Taxonomic and functional analyses of intact microbial communities thriving in extreme, astrobiology-relevant, anoxic sites.

BACKGROUND: Extreme terrestrial, analogue environments are widely used models to study the limits of life and to infer habitability of extraterrestrial settings. In contrast to Earth's ecosystems, potential extraterrestrial biotopes are usually characterized by a lack of oxygen. METHODS: In the MASE project (Mars Analogues for Space Exploration), we selected representative anoxic analogue environments (permafrost, salt-mine, acidic lake and river, sulfur springs) for the comprehensive analysis of their microbial communities. We assessed the microbiome profile of intact cells by propidium monoazide-based amplicon and shotgun metagenome sequencing, supplemented with an extensive cultivation effort. RESULTS: The information retrieved from microbiome analyses on the intact microbial community thriving in the MASE sites, together with the isolation of 31 model microorganisms and successful binning of 15 high-quality genomes allowed us to observe principle pathways, which pinpoint specific microbial functions in the MASE sites compared to moderate environments. The microorganisms were characterized by an impressive machinery to withstand physical and chemical pressures. All levels of our analyses revealed the strong and omnipresent dependency of the microbial communities on complex organic matter. Moreover, we identified an extremotolerant cosmopolitan group of 34 poly-extremophiles thriving in all sites. CONCLUSIONS: Our results reveal the presence of a core microbiome and microbial taxonomic similarities between saline and acidic anoxic environments. Our work further emphasizes the importance of the environmental, terrestrial parameters for the functionality of a microbial community, but also reveals a high proportion of living microorganisms in extreme environments with a high adaptation potential within habitability borders. Video abstract.

Biomarkers for oralization during long-term proton pump inhibitor therapy predict survival in cirrhosis.

Proton pump inhibitors (PPI) are an invaluable therapy option for acid related diseases; however, PPI therapy is also linked to a series of side effects in cirrhosis, such as microbiome alterations, spontaneous bacterial peritonitis and hepatic encephalopathy. Decision tools to balance benefits and risks of PPI therapy are largely missing. In this study, we tested gut-derived biomarkers to identify PPI-associated dysbiosis, its association with gut barrier function and liver-related mortality. In this observational study, faecal microbiome composition data obtained from 16S rDNA sequencing of 90 cirrhotic patients with and without long-term PPI use and additional potential biomarkers identified from the literature were evaluated for their predictive value regarding PPI-associated dysbiosis and liver-related three-year mortality. In addition, faecal calprotectin, faecal zonulin and serum lipopolysaccharides were assessed as markers for intestinal inflammation, gut permeability and bacterial translocation. Streptococcus salivarius, Veillonella parvula and the genus Streptococcus were significantly increased in patients with long-term PPI therapy and performed well as biomarkers for PPI-associated dysbiosis (accuracy: 74%, 72% and 74%, respectively). The abundance of Streptococcus salivarius was linked to intestinal inflammation and gut barrier dysfunction, whereas the abundance of Veillonella parvula showed associations with liver disease severity; both were independent predictors for liver-related three-year mortality. Gut-derived biomarkers of PPI-associated dysbiosis are linked to worse outcome and a potential option to evaluate the risks of adverse events during long-term PPI therapy.

Reproductive and Behavior Dysfunction Induced by Maternal Androgen Exposure and Obesity Is Likely Not Gut Microbiome-Mediated.

Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder of unclear etiology in women and is characterized by androgen excess, insulin resistance, and mood disorders. The gut microbiome is known to influence conditions closely related with PCOS, and several recent studies have observed changes in the stool microbiome of women with PCOS. The mechanism by which the gut microbiome interacts with PCOS is still unknown. We used a mouse model to investigate if diet-induced maternal obesity and maternal DHT exposure, mimicking the lean and obese PCOS women, cause lasting changes in the gut microbiome of offspring. Fecal microbiome profiles were assessed using Illumina paired-end sequencing of 16S rRNA gene V4 amplicons. We found sex-specific effects of maternal and offspring diet, and maternal DHT exposure on fecal bacterial richness and taxonomic composition. Female offspring exposed to maternal obesity and DHT displayed reproductive dysfunction and anxietylike behavior. Fecal microbiota transplantation from DHT and diet-induced obesity exposed female offspring to wild-type mice did not transfer reproductive dysfunction and did not cause the expected increase in anxietylike behavior in recipients. Maternal obesity and androgen exposure affect the gut microbiome of offspring, but the disrupted estrous cycles and anxietylike behavior are likely not microbiome-mediated.

