Integrated next-generation sequencing of 16S rDNA and metaproteomics differentiate the healthy urine microbiome from asymptomatic bacteriuria in neuropathic bladder associated with spinal cord injury

Background Clinical dogma is that healthy urine is sterile and the presence of bacteria with an inflammatory response is indicative of urinary tract infection (UTI). Asymptomatic bacteriuria (ABU) represents the state in which bacteria are present but the inflammatory response is negligible. Differentiating ABU from UTI is diagnostically challenging, but critical because overtreatment of ABU can perpetuate antimicrobial resistance while undertreatment of UTI can result in increased morbidity and mortality. In this study, we describe key characteristics of the healthy and ABU urine microbiomes utilizing 16S rRNA gene (16S rDNA) sequencing and metaproteomics, with the future goal of utilizing this information to personalize the treatment of UTI based on key individual characteristics. Methods A cross-sectional study of 26 healthy controls and 27 healthy subjects at risk for ABU due to spinal cord injury-related neuropathic bladder (NB) was conducted. Of the 27 subjects with NB, 8 voided normally, 8 utilized intermittent catheterization, and 11 utilized indwelling Foley urethral catheterization for bladder drainage. Urine was obtained by clean catch in voiders, or directly from the catheter in subjects utilizing catheters. Urinalysis, urine culture and 16S rDNA sequencing were performed on all samples, with metaproteomic analysis performed on a subsample. Results A total of 589454 quality-filtered 16S rDNA sequence reads were processed through a NextGen 16S rDNA analysis pipeline. Urine microbiomes differ by normal bladder function vs. NB, gender, type of bladder catheter utilized, and duration of NB. The top ten bacterial taxa showing the most relative abundance and change among samples were Lactobacillales, Enterobacteriales, Actinomycetales, Bacillales, Clostridiales, Bacteroidales, Burkholderiales, Pseudomonadales, Bifidobacteriales and Coriobacteriales. Metaproteomics confirmed the 16S rDNA results, and functional human protein-pathogen interactions were noted in subjects where host defenses were initiated. Conclusions Counter to clinical belief, healthy urine is not sterile. The healthy urine microbiome is characterized by a preponderance of Lactobacillales in women and Corynebacterium in men. The presence and duration of NB and method of urinary catheterization alter the healthy urine microbiome. An integrated approach of 16S rDNA sequencing with metaproteomics improves our understanding of healthy urine and facilitates a more personalized approach to prevention and treatment of infection

Intestinal fungi contribute to development of alcoholic liver disease

This study was supported in part by NIH grants R01 AA020703, U01 AA021856 and by Award Number I01BX002213 from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development (to B.S.). K.H. was supported by a DFG (Deutsche Forschungsgemeinschaft) fellowship (HO/ 5690/1-1). S.B. was supported by a grant from the Swiss National Science Foundation (P2SKP3_158649). G.G. received funding from the Yale Liver Center NIH P30 DK34989 and R.B. from NIAAA grant U01 AA021908. A.K. received support from NIH grants RC2 AA019405, R01 AA020216 and R01 AA023417. G.D.B. is supported by funds from the Wellcome Trust. We acknowledge the Human Tissue and Cell Research (HTCR) Foundation for making human tissue available for research and Hepacult GmbH (Munich, Germany) for providing primary human hepatocytes for in vitro analyses. We thank Dr. Chien-Yu Lin Department of Medicine, Fu-Jen Catholic University, Taiwan for statistical analysis.Peer reviewedPublisher PD

Urethral Catheter Biofilms Reveal Plasticity in Bacterial Composition and Metabolism and Withstand Host Immune Defenses in Hypoxic Environment

