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September 22, 2019  |  

Multi-omics approach identifies novel pathogen-derived prognostic biomarkers in patients with Pseudomonas aeruginosa bloodstream infection

Pseudomonas aeruginosa is a human pathogen that causes health-care associated blood stream infections (BSI). Although P. aeruginosa BSI are associated with high mortality rates, the clinical relevance of pathogen-derived prognostic biomarker to identify patients at risk for unfavorable outcome remains largely unexplored. We found novel pathogen-derived prognostic biomarker candidates by applying a multi-omics approach on a multicenter sepsis patient cohort. Multi-level Cox regression was used to investigate the relation between patient characteristics and pathogen features (2298 accessory genes, 1078 core protein levels, 107 parsimony-informative variations in reported virulence factors) with 30-day mortality. Our analysis revealed that presence of the helP gene encoding a putative DEAD-box helicase was independently associated with a fatal outcome (hazard ratio 2.01, p = 0.05). helP is located within a region related to the pathogenicity island PAPI-1 in close proximity to a pil gene cluster, which has been associated with horizontal gene transfer. Besides helP, elevated protein levels of the bacterial flagellum protein FliL (hazard ratio 3.44, p < 0.001) and of a bacterioferritin-like protein (hazard ratio 1.74, p = 0.003) increased the risk of death, while high protein levels of a putative aminotransferase were associated with an improved outcome (hazard ratio 0.12, p < 0.001). The prognostic potential of biomarker candidates and clinical factors was confirmed with different machine learning approaches using training and hold-out datasets. The helP genotype appeared the most attractive biomarker for clinical risk stratification due to its relevant predictive power and ease of detection.

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September 22, 2019  |  

Clonal emergence of invasive multidrug-resistant Staphylococcus epidermidis deconvoluted via a combination of whole-genome sequencing and microbiome analyses.

Pathobionts, bacteria that are typically human commensals but can cause disease, contribute significantly to antimicrobial resistance. Staphylococcus epidermidis is a prototypical pathobiont as it is a ubiquitous human commensal but also a leading cause of healthcare-associated bacteremia. We sought to determine the etiology of a recent increase in invasive S. epidermidis isolates resistant to linezolid.Whole-genome sequencing (WGS) was performed on 176 S. epidermidis bloodstream isolates collected at the MD Anderson Cancer Center in Houston, Texas, between 2013 and 2016. Molecular relationships were assessed via complementary phylogenomic approaches. Abundance of the linezolid resistance determinant cfr was determined in stool samples via reverse-transcription quantitative polymerase chain reaction.Thirty-nine of the 176 strains were linezolid resistant (22%). Thirty-one of the 39 linezolid-resistant S. epidermidis infections were caused by a particular clone resistant to multiple antimicrobials that spread among leukemia patients and carried cfr on a 49-kb plasmid (herein called pMB151a). The 6 kb of pMB151a surrounding the cfr gene was nearly 100% identical to a cfr-containing plasmid isolated from livestock-associated staphylococci in China. Analysis of serial stool samples from leukemia patients revealed progressive staphylococcal domination of the intestinal microflora and an increase in cfr abundance following linezolid use.The combination of linezolid use plus transmission of a multidrug-resistant clone drove expansion of invasive, linezolid-resistant S. epidermidis. Our results lend support to the notion that a combination of antibiotic stewardship plus infection control measures may help to control the spread of a multidrug-resistant pathobiont.

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September 22, 2019  |  

Tumor-specific mitochondrial DNA variants are rarely detected in cell-free DNA.