The inflammatory cell landscape in the lungs of patients with idiopathic pulmonary arterial hypertension.

Increasing evidence points towards an inflammatory component underlying pulmonary hypertension. However, the conclusive characterisation of multiple inflammatory cell populations in the lung is challenging due to the complexity of marker specificity and tissue inaccessibility. We used an unbiased computational flow cytometry approach to delineate the inflammatory landscape of idiopathic pulmonary arterial hypertension (IPAH) and healthy donor lungs.Donor and IPAH samples were discriminated clearly using principal component analysis to reduce the multidimensional data obtained from single-cell flow cytometry analysis. In IPAH lungs, the predominant CD45+ cell type switched from neutrophils to CD3+ T-cells, with increases in CD4+, CD8+ and γδT-cell subsets. Additionally, diversely activated classical myeloid-derived dendritic cells (CD14-HLA-DR+CD11c+CD1a+/-) and nonclassical plasmacytoid dendritic cells (pDCs; CD14-CD11c-CD123+HLA-DR+), together with mast cells and basophils, were more abundant in IPAH samples. We describe, for the first time, the presence and regulation of two cell types in IPAH, γδT-cells and pDCs, which link innate and adaptive immunity.With our high-throughput flow cytometry with multidimensional dataset analysis, we have revealed the interactive interplay between multiple inflammatory cells is a crucial part of their integrative network. The identification of γδT-cells and pDCs in this disease potentially provides a missing link between IPAH, autoimmunity and inflammation.

Diacylglycerol triggers Rim101 pathway-dependent necrosis in yeast: a model for lipotoxicity.

The loss of lipid homeostasis can lead to lipid overload and is associated with a variety of disease states. However, little is known as to how the disruption of lipid regulation or lipid overload affects cell survival. In this study we investigated how excess diacylglycerol (DG), a cardinal metabolite suspected to mediate lipotoxicity, compromises the survival of yeast cells. We reveal that increased DG achieved by either genetic manipulation or pharmacological administration of 1,2-dioctanoyl-sn-glycerol (DOG) triggers necrotic cell death. The toxic effects of DG are linked to glucose metabolism and require a functional Rim101 signaling cascade involving the Rim21-dependent sensing complex and the activation of a calpain-like protease. The Rim101 cascade is an established pathway that triggers a transcriptional response to alkaline or lipid stress. We propose that the Rim101 pathway senses DG-induced lipid perturbation and conducts a signaling response that either facilitates cellular adaptation or triggers lipotoxic cell death. Using established models of lipotoxicity, i.e., high-fat diet in Drosophila and palmitic acid administration in cultured human endothelial cells, we present evidence that the core mechanism underlying this calpain-dependent lipotoxic cell death pathway is phylogenetically conserved.

Microbial Community and Biochemical Dynamics of Biological Soil Crusts across a Gradient of Surface Coverage in the Central Mojave Desert.

In this study, we expand upon the biogeography of biological soil crusts (BSCs) and provide molecular insights into the microbial community and biochemical dynamics along the vertical BSC column structure, and across a transect of increasing BSC surface coverage in the central Mojave Desert, CA, United States. Next generation sequencing reveals a bacterial community profile that is distinct among BSCs in the southwestern United States. Distribution of major phyla in the BSC topsoils included Cyanobacteria (33 ± 8%), Proteobacteria (26 ± 6%), and Chloroflexi (12 ± 4%), with Phormidium being the numerically dominant genus. Furthermore, BSC subsurfaces contained Proteobacteria (23 ± 5%), Actinobacteria (20 ± 5%), and Chloroflexi (18 ± 3%), with an unidentified genus from Chloroflexi (AKIW781, order) being numerically dominant. Across the transect, changes in distribution at the phylum (p < 0.0439) and genus (p < 0.006) levels, including multiple biochemical and geochemical trends (p < 0.05), positively correlated with increasing BSC surface coverage. This included increases in (a) Chloroflexi abundance, (b) abundance and diversity of Cyanobacteria, (b) OTU-level diversity in the topsoil, (c) OTU-level differentiation between the topsoil and subsurface, (d) intracellular ATP abundances and catalase activities, and (e) enrichments in clay, silt, and varying elements, including S, Mn, Co, As, and Pb, in the BSC topsoils. In sum, these studies suggest that BSCs from regions of differing surface coverage represent early successional stages, which exhibit increasing bacterial diversity, metabolic activities, and capacity to restructure the soil. Further, these trends suggest that BSC successional maturation and colonization across the transect are inhibited by metals/metalloids such as B, Ca, Ti, Mn, Co, Ni, Mo, and Pb.