Biofilms composed of multiple microorganisms colonize the surfaces of indwelling urethral catheters that are used serially by neurogenic bladder patients and cause chronic infections. Well-adapted pathogens in this niche are Escherichia coli, Proteus, and Enterococcus spp., species that cycle through adhesion and multilayered cell growth, trigger host immune responses, are starved off nutrients, and then disperse. Viable microbial foci retained in the urinary tract recolonize catheter surfaces. The molecular adaptations of bacteria in catheter biofilms (CBs) are not well-understood, promising new insights into this pathology based on host and microbial meta-omics analyses from clinical specimens. We examined catheters from nine neurogenic bladder patients longitudinally over up to 6 months. Taxonomic analyses from 16S rRNA gene sequencing and liquid chromatography–tandem mass spectrometry (LC-MS/MS)–based proteomics revealed that 95% of all catheter and corresponding urinary pellet (UP) samples contained bacteria. CB biomasses were dominated by Enterobacteriaceae spp. and often accompanied by lactic acid and anaerobic bacteria. Systemic antibiotic drug treatments of patients resulted in either transient or lasting microbial community perturbations. Neutrophil effector proteins were abundant not only in UP but also CB samples, indicating their penetration of biofilm surfaces. In the context of one patient who advanced to a kidney infection, Proteus mirabilis proteomic data suggested a combination of factors associated with this disease complication: CB biomasses were high; the bacteria produced urease alkalinizing the pH and triggering urinary salt deposition on luminal catheter surfaces; P. mirabilis utilized energy-producing respiratory systems more than in CBs from other patients. The NADH:quinone oxidoreductase II (Nqr), a Na+ translocating enzyme not operating as a proton pump, and the nitrate reductase A (Nar) equipped the pathogen with electron transport chains promoting growth under hypoxic conditions. Both P. mirabilis and E. coli featured repertoires of transition metal ion acquisition systems in response to human host-mediated iron and zinc sequestration. We discovered a new drug target, the Nqr respiratory system, whose deactivation may compromise P. mirabilis growth in a basic pH milieu. Animal models would not allow such molecular-level insights into polymicrobial biofilm metabolism and interactions because the complexity cannot be replicated

Characterization of the Fecal Microbiome from Non-Human Wild Primates Reveals Species Specific Microbial Communities

BACKGROUND: Host-associated microbes comprise an integral part of animal digestive systems and these interactions have a long evolutionary history. It has been hypothesized that the gastrointestinal microbiome of humans and other non-human primates may have played significant roles in host evolution by facilitating a range of dietary adaptations. We have undertaken a comparative sequencing survey of the gastrointestinal microbiomes of several non-human primate species, with the goal of better understanding how these microbiomes relate to the evolution of non-human primate diversity. Here we present a comparative analysis of gastrointestinal microbial communities from three different species of Old World wild monkeys. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed fecal samples from three different wild non-human primate species (black-and-white colobus [Colubus guereza], red colobus [Piliocolobus tephrosceles], and red-tailed guenon [Cercopithecus ascanius]). Three samples from each species were subjected to small subunit rRNA tag pyrosequencing. Firmicutes comprised the vast majority of the phyla in each sample. Other phyla represented were Bacterioidetes, Proteobacteria, Spirochaetes, Actinobacteria, Verrucomicrobia, Lentisphaerae, Tenericutes, Planctomycetes, Fibrobacateres, and TM7. Bray-Curtis similarity analysis of these microbiomes indicated that microbial community composition within the same primate species are more similar to each other than to those of different primate species. Comparison of fecal microbiota from non-human primates with microbiota of human stool samples obtained in previous studies revealed that the gut microbiota of these primates are distinct and reflect host phylogeny. CONCLUSION/SIGNIFICANCE: Our analysis provides evidence that the fecal microbiomes of wild primates co-vary with their hosts, and that this is manifested in higher intraspecies similarity among wild primate species, perhaps reflecting species specificity of the microbiome in addition to dietary influences. These results contribute to the limited body of primate microbiome studies and provide a framework for comparative microbiome analysis between human and non-human primates as well as a comparative evolutionary understanding of the human microbiome

Comparative Genomics of Gardnerella vaginalis Strains Reveals Substantial Differences in Metabolic and Virulence Potential

Gardnerella vaginalis is described as a common vaginal bacterial species whose presence correlates strongly with bacterial vaginosis (BV). Here we report the genome sequencing and comparative analyses of three strains of G. vaginalis. Strains 317 (ATCC 14019) and 594 (ATCC 14018) were isolated from the vaginal tracts of women with symptomatic BV, while Strain 409-05 was isolated from a healthy, asymptomatic individual with a Nugent score of 9.Substantial genomic rearrangement and heterogeneity were observed that appeared to have resulted from both mobile elements and substantial lateral gene transfer. These genomic differences translated to differences in metabolic potential. All strains are equipped with significant virulence potential, including genes encoding the previously described vaginolysin, pili for cytoadhesion, EPS biosynthetic genes for biofilm formation, and antimicrobial resistance systems, We also observed systems promoting multi-drug and lantibiotic extrusion. All G. vaginalis strains possess a large number of genes that may enhance their ability to compete with and exclude other vaginal colonists. These include up to six toxin-antitoxin systems and up to nine additional antitoxins lacking cognate toxins, several of which are clustered within each genome. All strains encode bacteriocidal toxins, including two lysozyme-like toxins produced uniquely by strain 409-05. Interestingly, the BV isolates encode numerous proteins not found in strain 409-05 that likely increase their pathogenic potential. These include enzymes enabling mucin degradation, a trait previously described to strongly correlate with BV, although commonly attributed to non-G. vaginalis species.Collectively, our results indicate that all three strains are able to thrive in vaginal environments, and therein the BV isolates are capable of occupying a niche that is unique from 409-05. Each strain has significant virulence potential, although genomic and metabolic differences, such as the ability to degrade mucin, indicate that the detection of G. vaginalis in the vaginal tract provides only partial information on the physiological potential of the organism

