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In the original PDF version of this Article, which was published on 16 October 2017, the publication date was incorrectly given as 10 October 2017. This has now been corrected in the PDF; the HTML version of the paper was correct from the time of publication.

The presence of advanced fibrosis in nonalcoholic fatty liver disease (NAFLD) is the most important predictor of liver mortality. There are limited data on the diagnostic accuracy of gut microbiota-derived signature for predicting the presence of advanced fibrosis. In this prospective study, we characterized the gut microbiome compositions using whole-genome shotgun sequencing of DNA extracted from stool samples. This study included 86 uniquely well-characterized patients with biopsy-proven NAFLD, of which 72 had mild/moderate (stage 0-2 fibrosis) NAFLD, and 14 had advanced fibrosis (stage 3 or 4 fibrosis). We identified a set of 40 features (p < 0.006), which included 37 bacterial species that were used to construct a Random Forest classifier model to distinguish mild/moderate NAFLD from advanced fibrosis. The model had a robust diagnostic accuracy (AUC 0.936) for detecting advanced fibrosis. This study provides preliminary evidence for a fecal-microbiome-derived metagenomic signature to detect advanced fibrosis in NAFLD.

BackgroundAntibiotics are a mainstay of treatment for bacterial infections worldwide, yet the effects of typical antibiotic prescriptions on human indigenous microbiota have not been thoroughly evaluated. We examined the effects of the two most commonly prescribed antibiotics (amoxicillin and azithromycin) in the USA to discern whether short-term antibiotic courses may have prolonged effects on human microbiota.ResultsWe sampled the feces, saliva, and skin specimens from a cohort of unrelated, cohabitating individuals over 6 months. An individual in each household was given an antibiotic, and the other a placebo to discern antibiotic impacts on microbiota, as well as determine whether antibiotic use might reshape the microbiota of each household. We observed household-specific patterns of microbiota on each body surface, which persevered despite antibiotic perturbations. While the gut microbiota within an individual became more dissimilar over time, there was no evidence that the use of antibiotics accelerated this process when compared to household members. There was a significant change in microbiota diversity in the gut and mouth in response to antibiotics, but analogous patterns were not observed on the skin. Those who received 7 days of amoxicillin generally had greater reductions in diversity compared to those who received 3 days, in contrast to those who received azithromycin.ConclusionsAs few as 3 days of treatment with the most commonly prescribed antibiotics can result in sustained reductions in microbiota diversity, which could have implications for the maintenance of human health and resilience to disease.

Nonalcoholic fatty liver disease is associated with metabolic risk factors including hypertension and dyslipidemia and may progress to liver fibrosis. Studies have shown that hepatic steatosis and fibrosis are heritable, but whether they have a significant shared gene effect is unknown. This study examined the shared gene effects between hepatic steatosis and fibrosis and their associations with metabolic risk factors. This was a cross-sectional analysis of a prospective cohort of well-characterized, community-dwelling twins (45 monozygotic, 20 dizygotic twin pairs, 130 total subjects) from southern California. Hepatic steatosis was assessed with magnetic resonance imaging-proton density fat fraction and hepatic fibrosis with magnetic resonance elastography. A standard bivariate twin additive genetics and unique environment effects model was used to estimate the proportion of phenotypic variance between two phenotypes accounted for by additive genetic effects and individual-specific environmental effects. Genetic correlations estimated from this model represent the degree to which the genetic determinants of two phenotypes overlap. Mean (± standard deviation) age and body mass index were 47.1 (±21.9) years and 26.2 (±5.8) kg/m2 , respectively. Among the cohort, 20% (26/130) had hepatic steatosis (magnetic resonance imaging-proton density fat fraction ≥5%), and 8.2% (10/122) had hepatic fibrosis (magnetic resonance elastography ≥3 kPa). Blood pressure (systolic and diastolic), triglycerides, glucose, homeostatic model assessment of insulin resistance, insulin, hemoglobin A1c, and low high-density lipoprotein had significant shared gene effects with hepatic steatosis. Triglycerides, glucose, homeostatic model assessment of insulin resistance, insulin, hemoglobin A1c, and low high-density lipoprotein had significant shared gene effects with hepatic fibrosis. Hepatic steatosis and fibrosis had a highly significant shared gene effect of 0.756 (95% confidence interval 0.716-1, P < 0.0001).ConclusionsGenes involved with steatosis pathogenesis may also be involved with fibrosis pathogenesis. (Hepatology 2016;64:1547-1558).

