Prevalence and Identification of Bacillus cereus in Some Dried Dairy Products Focuses on Its Toxigenic Genes

Yahya Anwar Sidiq; Othman Abdulrahman  Mohammed

doi:10.24017/science.2025.2.11

Authors

Yahya Anwar Sidiq Food Science & Quality Control Department, Halabja Technical College, Sulaimani Polytechnic University, Sulay-maniyah, Iraq https://orcid.org/0009-0002-6881-6770
Othman Abdulrahman Mohammed Medical Laboratory Science Department, Halabja Technical College of Applied Sciences, Sulaimani Polytechnic Uni-versity, Sulaymaniyah, Iraq https://orcid.org/0000-0001-8154-8665

Abstract

Bacillus cereus is a spore-forming, toxin-producing bacterium. It has significant food safety risks, especially in dried dairy products, including powdered and infant milk. This may pose a risk to the consumers. The current study aimed to show the prevalence and toxigenic potential of B. cereus in 134 milk samples. They were collected randomly from food stores and various sale points in the local markets from Sulaymaniyah and Halabja provinces between November 2024 and May 2025. Bacterial isolation was performed using Mannitol egg Yolk Polymyxin (MYP) agar. The isolates were then confirmed by biochemical assays, VITEK 2 (BCL), and Polymerase Chain Reaction (PCR) amplification of gyrB and 16S rRNA genes. Phylogenetic analysis revealed close genetic clustering of the isolates with reference strains of B. cereus, B. thuringiensis, and B. tropicus. Antimicrobial susceptibility testing using the disk diffusion method presented complete resistance to β-lactam antibiotics. However, all isolates remained susceptible to ciprofloxacin, tetracycline, erythromycin, and vancomycin. Of the tested samples, B. cereus was detected in 48% of different types of powdered milk and 11.7% of infant milk samples. Virulence gene analysis displayed high prevalence rates of enterotoxins: nheA (100%), nheB (80.76%), cytK (86.53%), hblA (75%), and hblC (88.46%). While the emetic toxin gene ces was not detected in any milk samples. In conclusion, the presenting of multi-virulent and β-lactam-resistant B. cereus in dried milk reinforce the need for improved hygiene during dairy processing. Future studies can employ whole-genome sequencing approaches to better understand the genetic diversity, and virulence mechanisms in B. cereus from dairy environments.

Keywords:

Powdered milk, Emetic toxin, Diarrheal toxin, Polymerase Chain Reaction, Mannitol egg Yolk Polymyxin

References

S. Yang et al., "The sources of Bacillus cereus contamination and their association with cereulide production in dairy and cooked rice processing lines," Food Quality and Safety, vol. 7, pp. 1-14, 2023. https://doi.org/10.1093/fqsafe/fyad023 . DOI: https://doi.org/10.1093/fqsafe/fyad023

S. R. Organji, H. H. Abulreesh, K. Elbanna, G. E. H. Osman, and M. Khider, "Occurrence and characterization of toxi-genic Bacillus cereus in food and infant feces," Asian Pacific Journal of Tropical Biomedicine, vol. 5, no. 7, pp. 515–520, 2015. https://doi.org/10.1016/j.apjtb.2015.04.004. DOI: https://doi.org/10.1016/j.apjtb.2015.04.004

L. Meng et al., "Characterization and spoilage potential of Bacillus cereus isolated from farm environment and raw milk," Frontiers in Microbiology, vol. 13, pp. 1-12, 2022. https://doi.org/10.3389/fmicb.2022.940611. DOI: https://doi.org/10.3389/fmicb.2022.940611

P. Adamski, Z. Byczkowska-Rostkowska, J. Gajewska, A. J. Zakrzewski, and L. Kłębukowska, "Prevalence and antibi-otic resistance of Bacillus sp. isolated from raw milk," Microorganisms, vol. 11, no. 4, pp. 1065, 2023. https://doi.org/10.3390/microorganisms11041065. DOI: https://doi.org/10.3390/microorganisms11041065