The use of blood-circulating cell-free DNA (cfDNA) as a “liquid biopsy” in oncology is being explored for its potential as a cancer biomarker. Mitochondria contain their own circular genomic entity (mitochondrial DNA, mtDNA), up to even thousands of copies per cell. The mutation rate of mtDNA is several orders of magnitude higher than that of the nuclear DNA. Tumor-specific variants have been identified in tumors along the entire mtDNA, and their number varies among and within tumors. The high mtDNA copy number per cell and the high mtDNA mutation rate make it worthwhile to explore the potential of tumor-specific cf-mtDNA variants as cancer marker in the blood of cancer patients. We used single-molecule real-time (SMRT) sequencing to profile the entire mtDNA of 19 tissue specimens (primary tumor and/or metastatic sites, and tumor-adjacent normal tissue) and 9 cfDNA samples, originating from 8 cancer patients (5 breast, 3 colon). For each patient, tumor-specific mtDNA variants were detected and traced in cfDNA by SMRT sequencing and/or digital PCR to explore their feasibility as cancer biomarker. As a reference, we measured other blood-circulating biomarkers for these patients, including driver mutations in nuclear-encoded cfDNA and cancer-antigen levels or circulating tumor cells. Four of the 24 (17%) tumor-specific mtDNA variants were detected in cfDNA, however at much lower allele frequencies compared to mutations in nuclear-encoded driver genes in the same samples. Also, extensive heterogeneity was observed among the heteroplasmic mtDNA variants present in an individual. We conclude that there is limited value in tracing tumor-specific mtDNA variants in blood-circulating cfDNA with the current methods available. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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September 22, 2019  |  

A molecular window into the biology and epidemiology of Pneumocystis spp.

Pneumocystis, a unique atypical fungus with an elusive lifestyle, has had an important medical history. It came to prominence as an opportunistic pathogen that not only can cause life-threatening pneumonia in patients with HIV infection and other immunodeficiencies but also can colonize the lungs of healthy individuals from a very early age. The genus Pneumocystis includes a group of closely related but heterogeneous organisms that have a worldwide distribution, have been detected in multiple mammalian species, are highly host species specific, inhabit the lungs almost exclusively, and have never convincingly been cultured in vitro, making Pneumocystis a fascinating but difficult-to-study organism. Improved molecular biologic methodologies have opened a new window into the biology and epidemiology of Pneumocystis. Advances include an improved taxonomic classification, identification of an extremely reduced genome and concomitant inability to metabolize and grow independent of the host lungs, insights into its transmission mode, recognition of its widespread colonization in both immunocompetent and immunodeficient hosts, and utilization of strain variation to study drug resistance, epidemiology, and outbreaks of infection among transplant patients. This review summarizes these advances and also identifies some major questions and challenges that need to be addressed to better understand Pneumocystis biology and its relevance to clinical care. Copyright © 2018 American Society for Microbiology.

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September 22, 2019  |  

Using XCAVATOR and EXCAVATOR2 to Identify CNVs from WGS, WES, and TS Data.

Copy Number Variants (CNVs) are structural rearrangements contributing to phenotypic variation but also associated with many disease states. In recent years, the identification of CNVs from high-throughput sequencing experiments has become a common practice for both research and clinical purposes. Several computational methods have been developed so far. In this unit, we describe and give instructions on how to run two read count-based tools, XCAVATOR and EXCAVATOR2, which are tailored for the detection of both germline and somatic CNVs from different sequencing experiments (whole-genome, whole-exome, and targeted) in various disease contexts and population genetic studies. © 2018 by John Wiley & Sons, Inc.© 2018 John Wiley & Sons, Inc.

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September 22, 2019  |  

Comprehensive evaluation of the host responses to infection with differentially virulent classical swine fever virus strains in pigs.

