scholarly journals Removal of Free Extracellular DNA from Environmental Samples by Ethidium Monoazide and Propidium Monoazide

2008 ◽  
Vol 74 (8) ◽  
pp. 2537-2539 ◽  
Author(s):  
Andreas O. Wagner ◽  
Cornelia Malin ◽  
Brigitte A. Knapp ◽  
Paul Illmer
Keyword(s):  
Dna Extraction ◽  
Environmental Samples ◽  
Extracellular Dna ◽  
Bacterial Cells ◽  
Propidium Monoazide ◽  
Extraction Techniques ◽  
Ethidium Monoazide ◽  
New Methods

ABSTRACT Recently, new DNA extraction techniques (using ethidium monoazide and propidium monoazide) have been developed to discriminate between alive and dead bacterial cells. Nevertheless, for complex environmental samples, no data are available yet. In the present study, these new methods were applied to anaerobic-fermentor sludge and the results were compared to a conventional microbiological approach.

Extracellular DNA in environmental samples: Occurrence, extraction, quantification, and impact on microbial biodiversity assessment

2021 ◽  
Author(s):  
Sakcham Bairoliya ◽  
Jonas Koh Zhi Xiang ◽  
Bin Cao
Keyword(s):  
Dna Sequencing ◽  
Microbial Communities ◽  
Dna Extraction ◽  
Environmental Samples ◽  
Environmental Dna ◽  
Extracellular Dna ◽  
Biodiversity Assessment ◽  
Microbial Biodiversity ◽  
Technical Details ◽  
The Impact

Environmental DNA, i.e., DNA directly extracted from environmental samples, has been applied to understand microbial communities in the environments and to monitor contemporary biodiversity in the conservation context. Environmental DNA often contains both intracellular DNA (iDNA) and extracellular DNA (eDNA). eDNA can persist in the environment and complicate environmental DNA sequencing-based analyses of microbial communities and biodiversity. Although several studies acknowledged the impact of eDNA on DNA-based profiling of environmental communities, eDNA is still being neglected or ignored in most studies dealing with environmental samples. In this article, we summarize key findings on eDNA in environmental samples and discuss the methods used to extract and quantify eDNA as well as the importance of eDNA on the interpretation of experimental results. We then suggest several factors to consider when designing experiments and analyzing data to negate or determine the contribution of eDNA to environmental DNA-based community analyses. This field of research will be driven forward by: (i) carefully designing environmental DNA extraction pipelines by taking into consideration technical details in methods for eDNA extraction/removal and membrane-based filtration and concentration; (ii) quantifying eDNA in extracted environmental DNA using multiple methods including qPCR and fluorescent DNA binding dyes; (iii) carefully interpretating effect of eDNA on DNA-based community analyses at different taxonomic levels; and (iv) when possible, removing eDNA from environmental samples for DNA-based community analyses.

scholarly journals Rapid and Accurate Detection of Bacteriophage Activity againstEscherichia coliO157:H7 by Propidium Monoazide Real-Time PCR

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Hui Liu ◽  
Yan D. Niu ◽  
Jinquan Li ◽  
Kim Stanford ◽  
Tim A. McAllister
Keyword(s):  
Cell Surface ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Extracellular Dna ◽  
Bacterial Cells ◽  
Propidium Monoazide ◽  
Direct Plating ◽  
E Coli ◽  
Polymerase Chain ◽  
Phage Activity

