Effect of a mouthrinse containing Malva sylvestris on the viability and activity of microcosm biofilm and on enamel demineralization compared to known antimicrobials mouthrinses

The purpose of this study was to evaluate the antimicrobial (anti-biofilm) and anti-caries (enamel demineralization prevention) effects of Malva sylvestris (Malvatricin^® Plus) compared with known antimicrobial mouthrinses. Microcosm biofilm was produced on enamel, using inoculum from pooled human saliva mixed with McBain saliva (0.2% sucrose) for 14 days. The biofilm was treated with mouthrinses for 1 min day^?1. Oral-B^® Complete, Listerine^® Zero and Malvatricin^® Plus had the greatest effect on the reduction of biofilm viability (p < 0.0001). On the other hand, lactic acid production was reduced significantly with PerioGard^®, Noplak^® Max and Listerine^® Zero compared with the control (p < 0.0001). No significant differences were found among the mouthrinses with respect to the colony-forming unit counting (total microorganisms, total streptococci, mutans streptococci and lactobacilli) and extracellular polysaccharide production. Enamel demineralization was reduced significantly with PerioGard^®, Noplak^® Max and Malvatricin^® Plus compared with the control (p < 0.0001). Malva sylvestris has a comparable anti-caries effect to chlorhexidine mouthrinses.     

Decontamination of polymerase chain reaction reagents for detection of low concentrations of 16S rRNA genes

We describe a polymerase chain reaction (PCR) that allowed detection of rRNA consensus sequences from the DNA extracted from a wide range of bacterial species in amounts as low as 10 fg. To avoid false-positive results with universal primers for 16S rRNA PCR, contaminating DNA had to be eliminated from the polymerase preparations. Decontamination was undertaken before PCR to optimize treatment with DNase I and was followed by DNase inactivation at 94°C for 50 min, which eliminated contaminating DNA at concentrations of up to 100 pg. After optimization of PCR conditions for each polymerase, Deep-Vent Exo-^®polymerase (New England Biolabs, Beverly, MA), and super-Taq^® polymerase (HT Biotechnology, Cambridge, UK) were more effective than Ampli-Taq^® polymerase (Perkin-Elmer Cetus, Norwalk, CT), Ampli-Taq LD^® polymerase (Perkin-Elmer Cetus) or Deep-vent^® polymerase (New England Biolabs). The technique described in this article might prove to be a universal method for PCR detection of small numbers of unidentified bacteria in usually sterile clinical sites, such as blood and cerebrospinal fluids, in which a broad spectrum of pathogens can be expected.     

Evaluation of DNA extraction methods of rumen microbial populations

The dynamism of microbial populations in the rumen has been studied with molecular methods that analyze single nucleotide polymorphisms of ribosomal RNA gene fragments (rDNA). Therefore DNA of good quality is needed for this kind of analysis. In this work we report the evaluation of four DNA extraction protocols (mechanical lysis or chemical lysis with CTAB, ethylxanthogenate or DNAzol^®) from ruminal fluid. The suitability of two of these protocols (mechanical lysis and DNAzol^®) was tested on single-strand conformation polymorphism (SSCP) of rDNA of rumen microbial populations. DNAzol^® was a simple method that rendered good integrity, yield and purity. With this method, subtle changes in protozoan populations were detected in young bulls fed with slightly different formulations of a supplement of multinutritional blocks of molasses and urea. Sequences related to Eudiplodinium maggi and a non-cultured Entodiniomorphid similar to Entodinium caudatum, were related to major fluctuating populations in an SSCP assay.     

An effective method for isolation of DNA from pig faeces and comparison of five different methods

Polymerase chain reaction (PCR) detection of microorganism in faecal specimens is hampered by poor recovery of DNA and by the presence of PCR inhibitors. In this paper, we describe a new modified method for extracting PCR-quality microbial community DNA from pig faecal samples, which combines the pretreatment with polyformaldehyde, and subsequent DNA lysis in the presence of CTAB, salt, PVP, and β-mercaptoethanol, followed by isolation of nucleic acids using chloroform (no phenol) based protocol. The method resulted in a 1.3- to 11-fold increase in DNA yield when compared to four other widely used methods. Genomic DNA extracted from all five methods was assessed by both agarose gel electrophoresis and polymerase chain reaction for amplification of 16S rDNA specific fragments. The results showed that the improved method represented a reproducible, simple, and rapid technique for routine DNA extraction from faecal specimens and was notably better than using the QIAamp^® DNA Stool Mini Kit.     

