Deep Metagenomic Sequencing Research Articles

Mass Azithromycin Distribution and Community Microbiome: A Cluster-Randomized Trial.

BackgroundMass distributions of oral azithromycin have long been used to eliminate trachoma, and they are now being proposed to reduce childhood mortality. The observed benefit appears to be augmented with each additional treatment, suggesting a possible community-level effect. Here, we assess whether 2 biannual mass treatments of preschool children affect the community’s gut microbiome at 6 months after the last distribution.MethodsIn this cluster-randomized controlled trial, children aged 1–60 months in the Dossa region of Niger were randomized at the village level to receive a single dose of azithromycin or placebo every 6 months. Fecal samples were collected 6 months after the second treatment for metagenomic deep sequencing. The prespecified primary outcome was the Euclidean PERMANOVA of the gut microbiome, or effectively the distance between the genus-level centroid at the community level, with the secondary outcome being the Simpson’s α diversity.ResultsIn the azithromycin arm, the gut microbial structures were significantly different than in the placebo arm (Euclidean PERMANOVA, P < .001). Further, the diversity of the gut microbiome in the azithromycin arm was significantly lower than in the placebo arm (inverse Simpson’s index, P = .005).ConclusionsTwo mass azithromycin administrations, 6 months apart, in preschool children led to long-term alterations of the gut microbiome structure and community diversity. Here, long-term microbial alterations in the community did not imply disease but were associated with an improvement in childhood mortality.Clinical Trials RegistrationNCT02048007.

Microscopic and Molecular Evidence of the First Elasmobranch Adomavirus, the Cause of Skin Disease in a Giant Guitarfish, Rhynchobatus djiddensis.

ABSTRACTOnly eight families of double-stranded DNA (dsDNA) viruses are known to infect vertebrate animals. During an investigation of papillomatous skin disease in an elasmobranch species, the giant guitarfish (Rhynchobatus djiddensis), a novel virus, distinct from all known viral families in regard to particle size, morphology, genome organization, and helicase phylogeny was discovered. Large inclusion bodies containing 75-nm icosahedral viral particles were present within epithelial cell nuclei in the proliferative skin lesions. Deep metagenomic sequencing revealed a 22-kb circular dsDNA viral genome, tentatively named guitarfish “adomavirus” (GAdoV), with only distant homology to two other fish viruses, Japanese eel endothelial cell-infecting virus (JEECV) and a recently reported marbled eel virus. Phylogenetic analysis of the helicase domain places the guitarfish virus in a novel clade that is equidistant between members of the Papillomaviridae and Polyomaviridae families. Specific PCR, quantitative PCR, and in situ hybridization were used to detect, quantify, and confirm that GAdoV DNA was localized to affected epithelial cell nuclei. Changes in the viral titer, as well as the presence of a hybridization signal, coincided with the progression and then final resolution of gross and microscopic lesions. The results indicate that GAdoV is the causative agent of the proliferative skin lesions.

Metagenomic deep sequencing of aqueous fluid detects intraocular lymphomas

IntroductionCurrently, the detection of pathogens or mutations associated with intraocular lymphomas heavily relies on prespecified, directed PCRs. With metagenomic deep sequencing (MDS), an unbiased high-throughput sequencing approach, all pathogens as...

Insights into Microalga and Bacteria Interactions of Selected Phycosphere Biofilms Using Metagenomic, Transcriptomic, and Proteomic Approaches.

Microalga are of high relevance for the global carbon cycling and it is well-known that they are associated with a microbiota. However, it remains unclear, if the associated microbiota, often found in phycosphere biofilms, is specific for the microalga strains and which role individual bacterial taxa play. Here we provide experimental evidence that Chlorella saccharophila, Scenedesmus quadricauda, and Micrasterias crux-melitensis, maintained in strain collections, are associated with unique and specific microbial populations. Deep metagenome sequencing, binning approaches, secretome analyses in combination with RNA-Seq data implied fundamental differences in the gene expression profiles of the microbiota associated with the different microalga. Our metatranscriptome analyses indicates that the transcriptionally most active bacteria with respect to key genes commonly involved in plant–microbe interactions in the Chlorella (Trebouxiophyceae) and Scenedesmus (Chlorophyceae) strains belong to the phylum of the α-Proteobacteria. In contrast, in the Micrasterias (Zygnematophyceae) phycosphere biofilm bacteria affiliated with the phylum of the Bacteroidetes showed the highest gene expression rates. We furthermore show that effector molecules known from plant–microbe interactions as inducers for the innate immunity are already of relevance at this evolutionary early plant-microbiome level.

