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bioRxiv2017

Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid

Advances in genomics have the potential to revolutionize clinical diagnostics. Here, we examine the microbiome of vitreous (intraocular body fluid) from patients who developed endophthalmitis following cataract surgery or intravitreal injection. Endophthalmitis is an inflammation of the intraocular cavity and can lead to a permanent loss of vision. As controls, we included vitreous from endophthalmitis-negative patients, balanced salt solution used during vitrectomy, and DNA extraction blanks. We compared two DNA isolation procedures and found that an ultraclean production of reagents appeared to reduce background DNA in these low microbial biomass samples. We created a curated microbial genome database (>5700 genomes) and designed a metagenomics workflow with filtering steps to reduce DNA sequences originating from: i) human hosts, ii) ambiguousness/contaminants in public microbial reference genomes, and iii) the environment. Our metagenomic read classification revealed in nearly all cases the same microorganism than was determined in cultivation- and mass spectrometry-based analyses. For some patients, we identified the sequence type of the microorganism and antibiotic resistance genes through analyses of whole genome sequence (WGS) assemblies of isolates and metagenomic assemblies. Together, we conclude that genomics-based analyses of human ocular body fluid specimens can provide actionable information relevant to infectious disease management.

microbiology↗

bioRxiv2017

Interactions between species introduce spurious associations in microbiome studies

Microbiota contribute to many dimensions of host phenotype, including disease. To link specific microbes to specific phenotypes, microbiome-wide association studies compare microbial abundances between two groups of samples. Abundance differences, however, reflect not only direct associations with the phenotype, but also indirect effects due to microbial interactions. We found that microbial interactions could easily generate a large number of spurious associations that provide no mechanistic insight. Using techniques from statistical physics, we developed a method to remove indirect associations and applied it to the largest dataset on pediatric inflammatory bowel disease. Our method corrected the inflation of p-values in standard association tests and showed that only a small subset of associations is directly linked to the disease. Direct associations had a much higher accuracy in separating cases from controls and pointed to immunomodulation, butyrate production, and the brain-gut axis as important factors in the inflammatory bowel disease.

microbiology↗

bioRxiv2017

Discovery and whole genome sequencing of a human clinical isolate of the novel species Klebsiella quasivariicola sp. nov.

Originally thought to be a single species, Klebsiella pneumoniae has been divided into three distinct species: K. pneumoniae, K. quasipneumoniae and K. variicola. In a recent study of 1,777 extended-spectrum beta-lactamase (ESBL)-producing Klebsiella strains recovered from human infections in Houston, we discovered one strain (KPN1705) causing a wound infection that was phylogenetically distinct from all currently recognized Klebsiella species. Whole genome sequencing of strain KPN1705 revealed that it was single locus variant of the multilocus sequence type ST-1155. This sequence type was reported only once previously. To further investigate the phylogeny of these two organisms, we sequenced the genome of strain KPN1705 to closure and compared its genetic features to Klebsiella reference strains. Results demonstrated strain KPN1705 extensively shares core gene content, antimicrobial resistance genes, and plasmids with K. pneumoniae, K. quasipneumoniae and K. variicola. Since strain KPN1705 and the previously reported novel strain are phylogenetically most closely related to K. variicola, we propose the name K. quasivariicola sp. nov.

microbiology↗

bioRxiv2017

MicrobiomeDB: a systems biology platform for integrating, mining and analyzing microbiome experiments.

