Modeling the lag phase of Listeria monocytogenes

An estimate of the lag phase duration is an important component for predicting the growth of a bacterium and for creating process models and risk assessments. Most current research and data for predictive modeling programs initiated growth studies with cells grown to the stationary phase in a favorable pH, nutrient and temperature environment. In this work, Listeria monocytogenes Scott A cells were grown in brain heart infusion (BHI) broth at different temperatures from 4 to 37 degrees C to the exponential growth or stationary phases. Additional cells were suspended in a dilute broth, desiccated or frozen. These cells were then transferred to BHI broth at various temperatures from 4 to 37 degrees C and the lag phase durations were determined by enumerating cells at appropriate time intervals. Long lag phases were observed for cells initially grown at high temperatures and transferred to low temperatures. In general, exponential growth cells had the shortest lag phases, stationary phase and starved cells had longer, frozen cells had slightly longer and desiccated cells had the longest lag phases. These data were from immediate temperature transitions. When a computer-controlled water bath linearly changed the temperature from 37 to 5 degrees C over a 3.0- or 6.0-h period, the cells had short lags and grew continuously with declining growth rates. Transitions of 0.75 or 1.0 h had 20-h lag phases, essentially that of immediate transitions. When the transition was 1.5 h, an intermediate pattern of less than 1 log of growth followed by no additional growth for 20 h occurred.     

Modelling the individual cell lag phase: effect of temperature and pH on the individual cell lag distribution of Listeria monocytogenes

The individual-based approach of the lag phase is gaining interest, especially for pathogens that initially contaminate food products in low amounts. In this paper, the effect of temperature (30, 10, 7, 4 and 2 °C) and pH (7.4, 6.1, 5.5, 5.0, 4.7 and 4.4) on the individual cell lag phase of Listeria monocytogenes was examined in a factorial design, using OD measurements. Individual lag phases of about 100 individual cells per condition were examined and calculated using a linear extrapolation method. Generation times were calculated out of the slope.

The obtained data were analyzed at three different levels: in a first approach, the mean values were calculated for each set of environmental conditions and compared to predictions made by the USDA's Pathogen Modeling Program (PMP) for analogous growth conditions. The PMP predictions of the generation times were in the same order of magnitude as the obtained data, although a persistent underestimation could be observed. The observed individual cell lag data differed from lag phase predictions by PMP. Possible reasons for this discrepancy are discussed.

Secondly, histograms of individual lag phase measurements were constructed for the different temperature–pH combinations. In this way, the influence of both factors on the variability of individual lag phases could be estimated. At low stress levels, most individual cells showed a short lag phase resulting in a compression of the histograms at the zero-lag level, while, at high stress levels, the histograms shifted to longer lag phases with a significant increase in variability.

Thirdly, 37 different distribution types were fitted to the datasets to reveal the distributions that fitted best the obtained data. The gamma distribution was preferred at moderate stress levels, while the Weibull distribution was chosen for harsher growth conditions.     

Modelling the effect of a temperature shift on the lag phase duration of Listeria monocytogenes

The aim of this work is to study and model the effect of a temperature shift on h₀, the product of the growth rate by the lag phase duration (μλ). Our work is based on the data of Whiting and Bagi [Int. J. Food Microbiol. 73 (2002) 291], who studied the influence of both the pre-incubation temperature (T_prior) and the growth temperature (T_growth) on λ values of Listeria monocytogenes. We introduce a new model to describe the evolution of the parameter h₀ as a function of T_prior and T_growth, and compare it to Whiting and Bagi's published polynomial model that describes the influence of T_prior and T_growth on λ independently of μ. For exponential as well as stationary phase cells, h₀ increases almost linearly with the magnitude of the temperature shift. A simple linear model of h₀ turns out to be more suitable to predict λ values than a polynomial model of λ.     

A combined discrete-continuous model describing the lag phase of Listeria monocytogenes

Food microbiologists generally use continuous sigmoidal functions such as the empirical Gompertz equation to obtain the kinetic parameters specific growth rate (mu) and lag phase duration (lambda) from bacterial growth curves. This approach yields reliable information on mu; however, values for lambda are difficult to determine accurately due, in part, to our poor understanding of the physiological events taking place during adaptation of cells to new environments. Existing models also assume a homogeneous population of cells, thus there is a need to develop discrete event models which can account for the behavior of individual cells. Time to detection (t(d)) values were determined for Listeria monocytogenes using an automated turbidimetric instrument, and used to calculate mu. Mean individual cell lag times (tL) were calculated as the difference between the observed t(d) and the theoretical value estimated using mu. Variability in tL for individual cells in replicate wells was estimated using serial dilutions. A discrete stochastic model was applied to the individual cells, and combined with a deterministic population-level growth model. This discrete-continuous model incorporating tL and the variability in tL (expressed as standard deviation; S.D.(L)) predicted a reduced variability between wells with increased number of cells per well, in agreement with experimental findings. By combining the discrete adaptation step with a continuous growth function it was possible to generate a model which accurately described the transition from lag to exponential phase. This new model may serve as a useful tool for describing individual cell behavior, and thus increasing our knowledge of events occurring during the lag phase.     

