrangements of these interactions will result in different patterns of change in herbaceous plant diversity. We emphasized the need for careful interpretation of the levels of diversity (α, β) to evaluate the conservation status of forests.In recent years, there has been a growing awareness of the complex dependence of herbaceous plant diversity on forest structure and management. However, among the studies presented so far, those in which the chronosequence (approach based on the assumption of space-for-time substitution) was used, dominate. On the other hand, it is rare to find results based on long-term research on permanent or semi-permanent sampling plots. The aim of this study was to recognize the changes in the vegetation composition and dynamics of various indices of herbaceous plant diversity over 40 years of forest development, and their dependence on forest structure and management. Here we analyzed the temporal dynamics of herbaceous plant diversity in Carpathian fertile beech forests, based on datasets recorded on semi-permanent plots in three censuses (the 1970s, 2000s and 2010s). We checked the temporal changes in alpha, beta, and gamma diversity. Analyses of the plant diversity were performed on the background of changes in forest structure and management systems. We found that the within-plot (alfa diversity) and between-plot (beta diversity) herbaceous plant diversity metrics showed inconsistent patterns along with changes in the forest structure, management systems, and intensity of forest management, during the last 40 years. Temporal changes in the gamma diversity followed the changes in alpha diversity. Although the beta diversity after 40 years is greater than in the past, we argue that the conservation status of habitats typical for well-preserved fertile mountain beech forests has deteriorated due to a decline in the sharing of the diagnostic species of these forests. We showed the importance of the different temporal interactions between the forest structure and management for herbaceous plant diversity. We argue that, in view of the complexity of these processes, it would be a mistake to reject or prioritize alpha or beta diversity measurements to determine the real course of long-term changes in herbaceous plant diversity and to properly assess the state of the forest biodiversity, their conservation status, or conservation action plans. In addition, we need far more data from long-term observations to fully understand the possible relationship patterns between the factors controlling the forest structure and plant diversity.

Testing repeatability, testing repeatability, testing repeatability: harmonization of the DNA metabarcoding protocol for macrobenthos across Europe

Macrobenthos is a good indicator to evaluate the potential effects of human activities on the marine benthic ecosystem. In environmental impact assessments (EIAs), macrobenthic species identification is typically based on morphological characteristics, a time-consuming and labor-intensive process for which specific taxonomic knowledge and experts are needed. DNA metabarcoding can circumvent most of these shortcomings. However, to be applicable in EIAs and to be adapted by policy, a standardized protocol that allows for reproducible and reliable DNA metabarcoding results is a prerequisite. Here, three research questions were investigated as part of the international Interreg NSR project GEANS: 1) “How many replicates of DNA extractions and PCR products are needed to capture most of the macrobenthic species in a sample?”, 2) “Is a ‘fixed’ DNA metabarcoding protocol repeatable across different institutes?” and 3) “What is the impact of small changes in this DNA metabarcoding protocol on alpha diversity?”. These are important steps to convince stakeholders that this efficient and quick method generates reliable and comparable results. First, variation in macrobenthic species across technical replicates was investigated in three biological replicates from three macrobenthic communities in the Belgian Part of the North Sea (BPNS) with high, medium and low diversity. For each biological replicate, six DNA replicates were taken and one of these DNA replicates was used to assess variation between three replicates for PCR amplification. Three DNA replicates were needed in locations with a high and medium diversity to pick up at least 80% of the species diversity present in the six replicates, while four DNA replicates were needed in the location with low diversity. Variation in the detected species between PCR replicates was high, illustrating the importance of including at least three PCR replicates in the lab protocol. Second, we conducted a ring test where subsamples of 12 bulk macrobenthos samples, originating from four different macrobenthic communities in the BPNS (differing in species density and diversity), were distributed to four institutes located in Belgium, the Netherlands, Germany and Denmark. Samples were processed using the same standardized lab protocol and the resulting datasets were processed bioinformatically by one institute. The number of ASVs and the number of species reflected the morphological diversity patterns, i.e. highest values for the replicates from the highly diverse macrobenthic community, lowest numbers in the low diversity replicates and intermediate values in the samples from the medium diversity community. These patterns were identical between the four institutes, showing high repeatability for alpha diversity when using the same protocol. In total, 100 macrobenthic species were detected through DNA metabarcoding, of which 60 species were picked up by all four institutes, while 0-14 species were recorded by only one of the four institutes. Beta diversity patterns were also comparable between the four institutes, as the nMDS plot clearly showed clustering based on the macrobenthic communities, independent of the institute that conducted the work. Finally, small changes to the lab protocol (different DNA extraction kit, different high fidelity polymerases for PCR amplification, different reagents for clean-up) resulted in only minor changes in alpha diversity: similar number of species were detected as with the fixed protocol in all samples and 70% - 75% of the species were shared between the ‘fixed’ and adjusted protocols. This study shows for the first time that DNA metabarcoding offers a highly repeatable assessment of alpha and beta diversity patterns, which supports the suitability of DNA metabarcoding for monitoring of marine macrobenthos. These results are highly valuable to establish a harmonized and uniform DNA metabarcoding protocol, to be used by all institutions in Europe when implemented as a new standard method in EIAs of the benthic ecosystem.

The impact of Opisthorchis felineus infection and praziquantel treatment on the intestinal microbiota in children

The presence of some species of helminths is associated with changes in host microbiota composition and diversity, which varies widely depending on the infecting helminth species and other factors. We conducted a prospective case-control study to evaluate the gut microbiota in children with Opisthorchis felineus infection (n=50) before and after anthelmintic treatment and in uninfected children (n=49) in the endemic region. A total of 99 children and adolescents aged between 7 and 18 years were enrolled to the study. Helminth infection was assessed before and at 3 months after treatment with praziquantel. A complex examination for each participant was performed in the study, including an assessment of the clinical symptoms and an intestinal microbiota survey by 16S rRNA gene sequencing of stool samples. There was no change in alpha diversity between O. felineus-infected and control groups. We found significant changes in the abundances of bacterial taxa at different taxonomic levels between the infected and uninfected individuals. Enterobacteriaceae family was more abundant in infected participants compared to uninfected children. On the genus level, O. felineus-infected participants’ microbiota showed higher levels of Lachnospira, Escherichia-Shigella, Bacteroides, Eubacterium eligens group, Ruminiclostridium 6, Barnesiella, Oscillibacter, Faecalitalea and Anaerosporobacter and reduction of Blautia, Lachnospiraceae FCS020 and Eubacterium hallii group in comparison with the uninfected individuals. Following praziquantel therapy, there were significant differences in abundances of some microorganisms, including an increase of Faecalibacterium and decrease of Megasphaera, Roseburia. Enterobacteriaceae and Escherichia abundances were decreased up to the control group values. Our results highlight the importance of the host-parasite-microbiota interactions for the community health in the endemic regions.