How Do Advanced Molecular Tests Compare to Routine Clinical Laboratory Evaluation of CSF in Meningoencephalitis? A Study in 10 Urban Emergency Departments Across the USA

BackgroundThe EMERGEncy ID Net Study Group is investigating whether advanced molecular tests (AMT) increase the detection of causative agents in the CSF of patients presenting with meningoencephalitis (ME). We report findings from a pilot study using AMT on 18 CSF samples from 10 US Urban Emergency Departments. The purpose of the pilot was to compare the performance of these four AMT to established clinical laboratory methods.MethodsWe investigated four AMT: (1) BioFire FilmArray ME Panel targeting 14 causative agents; (2) an in-house target-directed next generation sequencing assay targeting 25 agents; (3) a microarray capable of detecting >2,500 agents; and (4) deep metagenomic next generation sequencing. For targeted sequencing, loci from 12 DNA-based and 13 RNA-based pathogens were amplified from the extracts by multiplex PCR. All sequencing was performed on an Illumina MiSeq using 500 cycle v2 Reagent Kits. Reads from the targeted sequencing were aligned to the 25 specific reference target sequences using Bowtie2 while metagenomics reads were processed with the taxonomic sequence classifying software Kraken. For microarray analysis, Lawrence Livermore Microbial Detection Array v2 arrays were hybridized with Cy3-labeled DNA or cDNA. Scanned images of arrays were analyzed by CLiMax 3.1.ResultsEight CSF samples had results positive for well-established causes of ME from prior testing (Table). The pilot study demonstrated none of the four AMT detected all causative agents in the eight CSF samples known to have well-established causes of ME. BioFire and targeted sequencing performed best, both detecting 6/8, metagenomics deep sequencing detected 3/8, and microarray detected 1/8.ConclusionDespite the sophistication of AMT, they cannot detect pathogens they do not target, that are present in small numbers, or that have been eliminated from the CSF by the immune response. Despite the theoretical potential for microarray and metagenomic sequencing to detect thousands of different agents, the agents probably must be present at high levels for detection.Clinical laboratory evaluationBioFire Film ArrayTargeted sequencingMicroarrayMetagenomic sequencing Cryptococcus spp.43302 Streptococcus pneumoniae 11111Enterovirus2220West Nile Virus1N/Aa000 aWest Nile Virus is not on the BioFire Panel.Disclosures All authors: No reported disclosures.

Characterization of the Genomic Diversity of Norovirus in Linked Patients Using a Metagenomic Deep Sequencing Approach

Norovirus (NoV) is the leading cause of gastroenteritis worldwide. A robust cell culture system does not exist for NoV and therefore detailed characterization of outbreak and sporadic strains relies on molecular techniques. In this study, we employed a metagenomic approach that uses non-specific amplification followed by next-generation sequencing to whole genome sequence NoV genomes directly from clinical samples obtained from 8 linked patients. Enough sequencing depth was obtained for each sample to use a de novo assembly of near-complete genome sequences. The resultant consensus sequences were then used to identify inter-host nucleotide variations that occur after direct transmission, analyze amino acid variations in the major capsid protein, and provide evidence of recombination events. The analysis of intra-host quasispecies diversity was possible due to high coverage-depth. We also observed a linear relationship between NoV viral load in the clinical sample and the number of sequence reads that could be attributed to NoV. The method demonstrated here has the potential for future use in whole genome sequence analyses of other RNA viruses isolated from clinical, environmental, and food specimens.