MicrobiomeDB (http://microbiomeDB.org) is a data discovery and analysis platform that empowers researchers to fully leverage experimental variables to interrogate microbiome datasets. MicrobiomeDB was developed in collaboration with the Eukaryotic Pathogens Bioinformatics Resource Center (http://EuPathDB.org) and leverages the infrastructure and user interface of EuPathDB, which allows users to construct in silico experiments using an intuitive graphical strategy approach. The current release of the database integrates microbial census data with sample details for nearly 14,000 samples originating from human, animal and environmental sources, including over 9,000 samples from healthy human subjects in the Human Microbiome Project (http://portal.ihmpdcc.org/). Query results can be statistically analyzed and graphically visualized via interactive web applications launched directly in the browser, providing insight into microbial community diversity and allowing users to identify taxa associated with any experimental covariate.

microbiology↗

bioRxiv2017

Associations Between Nutrition, Gut Microbiome, and Health in A Novel Nonhuman Primate Model

Red-shanked doucs (Pygathrix nemaeus) are endangered, foregut-fermenting colobine primates which are difficult to maintain in captivity. There are critical gaps in our understanding of their natural dietary habits including consumption of leaves, unripe fruit, flowers, seeds, and other plant parts. There is also a lack of understanding of enteric adaptations, including their unique microflora. To address these knowledge gaps, we used the douc as a model to study relationships between gastrointestinal microbial community structure, diet, and health. We analyzed published fecal samples as well as detailed dietary history from doucs with four distinct lifestyles (wild, semi-wild, semi-captive, and captive) and determined gastrointestinal bacterial microbiome composition using 16S rRNA sequencing. A clear gradient of microbiome composition was revealed along an axis of natural lifestyle disruption, including significant associations with diet, health, biodiversity, and microbial function. We identified potential microbial biomarkers of douc dysbiosis, including Bacteroides and Prevotella. Our results suggest a gradient-like shift in captivity causes an attendant shift to severe gut dysbiosis, thereby resulting in gastrointestinal issues.

microbiology↗

bioRxiv2017

The Epstein-Barr virus episome maneuvers between nuclear chromatin compartments during reactivation

The human genome is structurally organized in three-dimensional space to facilitate functional partitioning of transcription. We learned that the latent episome of the human Epstein-Barr virus (EBV) preferentially associates with gene-poor chromosomes and avoids gene-rich chromosomes. Kaposis sarcoma-associated herpesvirus behaves similarly, but human papillomavirus does not. Contacts localize on the EBV side to OriP, the latent origin of replication. This genetic element, and the EBNA1 protein that binds there, are sufficient to reconstitute chromosome association preferences of the entire episome. Contacts localize on the human side to gene-poor regions of chromatin distant from transcription start sites. Upon reactivation from latency, however, the episome moves away from repressive heterochromatin and toward active euchromatin. Our work adds three-dimensional relocalization to the molecular events that occur during reactivation. Involvement of a myriad of interchromosomal associations also suggests a role for this type of long - range association in gene regulation.\n\nIMPORTANCEThe human genome is structurally organized in three-dimensional space, and this structure functionally affects transcriptional activity. We set out to investigate whether a double stranded DNA virus, Epstein-Barr virus (EBV), uses similar mechanisms as the human genome to regulate transcription. We found that the EBV genome associates with repressive compartments of the nucleus during latency and active compartments during reactivation. This study is advances our knowledge of the EBV life cycle, adding three-dimensional re-localization as a novel component to the molecular events that occur during reactivation. Furthermore, the data adds to our understanding of nuclear compartments, showing that disperse interchromosomal interactions may be important for regulating transcription.

microbiology↗

bioRxiv2017

A Zebrafish Model For Ocular Tuberculosis

Ocular tuberculosis (TB) commonly causes severe inflammation and vision loss in TB-endemic countries. The mechanism by which tuberculous infection becomes established in the eye is poorly understood. We used Mycobacterium marinum-infected zebrafish larvae to study the early pathogenesis of ocular TB and found hematogenous bacterial seeding of the eye despite a functional blood retinal barrier. Prototypical early granulomas formed that involved the retinal vasculature and retinal pigment epithelium-choroid complex; characteristic locations for human ocular TB. Peripheral blood monocytes were recruited to the growing granuloma suggesting that the immune privileged nature of the eye is breached by this inflammatory focus.\n\nConflict of interestnone disclosed\n\nFundingThis work was supported in part by a Short-term fellowship to SB by Department of Health Research, Government of India.

