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1.  Heritability of Hepatic Fibrosis and Steatosis Based on a Prospective Twin Study 
Gastroenterology  2015;149(7):1784-1793.
Background & Aims
Little is known about the heritability of hepatic fibrosis, and the heritability of hepatic steatosis has not been systematically assessed in adults. We investigated the heritability of hepatic fibrosis and steatosis in a community-dwelling twin cohort.
We 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 years; average body mass index, 26.4±5.7 kg/m2). 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 non-invasively 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.
Twenty-six of the 120 subjects (21.7%) had non-alcoholic 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 (r2=0.70, P<.0001) but not between di-zygotic twins (r2=0.36, P=0.2). The level of liver fibrosis also correlated between monozygotic twins (r2=0.48, P<.002) but not between dizygotic twins (r2=.12, P=.7). In multivariable models adjusted for age, sex, and ethnicity, the heritability of hepatic steatosis (based on MRI-PDFF) was 0.52 (95% confidence interval, 0.31–0.73; P<1.1x10−11) and the heritability of hepatic fibrosis (based on liver stiffness) was 0.5 (95% confidence interval, 0.28–0.72; P<6.1 x 10−11).
A study of twins provides evidence that hepatic steatosis and hepatic fibrosis are heritable traits.
PMCID: PMC4663110  PMID: 26299412
genetic factors; fatty liver; NASH; NAFLD
3.  Comprehensive Metaproteomic Analyses of Urine in the Presence and Absence of Neutrophil-Associated Inflammation in the Urinary Tract 
Theranostics  2017;7(2):238-252.
Inflammation in the urinary tract results in a urinary proteome characterized by a high dynamic range of protein concentrations and high variability in protein content. This proteome encompasses plasma proteins not resorbed by renal tubular uptake, renal secretion products, proteins of immune cells and erythrocytes derived from trans-urothelial migration and vascular leakage, respectively, and exfoliating urothelial and squamous epithelial cells. We examined how such proteins partition into soluble urine (SU) and urinary pellet (UP) fractions by analyzing 33 urine specimens 12 of which were associated with a urinary tract infection (UTI). Using mass spectrometry-based metaproteomic approaches, we identified 5,327 non-redundant human proteins, 2,638 and 4,379 of which were associated with SU and UP fractions, respectively, and 1,206 non-redundant protein orthology groups derived from pathogenic and commensal organisms of the urogenital tract. Differences between the SU and UP proteomes were influenced by local inflammation, supported by respective comparisons with 12 healthy control urine proteomes. Clustering analyses showed that SU and UP fractions had proteomic signatures discerning UTIs, vascular injury, and epithelial cell exfoliation from the control group to varying degrees. Cases of UTI revealed clusters of proteins produced by activated neutrophils. Network analysis supported the central role of neutrophil effector proteins in the defense against invading pathogens associated with subsequent coagulation and wound repair processes. Our study expands the existing knowledge of the urinary proteome under perturbed conditions, and should be useful as reference dataset in the search of biomarkers.
PMCID: PMC5197061  PMID: 28042331
urine; clinical proteomics; urothelial cells; label-free quantitation; urinary tract infection; neutrophils; inflammation; network analysis; shotgun proteomics; urine sediment
4.  Minimally invasive sampling method identifies differences in taxonomic richness of nasal microbiomes in young infants associated with mode of delivery 
Microbial ecology  2015;71(1):233-242.
To date, there is a limited understanding of the role of the airway microbiome in the early-life development of respiratory diseases such as asthma, partly due to a lack of simple and minimally invasive sample collection methods. In order to characterize the baseline microbiome of the upper respiratory tract (URT) in infants, a comparatively non-invasive method for sampling the URT microbiome suitable for use in infants was developed. Microbiome samples were collected by placing filter paper in the nostrils of thirty-three healthy, term infants enrolled as part of the Infant Susceptibility to Pulmonary Infections and Asthma Following RSV Exposure (INSPIRE) study. After bacterial genomic DNA was extracted from the filters, amplicons were generated with universal primers targeting the V1 – V3 region of the 16S rRNA gene. This method was capable of capturing a wide variety of taxa expected to inhabit the nasal cavity. Analyses stratifying subjects by demographic and environmental factors previously observed or predicted to influence microbial communities were performed. Microbial community richness was found to be higher in infants who had been delivered via Cesarean section and in those who had been formula-fed; an association was observed between diet and delivery, which confounds this analysis. We have established a baseline URT microbiome using a non-invasive filter paper nasal sampling for this population and future studies will be performed in this large observational cohort of infants to investigate the relationship between viral infections, the URT microbiota, and the development of childhood wheezing illnesses.
