Population Diversity of Listeria monocytogenes LO28: Phenotypic and Genotypic Characterization of Variants Resistant to High Hydrostatic Pressure

Abstract

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...

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

“…In previous research, stable HHPresistant L. monocytogenes LO28 variants were isolated (32). Most of these variants also appeared to be heat resistant (31). Furthermore, biphasic inactivation was found not only for HHP but also for heat inactivation of WT LO28.…”

“…With this information, the best possible conditions can be established for the isolation of resistant variants. We selected representatives of the previously isolated stably resistant HHP variants that showed increased resistance to heat (31). Variant number 6 (a ctsR variant) and variant number 17 (an immotile variant) were investigated for their heat and HHP survival compared to the survival of the wild type under these conditions (Fig.…”

“…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 increased survival to heat (17,20,31). Further characterization of the LO28 HHP-resistant variants without mutations in ctsR also revealed increased resistance to heat for most of these variants (31).…”

“…The observed alterations in ctsR resulted not only in increased resistance to high pressure but also in increased survival to heat (17,20,31). Further characterization of the LO28 HHP-resistant variants without mutations in ctsR also revealed increased resistance to heat for most of these variants (31). In addition, thermal inactivation of L. monocytogenes was previously fitted with a biphasic model, indicating the presence of a heat-resistant fraction (1,3,5).…”

“…Therefore, the initial number of variants was calculated from the fraction of variants that survived HHP and by extrapolation of this fraction's survival curve, as follows: (2) where N t,var equals the number of variants (log CFU/ml) at time t (min) and D var the fitted decimal reduction time or D-value (min) of the HHP-inactivated variants (31,32). The inactivation data were fitted with an appropriate model.…”