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 biofilm-forming 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.

FIG. 1.

Reduction (in log CFU/ml) of Listeria monocytogenes LO28 WT and 24 HHP-resistant variants after HHP treatment of exponentially growing cells at 350 MPa, 20°C, for 20 min in ACES buffer (a), HHP treatment of stationary growing cells at 350 MPa, 20°C, for 20 min in ACES buffer (b); heat treatment of exponentially growing cells at 60°C for 1 min in ACES buffer (c); and low-pH treatment of exponentially growing cells at pH 2.5, 37°C, for 3 min in BHI broth (d). Reduction was determined by subtracting the number of surviving cells (log CFU/ml) after the treatment from the number of unstressed cells (log CFU/ml). Each inactivation experiment was reproduced at least two times on different days. The error bars show 1 standard deviation. Results in gray bars are not statistically different, whereas white bars show significant differences compared to the wild type.

FIG. 2.

Maximum specific growth rates of Listeria monocytogenes LO28 WT and 24 HHP-resistant variants at 30°C in BHI under aerobic conditions (a); at 30°C in BHI under anaerobic conditions (b); or at 7°C in BHI under aerobic conditions (c). (d) Colony size (in mm) was determined by measuring the diameters of colonies after 2 days of growth at 30°C on BHI agar under aerobic conditions. Each growth experiment was reproduced at least two times on different days. The error bars show 1 standard deviation. Results in gray bars are not statistically different, whereas white bars show significant differences compared to the wild type.

FIG. 3.

Biofilm formation (measured based on the OD₅₉₅) after 46 h of incubation in HTM in polystyrene microtiter plates at 30°C under aerobic (a) or anaerobic (b) conditions. Each measurement was reproduced at least three times on different days. The error bars show 1 standard deviation. Results in gray bars are not statistically different, whereas white bars show significant differences compared to the wild type.

FIG. 4.

Gene expression ratio (log₂) of ctsR, clpC, clpB, clpE, and clpP of exponentially growing unstressed cells of variant 6 with a deletion in the ctsR gene (ΔctsR) and variants 4 and 20, with a deletion in the promoter region upstream of the ctsR gene compared to the wild type. The genes ctsR and clpC are part of the same operon. Results in white bars show significant differences compared to WT (calculated with the pairwise fixed reallocation randomization test, using the relative expression software tool, version 2).

FIG. 5.

Cluster analysis of phenotypic characteristics of WT and HHP-resistant variants revealed three clusters formed by 14 variants, of which one cluster consisted of 7 variants having a mutation in the CtsR regulator. The two other clusters contained two variants with aerobic biofilm formation similar to the wild type and five immotile variants, respectively. The remaining 10 variants did not cluster, signifying their unique characteristics.

FIG. 6.

Scanning electron microscopy images of LO28 WT and two HHP-resistant variants (8 and 17). The wild type and variant 8 both showed the presence of flagella, whereas variant 17 showed an absence of flagella. Both variant cell types showed normal size and morphology compared to the wild type.

FIG. 7.

Selection and clustering of stress-resistant L. monocytogenes LO28. Of 5 million L. monocytogenes LO28 cells, more than 100 cells were able to survive 20 min at 350 MPa. One portion of these HHP survivors was temporarily resistant due to phenotypic switching, whereas the other part was stably resistant because of genotypic heterogeneity (28). Ordering all tested phenotypic characteristics of these stable HHP-resistant variants by cluster analysis resulted in 13 groups of variants with (a combination of) unique characteristics.