The effect of inoculum size on the lag phase of Listeria monocytogenes.

The effect of inoculum size on population lag times of Listeria monocytogenes was investigated using the Bioscreen automated microtitre plate incubator and reader. Under optimum conditions, lag times were little affected by inoculum size and there was little variation between replicate inocula even at very low cell numbers. However, in media containing inhibitory concentrations of NaCl, both the mean lag time and variation between replicate inocula increased as the inoculum size became smaller. The variation in lag time of cells within a population was investigated in more detail by measuring the distribution of detection times from 64 replicate inocula containing only one or two cells capable of initiating growth. The variance of the lag time distribution increased with increasing salt concentration and was greater in exponential than in stationary phase inocula. The number of cells required to initiate growth increased from one cell under optimum conditions to 10(5) cells in medium with 1.8 M NaCl. The addition of spent medium from a stationary phase culture reduced the variance and decreased lag times. The ability to initiate growth under severe salt stress appears to depend on the presence of a resistant sub-fraction of the population, although high cell densities assist adaptation of those resistant cells to the unfavourable growth conditions by some unspecified medium conditioning effect. These results are relevant to the prediction of lag times and probability of growth from low numbers of stressed cells in food.     

Effect of preincubation temperature and pH on the individual cell lag phase of Listeria monocytogenes, cultured at refrigeration temperatures

The impact of precultural temperature and pH on the distribution of the lag phase of individual Listeria monocytogenes cells was assessed during preincubation at 7 degrees C, using a dilution protocol to obtain single cells, and optical density measurements to estimate the individual lag phase. Firstly, the pure temperature effect (37, 15, 10, 7, 4 and 2 degrees C) was investigated on a subsequent growth at 7 degrees C and pH 7.4. Secondly, low precultural temperatures (10, 7 and 4 degrees C) were combined with a controlled pH at 7.4 and 5.7 with a subsequent growth at 7 degrees C and at different pH values (7.4, 6.0 and 5.5). For all temperature-pH combinations, the individual cell lag phase was determined using a three-phase linear growth model. It was observed that at low precultural temperatures (2, 4 and 7 degrees C), a high proportion of L. monocytogenes cells were able to grow at 7 degrees C with almost no lag phase, consequently, the resulting distributions were positively skewed. Beside this, the variability observed was lower than at higher precultural temperatures. Regarding the precultural pH effect, at pH 7.4 the mean values of the lag phases were shorter at lower preincubation temperatures; while at pH 5.7 small pH transitions produced shorter individual lag phases at all precultural temperatures. The quantification of the effect of precultural conditions on the individual cell lag phase duration would improve the accuracy of the existing growth models, especially when a series of processing and storage steps are linked together in a process model or exposure assessment. Distributions will be fitted to the data for every set of conditions, generating useful tools for further risk assessment purposes.     

Influence of different histories of the inoculum on lag phase and growth of Listeria monocytogenes in meat models

The aim of this study was to determine the effect of history of inoculum and preservatives on the lag phase and growth rate of Listeria monocytogenes strains in meat products packaged under modified atmosphere conditions. Inocula with different histories were added to meat models, and growth rate and lag phase of two strains of L. monocytogenes were measured at 5 and 10 degrees C. The meat model stored at 10 degrees C contained sodium lactate, but the model stored at 5 degrees C did not. The five different histories of the inocula included cold propagation, biofilm formation, and starvation. The lag phase ranged from 1 to 10 days and was affected by the history of the inoculum, whereas the growth rate was constant except for one combination of history of inoculum and strain, where growth did not start during the incubation period. In a second series of experiments, the growth rate and lag phase of the two Listeria strains and the effects of two different histories of inoculum were tested in meat models with pH 5.7 or 6.5 and increasing amounts of NaCl. The growth rate depended on salt concentration, bacterial strain, and pH, whereas lag phase duration depended on history of inoculum, salt concentration, and pH. The lag phase duration was highly dependent on the history of the inoculum, and higher amounts of preservative (NaCl) made these effects even more noticeable. The results of this study underline the importance of the effects of the history of the inoculum on lag phase duration and could be used to predict lag phase in industrial meat products.     