Crimean congo hemorrhagic fever, 2013 and 2014 Sudan

Purpose: The German Partnership Program for Excellence in Biological and Health Security was launched in 2013 and is funded by the German Federal Foreign Office. Currently, the program funds projects in 18 countries in the fields of infectious disease surveillance, detection & diagnostics, biosafety & biosecurity, capacity building and networking. In Sudan one focus of the partnership is the detection of highly pathogenic viruses and identification of known and yet unknown etiological agents in outbreak situations. Methods & Materials: In 2014 an outbreak of hemorrhagic fever in humans was reported from different states of Sudan (South Darfur, West Kordofan, South Kordofan). The NPHL investigated the cases and forwarded 29 sera samples from patients suffering from hemorrhagic fever to the RKI. The sample-set included a panel of 10 sera collected during former hemorrhagic fever outbreaks in the same region in 2013. All sera were tested with qPCR assays for Marburg virus, Ebola virus and CCHFV. Additionally all samples were subjected to metagenomic deep sequencing on an Illumina MiSeq sequencer. Results: CCHF was identified by two independent qPCR assays in a sample from November 2013 and November 2014, respectively. Deep sequencing confirmed these results. Based on the available sequences the novel CCHFV strain ‘Sudan 2014’ shares 96% identity (na) with its closest relative CCHFV SPU 187/90 from South Africa. Conclusion: CCHFV is reported to be transmitted by ticks in Europe, Asia and Africa and known as etiological agent of severe hemorrhagic fever in humans and livestock. Beside insect-repellent no preventive measures are available. The pathogenicity and characteristics of this novel strain have yet to be determined by cell-culture isolation and serology. Further molecular analysis will contribute to clarify the divergence of the CCHFV strains detected in 2013 and 2014. First results including possible co-infections will be presented.

Erratum to: Illuminating uveitis: metagenomic deep sequencing identifies common and rare pathogens.

Doan et al. Genome Medicine (2016) 8:123 DOI 10.1186/s13073-016-0377-x ERRATUM Open Access Erratum to: Illuminating uveitis: metagenomic deep sequencing identifies common and rare pathogens Thuy Doan 1,2† , Michael R. Wilson 3,4† , Emily D. Crawford 3,5 , Eric D. Chow 3 , Lillian M. Khan 3 , Kristeene A. Knopp 3 , Brian D. O’Donovan 3 , Dongxiang Xia 6 , Jill K. Hacker 6 , Jay M. Stewart 2 , John A. Gonzales 1,2 , Nisha R. Acharya 1,2 and Joseph L. DeRisi 3* Erratum It has come to our attention that there is an error in Fig. 3a for this article [1]. The correct version of Fig. 3a can be found below. The red markers now reflect the se- quence differences. The text is correct. There was also a row omitted in Additional file 1: Table S1. The revised version can be found below. Additional file Additional file 1: Table S1. List of nucleotide substitutions identified in subject 6’s RV genome. The patient’s RV genome was aligned with the Stuttgart strain (GenBank DQ388280.1). A nucleotide change was considered a substitution only if the change was present in ≥ 4 reads or in 80% of the total reads at that nucleotide position. (PDF 120 kb) Author details Francis I. Proctor Foundation, University of California San Francisco, San Francisco, CA, USA. 2 Department of Ophthalmology, University of California San Francisco, San Francisco, CA, USA. 3 Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, USA. Department of Neurology, University of California San Francisco, San Francisco, CA, USA. 5 Howard Hughes Medical Institute, Chevy Chase, MD, USA. 6 California Department of Public Health, Richmond, CA, USA. Received: 7 November 2016 Accepted: 7 November 2016 Reference 1. Doan T, Wilson MR, Crawford ED, Chow ED, Khan LM, Knopp KA, O’Donovan BD, Xia D, Hacker JK, Stewart JM, Gonzales JA, Archarya NR, DeRisi JL. Illuminating uveitis: metagenomic deep sequencing identifies common and rare pathogens. Genome Med. 2016;8:106. * Correspondence: joe@derisilab.ucsf.edu Equal contributors Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, USA © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Current and future molecular diagnostics for ocular infectious diseases.

Confirmation of ocular infections can pose great challenges to the clinician. A fundamental limitation is the small amounts of specimen that can be obtained from the eye. Molecular diagnostics can circumvent this limitation and have been shown to be more sensitive than conventional culture. The purpose of this review is to describe new molecular methods and to discuss the applications of next-generation sequencing-based approaches in the diagnosis of ocular infections. Efforts have focused on improving the sensitivity of pathogen detection using molecular methods. This review describes a new molecular target for Toxoplasma gondii-directed polymerase chain reaction assays. Molecular diagnostics for Chlamydia trachomatis and Acanthamoeba species are also discussed. Finally, we describe a hypothesis-free approach, metagenomic deep sequencing, which can detect DNA and RNA pathogens from a single specimen in one test. In some cases, this method can provide the geographic location and timing of the infection. Pathogen-directed PCRs have been powerful tools in the diagnosis of ocular infections for over 20 years. The use of next-generation sequencing-based approaches, when available, will further improve sensitivity of detection with the potential to improve patient care.