PMCID: PMC4688197  PMID: 26370110
microbiome; 16S rRNA; next-generation sequencing; upper respiratory tract
5.  A robust ambient temperature collection and stabilization strategy: Enabling worldwide functional studies of the human microbiome 
Scientific Reports  2016;6:31731.
As reports on possible associations between microbes and the host increase in number, more meaningful interpretations of this information require an ability to compare data sets across studies. This is dependent upon standardization of workflows to ensure comparability both within and between studies. Here we propose the standard use of an alternate collection and stabilization method that would facilitate such comparisons. The DNA Genotek OMNIgene∙Gut Stool Microbiome Kit was compared to the currently accepted community standard of freezing to store human stool samples prior to whole genome sequencing (WGS) for microbiome studies. This stabilization and collection device allows for ambient temperature storage, automation, and ease of shipping/transfer of samples. The device permitted the same data reproducibility as with frozen samples, and yielded higher recovery of nucleic acids. Collection and stabilization of stool microbiome samples with the DNA Genotek collection device, combined with our extraction and WGS, provides a robust, reproducible workflow that enables standardized global collection, storage, and analysis of stool for microbiome studies.
PMCID: PMC4997331  PMID: 27558918
6.  Gastrointestinal microbial populations can distinguish pediatric and adolescent Acute Lymphoblastic Leukemia (ALL) at the time of disease diagnosis 
BMC Genomics  2016;17:635.
An estimated 15,000 children and adolescents under the age of 19 years are diagnosed with leukemia, lymphoma and other tumors in the USA every year. All children and adolescent acute leukemia patients will undergo chemotherapy as part of their treatment regimen. Fortunately, survival rates for most pediatric cancers have improved at a remarkable pace over the past three decades, and the overall survival rate is greater than 90 % today. However, significant differences in survival rate have been found in different age groups (94 % in 1–9.99 years, 82 % in ≥10 years and 76 % in ≥15 years). ALL accounts for about three out of four cases of childhood leukemia. Intensive chemotherapy treatment coupled with prophylactic or therapeutic antibiotic use could potentially have a long-term effect on the resident gastrointestinal (GI) microbiome. The composition of GI microbiome and its changes upon chemotherapy in pediatric and adolescent leukemia patients is poorly understood. In this study, using 16S rRNA marker gene sequences we profile the GI microbial communities of pediatric and adolescent acute leukemia patients before and after chemotherapy treatment and compare with the microbiota of their healthy siblings.
Our study cohort consisted of 51 participants, made up of matched pediatric and adolescent patients with ALL and a healthy sibling. We elucidated and compared the GI microbiota profiles of patients and their healthy sibling controls via analysis of 16S rRNA gene sequencing data. We assessed the GI microbiota composition in pediatric and adolescent patients with ALL during the course of chemotherapy by comparing stool samples taken before chemotherapy with stool samples collected at varying time points during the chemotherapeutic treatment. The microbiota profiles of both patients and control sibling groups are dominated by members of Bacteroides, Prevotella, and Faecalibacterium. At the genus level, both groups share many taxa in common, but the microbiota diversity of the patient group is significantly lower than that of the control group. It was possible to distinguish between the patient and control groups based on their microbiota profiles. The top taxa include Anaerostipes, Coprococcus, Roseburia, and Ruminococcus2 with relatively higher abundance in the control group. The observed microbiota changes are likely the result of several factors including a direct influence of therapeutic compounds on the gut flora and an indirect effect of chemotherapy on the immune system, which, in turn, affects the microbiota.