A. S. Ibrahim, N. M. Hafiz, and M. Saad, "Prevalence of Bacillus cereus in dairy powders focusing on its toxigenic genes and antimicrobial resistance," Archives of Microbiology, vol. 204, no. 6, pp. 339, 2022. https://doi.org/10.1007/s00203-022-02945-3. DOI: https://doi.org/10.1007/s00203-022-02945-3

M. A. Abou Zeid and S. A. Yassin, "Detection of Some Virulence Factors of Bacillus cereus in Heat-Treated Milk," Alexan-dria Journal of Veterinary Sciences, vol. 53 ,no 1 ,pp. 72-78, 2017. 10.5455/ajvs.261157. DOI: https://doi.org/10.5455/ajvs.261157

R. Majed, C. Faille, M. Kallassy, and M. Gohar, "Bacillus cereus biofilms—same, only different," Frontiers in microbiology, vol. 7, pp. 1054, 2016. 10.5455/ajvs.261157. DOI: https://doi.org/10.3389/fmicb.2016.01054

J. Huang, M. Zhang, and Z. Fang, "Perspectives on novel technologies of processing and monitoring the safety and quality of prepared food products," Foods, vol. 12, no. 16, pp. 3052, 2023. https://doi.org/10.3390/foods12163052. DOI: https://doi.org/10.3390/foods12163052

Y. Huang, S. H. Flint, T. S. Loo, andmetic toxin production of Bacillus cereus in a biofilm,"Ffood Science and Technology, vol. 154, pp. 112840, 2022. https://doi.org/10.1016/j.lwt.2021.112840. DOI: https://doi.org/10.1016/j.lwt.2021.112840

K. Rouzeau-Szynalski, K. Stollewerk, U. Messelhäusser, and M. Ehling-Schulz, "Why be serious about emetic Bacillus cereus: Cereulide production and industrial challenges," Food Microbiology, vol. 85, pp. 103279, 2020. https://doi.org/10.1016/j.fm.2019.103279. DOI: https://doi.org/10.1016/j.fm.2019.103279

D. Saha, S. K. Nanda, and D. N. Yadav, "Shelf‐life study of spray‐dried groundnut milk powder," Journal of Food Process Engineering, vol. 43, no. 3, pp. e13259, 2020. https://doi.org/10.1111/jfpe.13259. DOI: https://doi.org/10.1111/jfpe.13259

J. Vidic, C. Chaix, M. Manzano, and M. Heyndrickx, "Food sensing: Detection of Bacillus cereus spores in dairy prod-ucts," Biosensors, vol. 10, no. 3, pp. 15, 2020. https://doi.org/10.3390/bios10030015. DOI: https://doi.org/10.3390/bios10030015

E. Tirloni, S. Stella, F. Celandroni, D. Mazzantini, C. Bernardi, and E. Ghelardi, "Bacillus cereus in dairy products and production plants," Foods, vol. 11, no. 17, pp. 2572, 2022. https://doi.org/10.3390/foods11172572. DOI: https://doi.org/10.3390/foods11172572

N. Jessberger, R. Dietrich, P. E. Granum, and E. Märtlbauer, "The Bacillus cereus food infection as multifactorial process," Toxins, vol. 12, no. 11, pp. 1-37, 2020. https://doi.org/10.3390/toxins12110701. DOI: https://doi.org/10.3390/toxins12110701

A. Hefny, H. Mohamed, E. I. Etokhy, and M. W. Abd El-Azeem, "Characterization of Bacillus cereus isolated from raw milk and milk products," Journal of Veterinary and Animal Research, vol. 3, pp. 1-18, 2020. doi: 10.3390/toxins12070454. DOI: https://doi.org/10.3390/toxins12070454

A. G. Dimri, S. Chaudhary, D. Singh, A. Chauhan, and M. Aggarwal, "Morphological and biochemical characterization of food borne gram-positive and gram-negative bacteria," Science Archives, vol. 1, no. 1, pp. 16–23, 2020.