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb¹. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits². During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel''s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight³. The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along⁴. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation⁵; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 1. Understand the mechanism by which DNA fragments are separated within a gel matrix 2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 3. Identify an agarose solution of appropriate concentration for their needs 4. Prepare an agarose gel for electrophoresis of DNA samples 5. Set up the gel electrophoresis apparatus and power supply 6. Select an appropriate voltage for the separation of DNA fragments 7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 8. Determine the sizes of separated DNA fragments       

Effect of commercial herbal toothpastes and mouth rinses on the prevention of enamel demineralization using a microcosm biofilm model

Abstract

This work evaluated the effects of commercial toothpastes and mouth rinses containing natural/herbal agents on biofilm viability, extracellular polysaccharide (EPS) production and on enamel demineralization in vitro. Microcosm biofilm was produced on bovine enamel for 5?days and treated daily with: Orgânico natural^® (toothpaste/mouth rinse), Boni Natural Menta &; Malaleuca^® (toothpaste/mouth rinse), Propolis &; Myrrh^® (toothpaste), Colgate Total 12 Clean Mint^® (toothpaste, positive control), Malvatricin^® Plus (mouth rinse), PerioGard^® (mouth rinse, positive control) or PBS (negative control). Tom’s Propolis &; Myrrh^® and Colgate Total 12^® toothpastes and Malvatricin^® Plus and PerioGard^® mouth rinses significantly reduced biofilm viability (p?® had significant effects on biofilm thickness and EPS. Despite the indication that Tom’s Propolis &; Myrrh^® significantly reduced lesion depth, only Colgate Total 12^® significantly reduced mineral loss. Malvatricin^® Plus significantly reduced mineral loss and lesion depth, as did PerioGard^®. Some herbal products, Malvatricin^® Plus and Tom’s Propolis &; Myrrh^®, showed anticaries effects.     

An optimized DNA extraction and purification method from dairy manure compost for genetic diversity analysis

An unbiased DNA extraction protocol is necessary for analysis of genetic diversity, particularly, of genes in complex environmental samples by nucleic acid techniques. In the present study, three manual extraction methods and two commonly used commercial kits, which were accompanied by two DNA purification strategies, were compared based on cell lysis efficiency, DNA and humic acid yields, PCR amplification and denaturing gradient gel electrophoresis (DGGE) analysis. The results show that in spite of higher cell lysis efficiencies of the two commercial kits, the purified DNA yields were only one-third of that obtained by the two manual methods of FTSP (Freeze–thaw–SDS–Protein K) and FTSPP (Freeze–thaw–SDS–Protein K-Polyvinylpolypyrrolidone). The purified DNA from all five methods was pure enough for successful PCR and real-time PCR amplifications in the presence of 1 μg μL^?1 BSA. However, the FTSPP extraction method with DNA purification by a Wizard^® kit yielded the largest number of 16S rRNA gene copies and ribotypes or bands in DGGE profiles, which indicated a superiority over the other four methods. The development of this optimized DNA extraction and purification method may provide a valuable tool for further molecular analysis of compost.     

An evaluation of commercial DNA extraction kits for the isolation of bacterial spore DNA from soil

Aims: To evaluate six commercial DNA extraction kits for their ability to isolate PCR‐quality DNA from Bacillus spores in various soil samples. Methods and Results: Three soils were inoculated with various amounts of Bacillus cereus spores to simulate an outbreak or intentional release of the threat agent Bacillus anthracis. DNA was isolated from soil samples using six commercial DNA extraction kits. Extraction and purification efficiencies were assessed using a duplex real‐time PCR assay that included an internal positive control. The FastDNA^® SPIN kit for Soil showed the highest DNA extraction yield, while the E.Z.N.A.^® Soil DNA and PowerSoil^® DNA Isolation kits showed the highest efficiencies in removing PCR inhibitors from loam soil extracts. Conclusions: The results of this study suggest that commercially available extraction kits can be used to extract PCR‐quality DNA from bacterial spores in soil. The selection of an appropriate extraction kit should depend on the characteristics of the soil sample and the intended downstream application. Significance and Impact of the Study: The results of this study aid in the selection of an appropriate DNA extraction kit for a given soil sample. Its application could expedite sample processing for real‐time PCR detection of a pathogen in soil.     

Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis Monitoring of Fecal Bifidobacterium Populations in a Prebiotic and Probiotic Feeding Trial

A culture-independent approach based on genus-specific PCR and denaturing gradient gel electrophoresis (DGGE) was used to monitor qualitative changes in fecal bifidobacterial communities in a human feeding trial. DNA was extracted directly from feces and bifidobacterial 16S rDNA sequences were amplified using genus-specific PCR. The PCR fragments were subsequently separated in a sequence-specific manner by DGGE in order to obtain a profile of bifidobacterial fragments. The DGGE profiles revealed that in general, administration for two weeks of galactooligosaccharide and/or Bifidobacterium lactis Bb-12 (8 g and 3 × 10¹⁰ cfu per day, respectively) did not affect the qualitative composition of the indigenous Bifidobacterium population, while B. lactis Bb-12 transiently colonised the gut.     

DNA Extraction from small blood volumes and the processing of cellulose blood cards for use in polymerase chain reaction

This article describes two procedures for the purification of genomic DNA from small blood volumes of whole blood using DNAzol^®BD. In the first procedure, DNA is isolated from 1–20 μL of whole blood using a fast and simple protocol that is appropriate for the simultaneous extraction of a large number of samples. The isolated DNA is suitable for gel electrophoresis and polymerase chain reaction (PCR). In the second procedure, cellulose blood cards containing approx 5 μL of dried blood are treated with DNAzol BD in order to retain DNA on the cellulose matrix while removing other cellular components. The blood card with DNA subsequently serves as template in PCR. The blood card processing and amplification procedures are performed in the same PCR tube without any centrifugation steps, making the combined procedures amenable for automated DNA preparation and amplification in a single tube.     

A method for separating DNA fragments by electrophoresis in polyacrylamide concentration gradient slab gels

Electrophoresis on slab gels containing a linear gradient of polyacrylamide concentration has been used to separate DNA fragments obtained by restriction of viral DNAs. A simple method of preparing gradient gels using a sucrose density-gradient mixer and preexisting slab gel apparatus is described. DNA fragments of molecular weights 7 × 10⁴–14 × 10⁶ have been fractionated on gels of 3.5–7.5% and 2.5–7.5% acrylamide concentration. In addition to the wide range of fragment sizes which may be run on a single gel, a further advantage of the system is that much sharper bands are obtained compared to conventional constant concentration gels, thus improving resolution.In the molecular-weight range below 5 × 10⁶, for bands whose terminal velocities in the polyacrylamide concentration gradient approach zero, an approximately linear relationship holds between the logarithms of the molecular weights of the fragments and the logarithms of the distances they have migrated in the gel. Thus, by choosing a suitable upper limit to the concentration gradient, the gel system provides a method for estimating approximate molecular weights of unknown DNA fragments, by comparing their mobilities to known standards.     

Fractionation and molecular weight determination of deoxyribonucleic acid fragments using agarose column chromatography

DNA fragments of several sizes have been produced by shearing E. coli DNA under different pressures. These fragments have been used to demonstrate that column chromatography on agarose Bio-Gel A-15M can provide a rapid, inexpensive fractionation and sizing method for single-stranded nucleic acids having masses between 10⁵ and 10⁶ daltons. Both chromatographic and electrophoretic analysis of the sheared DNA indicated that discrete fragment populations were produced at each shearing pressure and that these fragments were distributed essentially symmetrically around a mean piece size. The average molecular weight of the several DNA fragment distributions was determined electrophoretically by comparison with standard DNA fragments obtained from restriction endonuclease cleavage of SV40 viral DNA. The molecular weights of the denatured, sheared fragments (single-stranded) ranged from 1.25 × 10⁵ to 7.4 × 10⁵. The single-stranded DNA fragments were chromatographed over agarose Bio-Gel A-15M and a linear relationship was found to exist between the mobilities and logarithms of the molecular weights. Readily available tRNA, 5s RNA, and φX174 single-stranded circular DNA chromatographed at the extremes of the linear relationship and could be used to calibrate the column chromatography.     

Restriction endonuclease map of the chloroplast DNA of Chlamydomonas reinhardii

The chloroplast DNA of Chlamydomonas reinhardii has been examined by restriction endonuclease analysis. EcoRI, BamHI and BglII produce 30, 17 and 12 fragments, respectively, whose sites have been determined by electron microscopy and by comparative gel electrophoresis. These fragments have been ordered into a circular map which corresponds to a genome size of M_r = 126 × 10⁶. The map was established by comparing the double digests of individual restriction fragments and by hybridizing purified labelled fragments to restriction enzyme digests of chloroplast DNA. The restriction fragments were isolated by molecular cloning or by preparative agarose gel electrophoresis.The two sets of chloroplast ribosomal RNA genes are contained within two inverted repeats of 13 × 10⁶ molecular weight, which are located nearly at opposite sides of the map. In addition, the mapping studies have revealed the presence of short repeated base sequences which are interspersed throughout the chloroplast genome.     