A reference gene catalogue of the pig gut microbiome.

The pig is a major species for livestock production and is also extensively used as the preferred model species for analyses of a wide range of human physiological functions and diseases1. The importance of the gut microbiota in complementing the physiology and genome of the host is now well recognized2. Knowledge of the functional interplay between the gut microbiota and host physiology in humans has been advanced by the human gut reference catalogue3,4. Thus, establishment of a comprehensive pig gut microbiome gene reference catalogue constitutes a logical continuation of the recently published pig genome5. By deep metagenome sequencing of faecal DNA from 287 pigs, we identified 7.7 million non-redundant genes representing 719 metagenomic species. Of the functional pathways found in the human catalogue, 96% are present in the pig catalogue, supporting the potential use of pigs for biomedical research. We show that sex, age and host genetics are likely to influence the pig gut microbiome. Analysis of the prevalence of antibiotic resistance genes demonstrated the effect of eliminating antibiotics from animal diets and thereby reducing the risk of spreading antibiotic resistance associated with farming systems.

Illuminating uveitis: metagenomic deep sequencing identifies common and rare pathogens.

BackgroundOcular infections remain a major cause of blindness and morbidity worldwide. While prognosis is dependent on the timing and accuracy of diagnosis, the etiology remains elusive in ~50 % of presumed infectious uveitis cases. The objective of this study is to determine if unbiased metagenomic deep sequencing (MDS) can accurately detect pathogens in intraocular fluid samples of patients with uveitis.MethodsThis is a proof-of-concept study, in which intraocular fluid samples were obtained from five subjects with known diagnoses, and one subject with bilateral chronic uveitis without a known etiology. Samples were subjected to MDS, and results were compared with those from conventional diagnostic tests. Pathogens were identified using a rapid computational pipeline to analyze the non-host sequences obtained from MDS.ResultsUnbiased MDS of intraocular fluid produced results concordant with known diagnoses in subjects with (n = 4) and without (n = 1) uveitis. Samples positive for Cryptococcus neoformans, Toxoplasma gondii, and herpes simplex virus 1 as tested by a Clinical Laboratory Improvement Amendments-certified laboratory were correctly identified with MDS. Rubella virus was identified in one case of chronic bilateral idiopathic uveitis. The subject’s strain was most closely related to a German rubella virus strain isolated in 1992, one year before he developed a fever and rash while living in Germany. The pattern and the number of viral identified mutations present in the patient’s strain were consistent with long-term viral replication in the eye.ConclusionsMDS can identify fungi, parasites, and DNA and RNA viruses in minute volumes of intraocular fluid samples. The identification of chronic intraocular rubella virus infection highlights the eye’s role as a long-term pathogen reservoir, which has implications for virus eradication and emerging global epidemics.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-016-0344-6) contains supplementary material, which is available to authorized users.

Metagenomic recovery of phage genomes of uncultured freshwater actinobacteria.

Low-GC Actinobacteria are among the most abundant and widespread microbes in freshwaters and have largely resisted all cultivation efforts. Consequently, their phages have remained totally unknown. In this work, we have used deep metagenomic sequencing to assemble eight complete genomes of the first tailed phages that infect freshwater Actinobacteria. Their genomes encode the actinobacterial-specific transcription factor whiB, frequently found in mycobacteriophages and also in phages infecting marine pelagic Actinobacteria. Its presence suggests a common and widespread strategy of modulation of host transcriptional machinery upon infection via this transcriptional switch. We present evidence that some whiB-carrying phages infect the acI lineage of Actinobacteria. At least one of them encodes the ADP-ribosylating component of the widespread bacterial AB toxins family (for example, clostridial toxin). We posit that the presence of this toxin reflects a 'trojan horse' strategy, providing protection at the population level to the abundant host microbes against eukaryotic predators.

Multiplexed Metagenomic Deep Sequencing To Analyze the Composition of High-Priority Pathogen Reagents.

