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

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 anaerobicfermentor sludge and the results were compared to a conventional microbiological approach.For pathogen risk assessment and hygienic safety control in anaerobic digesters, various culture-based microbiological methods are in use. However, with the application of classical methods, a number of problems arise: long cultivation times for some microorganisms, the complexity of anaerobic cultivation, and timeconsuming lab work (enrichment of selected organisms, selective cultivation, and subsequent systematic differentiation). Alternatively, molecular tools could be used, but fast and easy methods, such as PCR amplification after conventional DNA extraction, do not always guarantee the amplification of viable cells' DNA only (6), which might result in false-positive data (9). On the other hand, RNA-based approaches, which would target the active part of a microbial community, thus enabling discrimination between living and dead cells, encounter problems with the high RNA decay rates after the loss of cell viability (1) and are also expensive and laborious.A new DNA extraction technique including an additional step to remove free, extracellular DNA and DNA of dead bacterial cells by using light-activated ethidium monoazide (EMA) or propidium monoazide (PMA) was described previously, noting the possibility of a selective suppression of DNA detection in dead cells (10,11,15). To our knowledge, these extraction procedures were tested successfully with a simple matrix (12), whereas an evaluation of environmental matrices, such as the sludge of an anaerobic digestion plant, has not yet been performed.The aim of this work was to test the suitability of EMA and PMA for the extraction of free DNA and DNA originating from dead cells in an environmental matrix. The extracted DNA was subsequently amplified via real-time PCR (quantitative PCR [qPCR]) using specific primers for selected pathogenic microorganisms (Clostridium perfringens, Listeria monocytogenes, and Salmonella enterica), and the results were compared to classical cultivation-based agar plating data.The following organisms, selected after an Austrian standard guideline (14), and an anaerobic spore-forming microorganism, were used after microscopic verification and selective plate counting: Clostridium perfringens (DSM 11780; German Collection of Microorganisms and Cell Cultures, http://www .dsmz.de), Listeria monocytogenes (DSM 15675), and Salmonella enterica subsp. enterica serovar Senftenberg (DSM 10062). Pure cultures of L. monocytogenes and C. perfringens were grown in DSM medium 92 (30.0 g Trypticase soy broth, 3.0 g yeast extract, 1,000 ml distilled water, pH 7.0), and pure cultures of S. enterica were grown in DSM medium 220 (15.0 g peptone from casein, 5.0 ...

Use of Propidium Monoazide in Reverse Transcriptase PCR To Distinguish between Infectious and Noninfectious Enteric Viruses in Water Samples

“…6). This is in agreement with the findings of other workers, who have reported that PMA is useful for removing nonspecific PCR products common in environmental water samples (17,46).…”

Survival of Host-Associated Bacteroidales Cells and Their Relationship with Enterococcus spp., Campylobacter jejuni, Salmonella enterica Serovar Typhimurium, and Adenovirus in Freshwater Microcosms as Measured by Propidium Monoazide-Quantitative PCR

“…The slopes from the cultivation-dependent method and PMA-qPCR were significantly different (P values ϭ 0.001 and 0.02 for sunlight exposure and dark condition, respectively), which was likely due to the presence of intact but noncultivable Enterococcus cells. The higher numbers of tENT compared to cENT in freshwater, estuarine water, and beach sand have previously been reported and attributed to greater persistence of noncultivable organisms and/or free DNA (10,11,46).…”

“…The potential contribution of sediments as secondary habitats of Bacteroidales or waterborne pathogens needs to be established considering that other enteric bacteria and waterborne pathogens can persist by colonizing surfaces and forming biofilms (6,9,46). The decay rates should be integrated into models of the fate of enteric bacteria in water to implement a remediation strategy, and longterm and large-scale field studies need to be conducted with the decay model to validate the potential of microbial source tracking.…”

Quantification of viable Candidatus Liberibacter asiaticus in hosts using quantitative PCR with the aid of ethidium monoazide (EMA)

“…This diagnostic DNAbased method combines the use of a live-dead discriminating dye with the speed and sensitivity of quantitative PCR. Viable and dead discrimination is obtained by covalent binding of EMA to DNA of dead cells by photoactivation (Wagner et al 2008). EMA penetrates only the dead cells with compromised membrane/cell systems.…”