Original Articles

Molecular Detection of blaTEM, blaCTX-M and blaSHV Beta-Lactamase Genes in Bacillus cereus Strains Isolated from Infant Dry Milk Samples


Background:     Bacillus cereus is one of the important pathogen, which can be found in food samples like milk and principally responsible for food poisoning. Metallo-beta-lactamase (MBL) genes present in the Bacillus cereus bacterium, which provide resistance to the bacteria against the extreme condition. The objective of this study is to carry out molecular detection of blaTEM, blaCTX-M and blaSHV MBL genes in B. cereus strains, isolated from infant dry milk samples.   Methods:     Total 50 samples of infant dry milk were collected from the drug store, and 19 samples were selected for this investigation. After morphological and biochemical characterization of suspected colonies which are obtained from infant dry milk samples, these isolates were confirmed for B. cereus. Antibiotic susceptibility tests were done as per criteria of Clinical and Laboratory Standards Institute (CLSI). The phenotypic confirmatory analysis was done in Mueller Hinton agar (MHA) plates with clavulanic acid. If inhibition diameter is ≥5 mm increases in the clavulanic acid (CA) containing plate than to a plate without CA, it confirmed the presence of MBL genes. PCR used for detection of MBL genes in the isolated strains. Results:     PCR detected the blaCTX-M (100%), blaSHV (4%) and blaTEM (84.2%) gene of B. cereus in the infant dry milk. Conclusion:     The study confirms that the infant dry milk is a good source of B. cereus, if dry milk has absorbed water content from the air and hence providing a perfect condition for the growth of the bacterium, so It should be kept airtight the dry milk and stored in the cold condition.
1. Rahimi E, Abdos F, Momtaz H, et al. Bacillus cereus in infant foods: prevalence study and distribution of enterotoxigenic virulence factors in Isfahan Province, Iran. Sci world J 2013; 2013:1-5.
2. Sadek ZI, Abdel-Rahman MA, Azab MS, et al. Microbiological evaluation of infant foods quality and molecular detection of Bacillus cereus toxins relating genes. Toxicol reports 2018; 5: 871-77.
3. Becker H, Schaller G, von Wiese W, et al. Bacillus cereus in infant foods and dried milk products. Int J food microbial 1994; 23(1):1-5.
4. Senesi S, Ghelardi E. Production, secretion and biological activity of Bacillus cereus enterotoxins. Toxins 2010; 2(7):1690-703.
5. Bottone EJ. Bacillus cereus, a volatile human pathogen. Clin Microbiol Rev 2010; 23(2):382-98.
6. Alanber MN, Alharbi NS, Khaled JM. Evaluation of multidrug-resistant Bacillus strains causing public health risks in powdered infant milk formulas. J Infect public Health 2019; 1-7.
7. Ojdana D, Sacha P, Wieczorek P, et al. The occurrence of blaCTX-M, blaSHV, and blaTEM genes in extended-Spectrum β-lactamase-positive strains of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis in Poland. Int J Antibiot 2014; 2014; 935842.
8. Tallent SM, Kotewicz KM, Strain EA, et al. Efficient isolation and identification of Bacillus cereus group. J AOAC Int 2012; 95(2): 446-51.
9. Banykó J, Vyletělová M. Determining the source of Bacillus cereus and B. licheniformis isolated from raw milk, pasteurized milk and yoghurt. Lett Appl Microbiol 2009; 48:318-23.
10. Tallent S, Rhodehamel E., Harmon, S. et al. Bacillus cereus, FDA bacteriological analytical manual online, https://www. fda.gov/food/laboratory-methods-food /bam-chapter-14-bacillus-cereus 2019.
11. Tewari A, Singh SP. Cultural and biochemical characterization of Bacillus cereus isolates collected from Pantnagar, J Vet Public Health 2015; 13(1):5-8.
12. Reiner K. Catalase Test Protocol, American Society For Microbiology, [Created on: 11 November 2010], Revised on 2016; 1-9.
13. Mahon CR, Lehman DC, Manuselis G. Textbook of diagnostic microbiology, 4th ed. W. B Saunders Co., Philadelphia, PA, 2011.
14. Mohamed AS, Alnakip MEA, Abd-El Aal SF. Occurrence of Bacillus cereus in raw milk and some dairy products in Egypt. Jpn Vet Res 2016; 64(2):S95-102.
