The effect of various environmental factors on the ethidium monazite and quantitative PCR method to detect viable bacteria

Abstract

Aims: Ethidium monoazide in combination with quantitative PCR (EMA–qPCR) has been considered as a promising method to enumerate viable cells; however, its efficacy can be significantly affected by disinfection conditions and various environments. In this study, thermal disinfection, osmotic pressure and acids with different pH values were systematically investigated to achieve the optimum conditions. Methods and Results: EMA treatment of pure cultures at low concentration (10 μg ml−1) for 20 min resulted in effective differentiation between viable and nonviable bacteria and had no effect on viable cells. Heating at 85°C for 35 min was the optimum condition that yields inactivated Escherichia coli (E. coli) cells that were not detected with EMA–qPCR. Performing EMA treatment in high‐salt ion environment (sodium chloride concentration ≥4%) could weaken EMA inhibition effect. Both strong and weak acid solutions could react with EMA, change its absorption spectra and influence EMA inhibition effect. Because of the sublethal acidification injury, underestimation of cell counts were found using EMA–qPCR method, and 40‐min incubation in Luria–Bertani medium could completely offset this error. Conclusion: Our results provided optimum EMA treatment, thermal disinfection and environment conditions for EMA–qPCR and demonstrated the feasibility of this method when enumerating viable cells under varied osmotic pressure and pH environment. Significance and Impact of the Study: Optimum EMA treatment, thermal disinfection and EMA‐treated environment will be successfully applied in EMA–qPCR. Osmotic pressure and acid‐induced injury can be detected by EMA–qPCR with optimization.

Progress in understanding preferential detection of live cells using viability dyes in combination with DNA amplification

“…Cawthorn and Witthuhn (2008) have found that the use of three different EMA concentrations with viable Enterobacter sakazakii cells − 100 (238 μM), 50 (119 μM), and 10 μg/ml (23.8 μM) -resulted in very different PCR product yields of 74, 82 and 92%, respectively (compared with 95, 96, and 97% after PMA treatment). A concentration of 10 μg/ml EMA was found to be suitable in various studies (Minami et al, 2010;Shi et al, 2011;Soejima et al, 2011a;Wang et al, 2009), whereas a higher concentration resulted in penetration of live cells. Minimum amounts of EMA required for effective signal suppression from dead cells on the other hand were reported to be 2.5 μg/ml for Vibrio vulnificus (Wang and Levin, 2006), 2.3 μg/ml for Legionella (Chen and Chang, 2010), 1.5 μg/ml for Bifidobacterium (Meng et al, 2010), and 1 μg/ml for bacterial flora from fish filet (Lee and Levin, 2009a).…”

“…Whereas the results of the above studies are based on the interaction of dye with purified DNA, a more recent study by Shi et al (2011) investigated the effect of different factors (including salt) on v-PCR results using cells exposed to different solutions. The experimental design was based on heat-killed cells that were exposed to different concentrations of NaCl (0.125 to 10%, corresponding to a range from approx.…”

“…The authors concluded that the presence of elevated salt concentrations should be avoided when EMA treatment is performed, and washing of cells is advisable. It is therefore likely that halophilic microorganisms will need customized procedures to maximize dye performance and minimize cell death during treatment as a consequence of osmotic alterations (Barth et al, 2012;Fittipaldi et al, 2011b;Shi et al, 2011). Recently Barth et al (2012) found that concentrations of 5% NaCl and above seemed to limit the suppression caused by PMA to DNA from dead cells.…”

New insights into Legionella pneumophila biofilm regulation by c-di-GMP signaling

“…vPCR allows the quanti cation of viable bacteria only. is method has been used successfully for the quanti cation of viable cells in several bacterial species (Shi et al 2011;Li & Chen 2013), including viable but not cultivable cells (VBNC), which can make up a high proportion of bio lm cells. e results were systematically compared with those of the WT bio lm by calculating the log-transformed ratio of the population of sessile bacteria in the mutant and the WT strains (log (mutant /WT)) ( Figure S2).…”

Establishment of a viable cell detection system for microorganisms in wine based on ethidium monoazide and quantitative PCR

“…oeni LAB and total AAB in wine (Hierro, Esteve-Zarzoso, Gonzá-lez, Mas, & Guillamón, 2006;Jara et al, 2008;Martorell et al, 2005;Neeley et al, 2005). An ethidium monoazide bromide (EMA) qPCR (EMA-qPCR) method has been described as a promising method to discriminate viable and non-viable cells (Nocker, Cheung, & Camper, 2006;Nogva, Drømtorp, Nissen, & Rudi, 2003;Shi et al, 2011). EMA, a DNA-intercalating dye that can only penetrate cells with compromised membranes, covalently binds to DNA through photoactivation, consequently inhibiting subsequent qPCR.…”