Protocols for dry DNA storage and shipment at room temperature

The globalization of DNA barcoding will require core analytical facilities to develop cost‐effective, efficient protocols for the shipment and archival storage of DNA extracts and PCR products. We evaluated three dry‐state DNA stabilization systems: commercial Biomatrica^® DNAstable^® plates, home‐made trehalose and polyvinyl alcohol (PVA) plates on 96‐well panels of insect DNA stored at 56 °C and at room temperature. Controls included unprotected samples that were stored dry at room temperature and at 56 °C, and diluted samples held at 4 °C and at ?20 °C. PCR and selective sequencing were performed over a 4‐year interval to test the condition of DNA extracts. Biomatrica^® provided better protection of DNA at 56 °C and at room temperature than trehalose and PVA, especially for diluted samples. PVA was the second best protectant after Biomatrica^® at room temperature, whereas trehalose was the second best protectant at 56 °C. In spite of lower PCR success, the DNA stored at ?20 °C yielded longer sequence reads and stronger signal, indicating that temperature is a crucial factor for DNA quality which has to be considered especially for long‐term storage. Although it is premature to advocate a transition to DNA storage at room temperature, dry storage provides an additional layer of security for frozen samples, protecting them from degradation in the event of freezer failure. All three forms of DNA preservation enable shipment of dry DNA and PCR products between barcoding facilities.     

Effect of the electric field on the apparent mobility of large DNA fragments in agarose gels

The electrophoresis of a series of DNA fragments ranging in size from 0.5 to 12 kilobase pairs, has been studied as a function of agarose gel concentration and electric field strength. The apparent mobility of all fragments decreased with decreasing electric field strength and with increasing gel concentration. When extrapolated to zero electric field strength and zero agarose concentration, the apparent mobility of all DNA fragments extrapolated to a common value (2.0 ± 0.1) × 10^?4 cm²/V s. The square roots of the retardation coefficients of the various fragments were found to be linearly related to the root-mean-square radii of gyration of the fragments, as predicted by pore-size distribution theory. As predicted by reptation theory, the molecular weights of the various fragments were found to be linearly related to the reciprocal of the apparent mobilities. An equation is given for estimating the apparent pore size of agarose gels between 0.25 and 1.5% in concentration.     

Evaluation of the in vitro differential protein adsorption patterns of didanosine-loaded nanostructured lipid carriers (NLCs) for potential targeting to the brain

The preferential in vitro adsorption of apolipoprotein E (Apo E) onto the surface of colloidal drug carriers may be used as a strategy to evaluate the in vivo potential for such systems to transport drugs to the brain. The aim of this research was to investigate the in vitro protein adsorption patterns of didanosine-loaded nanostructured lipid carriers (DDI-NLCs), using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), in order to establish the potential for NLCs to deliver DDI to the brain. NLC formulations were manufactured using high-pressure homogenization using a lipid matrix consisting of a mixture of Precirol^® ATO 5 and Transcutol^® HP. The 2-D PAGE analysis revealed that NLCs in formulations stabilized using Solutol^® HS 15 alone or with a ternary surfactant system consisting of Solutol^® HS 15, Tween^® 80, and Lutrol^® F68, preferentially adsorbed proteins, such as Apo E. Particles stabilized with Tween^® 80 and Lutrol^® F68 did not adsorb Apo E in these studies, which could be related to the relatively large particle size and hence small surface area observed for these NLCs. These findings have revealed that DDI-loaded NLCs may have the potential to deliver DDI to the brain in vivo and, in addition, to Tween^® 80, which has already been shown to have the ability to facilitate the targeting of colloidal drug delivery systems to the brain. Solutol^® HS 15–stabilized nanoparticles may also achieve a similar purpose.     

Electrophoretic mobility of lambda phage HIND III and HAE III DNA fragments in agarose gels: a detailed study

The electrophoretic mobility (μ) of DNA fragments from λ phage and ΦX 174, split by restriction enzyme to molecular lengths from 3 × 10² to 2.36 × 10⁴ base pairs, has been investigated in 0.6–4% agarose gels at various field strengths, ionic strengths, and temperatures. As already observed, μ is seen to be very sensitive to the field, increasing with field strength. The sensitivity increases with the molecular length of the DNA and decreases at high gel concentration. Our data are in qualitative agreement with recent theoretical predictions that concern the influence of the electric field on electrophoretic mobility. Mobility data have been extrapolated to zero field. This enables a comparison of our experimental results with theoretical predictions on the dependence of μ on the molecular weight of the DNA fragments. Our data fit, quite closely, a reptation model, where the tube path is described as a semiflexible entity with a persistence length equal to the pore diameter. The influence of the agarose concentration and the ionic strength of the buffer on the two parameters of the model—intrinsic electrophoretic mobility (μ₀) and the number of base pairs per element of the tube (g)—are well described by the model. The temperature dependence of the electrophoretic mobility, together with the influence of the agarose concentration on μ₀, indicate that the hydrodynamic drag is the leading frictional force on the DNA molecules in the gel.     

