Preparation and characterization of antibacterial cobalt-exchanged natural zeolite/poly(vinyl alcohol) hydrogels

In the present study, potential application of the local clinoptilolite-rich natural zeolite in formulation of antibacterial hydrogels was investigated. The zeolite powder exchanged with cobalt(II) ions was used in preparation of the zeolite/poly(vinyl alcohol) hydrogel films in different amounts. The films were physically crosslinked by the freezing-thawing method and characterized for their crystallinity, surface and cross sectional morphology, chemical composition, thermal behaviour, mechanical properties, swelling and dissolution behaviours, and antibacterial activities against a Gram-negative bacteria. The films with 0.48 wt% and higher cobalt-exchanged zeolite contents showed antibacterial activity. Addition of the zeolite powder in the formulations did not cause significant changes in the other properties of the films.Turkish Republic Prime Ministry State Planning Organization (DPT-2006 K120690

VNTR markers.

<p>*Data obtained in this study.</p>†<p>The individual marker diversity (D) was calculated as D = [1-∑(allele frequency)²].</p>††<p>Location within an open reading frame.</p><p>VNTR markers.</p

<i>PmeI</i> pulsed-field gel electrophoresis (PFGE) patterns for <i>F. tularensis</i> subsp. <i>holarectica</i>.

<p>Polymorphic band position 1 consists of two fragments in PFGE type 2. Polymorphic band position 2 and 3 consist of two fragments at the same position, both missing in PFGE type 2. Polymorphic band position 6 consists of two fragments in PFGE type 3.</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

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

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

Size differences in allele-specific PCR products.

<p>A) Schematic of PCR amplicons originating from ancestral and derived genomic templates. For both PCR amplicons, gray represents sequence originating from the primers (forward and reverse) and synthesized internal sequence from PCR extension. The primer sequence and synthesized internal sequence within the amplicon are indicated by the bracket. For the derived amplicon, the blue represents the incorporated GC-clamp which originates from the 5’end of the derived MAMA forward primer. The SNP region for each amplicon is represented in green (ancestral) and yellow (derived) and the deliberate antepenultimate mutation is represented as red (ancestral) and light blue (derived). B) Allele-specific PCR products migrate at different rates on a 2% agarose gel due to their size difference as conferred by the GC-clamp.</p

Agarose-MAMA primers targeting 18 previously published SNP positions.

<p>Agarose-MAMA primers targeting 18 previously published SNP positions.</p

Agarose-MAMA is capable of genotyping <i>Y</i>. <i>pestis</i> directly from complex clinical samples if pathogen targets are at sufficient levels.

<p>(A) Agarose-MAMA (Mad-43) gel showing the PCR products for two template controls at the expected size for each respective ancestral and derived allele state (#1 and #2, respectively). Three complex clinical samples (#3-#5) also yielded PCR products with the size expected of an ancestral genotype. Two other complex clinical samples (#6 & #7) showed no PCR products but displayed a banding pattern consistent with NTC negative controls. (B) To assess the relative quantity of the <i>Y</i>. <i>pestis</i> target in the five clinical samples (#3, #4, #5, #6, #7), we generated amplification plots of these clinical samples on a TaqMan 3a assay used to target <i>Y</i>. <i>pestis</i> chromosomal DNA. Three clinical samples (#3-#5) showed amplified at a mid-range cycle-time (Ct) value consistent with high copy numbers of template DNA [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006077#pntd.0006077.ref021" target="_blank">21</a>]. These same samples showed a robust signal of a PCR product on the MAMA gel. The two samples (#6 & #7) that failed on the MAMA gel showed a high Ct value and failed amplification, respectively, when tested by real-time PCR. The pairing of TaqMan 3a assay with MAMA gel results on the same templates provided insight to the genotyping capability of MAMA tools on complex clinical samples with low-level target template.</p

Simplified SNP phylogeny of <i>Y</i>. <i>pestis</i> depicting subgroups identified in Madagascar.

<p>Colored circles indicate phylogenetic groups as previously described [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006077#pntd.0006077.ref035" target="_blank">35</a>][<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006077#pntd.0006077.ref011" target="_blank">11</a>,<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006077#pntd.0006077.ref034" target="_blank">34</a>]. Group names are assigned as letters and sometimes followed by a number (e.g. s4). Black bars indicate the phylogenetic positions of the 18 SNPs targeted for MAMA PCR design in this study.</p