Leptospirosis, caused by spirochetes of the genus Leptospira, is a globally widespread, neglected and emerging zoonotic disease. While whole genome analysis of individual pathogenic, intermediately pathogenic and saprophytic Leptospira species has been reported, comprehensive cross-species genomic comparison of all known species of infectious and non-infectious Leptospira, with the goal of identifying genes related to pathogenesis and mammalian host adaptation, remains a key gap in the field. Infectious Leptospira, comprised of pathogenic and intermediately pathogenic Leptospira, evolutionarily diverged from non-infectious, saprophytic Leptospira, as demonstrated by the following computational biology analyses: 1) the definitive taxonomy and evolutionary relatedness among all known Leptospira species; 2) genomically-predicted metabolic reconstructions that indicate novel adaptation of infectious Leptospira to mammals, including sialic acid biosynthesis, pathogen-specific porphyrin metabolism and the first-time demonstration of cobalamin (B12) autotrophy as a bacterial virulence factor; 3) CRISPR/Cas systems demonstrated only to be present in pathogenic Leptospira, suggesting a potential mechanism for this clade's refractoriness to gene targeting; 4) finding Leptospira pathogen-specific specialized protein secretion systems; 5) novel virulence-related genes/gene families such as the Virulence Modifying (VM) (PF07598 paralogs) proteins and pathogen-specific adhesins; 6) discovery of novel, pathogen-specific protein modification and secretion mechanisms including unique lipoprotein signal peptide motifs, Sec-independent twin arginine protein secretion motifs, and the absence of certain canonical signal recognition particle proteins from all Leptospira; and 7) and demonstration of infectious Leptospira-specific signal-responsive gene expression, motility and chemotaxis systems. By identifying large scale changes in infectious (pathogenic and intermediately pathogenic) vs. non-infectious Leptospira, this work provides new insights into the evolution of a genus of bacterial pathogens. This work will be a comprehensive roadmap for understanding leptospirosis pathogenesis. More generally, it provides new insights into mechanisms by which bacterial pathogens adapt to mammalian hosts.

Chronic liver disease is rising in western countries and liver cirrhosis is the 12th leading cause of death worldwide. Simultaneously, use of gastric acid suppressive medications is increasing. Here, we show that proton pump inhibitors promote progression of alcoholic liver disease, non-alcoholic fatty liver disease, and non-alcoholic steatohepatitis in mice by increasing numbers of intestinal Enterococcus spp. Translocating enterococci lead to hepatic inflammation and hepatocyte death. Expansion of intestinal Enterococcus faecalis is sufficient to exacerbate ethanol-induced liver disease in mice. Proton pump inhibitor use increases the risk of developing alcoholic liver disease among alcohol-dependent patients. Reduction of gastric acid secretion therefore appears to promote overgrowth of intestinal Enterococcus, which promotes liver disease, based on data from mouse models and humans. Recent increases in the use of gastric acid-suppressive medications might contribute to the increasing incidence of chronic liver disease.

Dental caries, one of the most globally widespread infectious diseases, is intimately linked to pH dynamics. In supragingival plaque, after the addition of a carbohydrate source, bacterial metabolism decreases the pH which then subsequently recovers. Molecular mechanisms supporting this important homeostasis are poorly characterized in part due to the fact that there are hundreds of active species in dental plaque. Only a few mechanisms (for example, lactate fermentation, the arginine deiminase system) have been identified and studied in detail. Here, we conducted what is to our knowledge, the first full transcriptome and metabolome analysis of a diverse oral plaque community by using a functionally and taxonomically robust in vitro model system greater than 100 species. Differential gene expression analyses from the complete transcriptome of 14 key community members revealed highly varied regulation of both known and previously unassociated pH-neutralizing pathways as a response to the pH drop. Unique expression and metabolite signatures from 400 detected metabolites were found for each stage along the pH curve suggesting it may be possible to define healthy and diseased states of activity. Importantly, for the maintenance of healthy plaque pH, gene transcription activity of known and previously unrecognized pH-neutralizing pathways was associated with the genera Lactobacillus, Veillonella and Streptococcus during the pH recovery phase. Our in vitro study provides a baseline for defining healthy and disease-like states and highlights the power of moving beyond single and dual species applications to capture key players and their orchestrated metabolic activities within a complex human oral microbiome model.