microbiology↗

bioRxiv2017

Characterizing a thermostable Cas9 for bacterial genome editing and silencing

CRISPR-Cas9 based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here, we identify and characterize ThermoCas9: an RNA-guided DNA-endonuclease from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that ThermoCas9 is active in vitro between 20{degrees}C and 70{degrees}C, a temperature range much broader than that of the currently used Cas9 orthologues. Additionally, we demonstrate that ThermoCas9 activity at elevated temperatures is strongly associated with the structure of the employed sgRNA. Subsequently, we develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55{degrees}C in Bacillus smithii and for gene deletion at 37{degrees}C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish the first Cas9-based bacterial genome editing and silencing tool with a broad temperature range.

microbiology↗

bioRxiv2017

Characteristics of a Block to Entry via an HIV-2 Envelope, MCR

Successful viral infection depends not only on targeting the correct cell type, but also on the route taken into the cell. After entry, a retrovirus must reverse transcribe its genome and access the nucleus. Blocks to infection can arise at many different stages of the cycle; if they occur only in some cell types it can reveal hitherto unknown aspects of viral or cellular biology. A block to infection of a primary isolate of HIV-2 was previously identified in specific cell types. In this study, parameters of route of entry of the envelope protein from this primary isolate, MCR, were investigated. A critical component of the block acts at a pre-reverse transcription stage, as virions pseudotyped with MCR envelope did not undergo fusion and entry rates commensurate with productive infection. Furthermore, expression of p56lck, which regulates CD4 surface expression, partially rescued infection of MCR envelope-pseudotyped virus in restrictive cell types. Based on these findings, we propose that a part of this block results from poor cell-surface expression of CD4.

microbiology↗

bioRxiv2017

Replication of early and recent Zika virus isolates throughout mouse brain development

Fetal infection with Zika virus (ZIKV) can lead to congenital Zika virus syndrome (cZVS), which includes cortical malformations and microcephaly. The aspects of cortical development that are affected during virus infection are unknown. Using organotypic brain slice cultures generated from embryonic mice of various ages, sites of ZIKV replication including the neocortical proliferative zone and radial columns, as well as the developing midbrain, were identified. The infected radial units are surrounded by uninfected cells undergoing apoptosis, suggesting that programmed cell death may limit viral dissemination in the brain and may constrain virus associated injury. Therefore, a critical aspect of ZIKV induced neuropathology may be defined by death of uninfected cells. All ZIKV isolates assayed replicated efficiently in early and mid-gestation cultures, and two isolates examined replicated in late-gestation tissue. Alteration of neocortical cytoarchitecture, such as disruption of the highly-elongated basal processes of the radial glial progenitor cells, and impairment of postmitotic neuronal migration were also observed. These data suggest that all lineages of ZIKV tested are neurotropic, and that ZIKV infection interferes with multiple aspects of neurodevelopment that contribute to the complexity of cZVS.\n\nSignificanceZika virus infection has been associated with multiple pathologies of the central nervous system (CNS) including microcephaly, Guillain-Barre syndrome, lissencephaly, the loss of white and grey matter volume and acute myelitis. Using organotypic brain slice cultures, we determined that ZIKV replicates across different embryonic developmental stages, and viral infection can disrupt proper brain development leading to congenital CNS complications. These data illustrate that all lineages of ZIKV tested are neurotropic, and that infection may disrupt neuronal migration during brain development. The results expand our understanding of neuropathologies associated with congenital Zika virus syndrome.