J. Sánchez-Chica, M. M. Correa, A. E. Aceves-Diez, and L. M. Castaneda-Sandoval, "Genetic and toxigenic diversity of Bacillus cereus group isolated from powdered foods," Journal of Food Science and Technology, vol. 58, no. 5, pp. 1892–1899, 2021. https://doi.org/10.1007/s13197-020-04700-2. DOI: https://doi.org/10.1007/s13197-020-04700-2

R. Owusu-Darko, M. Allam, A. Ismail, C. A. Ferreira, S. D. d. Oliveira, and E. M. Buys, "Comparative genome analysis of Bacillus sporothermodurans with its closest phylogenetic neighbor, Bacillus oleronius, and Bacillus cereus and Bacillus subtilis groups," Microorganisms, vol. 8, no. 8, pp. 1185, 2020. https://doi.org/10.3390/microorganisms8081185. DOI: https://doi.org/10.3390/microorganisms8081185

M. Magnusson, A. Christiansson, and B. Svensson, "Bacillus cereus spores during housing of dairy cows: factors affect-ing contamination of raw milk," Journal of dairy science, vol. 90, no. 6, pp. 2745–2754, 2007. https://doi.org/10.3168/jds.2006-754. DOI: https://doi.org/10.3168/jds.2006-754

R. Dietrich, N. Jessberger, M. Ehling-Schulz, E. Märtlbauer, and P. E. Granum, "The food poisoning toxins of Bacillus cereus," Toxins, vol. 13, no. 2, pp. 98, 2021. https://doi.org/10.3390/toxins13020098. DOI: https://doi.org/10.3390/toxins13020098

J. Jovanovic, V. F. Ornelis, A. Madder, and A. Rajkovic, "Bacillus cereus food intoxication and toxicoinfection," Compre-hensive Reviews in Food Science and Food Safety, vol. 20, no. 4, pp. 3719–3761, 2021. https://doi.org/10.1111/1541-4337.12785. DOI: https://doi.org/10.1111/1541-4337.12785

J. Sánchez-Chica, M. M. Correa, A. E. Aceves-Diez, and L. M. Castañeda-Sandoval, "A novel method for direct detection of Bacillus cereus toxin genes in powdered dairy products," International Dairy Journal, vol. 103, pp. 104625, 2020. https://doi.org/10.1016/j.idairyj.2019.104625. DOI: https://doi.org/10.1016/j.idairyj.2019.104625

Y. T. Proroga et al., "Occurrence and toxin gene profile of Bacillus cereus in dairy products," Journal of Microbiology, Bio-technology & Food Sciences, vol. 9, no. 1, 2019. :10.15414/jmbfs.2019.9.1.58-62. DOI: https://doi.org/10.15414/jmbfs.2019.9.1.58-62

J. Sánchez-Chica, M. M. Correa, A. E. Aceves-Diez, and L. M. Castañeda-Sandoval, "Enterotoxin gene distribution and genotypes of Bacillus cereus sensu lato isolated from cassava starch," Toxins, vol. 13, no. 2, pp. 131, 2021. https://doi.org/10.3390/toxins13020131. DOI: https://doi.org/10.3390/toxins13020131

X. Pei et al., "Prevalence of Bacillus cereus in powdered infant and powdered follow-up formula in China," Food Control, vol. 93, pp. 101–105, 2018. https://doi.org/10.1016/j.foodcont.2018.05.049. DOI: https://doi.org/10.1016/j.foodcont.2018.05.049

I. M. Al Bulushi, Z. S. Al Kharousi, and M. S. Rahman, "Vitek: a platform for a better understanding of microbes," in Techniques to measure food safety and quality: microbial, chemical, and sensory: Springer, 2021, pp. 117–136. 10.1007/978-3-030-68636-9_6. DOI: https://doi.org/10.1007/978-3-030-68636-9_6

J. Kowalska, E. Maćkiw, D. Korsak, and J. Postupolski, "Characterization of the Bacillus cereus group isolated from ready-to-eat foods in Poland by whole-genome sequencing," Foods, vol. 13, no. 20, pp. 3266, 2024. https://doi.org/10.3390/foods13203266. DOI: https://doi.org/10.3390/foods13203266

