Molecular detection and toxigenic profiling of Bacillus cereus in milk-based sweets of Bikaner, Rajasthan: A public health perspective

Introduction

Traditional milk-based sweets constitute a vital part of the Indian diet and hold significant cultural importance. In Rajasthan, particularly in the city of Bikaner, these products are largely prepared from concentrated milk solids and distributed through small-scale, informal production and retail systems. Although highly popular, the manufacture of these sweets often lacks standardized processing and quality control measures and relies on traditional, open-air methods with extensive manual handling, rendering them susceptible to microbial contamination. Among the many pathogens, especially those causing foodborne illnesses, Bacillus cereus is one of the 22 foodborne pathogens identified by the World Health Organization as a key organism for evaluating the global burden of foodborne diseases (Kirk et al., 2015) [8] . Bacillus cereus is a widely distributed, Gram-positive, aerobic, sporeforming bacterium commonly present in soil, dust, water, and raw milk. The spores of this organism display exceptional resistance to heat and drying, enabling them to survive pasteurization and cooking. When conditions during storage become favourable, the spores may germinate and multiply, resulting in toxin production and the potential onset of foodborne disease. The pathogenicity of B. cereus is linked to its ability to produce a range of toxins responsible for two clinically distinct forms of food poisoning. The diarrheal form arises following ingestion of viable cells or spores that synthesize enterotoxins in the small intestine, including non-hemolytic enterotoxin (NHE), hemolysin BL (HBL), and cytotoxin K (CytK), encoded by the nheA, hblA, and cytK genes, respectively (Beecher and Macmillan, 1991) [3] (Lund and Granum, 1996) [12] (Lund et al., 2000) [13] . Conversely, the emetic form is caused by cereulide, a heatstable cyclic dodecadepsipeptide encoded by the ces gene cluster, which is pre-formed in food and remains biologically active despite exposure to high temperatures and resistance to gastric proteolysis (Ehling-Schulz and Scherer, 2004) [4] . Molecular identification targeting the gyrB gene offers greater discriminatory accuracy than conventional 16S rRNA-based methods, which lack sufficient resolution to reliably differentiate B. cereus from closely related species such as B. thuringiensis (Yamada et al., 1999) [18] . Accordingly, the present study employed gyrB (DNA gyrase subunit B) for precise species-level identification, while simultaneous detection of virulenceassociated genes enabled a more accurate evaluation of public health risk. Nevertheless, information on the molecular prevalence and toxigenic profiles of B. cereus in traditional milk-based sweets from Rajasthan is scarce. This study was therefore undertaken to address this gap by molecularly detecting B. cereus and characterizing its virulence gene distribution in milk-based sweets marketed in Bikaner.

Materials and Methods

Sample Collection

A total of 300 milk-based sweet samples were obtained from various sweet shops across Bikaner district, Rajasthan, comprising 150 mawa-based sweets and 150 chhana-based sweets. Samples were collected aseptically in sterile containers and subsequently transported and stored under refrigerated conditions until further analysis. Sweet shops were selected using a random sampling approach to encompass a broad geographic distribution across the city, thereby ensuring that the samples were representative (Acharya et al., 2013) [2] .

Isolation and Phenotypic Identification

One gram of each sample was aseptically homogenized in 9 mL of brain heart infusion broth and incubated at 37 °C for 24 h for enrichment. Following incubation, a loopful of the enriched culture was streaked onto Polymyxin Pyruvate Egg Yolk Mannitol Bromothymol Blue Agar (PEMBA) and incubated at 37 °C for a further 24 h. After incubation, the plates were inspected for the presence of characteristic peacock blue colonies, which were selected for subsequent analyses. The presumptive isolates were further examined by Gram staining and a series of biochemical tests to achieve preliminary identification and confirmation. (Wong et al., 1988) [17] Preparation of Bacterial Cell Culture Pure cultures of Bacillus cereus were grown overnight in 10 mL of Brain Heart Infusion (BHI) broth at 37 °C in a shaker incubator. Following incubation, the cultures were centrifuged at 13,000 rpm for 10 minutes to pellet the bacterial cells, after which the supernatant was carefully discarded.

