Selective PCR detection of viable Enterobacter sakazakii cells utilizing propidium monoazide or ethidium bromide monoazide.
OBJECTIVE
The detection of viable Enterobacter sakazakii cells is important due to the association of this pathogen with outbreaks of life-threatening neonatal infections. The aim of this study was to optimize a PCR-based method for selective detection of only viable Ent. sakazakii cells in the presence of dead cells, utilizing propidium monoazide (PMA) or ethidium bromide monoazide (EMA).


RESULTS
PMA or EMA was added to suspensions of viable and/or dead Ent. sakazakii cells at varying concentrations (10, 50 or 100 microg ml(-1)) prior to DNA isolation and PCR with Ent. sakazakii-specific primers. At concentrations of 50 and 100 microg ml(-1), PMA completely inhibited PCR amplification from dead cells, while causing no significant inhibition of the amplification from viable cells. PMA was also effective in allowing selective PCR detection of only viable cells in mixtures of varying ratios of viable and dead cells. EMA was equally effective in preventing amplification from dead cells, however, it also inhibited DNA amplification from viable cells.


CONCLUSIONS
This study demonstrated the efficiency of PMA for viable and dead differentiation of Ent. sakazakii, as well as the lack of selectivity of EMA for this purpose.


CONCLUSIONS
PMA-PCR, in particular, will be useful for monitoring the resistance, survival strategies and stress responses of Ent. sakazakii in foods and the environment.
Cawthorn
D-M
DM
Department of Food Science, University of Stellenbosch, Matieland, South Africa.
Witthuhn
R C
RC
D000345:Affinity Labels; D001386:Azides; D001431:Bacteriological Techniques; D044083:Cronobacter sakazakii / Q000235:genetics / Q000502:physiology*; D017931:DNA Primers / Q000235:genetics; D005516:Food Microbiology; D006801:Humans; D007223:Infant; D007225:Infant Food / Q000382:microbiology*; D007231:Infant, Newborn; D050296:Microbial Viability; D016133:Polymerase Chain Reaction / Q000379:methods*; D011419:Propidium / Q000031:analogs & derivatives*
D000345:Affinity Labels; D001386:Azides; D017931:DNA Primers; C533957:propidium monoazide; D011419:Propidium; C014535:8-azidoethidium