This study provides significant information on GI microbiota populations in immunocompromised children and opens up the potential for developing novel diagnostics based on stool tests and therapies to improve the dysbiotic condition of the microbiota at the time of diagnosis and in the earliest stages of chemotherapy.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-016-2965-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4986186  PMID: 27527070
Pediatric leukemia; Gastrointestinal microbiota; 16S rRNA gene sequencing; rRNA; Ribosomal RNA
7.  Microbial diversity in individuals and their household contacts following typical antibiotic courses 
Microbiome  2016;4:39.
Antibiotics 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.
We 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.
As 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.
Electronic supplementary material
The online version of this article (doi:10.1186/s40168-016-0187-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4967329  PMID: 27473422
Saliva; Gut; Skin; Microbiome; 16S rRNA; Antibiotic perturbations; Antibiotic courses; Antibiotics
8.  Similar Neutrophil-Driven Inflammatory and Antibacterial Responses in Elderly Patients with Symptomatic and Asymptomatic Bacteriuria 
Infection and Immunity  2015;83(10):4142-4153.
Differential diagnosis of asymptomatic bacteriuria (ASB) and urinary tract infection (UTI) is based on the presence of diverse symptoms, including fever (≥38.5°C), rigors, malaise, lethargy, flank pain, hematuria, suprapubic discomfort, dysuria, and urgent or frequent urination. There is consensus in the medical community that ASB warrants antibiotic treatment only for patients undergoing urological procedures that lead to mucosal bleeding, catheterized individuals whose ASB persists for more than 48 h after catheter removal, and pregnant women. Pyuria is associated with UTI and implicates host immune responses via release of antibacterial effectors and phagocytosis of pathogens by neutrophils. Such responses are not sufficiently described for ASB. Metaproteomic methods were used here to identify the pathogens and evaluate molecular evidence of distinct immune responses in cases of ASB compared to UTI in elderly patients who were hospitalized upon injury. Neutrophil-driven inflammatory responses to invading bacteria were not discernible in most patients diagnosed with ASB compared to those with UTI. In contrast, proteomic urine analysis for trauma patients with no evidence of bacteriuria, including those who suffered mucosal injuries via urethral catheterization, rarely showed evidence of neutrophil infiltration. The same enzymes contributing to the synthesis of leukotrienes LTB4 and LTC4, mediators of inflammation and pain, were found in the UTI and ASB cohorts. These data support the notion that the pathways mediating inflammation and pain in most elderly patients with ASB are not quantitatively different from those seen in most elderly patients with UTI and warrant larger clinical studies to assess whether a common antibiotic treatment strategy for elderly ASB and UTI patients is justified.
PMCID: PMC4567619  PMID: 26238715
9.  Where Next for Microbiome Research? 
PLoS Biology  2015;13(1):e1002050.
The last decade has seen a staggering transformation in our knowledge of microbial communities. Here, seven short pieces speculate as to what the next ten years might hold in store.
The development of high-throughput sequencing technologies has transformed our capacity to investigate the composition and dynamics of the microbial communities that populate diverse habitats. Over the past decade, these advances have yielded an avalanche of metagenomic data. The current stage of “van Leeuwenhoek”–like cataloguing, as well as functional analyses, will likely accelerate as DNA and RNA sequencing, plus protein and metabolic profiling capacities and computational tools, continue to improve. However, it is time to consider: what’s next for microbiome research? The short pieces included here briefly consider the challenges and opportunities awaiting microbiome research.
PMCID: PMC4300079  PMID: 25602283
10.  What Makes a Bacterial Species Pathogenic?:Comparative Genomic Analysis of the Genus Leptospira 
PLoS Neglected Tropical Diseases  2016;10(2):e0004403.
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.
