Bacillus Cereus

Introduction/ About the Microbe

Bacillus cereus: A Brief History

Bacillus cereus (B. cereus) was first isolated by Grace and Percy Frankland in 1887, as indicated in their publication titled “Studies on some New-Microorganisms obtained from Air”. It was noted as being 3.4- 12 µm long and 1.7 µm wide with noted spore formation. In 1916, the microorganism was further studied in Baltimore milk by Lawrence and Ford. It was concluded in the early studies that B. cereus is found inhabiting numerous environments including water, intestines, soil, and dust.

Handling, Safety, and Culturing

B. cereus has a BSL level of 1, indicating that it is not likely to cause disease in healthy adults and poses a minimal laboratory risk. When handling, it is advised to follow standard Microbial lab safety procedures and wear PPE. It can be handled in an open lab; however, the lab area must have sinks and a door separating the lab from the rest of the building. B. cereus may arrive in a freeze-dried format and should be stored between 2◦ C and 8◦C. Prior to use, the bacteria needs to be rehydrated first in molecular-grade water and then placed at room temperature (37◦F or 2◦C) for one hour overnight. For better results, an alternative rehydration tactic would be to incubate it overnight at 4◦C and then incubate it in a 65◦C environment for one hour.

Ideal growing conditions for B. cereus are at 30◦C in an aerobic environment. To grow B. cereus, MYP (Methanol-Egg-Yolk-Polymyxin) Agar is needed with a mixture of Antimicrobial Vial P. B. cereus has resistance to polymyxin, making it an ideal medium to isolate that specific microbe. ATCC also notes that is should be grown in “ATCC Medium 3: Nutrient Agar or Nutrient Broth”.

Taxonomy

Domain: Prokaryote
Kingdom: Bacteria
Phylum: Firmicutes
Class: Bacilli
Order: Bacillales
Family: Bacillaceae
Genus: Bacillus
Species: Cereus

Visualizing the Microbe

B. cereus is a gram-positive bacteria, meaning it has a cell wall with a thick layer of peptidoglycan and a thin layer of periplasm. Its purple color from the gram stain can be visualized in Figure 1 below. The cells are rod-shaped bacilli, sometimes appearing as diplobacilli. Below are some images portraying B. cereus for you to get a better understanding of the microbe!

Figure 1: This is an image of B. cereus in a gram stain using a light microscope.

Figure 2 is an image of B. cereus under an SEM; it is unclear if any staining was used.

Figure 3 shows B. cereus under TEM using negative staining.

Figure 4 is B. cereus on a polycarbonate membrane after Gram-staining under light microscope.

Figure 5 is B. cereus under a light microscope after “preculture media” was applied with varying amounts of chlorhexidine.

Diagnostic Tests

There are several tests that can be performed to distinguish B. cereus from other microbes. Using Gideon to make a comparison of diagnostic tests and their results, the following can be applied for B. cereus:

Positive test results:

Coccus, Bacillus, Spore formation, Facultative, Growth on ordinary blood agar, catalase, glucose fermenter, beta hemolysis, motile, casein hydrolysis, gelatin hydrolysis, Lecithinase, lipase, Tyrosine hydrolysis, growth at 6.5% NaCl, maltose, D-Mannose, salicin, trehalose

Negative test results:

coccus (other forms), branching filaments, acid fast, spirochete, curved bacilli, cell wall-deficient, aerobe, anaerobe, microaerophilic, growth on MacConkey agar, oxidase, glucose oxidizer, cholesterol needed for growth, x factor required, v factor required, capnophilic, indole, ONPG, citrate, hydrogen sulfide, nitrite to gas, yellow pigment, lysine decarboxylase, ornithine decarboxylase, phenylalanine deaminase, gas from glucose, Adonitol, L-arabinose, D-arabitol, dulcitol, Erthyritol, myo-Inositol, lactose, D-mannitol, melibiose, raffinose, L-Rhamnose, D-Sorbitol, D-Xylose

Can it be confused for something else?

B. cereus and B. anthracis are both very similar bacteria and can be easily confused for one another in lab analysis . However, using Gideon and doing a comparison of the two, notable differences can be drawn. B. cereus tests positive for Beta hemolysis, motility, D-Mannose, and Salicin. B. anthracis tests negative for those four tests.

In terms of symptoms, B.cereus resembles Staphylococcus aureus (S. aureus) as a food-borne pathogen. Comparing both pathogens, it can be observed that B. cereus tests positive for motility, while S. aureus tests negative for motility. B. cereus also tests negative for coccus morphology, D-Mannitol, and yellow pigment, while S. aureus tests positive for all three of them.