15. Khatun M, Bera P, Mitra D, et al. Estimation of Heavy metal tolerance and antibiotic susceptibility of Bacillus cereus isolated from municipal solid waste. Int J Pharm Bio Sci 2012; 3(4):819–29.
16. Griffiths MW, Scraft H. Bacillus cereus Food Poisoning. In Foodborne Diseases, Third Ed., Revised by Christine E R Dodd., 2017; 395-405.
17. Hwang JY, Park JH. Characteristics of enterotoxin distribution, hemolysis, lecithinase, and starch hydrolysis of Bacillus cereus isolated from infant formulas and ready-to-eat foods. J. Dairy Sci 2015; 98: 1652–1660.
18. CLSI guideline on methods for antimicrobial susceptibility testing for human Mycoplasmas (M43), 2011.
19. Oliwa‐Stasiak K, Molnar CI, Arshak K, et al. Development of a PCR assay for identification of the Bacillus cereus group species. J Appl Microbiol 2010; 108(1):266-73.
20. Senda K, Arakawa Y, Nakashima K, et al. PCR detection of metallo-beta-lactamase gene (blaIMP) in gram-negative rods resistant to broad-spectrum beta-lactams. J Clin Microbiol 1996 ; 34(12):2909-13.
21. Rahimifard N, Fatholahzadeh B, Noory Z, et al. Bacillus cereus contamination in infant formula: a study in food and drug control laboratory. Tehran Univ Med J TUMS Pub 2007; 65(8):64-8.
22. Gao T, Ding Y, Wu Q, et al. Prevalence, virulence genes, antimicrobial susceptibility, and genetic diversity of Bacillus cereus isolated from pasteurized milk in China. Front Microbiol 2018; 9:533.
23. Mansury D, Motamedifar M, Sarvari J, et al. Antibiotic susceptibility pattern and identification of extended spectrum β-lactamases (ESBLs) in clinical isolates of Klebsiella pneumoniae from Shiraz, Iran. Iran J Microbiol 2016; 8(1):55.
24. Ranjbar R, Shahreza MH. Prevalence, antibiotic-resistance properties and enterotoxin gene profile of Bacillus cereus strains isolated from milk-based baby foods. Trop J Pharm Res 2017; 16(8):1931-7.
25. Owusu-Kwarteng J, Wuni A, Akabanda F, et al. Prevalence, virulence factor genes and antibiotic resistance of Bacillus cereus sensu lato isolated from dairy farms and traditional dairy products. BMC microbiol 2017; 17(1):65.
26. Lesley MB, Ernie SR, Kasing A, et al. Detection of Bacillus cereus in formula milk and ultra-high temperature (UHT) treated milk products. Int Food Res J 2017; 24(3).
27. Soltan Dallal MM, Nezamabadi S, Mardaneh J, Rajabi Z, Sirdani A. Detection of toxigenic Bacillus cereus strains in powdered infant formula (PIF) milk by PCR assay. Tehran Med J TUMS Pub 2017; 75(3):179-86.
28. Cui Y, Liu X, Dietrich R, et al. Characterization of Bacillus cereus isolates from local dairy farms in China. FEMS Microbiol Lett 2016; 363(12):fnw096.
29. Zhang Z, Feng L, Xu H, et al. Detection of viable enterotoxin-producing Bacillus cereus and analysis of toxigenicity from ready-to-eat foods and infant formula milk powder by multiplex PCR. J Dairy Sci 2016; 99(2):1047-55.
30. Reis AL, Montanhini M, Bittencourt JV, et al. Gene detection and toxin production evaluation of hemolysin BL of Bacillus cereus isolated from milk and dairy products marketed in Brazil. Braz J Microbiol 2013; 44(4):1195-8.
31. Di Pinto A, Bonerba E, Bozzo G, et al. Occurence of potentially enterotoxigenic Bacillus cereus in infant milk powder. Europ Food Res Technol 2013; 237(2):275-9.
32. Shaheen R, Andersson MA, Apetroaie C, et al. Potential of selected infant food formulas for production of Bacillus cereus emetic toxin, cereulide. Int J Food Microbiol 2006; 107(3):287-94.
33. Rowan NJ, Anderson JG. Maltodextrin stimulates growth of Bacillus cereus and synthesis of diarrheal enterotoxin in infant milk formulae. Appl Environ Microbiol 1997; 63(3):1182-4.
34. Torkar KG, Bedenic B. Antimicrobial susceptibility and characterization of metallo-β-lactamases, extended-spectrum β-lactamases, and carbapenemases of Bacillus cereus isolates. Microb Pathog 2018; 1-19.
IssueVol 10 No 1-2 (2021) QRcode
SectionOriginal Articles
Antibiotic susceptibility B. cereus Metallo-beta-lactamase Milk Infants.

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How to Cite
Ghazaei C. Molecular Detection of blaTEM, blaCTX-M and blaSHV Beta-Lactamase Genes in Bacillus cereus Strains Isolated from Infant Dry Milk Samples. J Med Bacteriol. 2021;10(1,2):1-10.