Genomic DNA Extraction

Genomic DNA from the confirmed Bacillus cereus isolates was extracted using a commercial DNA extraction kit following the manufacturer's protocol with minor modifications. Briefly, 1.5 mL of an overnight-grown Brain Heart Infusion (BHI) broth culture, prepared from a single colony and exhibiting visible turbidity, was transferred into a capped microcentrifuge tube and centrifuged at 12,000 rpm for 2 minutes. The resulting cell pellet was retained, and the supernatant was carefully discarded. The pellet was resuspended in 180 µL of resuspension buffer, mixed thoroughly, and subsequently treated with 20 µL of Proteinase K. The mixture was incubated at 55 °C for 30 minutes in a thermomixer to facilitate enzymatic digestion. Following this, 200 µL of lysis buffer was added, and the tube was further incubated at 55 °C for 10 minutes. Subsequently, 180 µL of absolute ethanol was added, and the contents were mixed gently. The entire lysate was then transferred to a spin column and centrifuged at 8,000 rpm for 4 minutes at 4 °C. The flow-through was discarded, after which the column was washed with 500 µL of pre-wash buffer and centrifuged at 8,000 rpm for 1 minute. This step was followed by the addition of 500 µL of wash buffer and centrifugation at 8,000 rpm for 1-2 minutes. To remove residual ethanol, the column was subjected to a dry spin at 9,000 rpm for 4 minutes. The spin column was then placed into a fresh sterile microcentrifuge tube, and 100 µL of elution buffer was added directly onto the membrane. DNA was eluted by centrifugation at 8,000 rpm for 5 minutes. The eluted buffer containing genomic DNA was used as the template for PCR amplification. DNA purity was assessed by measuring the OD260/OD280 ratio using a spectrophotometer, and the extracted DNA samples were stored at-20 °C until further analysis.

PCR-Based Detection of Species-specific and Virulence

Genes PCR assays were employed for molecular confirmation of Bacillus cereus through amplification of the gyrB gene and for screening of virulence-related genes (nheA, hblA, cytK, and cesB). The primer sequences used for detecting the gyrB, nheA, hblA, cesB, and cytK genes in B. cereus isolates are presented in Table 1. Each PCR reaction was performed in a total volume of 15 µL, comprising 10 µL of 2× DreamTaq Green Master Mix, 0.5 µL each of forward and reverse primers (25 pM/µL), 2.0 µL of template DNA, and 2.0 µL of nuclease-free water, under the optimized cycling conditions detailed in Table 2. The amplified products were separated by electrophoresis on 1.5% agarose gels and visualized using a gel documentation system.

Electrophoresis of PCR products

A 1% agarose gel was prepared by dissolving 1.25 g of agarose in 100 ml of diluted 1× TBE buffer, followed by heating in a microwave oven until a clear and homogeneous solution was obtained. The molten agarose was then allowed to cool to approximately 50-60 °C before adding 4 µl of ethidium bromide to achieve the desired final concentration, and the solution was mixed thoroughly. The gel was poured into a suitable casting tray fitted with an acrylic comb and allowed to solidify. Once the gel had set, the comb was carefully removed. For electrophoresis, the gel was placed in a horizontal electrophoresis chamber filled with 1× TBE buffer, ensuring that the buffer level remained 2-3 mm above the gel surface and that the wells were oriented toward the cathode. Amplified PCR products (10 µl) were gently loaded into the wells using disposable micropipette tips. A 100 bp or 1 kb DNA ladder was loaded in a separate well to estimate the size of the amplified fragments, while a negative control was included in the final well.

Electrophoresis was performed at 70 V for 60 minutes, and the PCR amplicons were subsequently visualized using a UV transilluminator.