T. Gao et al., "Prevalence, virulence genes, antimicrobial susceptibility, and genetic diversity of Bacillus cereus isolated from pasteurized milk in China," Frontiers in Microbiology, vol. 9, pp. 533, 2018. https://doi.org/10.3389/fmicb.2018.00533. DOI: https://doi.org/10.3389/fmicb.2018.00533

N. Mostafa, R. Elkenany, and G. Younis, "Characterization of Bacillus cereus isolated from contaminated foods with sequencing of virulence genes in Egypt," Brazilian Journal of Biology, vol. 84, pp. e257516, 2022. https://doi.org/10.1590/1519-6984.257516. DOI: https://doi.org/10.1590/1519-6984.257516

R. Osama, M. Ahmed, A. Abdulmawjood, and M. Al-Ashmawy, "Prevalence and antimicrobial resistance of Bacillus cereus in milk and dairy products," Mansoura veterinary medical journal, vol. 21, no. 2, pp. 11–18, 2020. https://doi.org/10.35943/mvmj.2020.2.202. DOI: https://doi.org/10.21608/mvmj.2020.2.202

S. Kumari and P. K. Sarkar, "Prevalence and characterization of Bacillus cereus group from various marketed dairy products in India," Dairy science & technology, vol. 94, no. 5, pp. 483–497, 2014. https://doi.org/10.1007/s13594-014-0174-5. DOI: https://doi.org/10.1007/s13594-014-0174-5

M. t. Giffel, R. Beumer, M. Bonestroo, and F. Rombouts, "Incidence and characterization of Bacillus cereus in two dairy processing plants," 1996.

Y. Huang, S. H. Flint, and J. S. Palmer, "Bacillus cereus spores and toxins–The potential role of biofilms," Food microbiolo-gy, vol. 90, pp. 103493, 2020. https://doi.org/10.1016/j.fm.2020.103493. DOI: https://doi.org/10.1016/j.fm.2020.103493

R. Shaheen, B. Svensson, M. A. Andersson, A. Christiansson, and M. Salkinoja-Salonen, "Persistence strategies of Bacil-lus cereus spores isolated from dairy silo tanks," Food microbiology, vol. 27, no. 3, pp. 347–355, 2010. https://doi.org/10.1016/j.fm.2009.11.004. DOI: https://doi.org/10.1016/j.fm.2009.11.004

E. F. S. Authority, "Opinion of the scientific panel on biological hazards (BIOHAZ) on Bacillus cereus and other Bacillus spp in foodstuffs," EFSA Journal, vol. 3, no. 4, pp. 175, 2005. https://doi.org/10.2903/j.efsa.2005.175. DOI: https://doi.org/10.2903/j.efsa.2005.175

M. Hashemi, F. A. Touranlou, S. Adibi, A. Afshari, and G. Shakeri, "Foodborne Bacteria in Iran: A 23-year Systematic Review of High-risk Foods," Iranian Journal of Veterinary Science & Technology, vol. 16, no. 3, pp. 1-47, 2024. 10.22067/ijvst.2024.87069.1357.

J.-Y. Hwang and J.-H. Park, "Characteristics of enterotoxin distribution, hemolysis, lecithinase, and starch hydrolysis of Bacillus cereus isolated from infant formulas and ready-to-eat foods," Journal of Dairy Science, vol. 98, no. 3, pp. 1652–1660, 2015. https://doi.org/10.3168/jds.2014-9042. DOI: https://doi.org/10.3168/jds.2014-9042

M. A. El-Zamkan and A. G. Mubarak, "Detection of B. cereus and some of its virulence genes in some dairy desserts and children diarrhea," Alexandria Journal of Veterinary Sciences, vol. 53, no. 1, pp. 28-38, 2017. 10.5455/ajvs.259758. DOI: https://doi.org/10.5455/ajvs.259758