microbiology↗

bioRxiv2017

Translated Blast of L Polymerase as a Hit for Novel Arenaviruses Species

Many pathogenic viruses can transmit between human and animals as zoonotic viruses and cause dangerous diseases with obvious clinical signs globally. However, the world deals seriously with these viruses when the viruses infected either human or animals especially if the infection were confirmed that classified as zoonotic (Lal et al. 2005). There are many viruses distribution in many countries around the world including Ebolavirus, Marburgvirus, SARS and MERS coronaviruses, Hendra, Nipah and arenavirus haemorrhagic fever viruses were categorized as zoonotic RNA viruses that cause epidemic in some regions such as African countries (Fichet-Calvet & Rogers 2009) (Ehichioya et al. 2010). Consequently, structural bioinformatics of virus protein like L polymerase of arenaviruses was used for monitor the future outbreak that could be happens by new species of viruses. At this research, significant similarities with hemorrhagic fever viruses including arenaviruses were found on GenBank database. Translated blast (tBLASTn) available on https://blast.ncbi.nlm.nih.gov/Blast.cgi was used for searching translated nucleotide databases using a protein query of arenavirus L polymerase (McGinnis & Madden 2004). At this research, the new and archival metazoan transcriptome sequence data of the new TSA species that available on NCBI was used for identification with arenaviruses genes. Therefore, structure bioinformatics was utilized for better understanding and predication the evolution and natural history of the pools of uncharacterized virus on Genbank database that have led to emerging haemorrhagic fever in near future around the world.

microbiology↗

bioRxiv2017

Metagenomic sequencing to replace semi-quantitative urine culture for detection of urinary tract infections: a proof of concept

ABSTRACTSemi-quantitative bacterial culture is the standard method to diagnose urinary tract infections (UTI), but bacterial growth rate limits diagnostic speed and it is unreliable when patients have been pre-treated with antibiotics. Metagenomics could increase diagnostic speed and accuracy by sequencing the microbiome and resistome directly from urine samples, bypassing culture. However, a semi-quantitative approach - as needed for diagnosing UTIs - has not been established.\n\nMetagenomics was deployed to identify and semi-quantify bacterial presence indicative of UTI, predict antimicrobial susceptibility (AMR), and results were compared to semi-quantitative culture. Whole genome sequencing of the corresponding uropathogens was done for comparison. Analysis time and cost were tracked.\n\nForty-one consecutive urine samples underwent metagenomic analysis. All culture positive samples contained >200ng of DNA, suggestive of a threshold below which UTI could be ruled out solely based on DNA quantity. A semi-quantitative Diagnostic Index (DI) was created by multiplying the total DNA quantity by the relative abundance of uropathogens per urine sample. The DI allowed discrimination of UTI from non-UTI samples in all but 1 case. Metagenomic detection of AMR determinants correctly predicted the phenotype of uropathogens in 20 of 32 cases. The metagenomic work-flow was 31h and cost {euro}116 per sample, but could be reduced to 4.5h and {euro}5 for low-DNA-yield non-UTI samples.\n\nThe genomic determinants of AMR and their distribution across uropathogens need to be better understood for prediction of AMR phenotypes by metagenomics. The introduction of the DI demonstrates the potential of semi-quantitative metagenomics to replace culture as rapid diagnostic method for UTI.

microbiology↗

bioRxiv2017

Labeling of prokaryotic mRNA in live cells using fluorescent in situ hybridization of transcript-annealing molecular beacons (FISH-TAMB)

High-throughput sequencing and cellular imaging have expanded our knowledge of microbial diversity and expression of cellular activity. However, it remains challenging to characterize low-abundance, slow-growing microorganisms that play key roles in biogeochemical cycling. With the goal of isolating transcriptionally active cells of these microorganisms from environmental samples, we developed fluorescent in situ hybridization of transcript-annealing molecular beacons (FISH-TAMB) to label living prokaryotic cells. FISH-TAMB utilizes polyarginine cell-penetrating peptides to deliver molecular beacons across cell walls and membranes. Target cells are fluorescently labeled via hybridization between molecular beacons and messenger RNA of targeted functional genes. FISH-TAMBs target specificity and deliverance into both bacterial and archaeal cells were demonstrated by labeling intracellular methyl-coenzyme M reductase A (mcrA) transcripts expressed by Escherichia coli mcrA+, Methanosarcina barkeri, and a methanogenic enrichment of deep continental fracture fluid. Growth curve analysis supported sustained cellular viability following FISH-TAMB treatment. Flow cytometry and confocal microscopy detected labeled single cells and single cells in aggregates with unlabeled cells. As FISH-TAMB is amenable to target any functional gene of interest, when coupled with cell sorting, imaging, and sequencing techniques, FISH-TAMB will enable characterization of key uncharacterized rare biosphere microorganisms and of the syntrophically activated metabolic pathways between physically associated microorganisms.