On the Rocks: Microbiological Quality and Microbial Diversity of Packaged Ice in Southern California.

Ice is defined as a food and is frequently used in direct contact with food and beverages. Packaged ice is commercially produced and can be easily found in grocery and convenience stores. However, the quality and safety of packaged ice products is not consistent. The Packaged Ice Quality Control Standards manual (PIQCS) published by the International Packaged Ice Association provides the quality and processing standards for packaged ice produced by its members. Packaged ice produced on the premise of stores (on-site packaged ice) is not required to be in compliance with these standards. In this study, packaged ice produced by manufacturing plants or by in-store bagger (ISB) machines and on-site packaged ice were compared for their microbiological quality and microbial diversity. Our results revealed that 19% of the 120 on-site packaged ice samples did not meet the PIQCS microbial limit of 500 CFU/mL (or g) and also the absence of coliforms and Escherichia coli . Staphylococci were found in 34% of the on-site packaged ice samples, most likely through contamination from the packaging workers. None of the ISB and manufactured packaged ice samples had unacceptable microbial levels, and all were devoid of staphylococci. Salmonella was absent in all samples analyzed in this study. Microbial community analysis of ice based on 16S/18S rRNA targeted sequencing revealed a much higher microbial diversity and abundance in the on-site packaged ice than in the ISB ice. Proteobacteria, especially Alphaproteobacteria and Betaproteobacteria, were the dominant bacterial groups in all samples tested. Most of these bacteria were oligotrophic; however, a few opportunistic or potential pathogens were found at low levels in the on-site packaged ice but not in the ISB packaged ice. The types of microbes identified may provide information needed to investigate potential sources of contamination. Our data also suggest a need for enforcement of processing standards during the on-site packaging of ice.

Antibiotic-Associated Apoptotic Enterocolitis in the Absence of a Defined Pathogen: The Role of Intestinal Microbiota Depletion.

OBJECTIVE: Antibiotic therapy is a major risk factor for the development of diarrhea and colitis with varying severity. Often the origin of antibiotic-associated gastrointestinal deterioration remains elusive and no specific infectious agents could be discerned. PATIENTS: We represent three cases of intractable high-volume diarrhea associated with combined antibiotic and steroid therapy in critically ill patients not fitting into established disease entities. Cases presented with severe apoptotic enterocolitis resembling acute intestinal graft-versus-host-disease. Microbiologic workup precluded known enteropathogens, but microbiota analysis revealed a severely depleted gut microbiota with concomitant opportunistic pathogen overgrowth. INTERVENTIONS: Fecal microbiota transplantation, performed in one patient, was associated with correction of dysbiosis, rapid clinical improvement, and healing of enterocolitis. CONCLUSIONS: Our series represents a severe form of antibiotic-associated colitis in critically ill patients signified by microbiota depletion, and reestablishment of a physiologic gastrointestinal microbiota might be beneficial for this condition.

Alterations in Gut Microbiome Composition and Barrier Function Are Associated with Reproductive and Metabolic Defects in Women with Polycystic Ovary Syndrome (PCOS): A Pilot Study.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common female endocrinopathy of unclear origin characterized by hyperandrogenism, oligo-/anovulation, and ovarian cysts. Women with PCOS frequently display overweight, insulin resistance, and systemic low-grade inflammation. We hypothesized that endotoxemia resulting from a leaky gut is associated with inflammation, insulin resistance, fat accumulation, and hyperandrogenemia in PCOS. In this pilot study, we compared the stool microbiome, gut permeability, and inflammatory status of women with PCOS and healthy controls. METHODS: 16S rRNA gene amplicon sequencing was performed on stool samples from 24 PCOS patients and 19 healthy controls. Data processing and microbiome analysis were conducted in mothur and QIIME using different relative abundance cut-offs. Gut barrier integrity, endotoxemia, and inflammatory status were evaluated using serum and stool markers and associations with reproductive, metabolic, and anthropometric parameters were investigated. RESULTS: The stool microbiome of PCOS patients showed a lower diversity and an altered phylogenetic composition compared to controls. We did not observe significant differences in any taxa with a relative abundance>1%. When looking at rare taxa, the relative abundance of bacteria from the phylum Tenericutes, the order ML615J-28 (phylum Tenericutes) and the family S24-7 (phylum Bacteroidetes) was significantly lower and associated with reproductive parameters in PCOS patients. Patients showed alterations in some, but not all markers of gut barrier function and endotoxemia. CONCLUSION: Patients with PCOS have a lower diversity and an altered phylogenetic profile in their stool microbiome, which is associated with clinical parameters. Gut barrier dysfunction and endotoxemia were not driving factors in this patient cohort, but may contribute to the clinical phenotype in certain PCOS patients.

