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

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

Genotyping over a broad range of DNA amounts.

<p>Melt-MAMA sensitivity to low level DNA amounts varies greatly among different assays. <i>B. anthracis</i> melt-MAMA targeting the A.Br.006 clade <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032866#pone.0032866-VanErt1" target="_blank">[4]</a> accurately genotyped DNA at amount ∼19 copies. (A & B) The respective amplification plots of genomic DNA of ‘G’ and ‘A’ SNP allele templates show the amplification curves of templates titrated in ten-fold serial dilutions and in replicates of eight. The number assigned to each amplification curve (1–7) denotes the DNA amount for the starting template. (C & D) The temperature-dissociation (melt) curve derivatives for all initial template amounts are shown (numbers denote DNA amount shown). Panels C and D illustrates that genotyping accuracy is obtained across a broad range of DNA template amounts of ∼115 ng to 115 fg. Assay sensitivity to template is limited to ∼19 copies and above. An inherent characteristic of this assay is the occurrence of spurious amplification at extended cycle times (>35) in the absence of template as indicated by the NTCs. Melt-MAMAs detecting low level DNA are subject to stochastic sampling effects (<i>B. anthracis</i> single genome copy ∼6 fg), which is predictable using a Poisson distribution <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032866#pone.0032866-VanErt1" target="_blank">[4]</a>.</p

Competition between specific and non-specific amplification at extremely low level DNA amounts.

<p>With extremely low-level DNA amounts (<∼2 copies), stochastic, spurious, non-specific amplification could outcompete allele-specific amplification. The <i>Bacillus anthracis</i> melt-MAMA targeting the A.Br.003 clade <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032866#pone.0032866-VanErt1" target="_blank">[4]</a> stochastically amplified allele-specific product and non-specific spurious products at amounts of less than a single copy (∼0.19 copies). (A and B) The respective amplification plots of genomic DNA of ‘G’ allele and ‘A’ SNP allele templates show the amplification curves of templates at 1.15 ng and at two low level ten-fold dilution series (near-single copy and less than a single copy) in replicates of eight. The number assigned to each amplification curve (3, 8–9) denotes the DNA amount for the starting template. (C & D) The temperature-dissociation (melt) curve derivatives for the 1.15 ng and lowest template amounts are shown. This panel illustrates that genotyping accuracy was not compromised at DNA template amounts near single copy level, but spurious amplification was observed in dilution points below this level. This spurious amplification had a unique melt-profile that did not match the profile of either allele types (red arrow).</p

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

Melt-MAMA validation work flow.

<p>This figure shows the sequential steps involved in validation of Melt-MAMA assays. After SNP selection (step I), Melt-MAMA are designed so that the amplicon is <100 bp in length (step II). Assays are screened across ancestral and derived DNA templates under 3 primer ratio conditions where 1∶1 represents equal primer ratio, 4∶1 represents ancestral primer 4x and derived primer 1x, and 1∶4 represents ancestral primer 1x and derived primer 4x (step III). Five outcomes are indicated (step III a–e). Based on the performance of <i>B. anthracis</i>, <i>F. tularensis</i>, and <i>Y. pestis</i> assays, 70–80% Melt-MAMAs accurately genotyped at one of the tested primer ratio condition (step IIIa). These successful assays were immediately screened on a diversity panel of DNA samples (step IV). The remaining assays (20–30%) resulted in one of the other four outcomes (step III b–e). Each outcome required additional specific validation steps to determine the optimal PCR conditions or the need to abandon the SNP altogether. Our overall design success rate increased from 46% to 87%.</p

Melt-MAMAs targeting specific groups within eight pathogen species.

a<p>First design attempt and altered primer ratio optimization.</p>b<p>Success after combining first or second design attempts and altered primer ratio optimization.</p>c<p>Failed after first design attempt.</p>d<p>Assays that required altered primer concentration ratios.</p

Real-time PCR amplification and dissociation (melt) curve plots.

<p><i>B. anthracis</i> Melt-MAMA SYBR® Green assay targeting the A.Br.004 genetic clade. (A & C) The amplification of two alleles are illustrated for haploid template (<i>Bacillus anthracis</i>) possessing an ‘A’ polymorphic SNP-state or ‘G’ state. Each amplification plot represents a single PCR reaction containing a reverse “common” primer and two allele-specific MAMA primers. The AS-MAMA primers anneal to the same template target and then compete for extension across the SNP position. The polymerase-mediated extension rate of the 3′match AS-MAMA primer (perfect primer-template complex) exceeds that of the 3′mismatched MAMA primer (mismatched primer-template complex), thus the perfect match primer-template complex outcompetes the mismatched primer-template complex and dominates the PCR amplification. (B & D) Plots of the temperature-dissociation (melt) curve of the final PCR products for the two allele templates are shown next to their respective amplification plots (green arrows). Allele-specific PCR products are easily differentiated through temperature-dissociation (melt) curve analysis, which is conferred by the GC-clamp engineered on one of the AS-MAMA primer.</p

Two-Locus (duplexed) Melt-MAMA development.

<p>(A) A phylogenetic topology of the three subspecies of <i>F. tularensis</i> rooted with <i>F. novicida</i>. The SNP-signatures specific for the two pathogenic subspecies of <i>F. tularensis</i> (indicated by black bars) were incorporated into Melt-MAMAs. The table (right) indicates expected allele states (derived and ancestral) for strains from each <i>F. tularensis</i> subspecies represented on the topology; <i>F. novicida</i> would have the same allelic states as <i>F. tularensis</i> subsp. <i>mediasiatica</i>. (Bi–iv) Temperature-dissociation (melt) curves (derivative) of allele-specific PCR products from <i>F. tularensis</i> strains amplified in the duplexed assay (Type A and Type B). Each profile show two melt-curve peaks generated from a single <i>F. tularensis</i> strain. Each peak corresponds to the allele-specific PCR product for a single SNP-locus in the duplexed assay.</p