Effect of Sublethal Concentrations of Zinc Oxide Nanoparticles on Bacillus cereus

**Background:** Bacillus cereus is a Gram-positive, spore-forming bacterium widespread in soil, water, and food products. It is an opportunistic pathogen causing food poisoning and, rarely, severe eye infections such as endophthalmitis and keratitis. Its ability to form spores and biofilms makes it resistant to standard cleaning methods. Zinc oxide nanoparticles (ZnONPs) are used in many consumer products (cosmetics, sunscreens, food packaging) and are known to have antimicrobial properties. However, most studies focus on lethal concentrations; the effects of sublethal concentrations on bacterial physiology are less understood. This study investigates how sublethal concentrations of commercially produced ZnONPs affect growth, oxidative stress, sporulation, biofilm formation, and the ability to degrade the azo dye Evans Blue in B. cereus. It also tests the combined effect of ZnONPs with phenolic compounds commonly found in cosmetics.

**Methods:** Commercially produced ZnONPs (Sigma Aldrich, <100 nm, average 67 nm, zeta potential +46.1 mV) were used. The MIC and MBC against B. cereus PCM 1948 were determined by microdilution. Sublethal concentrations (0.2, 0.4, 0.6, 0.8 mg/mL) were tested. After 48 h incubation at 37°C, planktonic growth (OD600), superoxide radical production (NBT assay, absorbance at 560 nm), sporulation (Schaeffer–Fulton staining, % spores), biofilm formation (crystal violet assay, absorbance at 600 nm), and Evans Blue decolouration (absorbance at 606 nm) were measured. The combined effect of ZnONPs with seven phenolic compounds (4-hydroxybenzoic acid, sodium salicylate, trans-cinnamic acid, quercetin, gallic acid, coumarin, p-coumaric acid) was assessed by agar well diffusion. All experiments were performed in triplicate.

**Key Results:** The MIC of ZnONPs against B. cereus was 1.6 mg/mL, and the MBC was 1.8 mg/mL. Sublethal concentrations inhibited planktonic growth in a dose-dependent manner: 0.2 mg/mL reduced growth by ~14%, and 0.8 mg/mL (50% MIC) reduced growth by ~42% (OD from 1.447 to 0.839). Sporulation increased from 15% in control to 22.5% at 0.8 mg/mL, though differences were not statistically significant. Superoxide radical production increased with ZnONP concentration; formazan absorbance rose from 0.127 (control) to 0.289 at 0.8 mg/mL (227% of control). Biofilm formation increased by 80% at 0.8 mg/mL (crystal violet absorbance from 0.324 to 0.586). Evans Blue decolouration decreased from 42.67% (control) to 23.50% at 0.8 mg/mL. In the well diffusion assay, ZnONPs alone produced inhibition zones of 8–13 mm (0–0.8 mg). Phenolic compounds alone showed no inhibition. When combined with 0.2 or 0.4 mg ZnONPs, inhibition zones increased by 50–90% (e.g., p-coumaric acid + 0.4 mg ZnONPs gave 19 mm vs. 10 mm for ZnONPs alone). Higher ZnONP concentrations (0.6, 0.8 mg) did not further increase inhibition.

**Clinical Implications:** This study reveals that sublethal concentrations of ZnONPs, which may occur in the environment or in products, can paradoxically enhance the survival and persistence of B. cereus by stimulating biofilm formation and sporulation. This is concerning for food safety and healthcare settings, as it may promote contamination and resistance to cleaning. The increased oxidative stress and reduced planktonic growth are beneficial, but the stimulation of protective mechanisms could outweigh these effects. The synergistic effect with phenolic compounds suggests that formulations combining ZnONPs with natural plant extracts could be more effective antimicrobials, potentially useful in cosmetics and food packaging. However, the inhibition of azo dye degradation indicates that ZnONPs may impair the beneficial environmental role of B. cereus in bioremediation. These findings highlight the need for careful risk assessment of ZnONP contamination and for considering sublethal effects when designing antimicrobial strategies.