Z. I. Sadek, M. A. Abdel-Rahman, M. S. Azab, O. M. Darwesh, and M. S. Hassan, "Microbiological evaluation of infant foods quality and molecular detection of Bacillus cereus toxins relating genes," Toxicology reports, vol. 5, pp. 871–877, 2018. https://doi.org/10.1016/j.toxrep.2018.08.013. DOI: https://doi.org/10.1016/j.toxrep.2018.08.013

P. Ngamwongsatit et al., "Broad distribution of enterotoxin genes (hblCDA, nheABC, cytK, and entFM) among Bacillus thuringiensis and Bacillus cereus as shown by novel primers," International journal of food microbiology, vol. 121, no. 3, pp. 352–356, 2008. https://doi.org/10.1016/j.ijfoodmicro.2007.11.013. DOI: https://doi.org/10.1016/j.ijfoodmicro.2007.11.013

M. S. Al-Khatib, H. Khyami-Horani, E. Badran, and A. A. Shehabi, "Incidence and characterization of diarrheal entero-toxins of fecal Bacillus cereus isolates associated with diarrhea," Diagnostic microbiology and infectious disease, vol. 59, no. 4, pp. 383–387, 2007. https://doi.org/10.1016/j.diagmicrobio.2007.06.014. DOI: https://doi.org/10.1016/j.diagmicrobio.2007.06.014

T. Lindback, A. Fagerlund, M. S. Rødland, and P. E. Granum, "Characterization of the Bacillus cereus Nhe enterotoxin," Microbiology, vol. 150, no. 12, pp. 3959–3967, 2004. https://doi.org/10.1099/mic.0.27359-0. DOI: https://doi.org/10.1099/mic.0.27359-0

B. Svensson, A. Monthán, R. Shaheen, M. A. Andersson, M. Salkinoja-Salonen, and A. Christiansson, "Occurrence of emetic toxin producing Bacillus cereus in the dairy production chain," International dairy journal, vol. 16, no. 7, pp. 740–749, 2006. https://doi.org/10.1016/j.idairyj.2005.07.002. DOI: https://doi.org/10.1016/j.idairyj.2005.07.002

J. Owusu-Kwarteng, A. Wuni, F. Akabanda, K. Tano-Debrah, and L. Jespersen, "Prevalence, virulence factor genes and antibiotic resistance of Bacillus cereus sensu lato isolated from dairy farms and traditional dairy products," BMC micro-biology, vol. 17, no. 1, p. 65, 2017. https://doi.org/10.1186/s12866-017-0975-9. DOI: https://doi.org/10.1186/s12866-017-0975-9

D. Savić et al., "Antimicrobial susceptibility and β-lactamase production in Bacillus cereus isolates from stool of patients, food and environment samples," Vojnosanitetski pregled, vol. 73, no. 10, pp. 904–909, 2016. https://doi.org/10.2298/VSP150415134S. DOI: https://doi.org/10.2298/VSP150415134S

K. Godič Torkar, B. Bedenić, and V. Plečko, "Antimicrobial susceptibility and the in vitro postantibiotic effects of van-comycin and ciprofloxacin against Bacillus cereus isolates," Journal of chemotherapy, vol. 28, no. 3, pp. 151–158, 2016. https://doi.org/10.1179/1973947815Y.0000000069. DOI: https://doi.org/10.1179/1973947815Y.0000000069

D. J. Beecher, "The Bacillus cereus group," in Molecular medical microbiology: Elsevier, 2002, pp. 1161–1190. https://doi.org/10.1016/B978-012677530-3/50276-2. DOI: https://doi.org/10.1016/B978-012677530-3/50276-2

I. Fernández-No, M. Guarddon, K. Böhme, A. Cepeda, P. Calo-Mata, and J. Barros-Velázquez, "Detection and quantifica-tion of spoilage and pathogenic Bacillus cereus, Bacillus subtilis and Bacillus licheniformis by real-time PCR," Food micro-biology, vol. 28, no. 3, pp. 605–610, 2011. https://doi.org/10.1016/j.fm.2010.10.014. DOI: https://doi.org/10.1016/j.fm.2010.10.014