Hercules, CA, United States
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Kato-Maeda M.,University of California at San Francisco | Shanley C.A.,Colorado State University | Ackart D.,Colorado State University | Jarlsberg L.G.,University of California at San Francisco | And 16 more authors.
Clinical and Vaccine Immunology | Year: 2012

The Beijing family of Mycobacterium tuberculosis strains is part of lineage 2 (also known as the East Asian lineage). In clinical studies, we have observed that isolates from the sublineage RD207 of lineage 2 were more readily transmitted among humans. To investigate the basis for this difference, we tested representative strains with the characteristic Beijing spoligotype from four of the five sublineages of lineage 2 in the guinea pig model and subjected these strains to comparative whole-genome sequencing. The results of these studies showed that all of the clinical strains were capable of growing and causing lung pathology in guinea pigs after low-dose aerosol exposure. Differences between the abilities of the four sublineages to grow in the lungs of these animals were not overt, but members of RD207 were significantly more pathogenic, resulting in severe lung damage. The RD207 strains also induced much higher levels of markers associated with regulatory T cells and showed a significant loss of activated T cells in the lungs over the course of the infections. Whole-genome sequencing of the strains revealed mutations specific for RD207 which may explain this difference. Based on these data, we hypothesize that the sublineages of M. tuberculosis are associated with distinct pathological and clinical phenotypes and that these differences influence the transmissibility of particular M. tuberculosis strains in human populations. Copyright © 2012, American Society for Microbiology. All Rights Reserved.

Coscolla M.,Swiss Tropical and Public Health Institute | Coscolla M.,University of Basel | Barry P.M.,Center for Infectious Diseases | Oeltmann J.E.,Centers for Disease Control and Prevention | And 11 more authors.
Journal of Infectious Diseases | Year: 2015

The transcontinental spread of multidrug-resistant (MDR) tuberculosis is poorly characterized in molecular epidemiologic studies. We used genomic sequencing to understand the establishment and dispersion of MDR Mycobacterium tuberculosis within a group of immigrants to the United States. We used a genomic epidemiology approach to study a genotypically matched (by spoligotype, IS6110 restriction fragment length polymorphism, and mycobacterial interspersed repetitive units-variable number of tandem repeat signature) lineage 2/Beijing MDR strain implicated in an outbreak of tuberculosis among refugees in Thailand and consecutive cases within California. All 46 MDR M. tuberculosis genomes from both Thailand and California were highly related, with a median difference of 10 single-nucleotide polymorphisms (SNPs). The Wat Tham Krabok (WTK) strain is a new sequence type distinguished from all known Beijing strains by 55 SNPs and a genomic deletion (Rv1267c) associated with increased fitness. Sequence data revealed a highly prevalent MDR strain that included several closely related but distinct allelic variants within Thailand, rather than the occurrence of a single outbreak. In California, sequencing data supported multiple independent introductions of WTK with subsequent transmission and reactivation within the state, as well as a potential super spreader with a prolonged infectious period. Twenty-seven drug resistance-conferring mutations and 4 putative compensatory mutations were found within WTK strains. Genomic sequencing has substantial epidemiologic value in both low- and high-burden settings in understanding transmission chains of highly prevalent MDR strains. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.

Kyle J.L.,U.S. Department of Agriculture | Kyle J.L.,Eureka Genomics | Cummings C.A.,Life Technologies | Parker C.T.,U.S. Department of Agriculture | And 17 more authors.
Journal of Bacteriology | Year: 2012

Enteropathogenic Escherichia coli (EPEC) continues to be a leading cause of mortality and morbidity in children around the world. Two EPEC genomes have been fully sequenced: those of EPEC O127:H6 strain E2348/69 (United Kingdom, 1969) and EPEC O55:H7 strain CB9615 (Germany, 2003). The O55:H7 serotype is a recent precursor to the virulent enterohemorrhagic E. coli O157:H7. To explore the diversity of O55:H7 and better understand the clonal evolution of O157:H7, we fully sequenced EPEC O55:H7 strain RM12579 (California, 1974), which was collected 1 year before the first U.S. isolate of O157:H7 was identified in California. Phage-related sequences accounted for nearly all differences between the two O55:H7 strains. Additionally, O55:H7 and O157:H7 strains were tested for the presence and insertion sites of Shiga toxin gene (stx)-containing bacteriophages. Analysis of non-phage-associated genes supported core elements of previous O157:H7 stepwise evolutionary models, whereas phage composition and insertion analyses suggested a key refinement. Specifically, the placement and presence of lambda-like bacteriophages (including those containing stx) should not be considered stable evolutionary markers or be required in placing O55:H7 and O157:H7 strains within the stepwise evolutionary models. Additionally, we suggest that a 10.9-kb region (block 172) previously believed unique to O55:H7 strains can be used to identify early O157:H7 strains. Finally, we defined two subsets of O55:H7 strains that share an as-yet-unobserved or extinct common ancestor with O157:H7 strains. Exploration of O55:H7 diversity improved our understanding of the evolution of E. coli O157:H7 and suggested a key revision to accommodate existing and future configurations of stx-containing bacteriophages into current models. © 2012, American Society for Microbiology.