microbiology↗

bioRxiv2017

Identification and kinetics characterization of a wax ester hydrolase from a feather-degrading actinomycete

Streptomyces fradiae var. k11 is a Gram-positive soil microorganism capable of degrading chicken feathers. Apart from being mostly protein, chicken feathers have a considerable level of lipids, with wax esters being the largest lipid class. The waxes may pose a challenge while rendering the feathers into coproducts, such as feather meal, and so the identification of a wax-ester hydrolase is warranted. A draft genome sequence of S. fradiae var. k11 was used to identify 14 gene sequences of potential lipid-degrading enzymes. The genes were expressed in E. coli BL21(DE3) cells on a pET vector and screened for activity. Four of the 14 enzymes had detectable activity, with two of the enzymes, SFK3309 and SFK3087, active against p-nitrophenyl palmitate, a representative water-insoluble substrate. A modified enzymatic assay was designed to measure activity against three model wax substrates: jojoba oil, beeswax, and cetyl-palmitate. SFK3309 was characterized to hydrolyze all three wax substrates. Kinetic experiments for SFK3309 were performed with cetyl-palmitate at 37{degrees}C, pH 8.0. The Km was determined to be 850 {micro}M and the Kcat was 11.63 s-1. Through the characterization of SFK3309 as a wax-ester hydrolase, biotechnological implications of wax ester hydrolases in the rendering of many industrial wastes can be substantiated for further studies.

microbiology↗

bioRxiv2017

Acute Hepatitis E Virus infection in two geographical regions of Nigeria

Hepatitis E virus (HEV) remains a major public health concern in resource limited regions of the world. Yet data reporting is suboptimal and surveillance system inadequate. In Nigeria, there is dearth of information on prevalence of acute HEV infection. This study was therefore designed to describe acute HEV infection among antenatal clinic attendees and asymptomatic community dwellers from two geographical regions in Nigeria.\n\nIn this study 750 plasma samples were tested for HEV IgM by Enzyme Linked lmmunosorbent Assay (ELISA) technique. The tested samples were randomly selected from a pool of 1,115 samples previously collected from selected populations (pregnant women - 272, Oyo community dwellers - 438, Anambra community dwellers - 405) for viral hepatitis studies between September 2012 and August 2013.\n\nOne (0.4%) pregnant woman in her 3rd trimester had detectable HEV IgM, while community dwellers from the two study locations had zero prevalence rates of HEV IgM.\n\nDetection of HEV IgM in a pregnant woman, especially in her 3rd trimester is of clinical and epidemiological significance. The need therefore exists for establishment of a robust HEV surveillance system in Nigeria, and especially amidst the pregnant population in a bid to improve maternal and child health.

microbiology↗

bioRxiv2017

Global transcriptome analysis of Aedes aegypti mosquitoes in response to Zika virus infection

