Thesis:

Title : Investigation of Bacillus cereus toxins in infant formula in Sabzevar city using PCR

Abstract(include summary,goals,executive ways and results): Background and Objective: Bacillus cereus is a spore-forming bacterium that causes food poisoning by producing diarrheal and emetic toxins. Contamination of infant formula with this bacterium and its toxigenic genes is of particular importance because of the immaturity of the infant immune system. The present study aimed to determine the frequency of contamination with B. cereus and its toxigenic genes in infant formula marketed in Sabzevar County using culture and PCR methods. Materials and Methods: In this descriptive cross-sectional study conducted in 2025–2026 (1404 SH), 75 infant formula samples from three brands (25 samples per brand) were examined. Samples were cultured on MYP agar, and suspected colonies were examined by Gram staining and biochemical tests. Independently of the culture result, DNA was extracted from the initial suspension of each of the 75 samples, and PCR was performed on it for the 16S rRNA gene (universal primer; indicating bacterial DNA) and the toxigenic genes nheA, hblA, cytK and ces. Because the 16S rRNA primer is universal, a positive result does not by itself confirm the species; "PCR-positive" here means a positive 16S rRNA PCR. Data were analyzed using SPSS version 26 with the chi-square, Fisher's exact and McNemar tests. Results: The contamination rate was 17.3% (13 samples) by culture and 16% (12 samples) by PCR (16S rRNA). There was no significant difference among the three brands (p > 0.05). No significant difference was observed between culture and PCR results, but overall agreement was 90.7% (kappa 0.66), with positive and negative percent agreement of 75% and 93.7%, respectively; because the 16S rRNA primer is universal, PCR was treated as a comparator rather than a reference standard. Among all 75 samples, nhe had the highest frequency (10.7%, 8 samples) and ces the lowest (1.3%, 1 sample); the frequencies of hbl and cytK were 5.3% and 4.0%, respectively, and 9 samples (12%) carried at least one of the four genes (7 culture-positive and 2 culture-negative). In brand 3, one sample carried all four toxigenic genes simultaneously. Conclusion: Presumptive B. cereus and its toxigenic genes are present in a portion of the infant formulas marketed in Sabzevar. Given the discordance in 7 samples between the two methods, using culture and PCR together provides complementary information. Gene presence alone does not imply toxin production, and continuous monitoring of infant formula is recommended.

Keywords: Keywords: Bacillus cereus, infant formula, PCR, toxigenic genes, Sabzevar