Author Summary
Leptospirosis is an emerging and re-emerging globally important zoonotic infectious disease caused by spirochetes of the genus Leptospira. This genus is complex, with members that cause lethal human disease, yet mechanisms that underlie pathogenesis remain obscure. Leptospira species are divided into those that are infectious for mammals, and those that are non-infectious environmental saprophytes. Based on biological characteristics and molecular phylogeny, infectious Leptospira are further divided into pathogenic and intermediately pathogenic members. The pan-genus genomic analysis of 20 Leptospira species reported here shows the evolutionary relationship of the different Leptospira clades, and various genetic factors related to virulence and pathogenesis. Infectious Leptospira show key adaptations to mammals, for example sialic acid biosynthesis, pathogen-specific porphyrin metabolism, and the observation that pathogenic Leptospira are vitamin B12 autotrophs, able to synthesize it from a simple amino acid precursor, L-glutamine. A large novel protein family of unknown function—the Virulence Modifying proteins—is found uniquely in pathogenic Leptospira. Similarly, the CRISPR/Cas system was only found in pathogenic Leptospira. A comparative genomic analysis of a complex bacterial genus allowed us to identify large-scale changes that provides new insights into general processes by which bacteria evolve to become pathogenic.
PMCID: PMC4758666  PMID: 26890609
11.  Genomic insights into the Ixodes scapularis tick vector of Lyme disease 
Gulia-Nuss, Monika | Nuss, Andrew B. | Meyer, Jason M. | Sonenshine, Daniel E. | Roe, R. Michael | Waterhouse, Robert M. | Sattelle, David B. | de la Fuente, José | Ribeiro, Jose M. | Megy, Karine | Thimmapuram, Jyothi | Miller, Jason R. | Walenz, Brian P. | Koren, Sergey | Hostetler, Jessica B. | Thiagarajan, Mathangi | Joardar, Vinita S. | Hannick, Linda I. | Bidwell, Shelby | Hammond, Martin P. | Young, Sarah | Zeng, Qiandong | Abrudan, Jenica L. | Almeida, Francisca C. | Ayllón, Nieves | Bhide, Ketaki | Bissinger, Brooke W. | Bonzon-Kulichenko, Elena | Buckingham, Steven D. | Caffrey, Daniel R. | Caimano, Melissa J. | Croset, Vincent | Driscoll, Timothy | Gilbert, Don | Gillespie, Joseph J. | Giraldo-Calderón, Gloria I. | Grabowski, Jeffrey M. | Jiang, David | Khalil, Sayed M. S. | Kim, Donghun | Kocan, Katherine M. | Koči, Juraj | Kuhn, Richard J. | Kurtti, Timothy J. | Lees, Kristin | Lang, Emma G. | Kennedy, Ryan C. | Kwon, Hyeogsun | Perera, Rushika | Qi, Yumin | Radolf, Justin D. | Sakamoto, Joyce M. | Sánchez-Gracia, Alejandro | Severo, Maiara S. | Silverman, Neal | Šimo, Ladislav | Tojo, Marta | Tornador, Cristian | Van Zee, Janice P. | Vázquez, Jesús | Vieira, Filipe G. | Villar, Margarita | Wespiser, Adam R. | Yang, Yunlong | Zhu, Jiwei | Arensburger, Peter | Pietrantonio, Patricia V. | Barker, Stephen C. | Shao, Renfu | Zdobnov, Evgeny M. | Hauser, Frank | Grimmelikhuijzen, Cornelis J. P. | Park, Yoonseong | Rozas, Julio | Benton, Richard | Pedra, Joao H. F. | Nelson, David R. | Unger, Maria F. | Tubio, Jose M. C. | Tu, Zhijian | Robertson, Hugh M. | Shumway, Martin | Sutton, Granger | Wortman, Jennifer R. | Lawson, Daniel | Wikel, Stephen K. | Nene, Vishvanath M. | Fraser, Claire M. | Collins, Frank H. | Birren, Bruce | Nelson, Karen E. | Caler, Elisabet | Hill, Catherine A.
Nature Communications  2016;7:10507.
Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick–host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host ‘questing', prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent.
Ticks transmit a large number of pathogens that cause human diseases. Here, the authors sequence the genome of the tick Ixodes scapularis and uncover expansion of genes associated with parasitic processes unique to ticks and tick-host interactions.
PMCID: PMC4748124  PMID: 26856261
12.  Supplementation of Saturated Long-chain Fatty Acids Maintains Intestinal Eubiosis and Reduces Ethanol-induced Liver Injury in Mice 
Gastroenterology  2014;148(1):203-214.e16.
