Francisella tularensis subsp. novicida isolated from a human in Arizona

<p>Abstract</p> <p>Background</p> <p><it>Francisella tularensis </it>is the etiologic agent of tularemia and is classified as a select agent by the Centers for Disease Control and Prevention. Currently four known subspecies of <it>F. tularensis </it>that differ in virulence and geographical distribution are recognized:<it>tularensis </it>(type A), <it>holarctica </it>(type B), <it>mediasiatica</it>, and <it>novicida</it>. Because of the Select Agent status and differences in virulence and geographical location, the molecular analysis of any clinical case of tularemia is of particular interest. We analyzed an unusual <it>Francisella </it>clinical isolate from a human infection in Arizona using multiple DNA-based approaches.</p> <p>Findings</p> <p>We report that the isolate is <it>F. tularensis </it>subsp. <it>novicida</it>, a subspecies that is rarely isolated.</p> <p>Conclusion</p> <p>The rarity of this <it>novicida </it>subspecies in clinical settings makes each case study important for our understanding of its role in disease and its genetic relationship with other <it>F. tularensis </it>subspecies.</p

Melt analysis of mismatch amplification mutation assays (melt-MAMA): a functional study of a cost-effective SNP genotyping assay in bacterial models.

Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and biologically informative markers extensively used across broad scientific disciplines. Newly identified SNP markers are publicly available at an ever-increasing rate due to advancements in sequencing technologies. Efficient, cost-effective SNP genotyping methods to screen sample populations are in great demand in well-equipped laboratories, but also in developing world situations. Dual Probe TaqMan assays are robust but can be cost-prohibitive and require specialized equipment. The Mismatch Amplification Mutation Assay, coupled with melt analysis (Melt-MAMA), is flexible, efficient and cost-effective. However, Melt-MAMA traditionally suffers from high rates of assay design failures and knowledge gaps on assay robustness and sensitivity. In this study, we identified strategies that improved the success of Melt-MAMA. We examined the performance of 185 Melt-MAMAs across eight different pathogens using various optimization parameters. We evaluated the effects of genome size and %GC content on assay development. When used collectively, specific strategies markedly improved the rate of successful assays at the first design attempt from ~50% to ~80%. We observed that Melt-MAMA accurately genotypes across a broad DNA range (~100 ng to ~0.1 pg). Genomic size and %GC content influence the rate of successful assay design in an independent manner. Finally, we demonstrated the versatility of these assays by the creation of a duplex Melt-MAMA real-time PCR (two SNPs) and conversion to a size-based genotyping system, which uses agarose gel electrophoresis. Melt-MAMA is comparable to Dual Probe TaqMan assays in terms of design success rate and accuracy. Although sensitivity is less robust than Dual Probe TaqMan assays, Melt-MAMA is superior in terms of cost-effectiveness, speed of development and versatility. We detail the parameters most important for the successful application of Melt-MAMA, which should prove useful to the wider scientific community

Multidrug-Resistant Staphylococcus aureus in US Meat and Poultry

We characterized the prevalence, antibiotic susceptibility profiles, and genotypes of Staphylococcus aureus among US meat and poultry samples (n = 136). S. aureus contaminated 47% of samples, and multidrug resistance was common among isolates (52%). S. aureus genotypes and resistance profiles differed significantly among sample types, suggesting food animal–specific contamination

TaqMan Real-Time PCR Assays for Single-Nucleotide Polymorphisms Which Identify <i>Francisella tularensis</i> and Its Subspecies and Subpopulations

