A comparative phenotype analysis of 24 Listeria monocytogenes LO28 stress-resistant variants obtained after high-pressure treatment was performed to assess their robustness and growth performance under a range of food-relevant conditions. In addition, genetic analysis was conducted to characterize the promoter regions and open reading frames of the class I and III transcriptional repressors CtsR and HrcA, which control production of specific sets of stress proteins. Analysis of stress survival capacity, motility, biofilm formation, and growth under various conditions showed all variants to be more resistant to pressure and heat than the wild type; however, differences among variants were observed in acid resistance, growth rate, motility, and biofilmforming capacity. Genetic analysis revealed no variation in the genetic make-up of hrcA and its upstream region, but two variants had deletions in the upstream region of ctsR and seven variants had mutations in the ctsR gene itself. The results of the characterization were cluster analyzed to obtain insight into the diversity of variants. Ten unique variants and three clusters with specific features could be identified: one cluster consisting of seven variants having a mutation in the CtsR regulator gene, one cluster containing two variants with an aerobic biofilm formation capacity similar to that of the wild type, and a cluster composed of five immotile variants. The large population diversity of L. monocytogenes stress-resistant variants signifies the organism's genetic flexibility, which in turn may contribute to the survival and persistence of this human pathogen in food-processing environments.The opportunistic pathogen Listeria monocytogenes causes listeriosis, a serious infection that most commonly affects newborns, pregnant women, seniors, and immune-compromised patients. Because L. monocytogenes is ubiquitous it may be introduced into food-processing plants through many different routes. L. monocytogenes has been shown to colonize processing environments and to contaminate products during processing. Certain strains may become persistent in a plant and cause continuous contamination (18,20,27). The ability of part of a population to survive in a certain environment because of heterogeneity is called persistence. However, there is a difference between survivors that are phenotypically switching between normal cells and persister cells and survivors that are mutated and therefore genetically different (7). Although the origin of persistence can be different, overall persisters can have specific qualities, such as acid and heat tolerance and adherence to surfaces, contributing to the establishment of house strains. A number of studies have shown persistence of L. monocytogenes in various food-processing plants (6,17,19,21,22). Some of these persistent strains dominated and persisted in a plant or production line for years and caused food contamination and human disease. The generation, occurrence, and selection of these persistent strains can have a significan...

High hydrostatic pressure (HHP) inactivation of three Listeria monocytogenes strains (EGDe, LO28, and Scott A) subjected to 350 MPa at 20 degrees C in ACES buffer resulted in survival curves with significant tailing for all three strains. A biphasic linear model could be fitted to the inactivation data, indicating the presence of an HHP-sensitive and an HHP-resistant fraction, which both showed inactivation according to first-order kinetics. Inactivation parameters of these subpopulations of the three strains were quantified in detail. EGDe showed the highest D-values for the sensitive and resistant fraction, whereas LO28 and Scott A showed lower HHP resistance for both fractions. Survivors isolated from the tail of LO28 and EGDe were analyzed, and it was revealed that the higher resistance of LO28 was a stable feature for 24% (24 of 102) of the resistant fraction. These HHP-resistant variants were 10 to 600,000 times more resistant than wild type when exposed to 350 MPa at 20 degrees C for 20 min. Contrary to these results, no stable HHP-resistant isolates were found for EGDe (0 of 102). The possible effect of HHP survival capacity of stress-resistant genotypic and phenotypic variants of L. monocytogenes on the safety of HHP-processed foods is discussed.

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...

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