Conventional methods to determine the efficacy of bacteriophage (phage) for biocontrol ofE. colirequire several days, due to the need to culture bacteria. Furthermore, cell surface-attached phage particles may lyse bacterial cells during experiments, leading to an overestimation of phage activity. DNA-based real-time quantitative polymerase chain reaction (qPCR) is a fast, sensitive, and highly specific means of enumerating pathogens. However, qPCR may underestimate phage activity due to its inability to distinguish viable from nonviable cells. In this study, we evaluated the suitability of propidium monoazide (PMA), a microbial membrane-impermeable dye that inhibits amplification of extracellular DNA and DNA within dead or membrane-compromised cells as a means of using qPCR to identify only intactE. colicells that survive phage exposure.Escherichia coliO157:H7 strain R508N and 4 phages (T5-like, T1-like, T4-like, and O1-like) were studied. Results compared PMA-qPCR and direct plating and confirmed that PMA could successfully inhibit amplification of DNA from compromised/damaged cellsE. coliO157:H7. Compared to PMA-qPCR, direct plating overestimated (P< 0.01) phage efficacy as cell surface-attached phage particles lysedE. coliO157:H7 during the plating process. Treatment of samples with PMA in combination with qPCR can therefore be considered beneficial when assessing the efficacy of bacteriophage for biocontrol ofE. coliO157:H7.

scholarly journals Comparative Analysis and Limitations of Ethidium Monoazide and Propidium Monoazide Treatments for the Differentiation of Viable and Nonviable Campylobacter Cells

2014 ◽  
Vol 80 (7) ◽  
pp. 2186-2192 ◽  
Author(s):  
Diana Seinige ◽  
Carsten Krischek ◽  
Günter Klein ◽  
Corinna Kehrenberg
Keyword(s):  
Correlation Coefficient ◽  
Campylobacter Coli ◽  
Bacterial Cells ◽  
Propidium Monoazide ◽  
Content Type ◽  
Field Isolates ◽  
Ethidium Monoazide ◽  
Cell Counts ◽  
Intercalating Dye ◽  
Diagnostic Applications

ABSTRACTThe lack of differentiation between viable and nonviable bacterial cells limits the implementation of PCR-based methods for routine diagnostic approaches. Recently, the combination of a quantitative real-time PCR (qPCR) and ethidium monoazide (EMA) or propidium monoazide (PMA) pretreatment has been described to circumvent this disadvantage. In regard to the suitability of this approach forCampylobacterspp., conflicting results have been reported. Thus, we compared the suitabilities of EMA and PMA in various concentrations for aCampylobacterviability qPCR method. The presence of either intercalating dye, EMA or PMA, leads to concentration-dependent shifts toward higher threshold cycle (CT) values, especially after EMA treatment. However, regression analysis resulted in high correlation coefficient (R2) values of 0.99 (EMA) and 0.98 (PMA) betweenCampylobactercounts determined by qPCR and culture-based enumeration. EMA (10 μg/ml) and PMA (51.10 μg/ml) removed DNA selectively from nonviable cells in mixed samples at viable/nonviable ratios of up to 1:1,000. The optimized EMA protocol was successfully applied to 16Campylobacter jejuniandCampylobacter colifield isolates from poultry and indicated the applicability for field isolates as well. EMA-qPCR and culture-based enumeration ofCampylobacterspiked chicken leg quarters resulted in comparable bacterial cell counts. The correlation coefficient between the two analytical methods was 0.95. Nevertheless, larger amounts of nonviable cells (>104) resulted in an incomplete qPCR signal reduction, representing a serious methodological limitation, but double staining with EMA considerably improved the signal inhibition. Hence, the proposedCampylobacterviability EMA-qPCR provides a promising rapid method for diagnostic applications, but further research is needed to circumvent the limitation.

The Evolution of Lead Extraction

2009 ◽  
Vol 5 (1) ◽  
pp. 32
Author(s):  
Melanie Maytin ◽  
Laurence M Epstein ◽  
◽  
Keyword(s):  
Natural Selection ◽  
Lead Extraction ◽  
Significant Morbidity ◽  
Extraction Techniques ◽  
Extraction Technology ◽  
Device Management ◽  
Future Developments ◽  
The Past ◽  
New Methods ◽  
Life Threatening