Growth pH does not affect the initial physiological state parameter (pO) of Listeria monocytogenes

It has proven difficult to develop adequate mathematical models for the lag phase (lambda) which characterizes the adaptation period prior to the initiation of exponential growth by microorganisms. This is due, in part, to our incomplete understanding of the nature of the initial physiological state of cells (defined as h0 or p0 depending on the model), and changes taking place during adaptation. The objectives of the present study were to characterize p0 using data from growth of Listeria monocytogenes in an automated turbidimetric instrument (Bioscreen), and to determine the influence of limiting growth pH. A model was developed for individual cells which combined a continuous adaptation phase (defined by p0) with a discrete step marking the transition to a continuous exponential growth phase (the CDC model). Parameters of the new model were: p0; the specific growth rate (mu); the initial cell number (N0); and the maximum cell density (Nmax). Progressive reduction of the growth pH in the Bioscreen to 4.7 decreased the p. It was noted that the regression lines for all trials at all pH values appeared to have a common x-intercept (20.086+/-1.092), and it was deduced that, when the Bioscreen detection limit (15.07 In cfu well(-1)) was subtracted, the resulting value represented the "true" value for the initial physiological state of the cells.     

Modeling the physiological state of the inoculum and CO2 atmosphere on the lag phase and growth rate of Listeria monocytogenes

In previous studies, the growth of L. monocytogenes has been modeled under different CO2 headspace concentrations; however, the inoculum cells were always in the stationary phase. In this study, the growth of L. monocytogenes under different CO2 concentrations as affected by the physiological state of the cells was investigated. Exponential-growth-phase, stationary-phase, dried, and starved cells were prepared and inoculated at 5 degrees C into brain heart infusion broths that had been preequilibrated under atmospheres of 0, 20, 40, 60, or 80% CO2 (the balance was N2). Lag-phase duration times (LDTs) and exponential growth rates were determined by enumerating cells at appropriate time intervals and by fitting the data to a three-phase linear function that has a lag period before the initiation of exponential growth. Longer LDTs were observed as the CO2 concentration increased, with no growth observed at 80% CO2. For example, the LDTs for exponential-phase, stationary-phase, starved, and dried cells were 2.21, 8.27, 9.17, and 9.67 days, respectively, under the 40% CO2 atmosphere. In general, exponential-growth-phase cells had the shortest LDT followed by starved cells and stationary-phase cells. Dried cells had the longest LDT. Exponential growth rates decreased as the CO2 concentrations increased. Once exponential growth was attained, no retained differences among the various initial physiological states of the cells for any of the atmospheres were observed in the exponential growth rates. The exponential growth rates under 0, 20, 40, 60, and 80% CO2 averaged 0.39, 0.37, 0.23, 0.23, and 0.0 log CFU/day, respectively. Dimensionless factors were calculated that describe the inhibitory action of CO2 on the LDTs and exponential growth rates for the various physiological states.     

Mathematical modelling of the growth rate and lag time for Listeria monocytogenes

Growth data for Listeria monocytogenes were collected from the literature and a global model built with existing secondary models describing independently the effects of environmental factors on the growth rate and lag time was based on these data. The growth rates calculated with this model were consistent with the published ones but the fit was poor near the limits of growth of the micro-organism. The model was also less accurate to describe the lag time. It seems then that reliable predictions of the growth rate of L. monocytogenes could be obtained in a wide range of growth conditions, but models should take into account interactions between environmental factors. Furthermore, it is necessary to better model the lag phase duration and particularly to model the effect of the history of the inoculum on the subsequent lag time.     

The influence of mayonnaise pH and storage temperature on the growth of Listeria monocytogenes in seafood salad

