Virulence Potential and Antimicrobial Resistance of Bacillus cereus Isolated from Infant formula

Powdered infant formulae may be contaminated by pathogenic microorganisms, in particular spore-forming bac-teria can survive production and shelf life [1]. Bacillus spp. is frequently isolated [2], and Bacillus cereus can com-promise product quality and safety through spoilage and toxin production or infection [3]. During the microbiological evaluation of 83 powdered formulae (23 infant formulae, 42 follow-on formulae, and 18 formulae for special medical purposes), 42 B. cereus strains were isolated. Three strains were recovered by direct enumeration on PEMBA agar, while 39 were isolated after enrichment in buffered peptone water followed by streak-ing onto PEMBA agar. For phenotypic characterization, hemolysin BL production was assessed by measuring hemolysis zones on Co-lumbia blood agar supplemented with 5% sheep blood (after incubation at 30°C for 18 h), phosphatidylcholine-specific phospholipase C (PC-PLC) activity was evaluated by measuring precipitation halos on Blood Agar Base sup-plemented with 0.15% L-α-phosphatidylcholine, while proteolytic activity was assessed by measuring clear zones around colonies on 1.2% agar plates containing 1.5% skim milk (37°C for 18 h). For toxin-related genes, strains were grown in liquid culture, DNA was extracted and purified, and conventional PCR assays were performed targeting genes encoding sphingomyelinase (sph), enterotoxins (entFM, entS, bceT), non-hemolytic enterotoxin components (nheA, nheB, nheC), cytotoxin K (cytK), and cereulide (ces). Antimicrobial susceptibility of the 42 strains was as-sessed by broth microdilution using the Sensititre™ automated system with the GPALL1F plate, and minimum in-hibitory concentrations were interpreted according to EUCAST 2025 criteria. Only two strain harbored all virulence factors except ces or cytK. All strains exhibited proteolytic activity, 50% showed PC-PLC activity, and 67% produced hemolysin BL. The prevalence of toxin-encoding genes was as follows: sph (97.7%), entFM (64.3%), entS (66.7%), bceT (45.2%), nheA/B/C (76.2%), cytK (7.1%), and ces (28.3%). Alt-hough the prevalence of ces was relatively low, cereulide is of particular concern due to its heat stability and pre-formed nature in food. Antimicrobial resistance profiles revealed high resistance rates to β-lactams and moderate resistance to eryth-romycin (26.2%). Overall, 69% of the strains were fully susceptible, and all resistant isolates showed resistance to on-ly one antibiotic class, with no multidrug-resistant phenotypes detected. In conclusion, the frequent detection of B. cereus with multiple virulence factors in powdered formulae high-lights safety concerns, emphasizing the need for continuous monitoring, strict hygiene, and proper handling during and after reconstitution of formula to protect vulnerable consumers. [1] M. Larbi, A. I. A. Chincha, A. Vecchione, E. Ghelardi, J. M. C. Bonatto, A. J. Marsaioli, P. H. Campelo, I. Benamar, M.A. Allah, A. S. Sant’Ana, and M.B. Boumediene, “Aerobic spore-forming bacteria in powdered infant formula: Enumeration, identification by MALDI-TOF mass spectrometry (MS), presence of toxin genes and rpoB gene typing” International Journal of Food Microbiology, vol. 368, 2022. [2] X. Pei, S. Yang, L. Zhan, J. Zhu, X. Song, X. Hu, G. Liu, G. Ma, N. Li and D. Yang, “Prevalence of Bacillus cereus in powdered infant and pow-dered follow-up formula in China” Food Control, vol. 93, pp. 101–105, 2018. [3] Š. Bursová, L. Necidová and D. Haruštiaková, “Growth and toxin production of Bacillus cereus strains in reconstituted initial infant milk formula” Food Control, vol. 93, pp. 334–343, 2018.