Laboratories studying high-priority pathogens need comprehensive methods to confirm microbial species and strains while also detecting contamination. Metagenomic deep sequencing (MDS) inventories nucleic acids present in laboratory stocks, providing an unbiased assessment of pathogen identity, the extent of genomic variation, and the presence of contaminants. Double-stranded cDNA MDS libraries were constructed from RNA extracted from in vitro-passaged stocks of six viruses (La Crosse virus, Ebola virus, canine distemper virus, measles virus, human respiratory syncytial virus, and vesicular stomatitis virus). Each library was dual indexed and pooled for sequencing. A custom bioinformatics pipeline determined the organisms present in each sample in a blinded fashion. Single nucleotide variant (SNV) analysis identified viral isolates. We confirmed that (i) each sample contained the expected microbe, (ii) dual indexing of the samples minimized false assignments of individual sequences, (iii) multiple viral and bacterial contaminants were present, and (iv) SNV analysis of the viral genomes allowed precise identification of the viral isolates. MDS can be multiplexed to allow simultaneous and unbiased interrogation of mixed microbial cultures and (i) confirm pathogen identity, (ii) characterize the extent of genomic variation, (iii) confirm the cell line used for virus propagation, and (iv) assess for contaminating microbes. These assessments ensure the true composition of these high-priority reagents and generate a comprehensive database of microbial genomes studied in each facility. MDS can serve as an integral part of a pathogen-tracking program which in turn will enhance sample security and increase experimental rigor and precision. IMPORTANCE Both the integrity and reproducibility of experiments using select agents depend in large part on unbiased validation to ensure the correct identity and purity of the species in question. Metagenomic deep sequencing (MDS) provides the required level of validation by allowing for an unbiased and comprehensive assessment of all the microbes in a laboratory stock.

Not All Particles Are Equal: The Selective Enrichment of Particle-Associated Bacteria from the Mediterranean Sea.

We have used two metagenomic approaches, direct sequencing of natural samples and sequencing after enrichment, to characterize communities of prokaryotes associated to particles. In the first approximation, different size filters (0.22 and 5 μm) were used to identify prokaryotic microbes of free-living and particle-attached bacterial communities in the Mediterranean water column. A subtractive metagenomic approach was used to characterize the dominant microbial groups in the large size fraction that were not present in the free-living one. They belonged mainly to Actinobacteria, Planctomycetes, Flavobacteria and Proteobacteria. In addition, marine microbial communities enriched by incubation with different kinds of particulate material have been studied by metagenomic assembly. Different particle kinds (diatomaceous earth, sand, chitin and cellulose) were colonized by very different communities of bacteria belonging to Roseobacter, Vibrio, Bacteriovorax, and Lacinutrix that were distant relatives of genomes already described from marine habitats. Besides, using assembly from deep metagenomic sequencing from the particle-specific enrichments we were able to determine a total of 20 groups of contigs (eight of them with >50% completeness) and reconstruct de novo five new genomes of novel species within marine clades (>79% completeness and <1.8% contamination). We also describe for the first time the genome of a marine Rhizobiales phage that seems to infect a broad range of Alphaproteobacteria and live in habitats as diverse as soil, marine sediment and water column. The metagenomic recruitment of the communities found by direct sequencing of the large size filter and by enrichment had nearly no overlap. These results indicate that these reconstructed genomes are part of the rare biosphere which exists at nominal levels under natural conditions.

Marine microbial community structure assessed from combined metagenomic analysis and ribosomal amplicon deep-sequencing

ABSTRACTThe microbial taxonomic composition of the three domains of life in two coastal plankton samples was assessed by random total community metagenomic sequencing and PCR-based rDNA amplicon deep-sequencing in order to compare the resulting diversity and investigate possible limitations and complementarities of each method. The various universal primer sets, used to amplify different hypervariable rDNA regions, revealed the same major high-level taxonomic groups in Bacteria and unicellular Eukaryota, and showed a scarce Archaea apparent richness. However, significant differences were found between the different primer sets (p-value < 0.05, with the Kolmogorov–Smirnov test), regarding both operational taxonomic unit (OTU) richness and relative abundance of the major high-level taxonomic groups detected. Based on the metagenomic approach, the phylum Bacteroidetes dominated the prokaryotic community, followed by Proteobacteria, while the detected eukaryotic unicellular taxa belonged to the groups of Alveolata, Fungi, Chlorophyta, Stramenopiles and Phaeophyceae. These groups were found to carry genes typically found in microbial communities, which are linked to DNA, RNA and protein metabolism and the synthesis of nucleotides, amino acids, carbohydrates and vitamins. Although our findings suggest that the total community metagenomic approach can provide a more comprehensive picture of the planktonic microbial community structure, a number of issues associated with this approach emerged. These issues include the still relatively high cost compared to amplicon sequencing, the possible low coverage of the full marine diversity, the insufficiency of databases for other gene markers than the small subunit gene, and the bias towards bacterial sequences because of their higher abundance relative to eukaryotes in marine environments.

