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


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.


CONCLUSIONS
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.
Kobayashi
H
H
Pathology and Laboratory Medicine Institute, The Cleveland Clinic, Cleveland, OH 44195, USA.
Oethinger
M
M
Tuohy
M J
MJ
Hall
G S
GS
Bauer
T W
TW
D001386:Azides / Q000737:chemistry*; D001431:Bacteriological Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D050296:Microbial Viability; D013211:Staphylococcus aureus / Q000737:chemistry* / Q000235:genetics; D013212:Staphylococcus epidermidis / Q000737:chemistry* / Q000235:genetics
D001386:Azides; D004269:DNA, Bacterial; C014535:8-azidoethidium