Background & Aims
Alcoholic liver disease is a leading cause of mortality. Chronic alcohol consumption is accompanied by intestinal dysbiosis, and development of alcoholic liver disease requires gut-derived bacterial products. However, little is known about how alterations to the microbiome contribute to pathogenesis of alcoholic liver disease.
We used the Tsukamoto-French mouse model which involves continuous intragastric feeding of isocaloric diet or alcohol for 3 weeks. Bacterial DNA from the cecum was extracted for deep metagenomic sequencing. Targeted metabolomics assessed concentrations of saturated fatty acids in cecal contents. To maintain intestinal metabolic homeostasis, diets of ethanol-fed and control mice were supplemented with saturated long-chain fatty acids (LCFA). Bacterial genes involved in fatty acid biosynthesis, amounts of lactobacilli, and saturated LCFA were measured in fecal samples of non-alcoholic individuals and patients with active alcohol abuse.
Analyses of intestinal contents from mice revealed alcohol-associated changes to the intestinal metagenome and metabolome, characterized by reduced synthesis of saturated LCFA. Maintaining intestinal levels of saturated fatty acids in mice resulted in eubiosis, stabilized the intestinal gut barrier and reduced ethanol-induced liver injury. Saturated LCFA are metabolized by commensal Lactobacillus and promote their growth. Proportions of bacterial genes involved in fatty acid biosynthesis were lower in feces from patients with active alcohol abuse than controls. Total levels of LCFA correlated with those of lactobacilli in fecal samples from patients with active alcohol abuse but not in controls.
In humans and mice, alcohol causes intestinal dysbiosis, reducing the capacity of the microbiome to synthesize saturated LCFA and the proportion of Lactobacillus species. Dietary approaches to restore levels of saturated fatty acids in the intestine might reduce ethanol-induced liver injury in patients with alcoholic liver disease.
PMCID: PMC4274236  PMID: 25239591
metagenomics; metabolomics; microbiome; microbiota
13.  Meta-omics uncover temporal regulation of pathways across oral microbiome genera during in vitro sugar metabolism 
The ISME Journal  2015;9(12):2605-2619.
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.
PMCID: PMC4817640  PMID: 26023872
14.  Variable responses of human and non-human primate gut microbiomes to a Western diet 
Microbiome  2015;3:53.
The 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.
Here, 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.
These 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.
Electronic supplementary material
The online version of this article (doi:10.1186/s40168-015-0120-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4645477  PMID: 26568112
Gut microbiome; Vervet; Chlorocebus aethiops; Western diet; Human evolution
15.  Stool microbiota composition is associated with the prospective risk of Plasmodium falciparum infection 
BMC Genomics  2015;16(1):631.
In humans it is unknown if the composition of the gut microbiota alters the risk of Plasmodium falciparum infection or the risk of developing febrile malaria once P. falciparum infection is established. Here we collected stool samples from a cohort composed of 195 Malian children and adults just prior to an intense P. falciparum transmission season. We assayed these samples using massively parallel sequencing of the 16S ribosomal RNA gene to identify the composition of the gut bacterial communities in these individuals. During the ensuing 6-month P. falciparum transmission season we examined the relationship between the stool microbiota composition of individuals in this cohort and their prospective risk of both P. falciparum infection and febrile malaria.
Consistent with prior studies, stool microbial diversity in the present cohort increased with age, although the overall microbiota profile was distinct from cohorts in other regions of Africa, Asia and North America. Age-adjusted Cox regression analysis revealed a significant association between microbiota composition and the prospective risk of P. falciparum infection; however, no relationship was observed between microbiota composition and the risk of developing febrile malaria once P. falciparum infection was established.