A framework for human microbiome research

A variety of microbial communities and their genes (the microbiome) exist throughout the human body, with fundamental roles in human health and disease. The National Institutes of Health (NIH)-funded Human Microbiome Project Consortium has established a population-scale framework to develop metagenomic protocols, resulting in a broad range of quality-controlled resources and data including standardized methods for creating, processing and interpreting distinct types of high-throughput metagenomic data available to the scientific community. Here we present resources from a population of 242 healthy adults sampled at 15 or 18 body sites up to three times, which have generated 5,177 microbial taxonomic profiles from 16S ribosomal RNA genes and over 3.5 terabases of metagenomic sequence so far. In parallel, approximately 800 reference strains isolated from the human body have been sequenced. Collectively, these data represent the largest resource describing the abundance and variety of the human microbiome, while providing a framework for current and future studies

Structure, function and diversity of the healthy human microbiome

Author Posting. © The Authors, 2012. This article is posted here by permission of Nature Publishing Group. The definitive version was published in Nature 486 (2012): 207-214, doi:10.1038/nature11234.Studies of the human microbiome have revealed that even healthy individuals differ remarkably in the microbes that occupy habitats such as the gut, skin and vagina. Much of this diversity remains unexplained, although diet, environment, host genetics and early microbial exposure have all been implicated. Accordingly, to characterize the ecology of human-associated microbial communities, the Human Microbiome Project has analysed the largest cohort and set of distinct, clinically relevant body habitats so far. We found the diversity and abundance of each habitat’s signature microbes to vary widely even among healthy subjects, with strong niche specialization both within and among individuals. The project encountered an estimated 81–99% of the genera, enzyme families and community configurations occupied by the healthy Western microbiome. Metagenomic carriage of metabolic pathways was stable among individuals despite variation in community structure, and ethnic/racial background proved to be one of the strongest associations of both pathways and microbes with clinical metadata. These results thus delineate the range of structural and functional configurations normal in the microbial communities of a healthy population, enabling future characterization of the epidemiology, ecology and translational applications of the human microbiome.This research was supported in part by National Institutes of Health grants U54HG004969 to B.W.B.; U54HG003273 to R.A.G.; U54HG004973 to R.A.G., S.K.H. and J.F.P.; U54HG003067 to E.S.Lander; U54AI084844 to K.E.N.; N01AI30071 to R.L.Strausberg; U54HG004968 to G.M.W.; U01HG004866 to O.R.W.; U54HG003079 to R.K.W.; R01HG005969 to C.H.; R01HG004872 to R.K.; R01HG004885 to M.P.; R01HG005975 to P.D.S.; R01HG004908 to Y.Y.; R01HG004900 to M.K.Cho and P. Sankar; R01HG005171 to D.E.H.; R01HG004853 to A.L.M.; R01HG004856 to R.R.; R01HG004877 to R.R.S. and R.F.; R01HG005172 to P. Spicer.; R01HG004857 to M.P.; R01HG004906 to T.M.S.; R21HG005811 to E.A.V.; M.J.B. was supported by UH2AR057506; G.A.B. was supported by UH2AI083263 and UH3AI083263 (G.A.B., C. N. Cornelissen, L. K. Eaves and J. F. Strauss); S.M.H. was supported by UH3DK083993 (V. B. Young, E. B. Chang, F. Meyer, T. M. S., M. L. Sogin, J. M. Tiedje); K.P.R. was supported by UH2DK083990 (J. V.); J.A.S. and H.H.K. were supported by UH2AR057504 and UH3AR057504 (J.A.S.); DP2OD001500 to K.M.A.; N01HG62088 to the Coriell Institute for Medical Research; U01DE016937 to F.E.D.; S.K.H. was supported by RC1DE0202098 and R01DE021574 (S.K.H. and H. Li); J.I. was supported by R21CA139193 (J.I. and D. S. Michaud); K.P.L. was supported by P30DE020751 (D. J. Smith); Army Research Office grant W911NF-11-1-0473 to C.H.; National Science Foundation grants NSF DBI-1053486 to C.H. and NSF IIS-0812111 to M.P.; The Office of Science of the US Department of Energy under Contract No. DE-AC02-05CH11231 for P.S. C.; LANL Laboratory-Directed Research and Development grant 20100034DR and the US Defense Threat Reduction Agency grants B104153I and B084531I to P.S.C.; Research Foundation - Flanders (FWO) grant to K.F. and J.Raes; R.K. is an HHMI Early Career Scientist; Gordon&BettyMoore Foundation funding and institutional funding fromthe J. David Gladstone Institutes to K.S.P.; A.M.S. was supported by fellowships provided by the Rackham Graduate School and the NIH Molecular Mechanisms in Microbial Pathogenesis Training Grant T32AI007528; a Crohn’s and Colitis Foundation of Canada Grant in Aid of Research to E.A.V.; 2010 IBM Faculty Award to K.C.W.; analysis of the HMPdata was performed using National Energy Research Scientific Computing resources, the BluBioU Computational Resource at Rice University