Classical swine fever virus (CSFV) infection causes most variable clinical syndromes from chronic or latent infection to acute death, and it is generally acknowledged that the course of disease is affected by both virus and host factors. To compare host immune responses to differentially virulent CSFV strains in pigs, fifteen 8-week-old specific-pathogen-free pigs were randomly divided into four groups and inoculated with the CSFV Shimen strain (a highly virulent strain), the HLJZZ2014 strain (a moderately virulent strains), C-strain (an avirulent strain), and DMEM (mock control), respectively. Infection with the Shimen or HLJZZ2014 strain resulted in fever, clinical signs and histopathological lesions, which were not observed in the C-strain-inoculated pigs, though low viral genome copies were detected in the peripheral blood and tissue samples. The data showed that the virulence of the strains affected the outcome of duration and intensity of the disease rather than the tissue tropism of the virus. Furthermore, leukopenia, lymphocytopenia, differentiation of T-cells, and the secretion of cytokines associated with inflammation or apoptosis such as interferon alpha (IFN-a), tumor necrosis factor alpha (TNF-a), interleukin 2 (IL-2), IL-4, IL-6, and IL-10 were induced by the virulent CSFV infection, the differences reflected in onset and extent of the regulation. Taken together, our results revealed that the major differences among the three strains resided in the kinetics of host response to the infection: severe and immediate with the highly virulent strain, while progressive and delayed with the moderately virulent one. This comparative study will help to dissect the pathogenesis of CSFV. Copyright © 2018 Elsevier B.V. All rights reserved.

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September 22, 2019  |  

Characterization of Haemophilus parasuis serovar 2 CL120103, a moderately virulent strain in China

Haemophilus parasuis is an important bacterium affecting pigs, causing Glässer’s disease. To further characterize this species, we determined the complete genomic sequence of H. parasuis CL120103, which was isolated from diseased pigs. The strain H. parasuis CL120103 was identified as serovar 2. The size of the largest scaffold is 2,326,318 bp and contains 145 large contigs, with the N50 contig being 20,573 bp in length. The complete genome of H. parasuis CL120103 is 2,305,354 bp in length with 39.97% GC content and contains 2227 protein-coding genes, 19 ribosomal rRNA operons and 60 tRNA genes. Sequence similarity of the genome of H. parasuis CL120103 to the previously sequenced genome of H. parasuis was up to 96% and query cover to 86%. Annotation of the genome of H. parasuis CL120103 identified a number of genes encoding potential virulence factors. These virulence factors are involved in metabolism, adhesion, secretion and LPS biosynthesis. These related genes pave the way to better understand mechanisms underlying metabolic capabilities. The comprehensive genetic and phylogenetic analysis shows that H. parasuis is closely related to Actinobacillus pleuropneumoniae and provides a foundation for future experimental confirmation of the virulence and pathogen-host interactions in H. parasuis.

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September 22, 2019  |  

Genomics of Corynebacterium striatum, an emerging multidrug-resistant pathogen of immunocompromised patients.

Corynebacterium striatum is an emerging multidrug-resistant (MDR) pathogen of immunocompromised and chronically ill patients. The objective of these studies was to provide a detailed genomic analysis of disease-causing C. striatum and determine the genomic drivers of resistance and resistance-gene transmission.A multi-institutional and prospective pathogen genomics programme flagged seven MDR C. striatum infections occurring close in time, and specifically in immunocompromised patients with underlying respiratory diseases. Whole genome sequencing was used to identify clonal relationships among strains, genetic causes of antimicrobial resistance, and their mobilization capacity. Matrix-assisted linear desorption/ionization-time-of-flight analyses of sequenced isolates provided curated content to improve rapid clinical identification in subsequent cases.Epidemiological and genomic analyses identified a related cluster of three out of seven C. striatum among lung transplant patients who had common procedures and exposures at an outlying institution. Genomic analyses further elucidated drivers of the MDR phenotypes, including resistance genes mobilized by IS3504 and ISCg9a-like insertion sequences. Seven mobilizable resistance genes were localized to a common chromosomal region bounded by unpaired insertion sequences, suggesting that a single recombination event could spread resistance to aminoglycosides, macrolides, lincosamides and tetracyclines to naive strains.In-depth genomic studies of MDR C. striatum reveal its capacity for clonal spread within and across healthcare institutions and identify novel vectors that can mobilize multiple forms of drug resistance, further complicating efforts to treat infections in immunocompromised populations. Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. All rights reserved.

