Isolation of Highly Heat-Resistant Listeria monocytogenes Variants by Use of a Kinetic Modeling-Based Sampling Scheme

Abstract

Stable high-hydrostatic-pressure (HHP)-resistant Listeria monocytogenes LO28 variants were previously isolated and characterized. These HHP variants were also more resistant to heat. In addition, nonlinear heat inactivation kinetics pointed toward the existence of heat-resistant variants, although these could not be isolated so far. In this study, we used kinetic modeling of inactivation curves of two isolated HHP variants and their wild type, and this revealed that the probability of finding resistant variants should depend on the nature of the inactivation treatment and the time of exposure. At specific heat and HHP conditions, resistant LO28 and EGDe variants were indeed isolated. Resistant LO28 variants were even isolated after a heat inactivation at 72°C in milk, and these variants showed high resistance to standard pasteurization conditions. The increased resistance of part of the isolated LO28 and EGDe variants was due to mutations in their ctsR genes. For the variants whose ctsR genes and upstream regions were not altered, the mechanisms leading to increased resistance remain to be elucidated. This research showed the strength of kinetic modeling in unraveling the causes of nonlinear inactivation and facilitating the isolation of heat-resistant L. monocytogenes variants.Listeria monocytogenes can cause listeriosis in animal and human populations. Human listeriosis is a rare but severe disease and is one of the leading causes of death among patients with food-borne diseases in the United States. The estimated annual rate of invasive listeriosis in the United States is 3 cases and for countries within the European Union is 2 to 10 cases per million people per year (33). Recently, several European countries experienced an apparent increase in the incidence of listeriosis (13).A specific characteristic of L. monocytogenes that appears to be critical to its ability to cause human food-borne illness is its capacity to survive under harsh conditions. The occurrence of variants and the generation of population heterogeneity are factors that may contribute to the survival capacity of L. monocytogenes. Previous research showed that heterogeneity in L. monocytogenes populations (strains EGDe, LO28, and Scott A) affects resistance to high hydrostatic pressure (HHP). Inactivation of such heterogeneous populations resulted in survival curves with significant tailing, indicating the presence of an HHP-sensitive and an HHP-resistant fraction (32). Analysis of the cells that survived such HHP treatments revealed that the higher resistance of LO28 (32) and Scott A (17, 18) was a stable feature for part of the resistant fraction. Contrary to these results, no stable HHP-resistant isolates were obtained for EGDe (32). A significant fraction of the stably resistant variants of both Scott A and LO28 had an altered ctsR gene. This gene encodes CtsR, a DNA binding protein that regulates class III heat shock genes (7). The observed alterations in ctsR resulted not only in increased resistance to high pressure but also in increas...

Modeling and Validation of the Ecological Behavior of Wild-Type Listeria monocytogenes and Stress-Resistant Variants

“…Genotypic heterogeneity refers to the presence of stable stressresistant variants with an inheritable stress-resistant phenotype caused by genomic alterations (6). These stable stress-resistant variants have been repeatedly isolated from the foodborne pathogen Listeria monocytogenes upon exposure to different types of stress, e.g., heat, high hydrostatic pressure (HHP), and low pH (7)(8)(9)(10)(11).…”

Impact of microbial variability on food safety and quality

“…But differences between strains (figures 3.3C, 3.3F, and 3.3I) were much higher than differences between replicates or between reproductions. Moreover, at all temperatures, we observed that strains L6, FBR14, and FBR16 had lower inactivation rates than the other strains, and strains ScottA and LO28, which are often used as model strains (Casadei et al, 1998;Chen and Hoover, 2003;Gaze et al, 1989;Stephens et al, 1994;Sumner et al, 1991;Van Boeijen et al, 2011), were amongst the most heat sensitive strains at 60C and 65C. To estimate the D-value, the inactivation curves were fitted using a modified Weibull model.…”

“…Although a higher protective effect was given by ham, the D 72 -value of 3 L. monocytogenes strains grown and/or inactivated in ham were calculated to range from 0.5 s to 2 s. When a similar thermal processing for 15 s is applied for ham, the heat treatment will eliminate more than 7 log 10 CFU/g of the bacterial cells. However, Van Boeijen et al (2011) also reported the occurrence of resistant variants with average D 72 value of 3.02 ± 0.21 s, indicating that the current pasteurization process might not be sufficient to inactivate all heat stress robust variants or to meet the 6 log 10 reduction of the performance criterion as described by ILSI Europe. In general, the most important factors to be considered for thermal inactivation, in which each having about equal impact, are strain variability, the presence of resistant fractions in microbial populations, the effect of food matrix and effect of growth history.…”

“…Certain pre-culturing conditions were known to also increase the heat resistance of exponentially grown cells Linton et al, 1990), but their heat resistance was still lower than that of stationary grown cells. On the other hand, exposing cells to severe conditions might select for stable resistant variants that have higher heat resistance than stationary grown cells Van Boeijen et al, 2011). Moreover, the strong and consistent protective effect of ham as heating matrix increased the heat resistance of microorganisms, and this effect should also be considered during thermal inactivation.…”

“…The correlation between growth and thermal inactivation features and other phenotypic characteristics, such as biofilm forming capability and virulence capacity, might also be relevant for understanding the fate of these microorganisms along the food production chain. Since variants from a heat sensitive strain LO28 showed highly increased heat resistance (Van Boeijen et al, 2011), further research on the occurrence of heat resistance fractions for the most heat resistant strains used in our study, such as L6 for L. monocytogenes, might also be of interest.…”