Effect of Macondo Prospect 252 Oil On Microbiota Associated With Pelagic Sargassum in the Northern Gulf of Mexico

The environmental impact of major oil spills on marine microorganisms has yet to be thoroughly investigated using molecular biology techniques. The Deepwater Horizon (DWH) drilling rig explosion of 2010 affected an approximately 176,000 km2 surface area of the Gulf of Mexico (GOM) when an estimated 210 million gallons of oil from the Macondo Prospect spilled into the environment. Pelagic Sargassum, a complex of two surface drifting species (Sargassum natans and Sargassum fluitans) of marine brown macroalgae and a critically important habitat in the GOM ecosystem, was suffused by Macondo Prospect 252 oil released during the DWH event. Using 16S rRNA PCR and Roche 454 pyrosequencing, the effect of the oil on the bacterial population associated with pelagic Sargassum and contiguous waters was examined by comparing sequence data generated from samples collected from oiled and non-oiled locations in the northern GOM. Sequence data showed similar microbial composition in Sargassum regardless of exposure to oil primarily dominated by five phyla; Proteobacteria, Bacteroidetes, Actinobacteria, Verrucomicrobia, and unclassified bacteria. The microbial composition in water samples was significantly less diverse than for Sargassum and consisted primarily of Proteobacteria, Firmicutes, and Bacteroidetes. Due to the evenly distributed abundance of microbial species on oiled and non-oiled pelagic Sargassum, study findings indicate that DWH spilled oil had minimal effect on the composition and diversity of the microbial community associated with Sargassum and contiguous waters. However, higher abundances of Sulfitobacter and one species of Psychrobacter were found in oiled water samples when compared to non-oiled water samples indicating some effect of DHW oil in the microbial composition of seawater. Though there are a number of marine studies using molecular biology approaches, this is the first molecular examination of the impact of the DWH oil spill on bacterial communities associated with pelagic Sargassum and contiguous waters from the GOM

Gut Microbial Changes in Diabetic db/db Mice and Recovery of Microbial Diversity upon Pirfenidone Treatment

The leptin receptor-deficient db/db mouse model is an accepted in vivo model to study obesity, type 2 diabetes, and diabetic kidney disease. Healthy gastrointestinal (GI) microbiota has been linked to weight loss, improved glycemic control, and physiological benefits. We investigated the effect of various drugs on the GI microbiota of db/db mice as compared to control db/m mice. Treatment with long-acting pirfenidone (PFD) increased gut microbial diversity in diabetic db/db mice. Firmicutes, the most abundant phylum in db/m mice, decreased significantly in abundance in db/db mice but showed increased abundance with long-acting PFD treatment. Several bacterial taxa, including Lactobacillus and some Bacteroides, were less abundant in db/db mice and more abundant in long-acting-PFD-treated db/db mice. Long-acting PFD treatment reduced the abundance of Akkermansia muciniphila (5%) as compared to db/db mice (~15%). We conclude that gut microbial dysbiosis observed in db/db mice was partially reversed by long-acting PFD treatment and hypothesize that PFD has beneficial effects, in part, via its influence on the gut microbial metabolite profile. In quantitatively assessing urine metabolites, we observed a high abundance of diabetic ketoacidosis biomarkers, including 3-hydroxybutyric acid and acetoacetic acid in db/db mice, which were less abundant in the long-acting-PFD-treated db/db mice