Zika virus (ZIKV) of the Flaviviridae family is a recently emerged mosquito-borne virus that has been implicated in the surge of the number of microcephaly instances in South America. The recent rapid spread of the virus led to its declaration as a global health emergency by the World Health Organization. The virus is transmitted mainly by the mosquito Aedes aegypti that also vectors dengue virus, however little is known about the interactions of the virus with the mosquito vector. In this study, we investigated the transcriptome profiles of whole Ae. aegypti mosquitoes in response to ZIKV infection at 2, 7, and 14 days post-infection using RNA-Seq. Results showed changes in the abundance of a large number of transcripts at each time point following infection, with 18 transcripts commonly changed among the three time points. Gene ontology analysis revealed that most of the altered genes are involved in metabolic process, cellular process and proteolysis. In addition, 486 long intergenic non-coding RNAs were identified that were altered upon ZIKV infection. Further, we found correlational changes of a number of potential mRNA target genes with that of altered host microRNAs. The outcomes provide a basic understanding of Ae. aegypti responses to ZIKV and helps to determine host factors involved in replication or mosquito host anti-viral response against the virus.\n\nImportanceVector-borne viruses pose great risks on human health. Zika virus has recently emerged as a global threat, rapidly expanding its distribution. Understanding the interactions of the virus with mosquito vectors at the molecular level is vital for devising new approaches in inhibiting virus transmission. In this study, we embarked on analyzing the transcriptional response of Aedes aegypti mosquitoes to Zika virus infection. Results showed large changes both in coding and long non-coding RNAs. Analysis of these genes showed similarities with other flaviviruses, including dengue virus, which is transmitted by the same mosquito vector. The outcomes provide a global picture of changes in the mosquito vector in response to Zika virus infection.

microbiology↗

bioRxiv2017

Genomic and structural features of the Yellow Fever virus from the 2016-2017 Brazilian outbreak

Brazil has been suffering a severe sylvatic epidemic of yellow fever virus (YFV) since late 2016. Analysis of full-length YFV genomes from all hosts involved in the Brazilian 2017 outbreak reveals that they belong to sub-lineage 1E within modern-lineage, but display several unique amino acid substitutions in highly conserved positions at NS3 and NS5 viral proteins. Evolutionary analyses indicate that YFV carrying that set of amino acid substitution circulates in the Southern Brazilian region for several months before being detected in December 2016. Structural and selection analyses support that some of these substitutions were under positive selection and could impact enzyme structure and function. Altogether, this evidence demonstrated that the current Brazilian YFV carries unique amino acid signatures in the non-structural proteins and support the hypothesis that those substitutions may be affecting the viral fitness and transmissibility.

microbiology↗

bioRxiv2017

Comparison of algorithms for the detection of enteroviruses in stool specimens from children diagnosed with Acute Flaccid Paralysis

With poliovirus eradication within reach, the WHO has included in its recommendations a cell-culture independent algorithm for enterovirus surveillance. This study was designed to compare both the cell culture dependent and independent algorithms and assess how either might impact our perception of the diversity of enterovirus types present in a sample.\n\nSixteen paired samples (16 isolates from RD cell culture and their corresponding stool suspension. i.e. 32 samples) from AFP cases in Nigeria were analyzed in this study. One of these 16 sample pairs (the control) was previously identified and confirmed as poliovirus 2 (PV-2). All the samples were subjected to RNA extraction, cDNA synthesis, RT-snPCR (the WHO recommended cell-culture independent algorithm) and its modifications for co-infection detection and resolution. Amplicons were sequenced and strains identified using the enterovirus genotyping tool and phylogenetic analysis.\n\nThe enterovirus diversity was shown to be the same between RD cell culture isolates and fecal suspension for the control and five (7, 10, 11, 12 & 14) of the samples analyzed. It was however, different for the remaining 10 (62.5%) samples analyzed. Fourteen different enterovirus types were identified in this study. To be precise, 9 (CV-B4, E6, E7, E13, E14, E19, E29, EV-B75 and EV-B77) and 5 (CV-A1, CV-A11, CV-A13, EV-C99 and PV2) EV-B and EV-C types, respectively where detected in this study. It is crucial to mention that E19 and EV-B75were only recovered from RD cell culture isolates while E14, EV-B77, CV-A11 and CV-A13 were only recovered from fecal suspension.\n\nThe results of this study show that both the cell culture dependent and independent protocols recommended by the WHO for enterovirus detection unavoidably bias our perception of the diversity of enterovirus types present in a sample. Hence, rather than jettison one for the other, effort should be directed at harmonizing both for increased sensitivity.

microbiology↗