The Salivary Microbiome in Polycystic Ovary Syndrome (PCOS) and Its Association with Disease-Related Parameters: A Pilot Study.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common female endocrine condition of unclear etiology characterized by hyperandrogenism, oligo/amenorrhoea, and polycystic ovarian morphology. PCOS is often complicated by infertility, overweight/obesity, insulin resistance, and low-grade inflammation. The gut microbiome is known to contribute to several of these conditions. Recently, an association between stool and saliva microbiome community profiles was shown, making saliva a possible convenient, non-invasive sample type for detecting gut microbiome changes in systemic disease. In this study, we describe the saliva microbiome of PCOS patients and the association of microbiome features with PCOS-related parameters. METHODS: 16S rRNA gene amplicon sequencing was performed on saliva samples from 24 PCOS patients and 20 healthy controls. Data processing and microbiome analyses were conducted in mothur and QIIME. All study subjects were characterized regarding reproductive, metabolic, and inflammatory parameters. RESULTS: PCOS patients showed a decrease in bacteria from the phylum Actinobacteria and a borderline significant shift in bacterial community composition in unweighted UniFrac analysis. No differences between patients and controls were found in alpha diversity, weighted UniFrac analysis, or on other taxonomic levels. We found no association of saliva alpha diversity, beta diversity, or taxonomic composition with serum testosterone, oligo/amenorrhoea, overweight, insulin resistance, inflammatory markers, age, or diet. CONCLUSIONS: In this pilot study, patients with PCOS showed a reduced salivary relative abundance of Actinobacteria. Reproductive and metabolic components of the syndrome were not associated with saliva microbiome parameters, indicating that the majority of between-subject variation in saliva microbiome profiles remains to be explained.

Functional Metagenomics of Spacecraft Assembly Cleanrooms: Presence of Virulence Factors Associated with Human Pathogens.

Strict planetary protection practices are implemented during spacecraft assembly to prevent inadvertent transfer of earth microorganisms to other planetary bodies. Therefore, spacecraft are assembled in cleanrooms, which undergo strict cleaning and decontamination procedures to reduce total microbial bioburden. We wanted to evaluate if these practices selectively favor survival and growth of hardy microorganisms, such as pathogens. Three geographically distinct cleanrooms were sampled during the assembly of three NASA spacecraft: The Lockheed Martin Aeronautics' Multiple Testing Facility during DAWN, the Kennedy Space Center's Payload Hazardous Servicing Facility (KSC-PHSF) during Phoenix, and the Jet Propulsion Laboratory's Spacecraft Assembly Facility during Mars Science Laboratory. Sample sets were collected from the KSC-PHSF cleanroom at three time points: before arrival of the Phoenix spacecraft, during the assembly and testing of the Phoenix spacecraft, and after removal of the spacecraft from the KSC-PHSF facility. All samples were subjected to metagenomic shotgun sequencing on an Illumina HiSeq 2500 platform. Strict decontamination procedures had a greater impact on microbial communities than sampling location Samples collected during spacecraft assembly were dominated by Acinetobacter spp. We found pathogens and potential virulence factors, which determine pathogenicity in all the samples tested during this study. Though the relative abundance of pathogens was lowest during the Phoenix assembly, potential virulence factors were higher during assembly compared to before and after assembly, indicating a survival advantage. Decreased phylogenetic and pathogenic diversity indicates that decontamination and preventative measures were effective against the majority of microorganisms and well implemented, however, pathogen abundance still increased over time. Four potential pathogens, Acinetobacter baumannii, Acinetobacter lwoffii, Escherichia coli and Legionella pneumophila, and their corresponding virulence factors were present in all cleanroom samples. This is the first functional metagenomics study describing presence of pathogens and their corresponding virulence factors in cleanroom environments. The results of this study should be considered for microbial monitoring of enclosed environments such as schools, homes, hospitals and more isolated habitation such the International Space Station and future manned missions to Mars.