Unique wastewater antibiotic resistome

Antibiotic Resistance![Figure][1] Genes that confer antibiotic resistance to microbiota present in wastewater treatment plants, like this treatment lagoon, are specific to that community, suggesting there is little risk that they could be transferred to the human gut microbiome PHOTO: GEORG GERSTER/SCIENCE SOURCE Antibiotic resistance is currently high on the list of alarms. Wastewater treatment plants essentially use microbiological bioremediation to clean up water. As such, they are an obvious potential source of mobile antibiotic resistance genes from human pathogens. Munck et al. have been investigating the risks wastewater plants pose for antibiotic resistance. They identified resistance genes in samples from 7 different years by screening with 15 antibiotics. Deep metagenomic sequencing revealed a stably maintained core set of novel genes that conferred resistance to all the antibiotics tested. This “resistome” was specific to this microbiological community, indicating that there is in fact little scope for transfer into the human gut microbiota. Although this is a reason to be cheerful, it is sobering to note that the unique wastewater genescape persists because of high levels of ambient antibiotics. Nat. Commun. 10.1038/ncomms9452 (2015) [1]: pending:yes

Archaeal ancestors of eukaryotes: not so elusive any more.

The origin of eukaryotes is one of the hardest problems in evolutionary biology and sometimes raises the ominous specter of irreducible complexity. Reconstruction of the gene repertoire of the last eukaryotic common ancestor (LECA) has revealed a highly complex organism with a variety of advanced features but no detectable evolutionary intermediates to explain their origin. Recently, however, genome analysis of diverse archaea led to the discovery of apparent ancestral versions of several signature eukaryotic systems, such as the actin cytoskeleton and the ubiquitin network, that are scattered among archaea. These findings inspired the hypothesis that the archaeal ancestor of eukaryotes was an unusually complex form with an elaborate intracellular organization. The latest striking discovery made by deep metagenomic sequencing vindicates this hypothesis by showing that in phylogenetic trees eukaryotes fall within a newly identified archaeal group, the Lokiarchaeota, which combine several eukaryotic signatures previously identified in different archaea. The discovery of complex archaea that are the closest living relatives of eukaryotes is most compatible with the symbiogenetic scenario for eukaryogenesis.

Perilipin-2 Modulates Lipid Absorption and Microbiome Responses in the Mouse Intestine.

Obesity and its co-morbidities, such as fatty liver disease, are increasingly prevalent worldwide health problems. Intestinal microorganisms have emerged as critical factors linking diet to host physiology and metabolic function, particularly in the context of lipid homeostasis. We previously demonstrated that deletion of the cytoplasmic lipid drop (CLD) protein Perilipin-2 (Plin2) in mice largely abrogates long-term deleterious effects of a high fat (HF) diet. Here we test the hypotheses that Plin2 function impacts the earliest steps of HF diet-mediated pathogenesis as well as the dynamics of diet-associated changes in gut microbiome diversity and function. WT and perilipin-2 null mice raised on a standard chow diet were randomized to either low fat (LF) or HF diets. After four days, animals were assessed for changes in physiological (body weight, energy balance, and fecal triglyceride levels), histochemical (enterocyte CLD content), and fecal microbiome parameters. Plin2-null mice had significantly lower respiratory exchange ratios, diminished frequencies of enterocyte CLDs, and increased fecal triglyceride levels compared with WT mice. Microbiome analyses, employing both 16S rRNA profiling and metagenomic deep sequencing, indicated that dietary fat content and Plin2 genotype were significantly and independently associated with gut microbiome composition, diversity, and functional differences. These data demonstrate that Plin2 modulates rapid effects of diet on fecal lipid levels, enterocyte CLD contents, and fuel utilization properties of mice that correlate with structural and functional differences in their gut microbial communities. Collectively, the data provide evidence of Plin2 regulated intestinal lipid uptake, which contributes to rapid changes in the gut microbial communities implicated in diet-induced obesity.

