Impact of manganese and heme on biofilm formation of <i>Bacillus cereus</i> food isolates

<div><p>The objective of this study was to determine the impact of manganese (Mn²⁺) and heme on the biofilm formation characteristics of six <i>B</i>. <i>cereus</i> food isolates and two reference strains (ATCC 10987 and ATCC 14579). The data obtained from the crystal violet assay revealed that addition of a combination of Mn²⁺ and heme to BHI growth medium induced <i>B</i>. <i>cereus</i> biofilm formation. However, the induction of biofilm formation was strictly strain-dependent. In all of the induced strains, the impact of Mn²⁺ was greater than that of heme. The impact of these two molecules on the phenotypic characteristics related to biofilm formation, such as cell density, sporulation and swarming ability, was determined in a selected food isolate (GIHE 72–5). Addition of Mn²⁺ and heme to BHI significantly (p < 0.05) increased the number of cells, which was correlated with the results of crystal violet assays as well as scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) analyses. In addition, induced biofilms showed higher numbers of spores and greater resistance to benzalkonium chloride. The swarming ability of <i>B</i>. <i>cereus</i> planktonic cells was increased in the presence of Mn²⁺ and heme in BHI. The expression levels of a number of selected genes, which are involved in mobility and extracellular polymeric substances (EPS) formation in <i>B</i>. <i>cereus</i>, were positively correlated with biofilm formation in the presence of Mn²⁺ and heme in BHI. These results further confirming the role of these molecules in swarming mobility and making matrix components related to <i>B</i>. <i>cereus</i> biofilm formation. These data indicate that signaling molecules present in the food environment might substantially trigger <i>B</i>. <i>cereus</i> biofilm formation, which could pose a threat to the food industry.</p></div

List of primers used in real-time PCR.

<p>List of primers used in real-time PCR.</p

Representative confocal laser scanning microscopy (CLSM) images of biofilm formation by <i>B</i>. <i>cereus</i> GIHE 72–5.

<p><i>B</i>. <i>cereus</i> 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.</p

Swarming motility of <i>B</i>. <i>cereus</i> food isolate GIHE 72–5 planktonic cells.

<p>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.</p

Representative scanning electron microscope (SEM) images of biofilms formed by <i>B</i>. <i>cereus</i> GIHE 72–5.

<p>Representative scanning electron microscope (SEM) images of biofilms formed by <i>B</i>. <i>cereus</i> GIHE 72–5.</p

Impact of Mn²⁺ and heme on biofilm formation by <i>B</i>. <i>cereus</i> food isolates.

<p>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.</p

List of <i>B</i>. <i>cereus</i> strains used in this study.

<p>List of <i>B</i>. <i>cereus</i> strains used in this study.</p

Impact of Mn²⁺ and heme on the resistance of <i>B</i>. <i>cereus</i> biofilm cells to benzalkonium chloride.

<p>Biofilms of the <i>B</i>. <i>cereus</i> 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.</p

Fecundity of STEC strains in <i>C</i>. <i>elegans</i> compared to that of the <i>E</i>. <i>coli</i> OP50 strain.

<p>Brood sizes of <i>C</i>. <i>elegans</i> were examined at 20°C. n>10 in each case.</p

Correlation between the life span and fertility of <i>C</i>. <i>elegans</i> infected with STEC strains.

<p>The STEC (E15, E18 and E22) strains showed an increase in colonisation, which correlates with a decrease in longevity and fertility of the worm (<i>C</i>. <i>elegans</i>). This is considered evidence of STEC pathogenicity in the <i>C</i>. <i>elegans</i> model.</p