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September 22, 2019  |  

Hepacivirus A infection in horses defines distinct envelope hypervariable regions and elucidates potential roles of viral strain and adaptive immune status in determining envelope diversity and infection outcome.

Hepacivirus A (also known as nonprimate hepacivirus and equine hepacivirus) is a hepatotropic virus that can cause both transient and persistent infections in horses. The evolution of intrahost viral populations (quasispecies) has not been studied in detail for hepacivirus A, and its roles in immune evasion and persistence are unknown. To address these knowledge gaps, we first evaluated the envelope gene (E1 and E2) diversity of two different hepacivirus A strains (WSU and CU) in longitudinal blood samples from experimentally infected adult horses, juvenile horses (foals), and foals with severe combined immunodeficiency (SCID). Persistent infection with the WSU strain was associated with significantly greater quasispecies diversity than that observed in horses who spontaneously cleared infection (P = 0.0002) or in SCID foals (P < 0.0001). In contrast, the CU strain was able to persist despite significantly lower (P < 0.0001) and relatively static envelope diversity. These findings indicate that envelope diversity is a poor predictor of hepacivirus A infection outcomes and could be dependent on strain-specific factors. Next, entropy analysis was performed on all E1/E2 genes entered into GenBank. This analysis defined three novel hypervariable regions (HVRs) in E2, at residues 391 to 402 (HVR1), 450 to 461 (HVR2), and 550 to 562 (HVR3). For the experimentally infected horses, entropy analysis focusing on the HVRs demonstrated that these regions were under increased selective pressure during persistent infection. Increased diversity in the HVRs was also temporally associated with seroconversion in some horses, suggesting that these regions may be targets of neutralizing antibody and may play a role in immune evasion.IMPORTANCE Hepacivirus C (hepatitis C virus) is estimated to infect 150 million people worldwide and is a leading cause of cirrhosis and hepatocellular carcinoma. In contrast, its closest relative, hepacivirus A, causes relatively mild disease in horses and is frequently cleared. The relationship between quasispecies evolution and infection outcome has not been explored for hepacivirus A. To address this knowledge gap, we examined envelope gene diversity in horses with resolving and persistent infections. Interestingly, two strain-specific patterns of quasispecies diversity emerged. Persistence of the WSU strain was associated with increased quasispecies diversity and the accumulation of amino acid changes within three novel hypervariable regions following seroconversion. These findings provided evidence that envelope gene mutation is influenced by adaptive immune pressure and may contribute to hepacivirus persistence. However, the CU strain persisted despite relative evolutionary stasis, suggesting that some hepacivirus strains may use alternative mechanisms to persist in the host. Copyright © 2018 American Society for Microbiology.

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September 22, 2019  |  

Full gene HLA class I sequences of 79 novel and 519 mostly uncommon alleles from a large United States registry population.

HLA class I assignments were obtained at single genotype, G-level resolution from 98?855 volunteers for an unrelated donor registry in the United States. In spite of the diverse ancestry of the volunteers, over 99% of the assignments at each locus are common. Within this population, 52 novel alleles differing in exons 2 and 3 are identified and characterized. Previously reported alleles with incomplete sequences in the IPD-IMGT/HLA database (n?=?519) were selected for full gene sequencing and, from this sampling, another 27 novel alleles are described.© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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September 22, 2019  |  

Cryptocurrencies and Zero Mode Wave guides: An unclouded path to a more contiguous Cannabis sativa L. genome assembly