These findings underscore the diversity of gut microbiota across geographic regions, and suggest that strategic modulation of gut microbiota composition could decrease the risk of P. falciparum infection in malaria-endemic areas, potentially as an adjunct to partially effective malaria vaccines.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1819-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4546150  PMID: 26296559
Stool microbiota; Gut microbiota; Malaria; 16S rRNA gene sequencing; Plasmodium falciparum; Human; Prospective cohort
16.  Oribacterium parvum sp. nov. and Oribacterium asaccharolyticum sp. nov., obligately anaerobic bacteria from the human oral cavity, and emended description of the genus Oribacterium 
Three strictly anaerobic, Gram-positive, non-spore-forming, rod-shaped, motile bacteria, designated strains ACB1T, ACB7T and ACB8, were isolated from human subgingival dental plaque. All strains required yeast extract for growth. Strains ACB1T and ACB8 were able to grow on glucose, lactose, maltose, maltodextrin and raffinose; strain ACB7T grew weakly on sucrose only. The growth temperature range was 30–42 °C with optimum growth at 37 °C. Major metabolic fermentation end products of strain ACB1T were acetate and lactate; the only product of strains ACB7T and ACB8 was acetate. Major fatty acids of strain ACB1T were C14 : 0, C16 : 0, C16 : 1ω7c dimethyl aldehyde (DMA) and C18 : 1ω7c DMA. Major fatty acids of strain ACB7T were C12 : 0, C14 : 0, C16 : 0, C16 : 1ω7c and C16 : 1ω7c DMA. The hydrolysate of the peptidoglycan contained meso-diaminopimelic acid, indicating peptidoglycan type A1γ. Genomic DNA G+C content varied from 42 to 43.3 % between strains. According to 16S rRNA gene sequence phylogeny, strains ACB1T, ACB8 and ACB7T formed two separate branches within the genus Oribacterium, with 98.1–98.6 % sequence similarity to the type strain of the type species, Oribacterium sinus. Predicted DNA–DNA hybridization values between strains ACB1T, ACB8, ACB7T and O. sinusF0268 were <70 %. Based on distinct genotypic and phenotypic characteristics, strains ACB1T and ACB8, and strain ACB7T are considered to represent two distinct species of the genus Oribacterium, for which the names Oribacterium parvum sp. nov. and Oribacterium asaccharolyticum sp. nov. are proposed. The type strains are ACB1T ( = DSM 24637T = HM-481T = ATCC BAA-2638T) and ACB7T ( = DSM 24638T = HM-482T = ATCC BAA-2639T), respectively.
PMCID: PMC4129163  PMID: 24824639
17.  Draft Genome Sequence of Enterococcus faecalis PC1.1, a Candidate Probiotic Strain Isolated from Human Feces 
Genome Announcements  2013;1(1):e00160-12.
Enterococcus faecalis is commonly isolated from the gastrointestinal tract of healthy infants and adults, where it contributes to host health and well-being. We describe here the draft genome sequence of E. faecalis PC1.1, a candidate probiotic strain isolated from human feces.
PMCID: PMC3587934  PMID: 23469340
18.  High-quality draft genome sequences of five anaerobic oral bacteria and description of Peptoanaerobacter stomatis gen. nov., sp. nov., a new member of the family Peptostreptococcaceae 
Here we report a summary classification and the features of five anaerobic oral bacteria from the family Peptostreptococcaceae. Bacterial strains were isolated from human subgingival plaque. Strains ACC19a, CM2, CM5, and OBRC8 represent the first known cultivable members of “yet uncultured” human oral taxon 081; strain AS15 belongs to “cultivable” human oral taxon 377. Based on 16S rRNA gene sequence comparisons, strains ACC19a, CM2, CM5, and OBRC8 are distantly related to Eubacteriumyurii subs. yurii and Filifactor alocis, with 93.2 – 94.4 % and 85.5 % of sequence identity, respectively. The genomes of strains ACC19a, CM2, CM5, OBRC8 and AS15 are 2,541,543; 2,312,592; 2,594,242; 2,553,276; and 2,654,638 bp long. The genomes are comprised of 2277, 1973, 2325, 2277, and 2308 protein-coding genes and 54, 57, 54, 36, and 28 RNA genes, respectively. Based on the distinct characteristics presented here, we suggest that strains ACC19a, CM2, CM5, and OBRC8 represent a novel genus and species within the family Peptostreptococcaceae, for which we propose the name Peptoanaerobacter stomatis gen. nov., sp. nov. The type strain is strain ACC19aT (=HM-483T; =DSM 28705T; =ATCC BAA-2665T).