Results and Discussion

Among the 300 milk-based sweet samples examined, all presumptive isolates recovered from mawa-based sweets (25; 100%) and chhana-based sweets (14; 100%) produced the specific 475 bp amplicon and were conclusively identified as Bacillus cereus through PCR targeting the gyrB gene, as detailed in Table 3 and Figure 1. The agarose gel electrophoresis profiles demonstrating gyrB gene amplification in B. cereus isolates are additionally illustrated in Plate 1. Overall, mawa-based sweets showed a higher prevalence of B. cereus contamination compared to chhanabased sweet products. These results are consistent with earlier studies, including that of Tewari et al. (2013) [15] , who reported amplification of the gyrB gene in all of their isolates (100%, 29/29), and Rather et al. (2011) [14] , who confirmed 98.3% (59/60) of isolates as Bacillus cereus using gyrB-specific primers. In addition, reliance on gyrB-based molecular assays improves the precision and reliability of pathogen detection and serves as an important tool for surveillance of foodborne bacteria, facilitating early identification and effective public health interventions.

Distribution of Virulence Genes

Detection of nheA gene Among the 39 isolates examined, 11 were found to be positive for the target virulence gene. Of these, 9 isolates (36%) originated from mawa-based sweets, while 2 isolates (14.28%) were recovered from chhana-based sweets, resulting in an overall prevalence of 28.20%. These findings are summarized in Table 4 and illustrated in Figure 2.

Additionally, the PCR agarose gel electrophoresis results demonstrating amplification of the gyrB gene specific to Bacillus cereus are depicted in Plate 2. Abdelaziz et al. (2024) [1] analysed a total of 179 samples comprising milk-derived products and raw chicken meat and reported that 22.34% were positive for Bacillus cereus sensu stricto. Of the recovered isolates, 17.5% harboured the nheABC enterotoxin gene cluster, with the nheA gene detected in 55% of isolates-a prevalence notably higher than that observed in the present investigation. In a related study, Elgushi et al. (2023) [6] evaluated 180 dairy samples, including raw cow milk, milk powder, and Ras cheese (60 samples from each category). Using PCR assays targeting the nheA gene, which encodes the non-hemolytic enterotoxin, they identified enterotoxigenic B. cereus in 10.5% of the samples. This prevalence closely aligns with the results reported in the current study.

Detection of hblA gene

Of the 39 isolates examined, 12 were found to be positive, comprising 10 isolates (40%) from mawa-based sweets and 2 isolates (14.28%) from chhana-based sweets. This corresponds to an overall prevalence of 30.76% for the targeted virulence gene, as detailed in Table 5 and illustrated in Figure 3. In addition, the PCR agarose gel electrophoresis results demonstrating amplification of the gyrB gene specific to Bacillus cereus are shown in Plate 3. Abdelaziz et al. (2024) [1] analysed 179 samples and reported that 22.34% were positive for Bacillus cereus sensu stricto. Notably, milk products and raw chicken meat exhibited a 50% prevalence of the hblA gene, which is substantially higher than that observed in the present investigation. Similarly, Thabet et al. (2023) [16] evaluated 100 samples comprising raw milk, pasteurized milk, Damietta cheese, and yoghurt, and detected the hblA geneassociated with emetic syndrome-in 60% of the samples, a markedly greater proportion than that recorded in the current study. In another study, Kowalska et al. (2022) [9] assessed 267 B. cereus isolates recovered from various food products and identified the hblA gene in 53.56% of the strains, which also considerably exceeds the prevalence reported in the present findings.

Detection of cesB gene

Of the 39 isolates analysed, only four were found to be positive for the cesB gene. All four positive isolates (16%) originated from mawa-based sweets, while none of the isolates recovered from chhana-based sweets harboured this gene, indicating that this virulence determinant was present in a limited proportion of the total isolates (10.25%), as detailed in Table 6 and Figure 4. Additionally, the PCR agarose gel electrophoresis results demonstrating the amplification of the gyrB gene specific to Bacillus cereus are illustrated in Plate 4. Abdelaziz et al. (2024) [1] analysed 179 samples and reported that 22.34% were positive for Bacillus cereus sensu stricto. Among the isolates recovered from milk products and raw chicken meat, only seven strains (17.5%) harboured the emetic toxin-encoding ces gene, a finding that is comparable with the results observed in the present investigation. Similarly, Thabet et al. (2023) [16] evaluated 100 samples comprising raw milk, pasteurized milk, Damietta cheese, and yogurt, with 25 samples from each category. The ces gene associated with emetic toxin production was detected in 5 of the 34 confirmed B. cereus isolates, corresponding to a prevalence of 14.7%. This detection rate is also consistent with the observations of the current study.