We describe the use ofa Decentralized Autonomous Organization (DAO) to crypto- fund the single molecule sequencing and publication ofa Type ll Cannabis plant. This resulted in the construction of the most contiguous Cannabis genome assembly to date. The combined use of the Dash cryptocurrency, DAOs, and Pacific Biosciences sequencing delivered a 1.03 Gb genome with a N50 of 665Kb in 77 days from funding to public upload. This represents a 230 fold improvement in the contiguity of the first cannabis assemblies in 2011 and a 4 fold improvement over all cannabis assemblies to date. 34Gb ofadditional sequencing pushed the assembly to a N50 of 3.8Mb. Hi-C data from Phase Genomics further scaffolded the assembly to 35 contigs at an N50 of 74Mb but requires additional curation. The genome is partially phased and larger than previously reported (2N : 1.33Gb). The CBCA, THCA and CBDA synthase gene clusters have been phased onto respective contigs demonstrating tandem repeat expansions.

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September 22, 2019  |  

Diagnostic and Therapeutic Strategies for Fluoropyrimidine Treatment of Patients Carrying Multiple DPYD Variants.

DPYD genotyping prior to fluoropyrimidine treatment is increasingly implemented in clinical care. Without phasing information (i.e., allelic location of variants), current genotype-based dosing guidelines cannot be applied to patients carrying multiple DPYD variants. The primary aim of this study is to examine diagnostic and therapeutic strategies for fluoropyrimidine treatment of patients carrying multiple DPYD variants. A case series of patients carrying multiple DPYD variants is presented. Different genotyping techniques were used to determine phasing information. Phenotyping was performed by dihydropyrimidine dehydrogenase (DPD) enzyme activity measurements. Publicly available databases were queried to explore the frequency and phasing of variants of patients carrying multiple DPYD variants. Four out of seven patients carrying multiple DPYD variants received a full dose of fluoropyrimidines and experienced severe toxicity. Phasing information could be retrieved for four patients. In three patients, variants were located on two different alleles, i.e., in trans. Recommended dose reductions based on the phased genotype differed from the phenotype-derived dose reductions in three out of four cases. Data from publicly available databases show that the frequency of patients carrying multiple DPYD variants is low (< 0.2%), but higher than the frequency of the commonly tested DPYD*13 variant (0.1%). Patients carrying multiple DPYD variants are at high risk of developing severe toxicity. Additional analyses are required to determine the correct dose of fluoropyrimidine treatment. In patients carrying multiple DPYD variants, we recommend that a DPD phenotyping assay be carried out to determine a safe starting dose.

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September 22, 2019  |  

Investigation of a cluster of Sphingomonas koreensis infections.

Plumbing systems are an infrequent but known reservoir for opportunistic microbial pathogens that can infect hospitalized patients. In 2016, a cluster of clinical sphingomonas infections prompted an investigation.We performed whole-genome DNA sequencing on clinical isolates of multidrug-resistant Sphingomonas koreensis identified from 2006 through 2016 at the National Institutes of Health (NIH) Clinical Center. We cultured S. koreensis from the sinks in patient rooms and performed both whole-genome and shotgun metagenomic sequencing to identify a reservoir within the infrastructure of the hospital. These isolates were compared with clinical and environmental S. koreensis isolates obtained from other institutions.The investigation showed that two isolates of S. koreensis obtained from the six patients identified in the 2016 cluster were unrelated, but four isolates shared more than 99.92% genetic similarity and were resistant to multiple antibiotic agents. Retrospective analysis of banked clinical isolates of sphingomonas from the NIH Clinical Center revealed the intermittent recovery of a clonal strain over the past decade. Unique single-nucleotide variants identified in strains of S. koreensis elucidated the existence of a reservoir in the hospital plumbing. Clinical S. koreensis isolates from other facilities were genetically distinct from the NIH isolates. Hospital remediation strategies were guided by results of microbiologic culturing and fine-scale genomic analyses.This genomic and epidemiologic investigation suggests that S. koreensis is an opportunistic human pathogen that both persisted in the NIH Clinical Center infrastructure across time and space and caused health care-associated infections. (Funded by the NIH Intramural Research Programs.).

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