Assessment of the risk associated with Bacillus cereus isolates and potential combat with methanolic Artemisia vulgaris extract

Mohamed Fareid^1*Gamal M. El-Sherbiny^2*Nancy M. Elafandy¹Nagat E. Eltoum³Mohamed S. Othman⁴Mohamed H. Sharaf⁵Mohammed Abu-Elghait⁵Amr Shehabeldine⁵Fatma A, Hamada⁶

• ¹Clinical Laboratory Science Department, Applied Medical Science College, University of Ha’il, Hail 2440, Saudi Arabia, Hail, Saudi Arabia
• ²Department of Botany and Microbiology, Faculty of Science, Al-Azhar University, Cairo, Egypt
• ³Clinical Nutrition Department, Applied Medical Science College, University of Ha’il, Hail 2440, Saudi Arabia., Ha’il, Saudi Arabia
• ⁴Biochemistry Department, College of Medicine, University of Ha’il, Hail 2440, Saudi Arabia, Hail, Saudi Arabia
• ⁵Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Cairo 11884, Egypt., Cairo, Egypt
• ⁶First Year of Health and Medical Colleges, Basic Sciences Department, University of Ha’il, Hail 2440, Saudi Arabia, Ha’il, Saudi Arabia

Bacillus cereus (B. cereus) is widely distributed in natural environments, particularly in soil and plant matter. It is frequently associated with foodborne illness outbreaks, accounting for an estimated 1.4% to 12% of food poisoning cases worldwide. Therefore, the aim of this study was to assess the presence of toxigenic and emetic genes among B. cereus isolates, evaluate their antibiotic susceptibility, and investigate the potential of Artemisia vulgaris (A. vulgaris) extract as an antimicrobial agent. PCR analysis was employed to detect the presence of toxigenic and emetic genes in B. cereus isolates. The results revealed that the isolates harbored hemolysin BL (HBL) genes hblA (8.62%) and hblB (20.68%) as well as non-hemolytic enterotoxin (NHE) genes nheA (20.68%) and nheB (22.41%). Additionally, 29.30% of the isolates carried the bceT gene, and 15.51% carried the ces gene, associated with emetic toxin production. Antibiotic susceptibility testing showed that B. cereus isolates were highly sensitive to ciprofloxacin (91.37%) and rifampicin (96.54%). In contrast, high resistance was observed against ampicillin (86.20%) and novobiocin (65.51%). The A. vulgaris extracts exhibited notable antibacterial activity, with inhibition zones ranging from 19.20 ± 0.25 mm to 27.10 ± 0.13 mm. The methanolic extract was the most effective, showing minimum inhibitory concentrations (MICs) between 62.5 and 250 μg/mL. Furthermore, the methanolic extract effectively suppressed biofilm formation and significantly downregulated the expression of toxigenic genes by −2.5 to −5.2 fold (***P < 0.0001). The methanolic extract also exhibited strong antioxidant activity, with IC₅₀ values of 12.7 µg/mL (DPPH assay) and 14.2 µg/mL (ABTS assay). Cytotoxicity assays indicated that the extract was relatively safe, with IC₅₀ values of 524.7 ± 1.23 µg/mL and 236.5 ± 1.74 µg/mL for Vero and HFB4 cell lines, respectively. GC-MS analysis identified dopamine N,N-dimethyl-dimethyl ether (40.31%) as the major compound, followed by n-hexadecanoic acid (16.57%). HPLC analysis revealed a high content of chlorogenic acid, luteolin, and quercitrin. Conclusion: These findings underscore the public health risks posed by B. cereus isolates in food contamination. A. vulgaris extract demonstrates promising antibacterial, antibiofilm, antioxidant, and gene-suppressing properties, positioning it as a potential alternative strategy for managing B. cereus and its associated