Insufficient differentiation of live and dead Campylobacter jejuni and Listeria monocytogenes cells by ethidium monoazide (EMA) compromises EMA/real-time PCR

Abstract

Recently, ethidium monoazide (EMA) has been proposed as a means of reducing the real-time PCR signal originating from free DNA and dead bacterial cells by selectively entering damaged cells and blocking the DNA for PCR amplification via photoactivation. The present study investigated the effect of EMA on viable and dead bacterial cells using real-time PCR, plate count method and microscopy. The foodborne pathogens Campylobacter jejuni and Listeria monocytogenes were used as a Gram-negative and a Gram-positive model organism, respectively. EMA/real-time PCR analysis of heat-treated cultures of C. jejuni and L. monocytogenes containing 2.6x10(5) and 4x10(5) viable and 3x10(6) and 2x10(6) dead cells/ml, respectively, yielded 2x10(3) and 5.2x10(4) bacterial cell equivalents/ml after EMA treatment, thus underestimating the viable cell count in the samples. Similar results were obtained when analyzing late exponential phase cultures of C. jejuni and L. monocytogenes. Inhibition of growth by EMA was observed. It depended on the concentration of the bacterial cells present in the sample and the EMA concentration used (100-1 microg/ml). An EMA concentration at which dead cells would stain brightly and viable cells would not stain at all or would be very pale was not identified, as revealed by comparison with the results of a commercial live/dead stain. The results suggest that EMA influences not only dead but also viable cells of C. jejuni and L. monocytogenes. Thus EMA/real-time PCR is a poor indicator of cell viability.