Characterization of the microbiome of nipple aspirate fluid of breast cancer survivors.

The microbiome impacts human health and disease. Until recently, human breast tissue and milk were presumed to be sterile. Here, we investigated the presence of microbes in the nipple aspirate fluid (NAF) and their potential association with breast cancer. We compared the NAF microbiome between women with a history of breast cancer (BC) and healthy control women (HC) using 16S rRNA gene amplicon sequencing. The NAF microbiome from BC and HC showed significant differences in community composition. Two Operational Taxonomic Units (OTUs) showed differences in relative abundances between NAF collected from BC and HC. In NAF collected from BC, there was relatively higher incidence of the genus Alistipes. By contrast, an unclassified genus from the Sphingomonadaceae family was relatively more abundant in NAF from HC. These findings reflect the ductal source DNA since there were no differences between areolar skin samples collected from BC and HC. Furthermore, the microbes associated with BC share an enzymatic activity, Beta-Glucuronidase, which may promote breast cancer. This is the first report of bacterial DNA in human breast ductal fluid and the differences between NAF from HC and BC. Further investigation of the ductal microbiome and its potential role in breast cancer are warranted.

Effects of high doses of vitamin D3 on mucosa-associated gut microbiome vary between regions of the human gastrointestinal tract.

PURPOSE: Vitamin D is well known for its effects on bone mineralisation but has also been attributed immunomodulatory properties. It positively influences human health, but in vivo data describing vitamin D effects on the human gut microbiome are missing. We aimed to investigate the effects of oral vitamin D3 supplementation on the human mucosa-associated and stool microbiome as well as CD8(+) T cells in healthy volunteers. METHODS: This was an interventional, open-label, pilot study. Sixteen healthy volunteers (7 females, 9 males) were endoscopically examined to access a total of 7 sites. We sampled stomach, small bowel, colon, and stools before and after 8 weeks of vitamin D3 supplementation. Bacterial composition was assessed by pyrosequencing the 16S rRNA gene (V1-2), and CD8(+) T cell counts were determined by flow cytometry. RESULTS: Vitamin D3 supplementation changed the gut microbiome in the upper GI tract (gastric corpus, antrum, and duodenum). We found a decreased relative abundance of Gammaproteobacteria including Pseudomonas spp. and Escherichia/Shigella spp. and increased bacterial richness. No major changes occurred in the terminal ileum, appendiceal orifice, ascending colon, and sigmoid colon or in stools, but the CD8(+) T cell fraction was significantly increased in the terminal ileum. CONCLUSION: Vitamin D3 modulates the gut microbiome of the upper GI tract which might explain its positive influence on gastrointestinal diseases, such as inflammatory bowel disease or bacterial infections. The local effects of vitamin D demonstrate pronounced regional differences in the response of the GI microbiome to external factors, which should be considered in future studies investigating the human microbiome.

Lactobacillus casei Shirota Supplementation Does Not Restore Gut Microbiota Composition and Gut Barrier in Metabolic Syndrome: A Randomized Pilot Study.

UNLABELLED: Metabolic syndrome is associated with disturbances in gut microbiota composition. We aimed to investigate the effect of Lactobacillus casei Shirota (LcS) on gut microbiota composition, gut barrier integrity, intestinal inflammation and serum bile acid profile in metabolic syndrome. In a single-centre, prospective, randomised controlled pilot study, 28 subjects with metabolic syndrome received either LcS for 12 weeks (n = 13) or no LcS (n = 15). Data were compared to healthy controls (n = 16). Gut microbiota composition was characterised from stool using 454 pyrosequencing of 16S rRNA genes. Serum bile acids were quantified by tandem mass spectrometry. Zonulin and calprotectin were measured in serum and stool by ELISA. Bacteroidetes/Firmicutes ratio was significantly higher in healthy controls compared to metabolic syndrome but was not influenced by LcS. LcS supplementation led to enrichment of Parabacteroides. Zonulin and calprotectin were increased in metabolic syndrome stool samples but not influenced by LcS supplementation. Serum bile acids were similar to controls and not influenced by LcS supplementation. Metabolic syndrome is associated with a higher Bacteroidetes/Firmicutes ratio and gut barrier dysfunction but LcS was not able to change this. LcS administration was associated with subtle microbiota changes at genus level. TRIAL REGISTRATION: ClinicalTrials.gov NCT01182844.