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

View Article: PubMed Central - PubMed

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 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.

No MeSH data available.


The test statistic values for a short region of the DNA gyrase A gene in one of the Cipro resistant F. tularensis isolates.The log likelihood ratio has a clear peak in this region. Candidate SNP positions were identified by looking for regions of the genome where the log likelihood ratio exceeds a fixed threshold.
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pone.0163458.g001: The test statistic values for a short region of the DNA gyrase A gene in one of the Cipro resistant F. tularensis isolates.The log likelihood ratio has a clear peak in this region. Candidate SNP positions were identified by looking for regions of the genome where the log likelihood ratio exceeds a fixed threshold.

Mentions: To identify candidate SNP loci, we computed log LR(z) for every position z in the reference genome and compared it to a threshold value, which we selected by analyzing data from the test arrays hybridized to DNA from F. tularensis strains with SNPs at known positions relative to the LVS strain, and choosing the threshold that gave the best tradeoff between false positive and false negative error rates. This threshold was 20 for the F. tularensis arrays. Typically SNPs were characterized by a contiguous series of position values with log LR scores above the threshold. The most likely SNP location within the series was identified by the position with the maximum score. As an example, Fig 1 plots the test statistic values for a short region of the DNA gyrase A gene in one of the Cipro resistant F. tularensis LVS isolates. The log likelihood ratio has an obvious peak in this region.


Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F . tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing
The test statistic values for a short region of the DNA gyrase A gene in one of the Cipro resistant F. tularensis isolates.The log likelihood ratio has a clear peak in this region. Candidate SNP positions were identified by looking for regions of the genome where the log likelihood ratio exceeds a fixed threshold.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036845&req=5

pone.0163458.g001: The test statistic values for a short region of the DNA gyrase A gene in one of the Cipro resistant F. tularensis isolates.The log likelihood ratio has a clear peak in this region. Candidate SNP positions were identified by looking for regions of the genome where the log likelihood ratio exceeds a fixed threshold.
Mentions: To identify candidate SNP loci, we computed log LR(z) for every position z in the reference genome and compared it to a threshold value, which we selected by analyzing data from the test arrays hybridized to DNA from F. tularensis strains with SNPs at known positions relative to the LVS strain, and choosing the threshold that gave the best tradeoff between false positive and false negative error rates. This threshold was 20 for the F. tularensis arrays. Typically SNPs were characterized by a contiguous series of position values with log LR scores above the threshold. The most likely SNP location within the series was identified by the position with the maximum score. As an example, Fig 1 plots the test statistic values for a short region of the DNA gyrase A gene in one of the Cipro resistant F. tularensis LVS isolates. The log likelihood ratio has an obvious peak in this region.

View Article: PubMed Central - PubMed

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 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.

No MeSH data available.