<div><p><i>Francisella tularensis</i>, the etiologic agent of tularemia and a Class A Select Agent, is divided into three subspecies and multiple subpopulations that differ in virulence and geographic distribution. Given these differences, there is a need to rapidly and accurately determine if a strain is <i>F. tularensis</i> and, if it is, assign it to subspecies and subpopulation. We designed TaqMan real-time PCR genotyping assays using eleven single nucleotide polymorphisms (SNPs) that were potentially specific to closely related groups within the genus <i>Francisella</i>, including numerous subpopulations within <i>F. tularensis</i> species. We performed extensive validation studies to test the specificity of these SNPs to particular populations by screening the assays across a set of 565 genetically and geographically diverse <i>F. tularensis</i> isolates and an additional 21 genetic near-neighbor (outgroup) isolates. All eleven assays correctly determined the genetic groups of all 565 <i>F. tularensis</i> isolates. One assay differentiates <i>F. tularensis</i>, <i>F. novicida</i>, and <i>F. hispaniensis</i> from the more genetically distant <i>F. philomiragia</i> and <i>Francisella-</i>like endosymbionts. Another assay differentiates <i>F. tularensis</i> isolates from near neighbors. The remaining nine assays classify <i>F. tularensis</i>-confirmed isolates into <i>F. tularensis</i> subspecies and subpopulations. The genotyping accuracy of these nine assays diminished when tested on outgroup isolates (i.e. non <i>F. tularensis</i>), therefore a hierarchical approach of assay usage is recommended wherein the <i>F. tularensis</i>-specific assay is used before the nine downstream assays. Among <i>F. tularensis</i> isolates, all eleven assays were highly sensitive, consistently amplifying very low concentrations of DNA. Altogether, these eleven TaqMan real-time PCR assays represent a highly accurate, rapid, and sensitive means of identifying the species, subspecies, and subpopulation of any <i>F. tularensis</i> isolate if used in a step-wise hierarchical scheme. These assays would be very useful in clinical, epidemiological, and/or forensic investigations involving <i>F. tularensis</i>.</p></div

Schematic evolutionary tree of <i>Francisella tularensis</i> and <i>Francisella</i> genetic near neighbor species.

<p>Black bars indicate the important canSNP signatures specific to major genetic groups among <i>Francisella</i> species and within <i>F. tularensis</i>. The three recognized subspecies*, as well as divisions within the two major subspecies, <i>tularensis</i> and <i>holarctica</i>, are indicated. The strain representing each genetic group is indicated in blue text.</p

Primers and TaqMan-MGB probes for <i>F. tularensis</i> canSNP assays.

<p>Primers and TaqMan-MGB probes for <i>F. tularensis</i> canSNP assays.</p

Detection of Low-Level Mixed-Population Drug Resistance in Mycobacterium tuberculosis Using High Fidelity Amplicon Sequencing.

Undetected and untreated, low-levels of drug resistant (DR) subpopulations in clinical Mycobacterium tuberculosis (Mtb) infections may lead to development of DR-tuberculosis, potentially resulting in treatment failure. Current phenotypic DR susceptibility testing has a theoretical potential for 1% sensitivity, is not quantitative, and requires several weeks to complete. The use of "single molecule-overlapping reads" (SMOR) analysis with next generation DNA sequencing for determination of ultra-rare target alleles in complex mixtures provides increased sensitivity over standard DNA sequencing. Ligation free amplicon sequencing with SMOR analysis enables the detection of resistant allele subpopulations at ≥0.1% of the total Mtb population in near real-time analysis. We describe the method using standardized mixtures of DNA from resistant and susceptible Mtb isolates and the assay's performance for detecting ultra-rare DR subpopulations in DNA extracted directly from clinical sputum samples. SMOR analysis enables rapid near real-time detection and tracking of previously undetectable DR sub-populations in clinical samples allowing for the evaluation of the clinical relevance of low-level DR subpopulations. This will provide insights into interventions aimed at suppressing minor DR subpopulations before they become clinically significant

Minor subpopulation detection in two sputum samples from Moldova.

<p>Resistant and erroneous allele frequencies from three resistance SNP loci in the <i>inhA</i> promoter are shown. Patient 21–0067 with 0.05% resistant allele and patient 22–0129 with 11.39% resistant allele at <i>inhA</i> -15, compared to erroneous and resistant alleles below 0.01% at the other two SNP positions.</p

SMOR minor subpopulation examination of a single replicate of 7 mixtures at six known differing resistance SNP loci.

<p>Each circle represents the percent SMOR call, where color represents allele state, for a single sample at the six resistant SNP loci. All 36 SNP loci were examined; however the seven different mixtures contain six known allelic differences in resistant conferring SNP loci.</p