Detection of cytK gene

Among the 39 isolates analysed, 14 were found to be positive for the virulence gene, including 12 isolates (48%) from mawa-based sweets and 2 isolates (14.28%) from chhana-based sweets, reflecting that this gene was present in a notable proportion of the isolates (35.89%), as shown in Table 7 and Figure 5. The PCR agarose gel results demonstrating the amplification of the gyrB gene in Bacillus cereus are presented in Plate 5. Thabet et al. (2023) [16] examined 100 samples comprising raw milk, pasteurized milk, Damietta cheese, and yogurt (25 samples of each type) and reported the presence of the cytK virulence gene in 80% of the samples, a detection rate considerably higher than that observed in the present study. Similarly, Mostafa et al. (2022) [10] investigated multiple virulence genes, including cytK, in 18 different Bacillus cereus strains and found a prevalence of 63.64%, which also surpasses the results obtained in the current research. These results reveal considerable variability in the distribution of virulence genes among the Bacillus cereus isolates. The nheA gene was the most frequently detected, suggesting a higher potential for causing diarrheal illness. The predominance of nheA and hblA supports previous findings that strains producing diarrheal toxins are commonly associated with dairy products. The detection of cytK further heightens public health concerns, as this gene is linked to severe gastrointestinal effects. Although the proportion of cesB-positive isolates was relatively low, the remarkable heat resistance of cereulide makes even minimal prevalence epidemiologically important. Several isolates carried combinations of multiple virulence genes, indicating an increased pathogenic versatility.

Overall, this study demonstrates that traditional milk-based sweets sold in Bikaner may act as reservoirs for toxigenic B. cereus. The prevalence observed is consistent with reports from other warm-climate regions, where insufficient temperature control and post-processing contamination facilitate bacterial survival. The co-occurrence of multiple toxin genes in single isolates highlights the importance of molecular surveillance, as reliance solely on conventional culture methods may underestimate the risk. From a veterinary public health perspective, these findings underscore the urgent need to enhance hygiene practices, strengthen cold chain management, and incorporate molecular diagnostic approaches into routine food safety monitoring programs. [15] 2. nheA F-TACGCTAAGGAGGGGCA R-GTTTTATTGCTTCATCGGCT 480 bp Fagerlund et al. (2004) [7] 3. hblA F-GTGCAGATGTTGATGCCGAT R-ATGCCACTGCGTGGACATAT 301 bp Fagerlund et al. (2004) [7] 4. cesB F-GGTGACACATTATCATATAAGGTG R-GTAAGCGAACCTGTCTGTAACAACA 1271 bp Ehling-Schulz et al. (2006) [5] 5. cytK F-ACAGATATCGGGTCAAAATGC R-TCCAACCCAGTTATGCCAGTTC 809 bp Kwarteng et al. (2017) [11]

Conclusion

The present study effectively employed a molecular approach to characterize the toxigenic profile of Bacillus cereus in Bikaner's popular milk sweets, confirming the presence of toxigenic strains in these widely consumed dairy products. PCR-based detection of the gyrB gene along with virulence-associated genes offers critical insights into the potential public health risks linked to these sweets. The observed high prevalence of enterotoxin genes and detection of emetic markers underscore the need for strengthened monitoring. Implementation of Good Manufacturing Practices (GMP), Hazard Analysis and Critical Control Point (HACCP) systems, and regular molecular screening is strongly recommended to safeguard consumer health.

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