Surviving host - and food relevant stresses: phenotype of L. monocytogenes strains isolated from food and clinical sources

Abstract

The aim of this study was to compare the phenotype of 40 strains of L. monocytogenes under food and host relevant stress conditions. The strains were chosen to represent food and clinical isolates and to be equally distributed between the most relevant clonal complexes for clinical and food isolates (CC1 and CC6 vs CC121 and CC9), plus one group of eight strains of rare clonal complexes. Human-associated CC1 had a faster maximal growth rate than the other major complexes, and the lag time of CC1 and CC6 was significantly less affected by the addition of 4% NaCl to the medium. Food-associated CC9 strains were hypohemolytic compared to other clonal complexes, and all strains found to be resistant to increased concentrations of benzalkonium chloride belonged to CC121 and were positive for Tn6188 carrying the qacH gene. Lactic acid affected the survival of L. monocytogenes more than HCl, and there was a distinct, strain specific pattern of acid tolerant and sensitive strains. Strains from CC6 and human clinical isolates are less resilient under acid stress than those from other complexes and from food. One strain isolated from a human patient exhibited significant growth defects across all conditions.

Figure 1

Maximal growth rate. The y axis represents the maximum growth rate (defined as the maximum slope calculated by a sliding window approach) in BHI (a) and BHI with 4% NaCl (b) plotted against clonal complex. The shaded area denotes the four larger CC included in the study. The white area contains CC which are represented by only one strain. Boxplots represent the mean, 25th and 75th quantile with 1.5*IQR whiskers. Dots represent outliers and *denotes a significant contribution of CC to the phenotype.

Figure 2

Relative lag time. The y axis represents the relative lag time (lag time in BHI supplemented with 4% NaCl/lag time in BHI), plotted against lineage (a) and clonal complex (b). The shaded area denotes the four larger CC included in the study. The white area contains CC which are represented by only one strain. Boxplots represent the mean, 25th and 75th quantile with 1.5*IQR whiskers. Dots represent outliers and *denotes a significant contribution of the qualifier to the phenotype.

Figure 3

(a) Log reduction of CFU/ml in individual strains between before and after 1 h acid exposure at pH3 for HCl, pH3.5 for LA. The upper panel represents data from strains that were pre-exposed to mild acid stress at pH5.5 for one hour prior to exposure the acid stress. The lower panel represents data from acid-shocked strains without prior adaptation to pH5.5. The horizontal line represents the delineation between “acid sensitive” or “acid resistant” phenotypes as determined by k-means. (b) Probabilities of belonging to the “acid sensitive” phenotype by source. (c) Probabilities of belonging to the “acid sensitive” phenotype by clonal complex. The shaded area denotes the four larger CC included in the study. The white area contains CC which are represented by only one strain. CCs with collapsed error bars had only acid-sensitive or acid-resistant observations, making a realistic estimate of the variance impossible. *Denotes a significant association of the qualifier with the phenotype.

Figure 4

The concentration of benzalkonium chloride (μg/ml) to which strains were resistant plotted against clonal complex. The y-axis represents the probability of a strain to be resistant to each concentration of BC. *Denotes CCs that were significantly associated with the phenotype.

Figure 5

Hemolysis. The y-axis represents the amount of hemolysis caused by the supernatant of each strain, as determined by a mixed effects linear model, plotted against clonal complex. The dots represent the least square means, the whiskers the upper and lower 95% confidence level. CCs that share a number above the bars are not significantly different from each other, as determined by a false discovery rate of <0.05.