Effect of gaseous ozone treatment on cells and biofilm of dairy Bacillus spp. isolates

Abstract

Bacillus spp. can produce biofilms and cause recurrent contamination in the food industry. The common clean-in-place (CIP) method is usually employed in sanitizing processing equipment. However, CIP is not always effective in removing biofilms. Ozone represents a promising "green" alternative to control biofilms. In this study, the effect of gaseous ozone (50 ppm) was evaluated in vitro against planktonic and sessile B. cereus and B. subtilis isolates collected from the dairy sector. Planktonic cells were enumerated by plate counts after 10 min, 1 h, and 6 h of ozone treatment. After a short-term (10 min) exposure, a slight reduction in microbial loads (0.66-2.27 ± 0.15 Log₁₀ CFU/mL) was observed for B. cereus strains, whereas a more pronounced reduction (2.90-3.81 ± 0.12 Log₁₀ CFU/mL) was noted in B. subtilis isolates. The microbial load further decreased after 1 h-treatments, around 1.5-3.46 ± 0.11 Log₁₀ CFU/mL for B. cereus strains, and 4.0-5.6 ± 0.11 Log₁₀ CFU/mL for B. subtilis isolates, until complete inactivation of bacterial cells after 6 h of exposure. Moreover, the effect of gaseous ozone treatment (50 ppm, 6 h) was evaluated for its ability to inhibit and eradicate biofilms formed on two common food-contact materials (polystyrene and stainless steel). Sessile B. subtilis cells were the more sensitive to the action of ozone, while a weak effect was highlighted on B. cereus isolates on both surface types. These results were further confirmed by scanning microscopy analysis. The number of cells in the biofilm state was also assessed, showing a not-complete correlation with a decrease in Biofilm Production Indices (BPIs). These findings highlighted the effectiveness of the sanitizing protocol using gaseous ozone in contrasting Bacillus free-living cells, but a not completely counteraction in biofilm formation (inhibition) or eradication of pre-formed biofilm. Thus, the application of ozone could be thought of not alone, but in combination with common sanitization practices to improve their effectiveness.

Keywords: Bacillus cereus; Bacillus subtilis; antimicrobial biofilm; food contact surfaces; gaseous ozone.

Figure 1

Schematic representation of 50 ppm gaseous ozone treatments on cells of dairy-related B. cereus and B. subtilis strains, including reference strains, at different exposure times. This image was created with BioRender (https://biorender.com/).

Figure 2

Graphic description of the experimental assay to evaluate the effect of ozone gas (50 ppm, 6 h) to inhibit and eradicate biofilm of dairy-related B. cereus and B. subtilis strains, and reference strains, at different exposure times. This image was created with BioRender (https://biorender.com/).

Figure 3

Effect of gaseous ozone at 50 ppm for 6 h on biofilm formation and eradication of (A) B. cereus and (B) B. subtilis isolates, including reference strains, in polystyrene wells. Error bars indicate standard deviation. Asterisks (*) indicate differences statistically significant according to Dunnett’s multiple comparisons. One asterisk (*) means p < 0.05. Four asterisks (****) indicate that the p < 0.0001.

Figure 4

Effect of gaseous ozone at 50 ppm for 6 h on biofilm formation and eradication of (A) B. cereus and (B) B. subtilis isolates, including reference strains, in stainless steel coupons. Error bars indicate the standard deviation. Asterisks (*) indicate differences statistically significant according to Dunnett’s multiple comparisons. One asterisk (*) means p < 0.05. Two asterisks (**) indicate p < 0.01. Four asterisks (****) mean p < 0.0001.

Figure 5

SEM micrographs of biofilms formed by B. cereus ATCC 14579 reference strain in (A) polystyrene and (B) stainless steel, after 6 h of exposure to 50 ppm of gaseous ozone. The control group (top of the figure) represents the untreated biofilm. The central/middle images represented the effect of ozone on inhibiting ATCC 14579 biofilm formation. The downward figures show the effect of gaseous ozone on preformed biofilm (eradication). Magnification 2000x.

Figure 6

SEM micrographs of biofilms formed by B. subtilis NCIB 3610 reference strain in (A) polystyrene and (B) stainless steel, after 6 h of exposure to 50 ppm of gaseous ozone. The control group (top of the figure) represents the untreated biofilm. The central/middle images represented the effect of ozone on inhibiting NCIB 3610 biofilm formation. The downward figures show the effect of gaseous ozone on preformed biofilm (eradication). Magnification 2000x.