BackgroundThe human gut microbiota interacts closely with human diet and physiology. To better understand the mechanisms behind this relationship, gut microbiome research relies on complementing human studies with manipulations of animal models, including non-human primates. However, due to unique aspects of human diet and physiology, it is likely that host-gut microbe interactions operate differently in humans and non-human primates.ResultsHere, we show that the human microbiome reacts differently to a high-protein, high-fat Western diet than that of a model primate, the African green monkey, or vervet (Chlorocebus aethiops sabaeus). Specifically, humans exhibit increased relative abundance of Firmicutes and reduced relative abundance of Prevotella on a Western diet while vervets show the opposite pattern. Predictive metagenomics demonstrate an increased relative abundance of genes associated with carbohydrate metabolism in the microbiome of only humans consuming a Western diet.ConclusionsThese results suggest that the human gut microbiota has unique properties that are a result of changes in human diet and physiology across evolution or that may have contributed to the evolution of human physiology. Therefore, the role of animal models for understanding the relationship between the human gut microbiota and host metabolism must be re-focused.

Background & aimsLittle is known about the heritability of hepatic fibrosis, and the heritability of hepatic steatosis has not been assessed systematically in adults. We investigated the heritability of hepatic fibrosis and steatosis in a community-dwelling twin cohort.MethodsWe performed a cross-sectional analysis of a cohort of well-characterized twins residing in Southern California including 60 pairs of twins (42 monozygotic and 18 dizygotic; average age, 45.7 ± 22.1 y; average body mass index, 26.4 ± 5.7 kg/m(2)). We collected data on medical history, physical examinations, fasting laboratory test results, and liver health; all participants underwent an advanced magnetic resonance imaging (MRI) examination of the liver from January 2012 through January 2015. Hepatic steatosis was quantified noninvasively by MRI and determined based on the proton-density fat fraction (MRI-PDFF); liver fibrosis was measured based on stiffness measured by magnetic resonance elastography.ResultsTwenty-six of the 120 subjects (21.7%) had nonalcoholic fatty liver disease (defined as MRI-PDFF ≥ 5% after exclusion of other causes of hepatic steatosis). The presence of hepatic steatosis correlated between monozygotic twins (r(2) = 0.70; P < .0001) but not between dizygotic twins (r(2) = 0.36; P = .2). The level of liver fibrosis also correlated between monozygotic twins (r(2) = 0.48; P

Observations from human microbiome studies are often conflicting or inconclusive. Many factors likely contribute to these issues including small cohort sizes, sample collection, and handling and processing differences. The field of microbiome research is moving from 16S rDNA gene sequencing to a more comprehensive genomic and functional representation through whole-genome sequencing (WGS) of complete communities. Here we performed quantitative and qualitative analyses comparing WGS metagenomic data from human stool specimens using the Illumina Nextera XT and Illumina TruSeq DNA PCR-free kits, and the KAPA Biosystems Hyper Prep PCR and PCR-free systems. Significant differences in taxonomy are observed among the four different next-generation sequencing library preparations using a DNA mock community and a cell control of known concentration. We also revealed biases in error profiles, duplication rates, and loss of reads representing organisms that have a high %G+C content that can significantly impact results. As with all methods, the use of benchmarking controls has revealed critical differences among methods that impact sequencing results and later would impact study interpretation. We recommend that the community adopt PCR-free-based approaches to reduce PCR bias that affects calculations of abundance and to improve assemblies for accurate taxonomic assignment. Furthermore, the inclusion of a known-input cell spike-in control provides accurate quantitation of organisms in clinical samples.

The candidate phylum TM7 is globally distributed and often associated with human inflammatory mucosal diseases. Despite its prevalence, the TM7 phylum remains recalcitrant to cultivation, making it one of the most enigmatic phyla known. In this study, we cultivated a TM7 phylotype (TM7x) from the human oral cavity. This extremely small coccus (200-300 nm) has a distinctive lifestyle not previously observed in human-associated microbes. It is an obligate epibiont of an Actinomyces odontolyticus strain (XH001) yet also has a parasitic phase, thereby killing its host. This first completed genome (705 kb) for a human-associated TM7 phylotype revealed a complete lack of amino acid biosynthetic capacity. Comparative genomics analyses with uncultivated environmental TM7 assemblies show remarkable conserved gene synteny and only minimal gene loss/gain that may have occurred as TM7x adapted to conditions within the human host. Transcriptomic and metabolomic profiles provided the first indications, to our knowledge, that there is signaling interaction between TM7x and XH001. Furthermore, the induction of TNF-α production in macrophages by XH001 was repressed in the presence of TM7x, suggesting its potential immune suppression ability. Overall, our data provide intriguing insights into the uncultivability, pathogenicity, and unique lifestyle of this previously uncharacterized oral TM7 phylotype.