Be N.A.,Lawrence Livermore National Laboratory | Thissen J.B.,Lawrence Livermore National Laboratory | Gardner S.N.,Lawrence Livermore National Laboratory | McLoughlin K.S.,Lawrence Livermore National Laboratory | And 6 more authors.
PLoS ONE | Year: 2013

Bacillus anthracis is the potentially lethal etiologic agent of anthrax disease, and is a significant concern in the realm of biodefense. One of the cornerstones of an effective biodefense strategy is the ability to detect infectious agents with a high degree of sensitivity and specificity in the context of a complex sample background. The nature of the B. anthracis genome, however, renders specific detection difficult, due to close homology with B. cereus and B. thuringiensis. We therefore elected to determine the efficacy of next-generation sequencing analysis and microarrays for detection of B. anthracis in an environmental background. We applied next-generation sequencing to titrated genome copy numbers of B. anthracis in the presence of background nucleic acid extracted from aerosol and soil samples. We found next-generation sequencing to be capable of detecting as few as 10 genomic equivalents of B. anthracis DNA per nanogram of background nucleic acid. Detection was accomplished by mapping reads to either a defined subset of reference genomes or to the full GenBank database. Moreover, sequence data obtained from B. anthracis could be reliably distinguished from sequence data mapping to either B. cereusB. thuringiensis. We also demonstrated the efficacy of a microbial census microarray in detecting B. anthracis in the same samples, representing a cost-effective and high-throughput approach, complementary to next-generation sequencing. Our results, in combination with the capacity of sequencing for providing insights into the genomic characteristics of complex and novel organisms, suggest that these platforms should be considered important components of a biosurveillance strategy. © 2013 Be et al.

Poojari S.,Washington State University | Alabi O.J.,Washington State University | Fofanov V.Y.,Eureka Genomics | Naidu R.A.,Washington State University
PLoS ONE | Year: 2013

A graft-transmissible disease displaying red veins, red blotches and total reddening of leaves in red-berried wine grape (Vitis vinifera L.) cultivars was observed in commercial vineyards. Next-generation sequencing technology was used to identify etiological agent(s) associated with this emerging disease, designated as grapevine redleaf disease (GRD). High quality RNA extracted from leaves of grape cultivars Merlot and Cabernet Franc with and without GRD symptoms was used to prepare cDNA libraries. Assembly of highly informative sequence reads generated from Illumina sequencing of cDNA libraries, followed by bioinformatic analyses of sequence contigs resulted in specific identification of taxonomically disparate viruses and viroids in samples with and without GRD symptoms. A single-stranded DNA virus, tentatively named Grapevine redleaf-associated virus (GRLaV), and Grapevine fanleaf virus were detected only in grapevines showing GRD symptoms. In contrast, Grapevine rupestris stem pitting-associated virus, Hop stunt viroid, Grapevine yellow speckle viroid 1, Citrus exocortis viroid and Citrus exocortis Yucatan viroid were present in both symptomatic and non-symptomatic grapevines. GRLaV was transmitted by the Virginia creeper leafhopper (Erythroneura ziczac Walsh) from grapevine-to-grapevine under greenhouse conditions. Molecular and phylogenetic analyses indicated that GRLaV, almost identical to recently reported Grapevine Cabernet Franc-associated virus from New York and Grapevine red blotch-associated virus from California, represents an evolutionarily distinct lineage in the family Geminiviridae with genome characteristics distinct from other leafhopper-transmitted geminiviruses. GRD significantly reduced fruit yield and affected berry quality parameters demonstrating negative impacts of the disease. Higher quantities of carbohydrates were present in symptomatic leaves suggesting their possible role in the expression of redleaf symptoms. © 2013 Poojari et al.

PubMed | Eureka Genomics and Lawrence Livermore National Laboratory
Type: Journal Article | Journal: PloS one | Year: 2016

Francisella tularensis is classified as a Class A bioterrorism agent by the U.S. government due to its high virulence and the ease with which it can be spread as an aerosol. It is a facultative intracellular pathogen and the causative agent of tularemia. Ciprofloxacin (Cipro) is a broad spectrum antibiotic effective against Gram-positive and Gram-negative bacteria. Increased Cipro resistance in pathogenic microbes is of serious concern when considering options for medical treatment of bacterial infections. Identification of genes and loci that are associated with Ciprofloxacin resistance will help advance the understanding of resistance mechanisms and may, in the future, provide better treatment options for patients. It may also provide information for development of assays that can rapidly identify Cipro-resistant isolates of this pathogen. In this study, we selected a large number of F. tularensis live vaccine strain (LVS) isolates that survived in progressively higher Ciprofloxacin concentrations, screened the isolates using a whole genome F. tularensis LVS tiling microarray and Illumina sequencing, and identified both known and novel mutations associated with resistance. Genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms.

White T.B.,Tulane Cancer Center | McCoy A.M.,Bio Rad Laboratories Inc. | McCoy A.M.,Eureka Genomics | Streva V.A.,Tulane Cancer Center | And 3 more authors.
Mobile DNA | Year: 2014

Background: The active human mobile element, long interspersed element 1 (L1) currently populates human genomes in excess of 500,000 copies per haploid genome. Through its mobility via a process called target primed reverse transcription (TPRT), L1 mobilization has resulted in over 100 de novo cases of human disease and has recently been associated with various cancer types. Large advances in high-Throughput sequencing (HTS) technology have allowed for an increased understanding of the role of L1 in human cancer; however, researchers are still limited by the ability to validate potentially rare L1 insertion events detected by HTS that may occur in only a small fraction of tumor cells. Additionally, HTS detection of rare events varies greatly as a function of read depth, and new tools for de novo element discovery are needed to fill in gaps created by HTS. Results: We have employed droplet digital PCR (ddPCR) to detect rare L1 loci in mosaic human genomes. Our assay allows for the detection of L1 insertions as rare as one cell in every 10,000. Conclusions: ddPCR represents a robust method to be used alongside HTS techniques for detecting, validating and quantitating rare L1 insertion events in tumors and other tissues. © 2014 White et al.

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