Electronic supplementary material
The online version of this article (doi:10.1186/s40793-015-0027-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4517659  PMID: 26221418
Peptostreptococcaceae; Peptoanaerobacter stomatis; Uncultivable bacteria; Anaerobic oral bacteria; Human oral taxa
19.  Urine Sample Preparation in 96-Well Filter Plates for Quantitative Clinical Proteomics 
Analytical Chemistry  2014;86(11):5470-5477.
Urine is an important, noninvasively collected body fluid source for the diagnosis and prognosis of human diseases. Liquid chromatography mass spectrometry (LC-MS) based shotgun proteomics has evolved as a sensitive and informative technique to discover candidate disease biomarkers from urine specimens. Filter-aided sample preparation (FASP) generates peptide samples from protein mixtures of cell lysate or body fluid origin. Here, we describe a FASP method adapted to 96-well filter plates, named 96FASP. Soluble urine concentrates containing ∼10 μg of total protein were processed by 96FASP and LC-MS resulting in 700–900 protein identifications at a 1% false discovery rate (FDR). The experimental repeatability, as assessed by label-free quantification and Pearson correlation analysis for shared proteins among replicates, was high (R ≥ 0.97). Application to urinary pellet lysates which is of particular interest in the context of urinary tract infection analysis was also demonstrated. On average, 1700 proteins (±398) were identified in five experiments. In a pilot study using 96FASP for analysis of eight soluble urine samples, we demonstrated that protein profiles of technical replicates invariably clustered; the protein profiles for distinct urine donors were very different from each other. Robust, highly parallel methods to generate peptide mixtures from urine and other body fluids are critical to increase cost-effectiveness in clinical proteomics projects. This 96FASP method has potential to become a gold standard for high-throughput quantitative clinical proteomics.
PMCID: PMC4045327  PMID: 24797144
20.  Diagnosing inflammation and infection in the urinary system via proteomics 
Current methodology for the diagnosis of diseases in the urinary system includes patient symptomology, urine analysis and urine culture. Asymptomatic bacteriuria from urethral colonization or indwelling catheters, sample contamination from perineal or vaginal sources, and non-infectious inflammatory conditions can mimic UTIs, leading to uncertainty on medical treatment decisions.
Innovative shotgun metaproteomic methods were used to analyze urine sediments from 120 patients also subjected to conventional urinalysis for various clinical reasons including suspected UTIs. The proteomic data were simultaneously searched for the presence of microbial agents, inflammation, immune responses against pathogens, and evidence of urothelial tissue injury. Hierarchical clustering analysis was performed to identify host protein patterns discerning UTI from urethral colonization and vaginal contamination of urine samples.
Organisms causing more than 98% of all UTIs and commensal microbes of the urogenital and perineal area were identified from 76 urine sediments with detection sensitivities estimated to be similar to urine culture. Proteomic data permitted a thorough evaluation of inflammatory and antimicrobial immune responses. Hierarchical clustering of the data revealed that high abundances of proteins from activated neutrophils were associated with pathogens in most cases, and correlated well with leukocyte esterase activities and leukocyte counts via microscopy. Proteomic data also allowed assessments of urothelial injury, by quantifying proteins highly expressed in red blood cells and contributing to the acute phase response. Lactobacillus and Gardnerella vaginalis were frequently identified suggesting urethral colonization and/or vaginal contamination of urine.
A metaproteomic approach of interest for routine urine clinical diagnostics is presented. As compared to urinalysis and urine culture methods, the data are derived from a single experiment for a given sample and provide additional insights into presence or absence of inflammatory responses and vaginal contamination of urine specimens.
Electronic supplementary material
The online version of this article (doi:10.1186/s12967-015-0475-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4396075  PMID: 25889401
Urinary tract infection; Innate immune response; Inflammation; Neutrophil infiltration; Urine diagnostics; Proteomics; Metaproteomics; Gardnerella; Klebsiella; Uropathogen
21.  Human Papillomavirus Community in Healthy Persons, Defined by Metagenomics Analysis of Human Microbiome Project Shotgun Sequencing Data Sets 
Journal of Virology  2014;88(9):4786-4797.
Human 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.
IMPORTANCE This 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.