Seafood salad has been identified as a ready-to-eat food with a relatively high incidence of contamination by Listeria monocytogenes; however, little is known about the behavior of this pathogen in seafood salad as a function of product pH and storage temperature. To produce data towards the development of a predictive growth model, a 6-strain cocktail of L. monocytogenes was inoculated onto the surface of a shrimp-crabmeat product, mixed with mayonnaise that was previously adjusted with NaOH to pH 3.7, 4.0, 4.4, 4.7 or 5.1, and then stored at 4 degrees , 8 degrees or 12 degrees C under both aerobic and vacuum conditions. At each storage temperature, L. monocytogenes was able to grow in the seafood salad under both aerobic and vacuum conditions. The slowest growth of L. monocytogenes was observed in seafood salad with a mayonnaise pH of 3.7 and a storage temperature of 4 degrees C under vacuum condition. In salad with the same mayonnaise pH, the growth rate (GR, log10 cfu/h) of L. monocytogenes increased as a function of storage temperature. At the same storage temperature, the lag phase duration (LPD, h) of L. monocytogenes decreased as mayonnaise pH increased. At the same mayonnaise pH and temperature, LPD of L. monocytogenes was greater under aerobic than under vacuum conditions. Regression analyses indicated that mayonnaise pH is the main effector on the LPD of L. monocytogenes in seafood salad, and storage temperature was the main effector on the GR. Secondary models that describe LPD and GR of L. monocytogenes in seafood salad as a function of mayonnaise pH and storage temperature were produced.     

A model describing the effect of temperature history on lag time for Listeria monocytogenes

A model was built to describe the influence of the temperature and the duration of pre-incubation on the lag time for regrowth of Listeria monocytogenes at low temperature. This model is consistent with the usual procedure used to calculate lag times of cultures growing under fluctuating temperatures. It also describes the effect of prolonged starvation conditions on the regrowth lag time and takes into account the influence of the physiological state of inocula in predictive models.     

Effect of temperature and pH on survival of Listeria monocytogenes in coleslaw

The survival and growth of Listeria monocytogenes in fresh coleslaw, pH 3.9, and in coleslaw adjusted to pH 4.0, 5.0, 6.0 or 7.0 before inoculation was studied at three temperatures (4, 15 and 25 degrees C). L. monocytogenes was not detectable after 5 days incubation in fresh coleslaw nor in coleslaw adjusted to pH 4.0. Coleslaw at pH 5.0 was also inhibitory to L. monocytogenes at all three temperatures studied. A decline in viable numbers of L. monocytogenes in coleslaw at pH 6.0 occurred at 4 degrees C and at 15 degrees C, whereas at 25 degrees C the viable count of L. monocytogenes increased initially and remained high after incubation for 25 days. L. monocytogenes grew rapidly in coleslaw at pH 7.0 at all three temperatures studied, followed by an equally rapid decline in viable count.     

Growth kinetics of Listeria monocytogenes in broth and beef frankfurters--determination of lag phase duration and exponential growth rate under isothermal conditions

ABSTRACT:  The objective of this study was to develop a new kinetic model to describe the isothermal growth of microorganisms. The new model was tested with Listeria monocytogenes in tryptic soy broth and frankfurters, and compared with 2 commonly used models—Baranyi and modified Gompertz models. Bias factor (BF), accuracy factor (AF), and root mean square errors (RMSE) were used to evaluate the 3 models. Either in broth or in frankfurter samples, there were no significant differences in BF (approximately 1.0) and AF (1.02 to 1.04) among the 3 models. In broth, the mean RMSE of the new model was very close to that of the Baranyi model, but significantly lower than that of the modified Gompertz model. However, in frankfurters, there were no significant differences in the mean RMSE values among the 3 models. These results suggest that these models are equally capable of describing isothermal bacterial growth curves. Almost identical to the Baranyi model in the exponential and stationary phases, the new model has a more identifiable lag phase and also suggests that the bacteria population would increase exponentially until the population approaches to within 1 to 2 logs from the stationary phase. In general, there is no significant difference in the means of the lag phase duration and specific growth rate between the new and Baranyi models, but both are significantly lower than those determined from the modified Gompertz models. The model developed in this study is directly derived from the isothermal growth characteristics and is more accurate in describing the kinetics of bacterial growth in foods.     

Effects of Temperature and Oxygen on the Growth of Listeria monocytogenes at pH 4.5

Growth effects were studied using tryptose phosphate broth adjusted with hydrochloric acid. The microorganism survived for extended periods at low incubation temperatures (5 and 10°C), and grew at intermediate temperatures (19 and 28°C). Aerobic incubation at 37°C resulted in relatively rapid inactivation of the organism; however, when oxygen was restricted the organism recovered and survived for extended periods. Oxygen restriction enhanced the growth rate at 19°C. Results demonstrated temperature and oxygen availability interacted to influence survival of L. monocytogenes in low pH environment.     

Attachment of Listeria monocytogenes to Stainless Steel Surfaces at Various Temperatures and pH Values

The attachment of Listeria monocytogenes isolates Scott A and Jalisco Cheese to stainless steel surfaces at 35°, 21°, and 10°C was investigated using scanning electron microscopy (SEM). Cells were found to adhere at all three temperatures, but cells with fibrils were observed only at 21°C. When grown at pH 8, the attachment matrix was more prevalent at 21°C than at 35°C and may also be related to the length of incubation time at 21°C. It appears that adherence may be related to the flagella and any exopolymer surrounding the cells.     

