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

Abstract

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 (r 2 ‫؍‬ 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 (r 2 ‫؍‬ 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.

Comparison of the Effects of Environmental Parameters on Growth Rates of Vibrio vulnificus Biotypes I, II, and III by Culture and Quantitative PCR Analysis

“…Findings by others support the existence of discrepancies between culture-dependent and culture-independent measurements, with concentrations determined by culture-dependent methods being smaller in each case (12,32). In our study, the greatest differences were always observed during the lag phase, and these discrepancies decreased as the cultures entered exponential phase.…”

“…Bacterial concentrations determined by qPCR are comparable to those measured by culturing during the exponential phase of growth (32). However, under less optimal conditions, such as during the stationary phase of growth, when nutrients are depleted and metabolites accumulate (22), culturable concentrations decrease, while qPCR-based concentrations remain unchanged (32).…”

“…Growth rates, determined as maximal rates of change of the fitted logistic curves (tangent lines at the point of inflection), were always larger when based on plate counts than when based on qPCR analysis. Whereas this is perhaps counterintuitive based on the fact that values of cell concentrations have the opposite relationship (estimates by qPCR are greater than those by culture methods), others have also observed differences between growth rates determined from culture-dependent and culture-independent estimates (32). Since the initial populations (lag phase and early exponential phase) likely contained relatively high proportions of nonculturable targets, the exponential phase of the respective logistic curves produced more shallow slopes.…”

Quantifying viable Vibrio parahaemolyticus and Listeria monocytogenes simultaneously in raw shrimp

“…The traditional predictive model was based on microbial growth data obtained by culture-dependent methods, where the process was rather time-consuming and labor-intensive (Ye et al 2013;Wang et al 2014). Recently, a few studies have reported that molecular methods could be used for construction of predictive models to describe the behavior of bacteria in food (Reichert-Schwillinsky et al 2009;Ye et al 2013Ye et al , 2014Liao et al 2015). As a novel molecular method, this PMA TaqMan-based multiplex real-time PCR method applied to construct predictive models had some remarkable advantages.…”

Development of a multiplex real-time PCR method for simultaneous detection of Salmonella enterica, Shigella flexneri and Listeria monocytogenes in processed food samples

“…1-2 m in length [10,11], and it is recognized worldwide as one of the most important food-borne pathogens of great concern for the food industries. This bacterium has the peculiarity that it can survive or even grow at low temperatures [12,13].…”