PMCID: PMC3993818  PMID: 24522917
22.  Genome Sequence of the Radioresistant Bacterium Deinococcus radiodurans R1 
Science (New York, N.Y.)  1999;286(5444):1571-1577.
The complete genome sequence of the radiation resistant bacterium Deinococcus radiodurans R1 is composed of two chromosomes (2,648,615 and 412,340 basepairs), a megaplasmid (177,466 basepairs), and a small plasmid (45,702 basepairs) yielding a total genome of 3,284,123 basepairs. Multiple components distributed on the chromosomes and megaplasmid that contribute to the ability of D. radiodurans to survive under conditions of starvation, oxidative stress, and high levels of DNA-damage have been identified. D. radiodurans represents an organism in which all systems for DNA repair, DNA damage export, desiccation and starvation recovery, and genetic redundancy are present in one cell.
PMCID: PMC4147723  PMID: 10567266
23.  Genomic Encyclopedia of Bacteria and Archaea: Sequencing a Myriad of Type Strains 
PLoS Biology  2014;12(8):e1001920.
This manuscript calls for an international effort to generate a comprehensive catalog from genome sequences of all the archaeal and bacterial type strains.
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.
PMCID: PMC4122341  PMID: 25093819
24.  Habitat degradation impacts black howler monkey (Alouatta pigra) gastrointestinal microbiomes 
The ISME Journal  2013;7(7):1344-1353.
The gastrointestinal (GI) microbiome contributes significantly to host nutrition and health. However, relationships involving GI microbes, their hosts and host macrohabitats remain to be established. Here, we define clear patterns of variation in the GI microbiomes of six groups of Mexican black howler monkeys (Alouatta pigra) occupying a gradation of habitats including a continuous evergreen rainforest, an evergreen rainforest fragment, a continuous semi-deciduous forest and captivity. High throughput microbial 16S ribosomal RNA gene sequencing indicated that diversity, richness and composition of howler GI microbiomes varied with host habitat in relation to diet. Howlers occupying suboptimal habitats consumed less diverse diets and correspondingly had less diverse gut microbiomes. Quantitative real-time PCR also revealed a reduction in the number of genes related to butyrate production and hydrogen metabolism in the microbiomes of howlers occupying suboptimal habitats, which may impact host health.
PMCID: PMC3695285  PMID: 23486247
Alouatta; gut microbiome; habitat; primate
25.  Standardized Metadata for Human Pathogen/Vector Genomic Sequences 
PLoS ONE  2014;9(6):e99979.
High throughput sequencing has accelerated the determination of genome sequences for thousands of human infectious disease pathogens and dozens of their vectors. The scale and scope of these data are enabling genotype-phenotype association studies to identify genetic determinants of pathogen virulence and drug/insecticide resistance, and phylogenetic studies to track the origin and spread of disease outbreaks. To maximize the utility of genomic sequences for these purposes, it is essential that metadata about the pathogen/vector isolate characteristics be collected and made available in organized, clear, and consistent formats. Here we report the development of the GSCID/BRC Project and Sample Application Standard, developed by representatives of the Genome Sequencing Centers for Infectious Diseases (GSCIDs), the Bioinformatics Resource Centers (BRCs) for Infectious Diseases, and the U.S. National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health (NIH), informed by interactions with numerous collaborating scientists. It includes mapping to terms from other data standards initiatives, including the Genomic Standards Consortium’s minimal information (MIxS) and NCBI’s BioSample/BioProjects checklists and the Ontology for Biomedical Investigations (OBI). The standard includes data fields about characteristics of the organism or environmental source of the specimen, spatial-temporal information about the specimen isolation event, phenotypic characteristics of the pathogen/vector isolated, and project leadership and support. By modeling metadata fields into an ontology-based semantic framework and reusing existing ontologies and minimum information checklists, the application standard can be extended to support additional project-specific data fields and integrated with other data represented with comparable standards. The use of this metadata standard by all ongoing and future GSCID sequencing projects will provide a consistent representation of these data in the BRC resources and other repositories that leverage these data, allowing investigators to identify relevant genomic sequences and perform comparative genomics analyses that are both statistically meaningful and biologically relevant.
PMCID: PMC4061050  PMID: 24936976

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