Modeling the lag phase and growth rate of Listeria monocytogenes in ground ham containing sodium lactate and sodium diacetate at various storage temperatures

ABSTRACT:  Refrigerated ready-to-eat (RTE) meats contaminated with Listeria monocytogenes were implicated in several listeriosis outbreaks. Lactate and diacetate have been shown to control L. monocytogenes in RTE meats. The objective of this study was to examine and model the effect of lactate (1.0% to 4.2%) and diacetate (0.05% to 0.2%) in ground ham on the lag phase duration (LPD, h) and growth rate (GR, log CFU/h) of L. monocytogenes at a range of temperatures (0 to 45 °C). A 6-strain mixture of L. monocytogenes was inoculated into ground ham containing lactate and diacetate, and stored at various temperatures. The LPD and GR of L. monocytogenes in ham as affected by lactate, diacetate, and storage temperature were analyzed and accurately represented with mathematical equations. Resulting LPD and GR equations for storage temperatures within the range of 0 to 36 °C significantly represented the experimental data with a regression coefficient of 0.97 and 0.96, respectively. Significant factors ( P < 0.05) that affected the LPD were temperature, lactate, diacetate, and the interactions of all three, whereas only temperature and the interactions between temperature and lactate and diacetate had a significant effect on GR. At suboptimal growth temperatures (≤12 °C) the increase of lactate and diacetate concentrations, individually or in combination, extended the LPD. The effect of higher concentrations of both additives on reducing the GR was observed only at temperatures that were more suitable for growth of L. monocytogenes , that is, 15 to 35 °C. These data may be used to assist in determining concentrations of lactate and diacetate in cooked ham products to control the growth of L. monocytogenes over a wide range of temperatures during manufacturing, distribution, and storage.     

Characterization of the novel Listeria monocytogenes PCR serogrouping profile IVb-v1

The World Health Organization Collaborating Centre for Listeria (WHOCCL) has developed in 2004 a multiplex PCR assay that separates the 4 major Listeria monocytogenes serovars (1/2a, 1/2b, 1/2c, and 4b) into distinct PCR serogroups. A new PCR profile has been recently identified, constituted of amplified DNA fragments of prs, ORF2819, ORF2110 and lmo0737. Here we characterize 22 L. monocytogenes isolates of the WHOCCL collection with this PCR IVb variant 1 (IVb-v1) profile. The 22 isolates belong to the clinically predominant serovar 4b, exhibit 6 distinct pulsed-field gel electrophoresis ApaI/AscI combined profiles, and belong to 2 unrelated multilocus sequence types, indicating that the novel profile does not correspond to a recent clonal emergence. We have updated the WHOCCL serogroup-related PCR typing scheme to include this new profile.     

A model describing the relationship between regrowth lag time and mild temperature increase for Listeria monocytogenes

In order to comply with the consumer demand for ready-to-eat and look 'fresh' products, mild heat treatment will be used more and more in the agrofood industry. Nonetheless there is no tool to define the most appropriate mild heat treatment. In order to build this tool, it is necessary to study and describe the response of a bacterial population to a mild increase in temperature, from the dynamic point of view. The response to a mild increase in temperature, defined by stress duration and temperature, consisted in a mortality phase followed by the lag time of the survivors and their exponential growth. The effect of the mild increase in temperature on the mortality phase was described in a previous paper (Bréand et al., Int. J. Food Microbiol., in press). The effect of the stress duration on the lag was presented in a previous paper (Bréand et al., Int. J. Food Microbiol. 38 (1997) 157-167). In particular, the biphasic relationship between the lag and the stress duration was observed and modelled with a four parameter nonlinear model: the primary model (Bréand et al., Int. J. Food Microbiol. 38 (1997) 157-167). The study presented in this paper deals with the effect of the stress temperature on the biphasic relationship between the lag time and the stress duration. The secondary models describing the effect of the stress temperature on this biphasic relationship, were empirically built from our experimental data concerning Listeria monocytogenes. This work pointed out that the higher the stress temperature, the narrower the range of stress duration for which the lag time increased. Since the primary and the secondary models of the lag time were available, the global model describing the effect of the mild increase duration and temperature directly on the lag was fitted. This model allowed an improvement of the parameter estimator precision. The potential contribution in mild heat treatment optimization of this global model and the one built for the mortality phase (Bréand et al., Int. J. Food Microbiol., in press) is discussed.