skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F. tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing

Abstract

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 then 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. For genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Finally, structural modeling performed on these proteinsmore » provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms.« less

Authors:
 [1];  [2];  [1];  [2];  [2];  [1];  [1];  [1];  [3];  [3];  [1];  [4]
  1. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States). Physical Life Sciences Directorate
  2. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States). Computations Directorate
  3. Eureka Genomics, Hercules, CA (United States)
  4. Mayo Clinic, Phoenix, AZ (United States)
Publication Date:
Research Org.:
Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1337000
Report Number(s):
LLNL-JRNL-698269
Journal ID: ISSN 1932-6203
Grant/Contract Number:
AC52-07NA27344
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Volume: 11; Journal Issue: 9; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Jaing, Crystal J., McLoughlin, Kevin S., Thissen, James B., Zemla, Adam, Gardner, Shea N., Vergez, Lisa M., Bourguet, Feliza, Mabery, Shalini, Fofanov, Viacheslav Y., Koshinsky, Heather, Jackson, Paul J., and Wang, Junwen. Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F. tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing. United States: N. p., 2016. Web. doi:10.1371/journal.pone.0163458.
Jaing, Crystal J., McLoughlin, Kevin S., Thissen, James B., Zemla, Adam, Gardner, Shea N., Vergez, Lisa M., Bourguet, Feliza, Mabery, Shalini, Fofanov, Viacheslav Y., Koshinsky, Heather, Jackson, Paul J., & Wang, Junwen. Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F. tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing. United States. doi:10.1371/journal.pone.0163458.
Jaing, Crystal J., McLoughlin, Kevin S., Thissen, James B., Zemla, Adam, Gardner, Shea N., Vergez, Lisa M., Bourguet, Feliza, Mabery, Shalini, Fofanov, Viacheslav Y., Koshinsky, Heather, Jackson, Paul J., and Wang, Junwen. 2016. "Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F. tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing". United States. doi:10.1371/journal.pone.0163458. https://www.osti.gov/servlets/purl/1337000.
@article{osti_1337000,
title = {Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F. tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing},
author = {Jaing, Crystal J. and McLoughlin, Kevin S. and Thissen, James B. and Zemla, Adam and Gardner, Shea N. and Vergez, Lisa M. and Bourguet, Feliza and Mabery, Shalini and Fofanov, Viacheslav Y. and Koshinsky, Heather and Jackson, Paul J. and Wang, Junwen},
abstractNote = {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 then 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. For genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Finally, structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms.},
doi = {10.1371/journal.pone.0163458},
journal = {PLoS ONE},
number = 9,
volume = 11,
place = {United States},
year = 2016,
month = 9
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 1work
Citation information provided by
Web of Science

Save / Share:
  • The objective of this project is to provide DHS a comprehensive evaluation of the current genomic technologies including genotyping, Taqman PCR, multiple locus variable tandem repeat analysis (MLVA), microarray and high-throughput DNA sequencing in the analysis of biothreat agents from complex environmental samples. As the result of a different DHS project, we have selected for and isolated a large number of ciprofloxacin resistant B. anthracis Sterne isolates. These isolates vary in the concentrations of ciprofloxacin that they can tolerate, suggesting multiple mutations in the samples. In collaboration with University of Houston, Eureka Genomics and Oak Ridge National Laboratory, we analyzedmore » the ciprofloxacin resistant B. anthracis Sterne isolates by microarray hybridization, Illumina and Roche 454 sequencing to understand the error rates and sensitivity of the different methods. The report provides an assessment of the results and a complete set of all protocols used and all data generated along with information to interpret the protocols and data sets.« less
  • Classical forward genetics has been foundational to modern biology, and has been the paradigm for characterizing the role of genes in shaping phenotypes for decades. In recent years, reverse genetics has been used to identify the functions of genes, via the intentional introduction of variation and subsequent evaluation in physiological, molecular, and even population contexts. These approaches are complementary and whole genome analysis serves as a bridge between the two. We report in this article the whole genome sequencing of eighteen classical mutant strains of Neurospora crassa and the putative identification of the mutations associated with corresponding mutant phenotypes. Althoughmore » some strains carry multiple unique nonsynonymous, nonsense, or frameshift mutations, the combined power of limiting the scope of the search based on genetic markers and of using a comparative analysis among the eighteen genomes provides strong support for the association between mutation and phenotype. For ten of the mutants, the mutant phenotype is recapitulated in classical or gene deletion mutants in Neurospora or other filamentous fungi. From thirteen to 137 nonsense mutations are present in each strain and indel sizes are shown to be highly skewed in gene coding sequence. Significant additional genetic variation was found in the eighteen mutant strains, and this variability defines multiple alleles of many genes. These alleles may be useful in further genetic and molecular analysis of known and yet-to-be-discovered functions and they invite new interpretations of molecular and genetic interactions in classical mutant strains.« less