Microbes hold the key to life. They hold the secrets to our past (as the descendants of the earliest forms of life) and the prospects for our future (as we mine their genes for solutions to some of the planet's most pressing problems, from global warming to antibiotic resistance). However, the piecemeal approach that has defined efforts to study microbial genetic diversity for over 20 years and in over 30,000 genome projects risks squandering that promise. These efforts have covered less than 20% of the diversity of the cultured archaeal and bacterial species, which represent just 15% of the overall known prokaryotic diversity. Here we call for the funding of a systematic effort to produce a comprehensive genomic catalog of all cultured Bacteria and Archaea by sequencing, where available, the type strain of each species with a validly published name (currently∼11,000). This effort will provide an unprecedented level of coverage of our planet's genetic diversity, allow for the large-scale discovery of novel genes and functions, and lead to an improved understanding of microbial evolution and function in the environment.

UnlabelledHuman papillomavirus (HPV) causes a number of neoplastic diseases in humans. Here, we show a complex normal HPV community in a cohort of 103 healthy human subjects, by metagenomics analysis of the shotgun sequencing data generated from the NIH Human Microbiome Project. The overall HPV prevalence was 68.9% and was highest in the skin (61.3%), followed by the vagina (41.5%), mouth (30%), and gut (17.3%). Of the 109 HPV types as well as additional unclassified types detected, most were undetectable by the widely used commercial kits targeting the vaginal/cervical HPV types. These HPVs likely represent true HPV infections rather than transitory exposure because of strong organ tropism and persistence of the same HPV types in repeat samples. Coexistence of multiple HPV types was found in 48.1% of the HPV-positive samples. Networking between HPV types, cooccurrence or exclusion, was detected in vaginal and skin samples. Large contigs assembled from short HPV reads were obtained from several samples, confirming their genuine HPV origin. This first large-scale survey of HPV using a shotgun sequencing approach yielded a comprehensive map of HPV infections among different body sites of healthy human subjects.ImportanceThis nonbiased survey indicates that the HPV community in healthy humans is much more complex than previously defined by widely used kits that are target selective for only a few high- and low-risk HPV types for cervical cancer. The importance of nononcogenic viruses in a mixed HPV infection could be for stimulating or inhibiting a coexisting oncogenic virus via viral interference or immune cross-reaction. Knowledge gained from this study will be helpful to guide the designing of epidemiological and clinical studies in the future to determine the impact of nononcogenic HPV types on the outcome of HPV infections.

Abstract Background Our knowledge of microbial diversity in the human oral cavity has vastly expanded during the last two decades of research. However, much of what is known about the behavior of oral species to date derives from pure culture approaches and the studies combining several cultivated species, which likely does not fully reflect their function in complex microbial communities. It has been shown in studies with a limited number of cultivated species that early oral biofilm development occurs in a successional manner and that continuous low pH can lead to an enrichment of aciduric species. Observations that in vitro grown plaque biofilm microcosms can maintain similar pH profiles in response to carbohydrate addition as plaque in vivo suggests a complex microbial community can be established in the laboratory. In light of this, our primary goal was to develop a robust in vitro biofilm-model system from a pooled saliva inoculum in order to study the stability, reproducibility, and development of the oral microbiome, and its dynamic response to environmental changes from the community to the molecular level. Results Comparative metagenomic analyses confirmed a high similarity of metabolic potential in biofilms to recently available oral metagenomes from healthy subjects as part of the Human Microbiome Project. A time-series metagenomic analysis of the taxonomic community composition in biofilms revealed that the proportions of major species at 3 hours of growth are maintained during 48 hours of biofilm development. By employing deep pyrosequencing of the 16S rRNA gene to investigate this biofilm model with regards to bacterial taxonomic diversity, we show a high reproducibility of the taxonomic carriage and proportions between: 1) individual biofilm samples; 2) biofilm batches grown at different dates; 3) DNA extraction techniques and 4) research laboratories. Conclusions Our study demonstrates that we now have the capability to grow stable oral microbial in vitro biofilms containing more than one hundred operational taxonomic units (OTU) which represent 60-80% of the original inoculum OTU richness. Previously uncultivated Human Oral Taxa (HOT) were identified in the biofilms and contributed to approximately one-third of the totally captured 16S rRNA gene diversity. To our knowledge, this represents the highest oral bacterial diversity reported for an in vitro model system so far. This robust model will help investigate currently uncultivated species and the known virulence properties for many oral pathogens not solely restricted to pure culture systems, but within multi-species biofilms.

This manuscript describes the NIH Human Microbiome Project, including a brief review of human microbiome research, a history of the project, and a comprehensive overview of the consortium's recent collection of publications analyzing the human microbiome.