Insufficient Distinction between DNA from Viable and Nonviable Staphylococcus aureus Cells in Wipe-Samples by Use of Propidium Monoazide-PCR

Abstract

Treatment of dead bacteria with propidium monoazide (PMA) prior to DNA extraction has been described to inhibit subsequent amplification of DNA by polymerase chain reaction (PCR) methods. As an enforcement authority involved in the surveillance of compliance with biosafety regulations, the State Laboratory of Basel City swabs surfaces to check for contaminations with microorganisms. To evaluate the PMA-PCR method for its use in distinguishing DNA from living and dead bacteria in swab samples, we used Staphylococcus aureus as a model organism. In this study PMA treatment was able to reduce amplification of DNA from dead bacteria by a factor of 1,000. However, PMA also entered living S. aureus to some extent, resulting in a reduction of DNA amplification by a factor of 10. When cells were spotted on a dry surface and collected again by wiping, cells were still viable on agar plates, but a distinction between DNA from living or dead bacteria was no longer possible using the PMA-PCR method. The process of drying and wiping seemed to interfere with the membrane of S. aureus resulting in diffusion of PMA into still viable cells. The authors conclude that this method offers a limited capacity to distinguish between living and dead S. aureus in liquid culture but does not allow determining the amount of living S. aureus cells in wipe-samples.