A novel outbreak enterovirus D68 strain associated with acute flaccid myelitis cases in the USA (2012–14): a retrospective cohort study

Enterovirus D68 was implicated in a widespread outbreak of severe respiratory illness across the USA in 2014 and has also been reported sporadically in patients with acute flaccid myelitis. We aimed to investigate the association between enterovirus D68 infection and acute flaccid myelitis during the 2014 enterovirus D68 respiratory outbreak in the USA. Patients with acute flaccid myelitis who presented to two hospitals in Colorado and California, USA, between Nov 24, 2013, and Oct 11, 2014, were included in the study. Additional cases identified from Jan 1, 2012, to Oct 4, 2014, via statewide surveillance were provided by the California Department of Public Health. We investigated the cause of these cases by metagenomic next-generation sequencing, viral genome recovery, and enterovirus D68 phylogenetic analysis. We compared patients with acute flaccid myelitis who were positive for enterovirus D68 with those with acute flaccid myelitis but negative for enterovirus D68 using the two-tailed Fisher's exact test, two-sample unpaired t test, and Mann-Whitney U test. 48 patients were included: 25 with acute flaccid myelitis, two with enterovirus-associated encephalitis, five with enterovirus-D68-associated upper respiratory illness, and 16 with aseptic meningitis or encephalitis who tested positive for enterovirus. Enterovirus D68 was detected in respiratory secretions from seven (64%) of 11 patients comprising two temporally and geographically linked acute flaccid myelitis clusters at the height of the 2014 outbreak, and from 12 (48%) of 25 patients with acute flaccid myelitis overall. Phylogenetic analysis revealed that all enterovirus D68 sequences associated with acute flaccid myelitis grouped into a clade B1 strain that emerged in 2010. Of six coding polymorphisms in the clade B1 enterovirus D68 polyprotein, five were present in neuropathogenic poliovirus or enterovirus D70, or both. One child with acute flaccid myelitis and a sibling with only upper respiratory illness were both infected by identical enterovirus D68 strains. Enterovirus D68 viraemia was identified in a child experiencing acute neurological progression of his paralytic illness. Deep metagenomic sequencing of cerebrospinal fluid from 14 patients with acute flaccid myelitis did not reveal evidence of an alternative infectious cause to enterovirus D68. These findings strengthen the putative association between enterovirus D68 and acute flaccid myelitis and the contention that acute flaccid myelitis is a rare yet severe clinical manifestation of enterovirus D68 infection in susceptible hosts. National Institutes of Health, University of California, Abbott Laboratories, and the Centers for Disease Control and Prevention.

Key roles for freshwater Actinobacteria revealed by deep metagenomic sequencing.

Freshwater ecosystems are critical but fragile environments directly affecting society and its welfare. However, our understanding of genuinely freshwater microbial communities, constrained by our capacity to manipulate its prokaryotic participants in axenic cultures, remains very rudimentary. Even the most abundant components, freshwater Actinobacteria, remain largely unknown. Here, applying deep metagenomic sequencing to the microbial community of a freshwater reservoir, we were able to circumvent this traditional bottleneck and reconstruct de novo seven distinct streamlined actinobacterial genomes. These genomes represent three new groups of photoheterotrophic, planktonic Actinobacteria. We describe for the first time genomes of two novel clades, acMicro (Micrococcineae, related to Luna2,) and acAMD (Actinomycetales, related to acTH1). Besides, an aggregate of contigs belonged to a new branch of the Acidimicrobiales. All are estimated to have small genomes (approximately 1.2 Mb), and their GC content varied from 40 to 61%. One of the Micrococcineae genomes encodes a proteorhodopsin, a rhodopsin type reported for the first time in Actinobacteria. The remarkable potential capacity of some of these genomes to transform recalcitrant plant detrital material, particularly lignin-derived compounds, suggests close linkages between the terrestrial and aquatic realms. Moreover, abundances of Actinobacteria correlate inversely to those of Cyanobacteria that are responsible for prolonged and frequently irretrievable damage to freshwater ecosystems. This suggests that they might serve as sentinels of impending ecological catastrophes.