Prior to the introduction of successful intravascular countertraction techniques, options for lead extraction were limited and dedicated tools were non-existent. The significant morbidity and mortality associated with these early extraction techniques limited their application to life-threatening situations such as infection and sepsis. The past 30 years have witnessed significant advances in lead extraction technology, resulting in safer and more efficacious techniques and tools. This evolution occurred out of necessity, similar to the pressure of natural selection weeding out the ineffective and highly morbid techniques while fostering the development of safe, successful and more simple methods. Future developments in lead extraction are likely to focus on new tools that will allow us to provide comprehensive device management and the design of new leads conceived to facilitate future extraction. With the development of these new methods and novel tools, the technique of lead extraction will continue to require operators that are well versed in several methods of extraction. Garnering new skills while remembering the lessons of the past will enable extraction technologies to advance without repeating previous mistakes.

Preparation of environmental samples for chemical speciation of metal/metalloids: A review of extraction techniques

Talanta ◽  
2021 ◽  
Vol 226 ◽  
pp. 122119
Author(s):  
José Lucas Martins Viana ◽  
Amauri Antônio Menegário ◽  
Anne Hélène Fostier
Keyword(s):  
Environmental Samples ◽  
Chemical Speciation ◽  
Extraction Techniques

scholarly journals Discrimination of Viable and Dead Fecal Bacteroidales Bacteria by Quantitative PCR with Propidium Monoazide

2009 ◽  
Vol 75 (9) ◽  
pp. 2940-2944 ◽  
Author(s):  
Sungwoo Bae ◽  
Stefan Wuertz
Keyword(s):  
Quantitative Pcr ◽  
Light Exposure ◽  
Bacteroides Fragilis ◽  
Extracellular Dna ◽  
Propidium Monoazide ◽  
Human Feces ◽  
Heat Treated ◽  
Pcr Conditions

ABSTRACT Propidium monoazide (PMA) was optimized to discriminate between viable and dead Bacteroides fragilis cells and extracellular DNA at different concentrations of solids using quantitative PCR. Conditions of 100 μM PMA and a 10-min light exposure also excluded DNA from heat-treated cells of nonculturable Bacteroidales in human feces and wastewater influent and effluent.

Pseudomonas aeruginosa Oligoribonuclease Controls Susceptibility to Polymyxin B by Regulating Pel Exopolysaccharide Production

2022 ◽  
Author(s):  
Baopeng Yang ◽  
Yujun Jiang ◽  
Yongxin Jin ◽  
Fang Bai ◽  
Zhihui Cheng ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Resistance ◽  
Defense Mechanism ◽  
Extracellular Polysaccharide ◽  
Opportunistic Pathogen ◽  
Polymyxin B ◽  
Guanosine Monophosphate ◽  
Extracellular Dna ◽  
Bacterial Cells ◽  
Bacterial Survival

Polymyxins are considered as the last resort antibiotics to treat infections caused by multidrug-resistant Gram negative pathogens. Pseudomonas aeruginosa is an opportunistic pathogen that causes various infections in humans. Proteins involved in lipopolysaccharide modification and maintaining inner and outer membrane integrities have been found to contribute to the bacterial resistance to polymyxins. Oligoribonuclease (Orn) is an exonuclease that regulates the homeostasis of intracellular (3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), thereby regulating the production of extracellular polysaccharide in P. aeruginosa . Previously, we demonstrated that Orn affects the bacterial resistance to fluoroquinolone, β-lactam and aminoglycoside antibiotics. In this study, we found that mutation of orn increased the bacterial survival following polymyxin B treatment in a wild type P. aeruginosa strain PA14. Overexpression of c-di-GMP degradation enzymes in the orn mutant reduced the bacterial survival. By using a fluorescence labeled polymyxin B, we found that mutation of orn increased the bacterial surface bound polymyxin B. Deletion of the Pel synthesis genes or treatment with a Pel hydrolase reduced the surface bound polymyxin B and bacterial survival. We further demonstrated that Pel binds to extracellular DNA (eDNA), which traps polymyxin B and thus protects the bacterial cells. Collectively, our results revealed a novel defense mechanism against polymyxin in P. aeruginosa .

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