Unsuitable distinction between viable and dead Staphylococcus aureus and Staphylococcus epidermidis by ethidium bromide monoazide

Authors


Thomas W. Bauer, Departments of Pathology, Orthopaedic Surgery and the Spine Center, L-25, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA. E-mail: osteoclast@aol.com

Abstract

Aims:  The DNA-intercalating dye ethidium bromide monoazide (EMA) has recently been used as a DNA binding agent to differentiate viable and dead bacterial cells by selectively penetrating through the damaged membrane of dead cells and blocking the DNA amplification during the polymerase chain reaction (PCR). We optimized and tested the assay in vitro using Staphylococcus aureus and Staphylococcus epidermidis cultures to distinguish viable from dead bacteria, with the goal of reducing false positive PCR results.

Methods and Results:  Viable and heat-inactivated bacteria were treated with EMA or left untreated before DNA extraction. A real-time PCR assay for the detection of the tuf gene in each DNA extract was used. Our results indicated that EMA influenced viable bacteria as well as dead bacteria, and the effect of EMA depended on the EMA concentration and bacterial number.

Conclusions:  EMA is not a suitable indicator of bacterial viability, at least with respect to Staphylococcus species.

Significance and Impact of the Study:  Determining the viability of pathogens has a major impact on interpreting the results of molecular tests for bacteria and subsequent clinical management of patients. To this end, several methods are being evaluated. One of these methods – intercalating DNA of dead bacteria by EMA – looked very promising, but our study found it unsatisfactory for S. aureus and coagulase-negative Staphylococci.

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