Table 1.
List of B. cereus strains used in this study.
Table 2.
List of primers used in real-time PCR.
Fig 1.
Impact of Mn2+ and heme on biofilm formation by B. cereus food isolates.
Biofilms were grown on SS coupons with or without Mn2+ and/or heme in BHI supplemented with glycerol at 30°C for 48 h. Established biofilms were quantified using the crystal violet assay. The threshold of biofilm formation (solid line) is equal to the background absorbance value plus three times the standard deviation (OD = 0.3). Each data point represents the average value obtained in three biological experiments for each strain. The error bars indicate the standard deviation. To compare effects on biofilm formation in each strain, one-way ANOVA and Tukey's post hoc test (p < 0.05) were performed. Groups marked with different letters in each strain display significant differences.
Fig 2.
Number of cells and spores in B. cereus biofilms.
B. cereus GIHE 72–5 was grown in BHI supplemented with glycerol with or without Mn2+ and heme at 30°C for 48 h. Number of cells in logCFU/cm2 (A); scatter plot showing the relationship between the number of cells and the results of crystal violet assays (B); number of spores in logCFU/cm2 (C); scatter plot showing the relationship between spore formation and the results of crystal violet assays (D). While, squares (C) show the percentage of spore formation compared to the number of cells in biofilms. Each data point represents the average value obtained in three biological experiments, and the standard deviation. To compare the number of cells and spores obtained among the conditions used, one-way ANOVA and Tukey's post hoc test (p < 0.05) were performed. Groups marked with different letters in each growth condition display significant differences.
Fig 3.
Representative scanning electron microscope (SEM) images of biofilms formed by B. cereus GIHE 72–5.
Fig 4.
Representative confocal laser scanning microscopy (CLSM) images of biofilm formation by B. cereus GIHE 72–5.
B. cereus GIHE 72–5 biofilms were grown on plastic coupons with or without Mn2+ andr heme in BHI supplemented with glycerol at 30°C for 48 h. Subsequently, the biofilms were stained using a LIVE/DEAD BacLight bacterial viability staining kit. The scale bar represents 50 μm.
Fig 5.
Swarming motility of B. cereus food isolate GIHE 72–5 planktonic cells.
Planktonic cells were grown in BHI with or without Mn2+ and/or heme in BHI supplemented with glycerol at 30°C overnight (18) h. The overnight cultures were washed in PBS, and their swarming mobility on BHI soft agar (0.5%) plates was examined. The data represent the average swarming mobility obtained in three biological experiments; the standard deviation is shown. To compare the swarming mobility under different conditions, one-way ANOVA and Tukey's post hoc test (p < 0.05) were performed. Groups marked with different letters display significant differences.
Fig 6.
Impact of Mn2+ and heme on the resistance of B. cereus biofilm cells to benzalkonium chloride.
Biofilms of the B. cereus food isolate GIHE 72–5 were grown on SS coupons with or without Mn2+ and/or heme in BHI supplemented with glycerol at 30°C for 48 h. After maturation, biofilms were washed with PBS and subsequently treated with BAC (200 μg/ml) for 5 min, and the number of surviving cells was determined (A). The average number of surviving cells in logCFU/cm2 (A) is shown as a scatter plot of the Log reduction versus the crystal violet assay results (OD values) (B). Each data point represents the average value obtained in three biological experiments; the standard deviation for each condition is shown. To compare the number of surviving cells obtained under different conditions, one-way ANOVA and Tukey's post hoc test (p < 0.05) were performed. Groups marked with different letters display significant differences.
Fig 7.
Impact of Mn2+ and heme on the expression of a number of selected genes in B. cereus in the biofilm growth phases.
The graphs show the expression of spoOA (A), abrB (B), sinI (C), sinR (D), sipW (E) and tasA (F) in the biofilm phase of GIHE 72–5. Biofilms were grown on SS coupons in Mn2+ and/or heme in BHI supplemented with glycerol at 30°C for 48 h. The fold change in expression relative to the expression in biofilm cells grown in BHI is shown. Each data point represents the average value obtained in at least two biological experiments; the error bars indicate standard deviation. To compare fold changes in expression under different conditions, one-way ANOVA and Tukey's post hoc test were performed. Groups marked with different letters in each growth condition display significant differences (p < 0.05).