Selective Removal of DNA from Dead Cells of Mixed Bacterial Communities by Use of Ethidium Monoazide

Abstract

The distinction between viable and dead bacterial cells poses a major challenge in microbial diagnostics. Due to the persistence of DNA in the environment after cells have lost viability, DNA-based quantification methods overestimate the number of viable cells in mixed populations or even lead to false-positive results in the absence of viable cells. On the other hand, RNA-based diagnostic methods, which circumvent this problem, are technically demanding and suffer from some drawbacks. A promising and easy-to-use alternative utilizing the DNA-intercalating dye ethidium monoazide bromide (EMA) was published recently. This chemical is known to penetrate only into "dead" cells with compromised cell membrane integrity. Subsequent photoinduced crosslinking was reported to inhibit PCR amplification of DNA from dead cells. We provide evidence here that in addition to inhibition of amplification, most of the DNA from dead cells is actually lost during the DNA extraction procedure, probably together with cell debris which goes into the pellet fraction. Exposure of bacteria to increasing stress and higher proportions of dead cells in defined populations led to increasing loss of genomic DNA. Experiments were performed using Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium as model pathogens and using real-time PCR for their quantification. Results showed that EMA treatment of mixed populations of these two species provides a valuable tool for selective removal of DNA of nonviable cells by using conventional extraction protocols. Furthermore, we provide evidence that prior to denaturing gradient gel electrophoresis, EMA treatment of a mature mixed-population drinking-water biofilm containing a substantial proportion of dead cells can result in community fingerprints dramatically different from those for an untreated biofilm. The interpretation of such fingerprints can have important implications in the field of microbial ecology.Whether bacterial cells are dead or alive is an important question with many implications for monitoring of food and water safety and for analysis of the sterility of pharmaceutical drugs. Due to the relatively long persistence of DNA after cell death, in the range of several days to 3 weeks (10, 14), quantitative analysis of total DNA can lead to a substantial overestimation of the presence of living microorganisms and the accompanying pathogenic threats. The lack of differentiation between DNA from viable and dead bacterial cells is therefore a major obstacle to broad-range application of DNA-based molecular diagnostics (10, 12). The most commonly used strategy to overcome this difficulty is to focus on the presence of the rapidly degrading RNA instead of the stable DNA (16,27,28,31). Due to its rapid turnover, the detection of RNA is far more indicative of the presence of viable cells (1, 2). Nevertheless, working with RNA is technically demanding, and RNA is prone to contamination with RNA-degrading enzymes, resulting in problems of reproducibility. Moreover, the RNA express...

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

“…EMA allows selective amplification of DNA by QPCR from only viable and not dead cells, which substantially increases the utility of PCR for rapidly determining and quantifying the presence of targeted viable microorganisms in environmental samples without enrichment (Rudi et al 2005). The discrimination of live versus dead cells occurs both during DNA extraction and PCR amplification (Nocker and Camper 2006). Our results are in accordance with earlier reports which have shown that quantitative analysis of total DNA can lead to a substantial overestimation of the presence of living microorganisms and the accompanying pathogenic threats (Luo et al 2008;Nocker andCamper 2006, Wang andLevin 2006).…”

“…These quantification methods significantly extrapolate the number of viable cells in mixed populations (Nocker and Camper 2006). Other methods for determination of viable cells such as those utilising RNA-based diagnostics are expensive and time-consuming (Alvarez 2004;Norton and Batt 1999).…”

“…The leaf and root samples were procured as described above. The method of EMA pretreatment was adopted from Nocker and Camper (2006) and modified partially as below. EMA (Molecular Probes, Invitrogen, Carlsbad, CA, USA) was dissolved in water (in the dark) to yield a stock solution of 5 mg ml −1 and stored at −20°C.…”

“…Ethidium monoazide (EMA) PCR has been reported to effectively discriminate between live and dead cells (Nocker and Camper 2006;Rudi et al 2005;Wang and Levin 2006). This diagnostic DNAbased method combines the use of a live-dead discriminating dye with the speed and sensitivity of quantitative PCR.…”

“…Subsequent photoinduced cross-linking inhibits the PCR amplification of DNA from dead cells (Soejima et al 2007). Nocker and Camper (2006) have reported that in addition to inhibition of amplification, DNA yield from the dead cells is affected during the DNA extraction procedure. Although EMA has been used to facilitate the quantitative real-time PCR amplification of targeted DNA sequences of various human pathogens (Nocker and Camper 2006;Rudi et al 2005;Wang and Levin 2006), there is just one report of using this technique to differentiate viable and dead cells of the plant pathogenic bacterium Clavibacter michiganensis subsp.…”