Rapid detection and identification of the bacterium Pantoea stewartii in maize by TaqMan real-time PCR assay targeting the cpsD gene

Aims: The development and evaluation of a sensitive and specific TaqMan^® real-time polymerase chain reaction (PCR) for the detection and identification of Pantoea stewartii on maize. Methods and Results: A TaqMan^®-based real-time PCR assay targeting the cpsD gene enabling specific detection of P. stewartii in maize leaves and seeds was developed. Under optimal conditions, the selected primers and probe were specific for the detection of all 14 reference P. stewartii strains by real-time PCR. The 32 non-Panteoa and eight other Pantoea strains tested negative. The TaqMan^® PCR assay detected 1 pg of purified DNA and 10⁴P. stewartii colony forming units per millilitre (10 cells per reaction) in pure cultures consisting of 92·0% intact (viable) cells. Direct processing of leaf lesions and seeds by the real-time PCR detected 10 and 50 P. stewartii cells per reaction respectively. TaqMan^® real-time PCR results were validated by dilution plating of macerates and PCR-based subcloning followed by DNA sequencing. Conclusions: The real-time PCR assay described is a rapid, reliable and more sensitive tool for the detection of P. stewartii. Significance and Impact of the study: This real-time PCR assay would avoid false-negative results and reduce the time required for certifying maize seed shipments.     

Cytotoxicity testing of burn wound dressings: first results

Topical antimicrobial therapy represents an essential part of burn wound care. In order to prevent and treat burn wound infection dressings with antimicrobial properties are applied directly on the wound surface. Not only the infection control but also promotion of healing is very important in burn wound management. It is well known, that a dressing in bactericidal concentration might also delay wound healing. This study was aimed to evaluate the potential toxic effect of topical antimicrobial agents on murine and human dermal cells. For toxicity testing the method by Vittekova et al. was used to evaluate potential toxic effects of 16 agents and 6 control samples on two in vitro cultured cell systems [3T3 cells and dermal fibroblasts] during the first 24 h. Following the 24 h cell culture with the tested agents the live cell counts were evaluated. According to results obtained on both cell systems, the tested samples were divided into three groups—nontoxic, semi-toxic and toxic. Nontoxic samples included Acetic acid 1%, Acticoat^®, Dermacyn^®, Framykoin^®, Silverlon^®, gauze, acellular human allodermis and acellular porcine xenodermis. Semi-toxic group included Algivon^®Plus, Aquacel^®Ag, Betadine^®, Nitrofurazone, Octenisept^®, Suprasorb^® A and a porcine dermal scaffold Xeno-Impl. Finally, the toxic group included Algivon^®, Dermazin^®, Ialugen^®Plus, Prontoderm^®, Suprasorb^® A Ag and 20% SDS. As the preliminary results of this study have shown, our findings may serve as a potential guide to selection of the most appropriate topical antimicrobial dressings for treatmet of burns. However before they can be translated into clinical practice recommendations, more research on antimicrobial dressings cytotoxicity testing will be necessary.     

Effect of the herbicide glyphosate on glyphosate‐tolerant maize rhizobacterial communities: a comparison with pre‐emergency applied herbicide consisting of a combination of acetochlor and terbuthylazine

A comparison was made of the effect of glyphosate (Roundup^®Plus), a post‐emergency applied herbicide, and of Harness^®GTZ, a pre‐emergency applied herbicide, on the rhizobacterial communities of genetically modified NK603 glyphosate‐tolerant maize. The potential effect was monitored by direct amplification, cloning and sequencing of soil DNA encoding 16S rRNA, rhizobacterial DNA hybridization to commercially available genome‐wide microarrays from the soil bacterium Streptomyces coelicolor, and high‐throughput DNA pyrosequencing of the bacterial DNA coding for 16S rRNA hypervariable V6 region. The results obtained strongly suggest that both herbicides do in fact affect the maize rhizobacterial communities, glyphosate being, to a great extent, the environmentally less aggressive herbicide.