Stress- and Growth Rate-Related Differences between Plate Count and Real-Time PCR Data during Growth of Listeria monocytogenes

• Published in: Applied and Environmental Microbiology 2009-04, Vol.75 (7), p.2132-2138
• Main Authors: Reichert-Schwillinsky, Franziska, Pin, Carmen, Dzieciol, Monika, Wagner, Martin, Hein, Ingeborg
• Format: Article
• Language: English
• Subjects: Acids - pharmacology; Anti-Bacterial Agents - pharmacology; Bacteria; Biochemistry; Biological and medical sciences; Cell culture; Cell growth; Cells; Colony Count, Microbial - methods; Correlation analysis; Food Microbiology; Fundamental and applied biological sciences. Psychology; Listeria monocytogenes; Listeria monocytogenes - drug effects; Listeria monocytogenes - growth & development; Microbial Viability; Microbiology; Osmotic Pressure; Polymerase Chain Reaction - methods; Statistics as Topic; Stress, Physiological
• ISSN: 0099-2240; 1098-5336; 1098-6596
• DOI: 10.1128/AEM.01796-08

To assess the overestimation of bacterial cell counts in real-time PCR in relation to stress and growth phase, four different strains of L. monocytogenes were exposed to combinations of osmotic stress (0.5 to 8% [vol/vol] NaCl) and acid stress (pH 5 to 7) in a culture model at a growth temperature of 10°C or were grown under optimal conditions. Growth curves obtained from real-time PCR, optical density, and viable count data were compared. As expected, optical density data revealed entirely different growth curves. Good to moderate growth conditions yielded good correlation of real-time PCR data and plate count data (r² = 0.96 and 0.99) with similar cell counts. When growth conditions became worse, the numbers of CFU decreased during the stationary phase, whereas real-time-PCR-derived bacterial cell equivalents differed in this regard; the correlation worsened (r² = 0.84). However, fitted growth curves revealed that maximum growth rates calculated from real-time PCR data were not significantly different from those derived from plate count data. The overestimation of bacterial cell counts by real-time PCR observed in the stationary phase under higher-stress conditions might be explained by the accumulation of viable but nonculturable bacteria or dead bacteria and extracellular DNA. Considering these results, real-time PCR data collected from naturally contaminated samples should be viewed with caution.