By categorizing tests preferences on Gideon, similar bacteria to B. cereus can be identified. Drawing characteristics from B. cereus, such as (+) Gram positive, (+) Bacillus or coccobacillus, and (+) spore formation, the first microbe identified is Bacillus subtilis. It makes sense that these two species would have similar test characteristics since they are from the same genus. However, once the facultative test is marked (positive for B. cereus), then B. cereus appears as the most comparable pathogen. Similarly, by selecting positive for Gram-positive, glucose fermenter, catalase, blood agar, and facultative, Gideon shows 44% match for S. aureus; however, when spore formation (+) is selected, B. cereus becomes a better match at 79%.

Molecular Diagnostics

In order to find a transcript of the 16S rRNA gene, an in-silico PCR can be done to find a partial sequence first. To perform the in-silico PCR, the following primers were utilized for amplification 67F (5´TGA AAA CTG AAC GAA ACA AAC 3´) and 1671R (5´CTC TCA AAA CTG AAC AAA ACG AAA) 3´. The melting temperature of 67F is 59◦C and the melting temperature of 1671R is 61◦C. The GC content for the 67F primer is 7/21 or 33.33%, and the GC content for 1671R is 8/24 or 33.33%.

After finding the partial sequence and running it through NCBI BLAST, the following 16S rRNA sequence was found:

>NR_115714.1_Bacillus cereus strain

AGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAATGGATTAAGAGCTTGCT

CTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGG

CTAATACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGT

CGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGG

GACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACG

CCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACC

TTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGG

AATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTG

GAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACA

CCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCT

GGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCAC

TCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTT

AATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGAAAACCCTAGAGATAGGGCTTCTCCTTCGGGAGC

AGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTG

ATCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAA

TCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCT

AATCTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGAT

CAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCG

GTGGGGTAACCTTTTTGGAGCCAGCCGCCTAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGG

AAGGTGCGGCTGGAT

Bacillus cereus would appear under the following tests...

Blood Agar (+)

This picture indicates β-hemolysis since a clearing of the medium occurs. B. cereus produces hemolysin toxins that destroyed both hemoglobin and the red blood cells in the media.

MacConkey (-)

MacConkey is selective for Gram-negative bacteria and differentiative for Enterobacteriaceae. B. cereus is Gram-positive; it does not grow on MacConkey media.

D-Mannitol (-)

This media is selective for Staphylococcus and measures pH through mannitol fermentation. B. cereus does not ferment the mannitol carbohydrate. Thus, as it plates, the pH depicts a more alkaline environment and will portray a red/pink hue as shown in the figure

DNAse (+)

B. cereus exhibits DNAse activity, breaking down the green methyl-DNA complex .

Anaerobic chamber (-); Facultative (+)

B. cereus is a facultative bacterium and would therefore grow in an anaerobic chamber.

Gram stain (+)

B. cereus is a Gram-positive bacteria, meaning it should stain purple from the crystal violet.

Phenol Red Broth: (+) glucose fermenter

Phenol red broth measures glucose fermentation by changing color from red to yellow. Additionally, it also can measure gas production during fermentation. B. cereus is a positive glucose fermenter, meaning that the color of the phenol red should turn yellow in the presence of a more acidic environment. B. cereus does not produce gas during fermentation, indicating a homofermentative result.

SIM Medium Deep Agar- Sulfur (-), (-) Indole, (+) motile

SIM agar tests for sulfur reduction, indole production, and motility. Indole reduction is tested by the addition of Kovac’s reagent to tryptophan and seeing if the media turs red. Sulfur reduction is shown by formation of a black precipitant. B. cereus tests negative for both sulfur and indole; however, it is motile in SIM agar

Catalase (+)

Catalase is an enzyme that breaks hydrogen peroxide into water and oxygen. B. cereus tests positive for catalase and thus forms gas bubbles in the presence of hydrogen peroxide.

Oxidase (v)

Bacteria with oxidase function can reduce Cytochrome C to oxygen and create a purple hue. B. cereus is oxidase negative, so it fails to reduce Cytochrome C, producing no color change.

Diseases and Treatment

Bacillus cereus Pathogenicity

B. cereus is a pathogen that causes a widespread of diseases including endophthalmitis and septicemia. Different strains of B. cereus can vary in toxicity; some strains are used as probiotics, while others are known to cause certain food-related fatalities. It is known to emit emetic and enterotoxins, which elicit emetic and diarrheal syndromes, respectively. Specifically, B. cereus also secretes four hemolysins, three phospholipases, an emesis-inducing toxin, and proteases that all act as toxins causing gastrointestinal problems. B. cereus is further known to induce central nervous system infections, fulminant sepsis, and anthrax-like pneumonia, especially in people with pre-existing health problems.

Disease Prevention

Since B. cereus primarily causes infection from foods, it is recommended that hot foods be kept 135◦F (57◦C) or higher and cold foods be kept at 41◦F (5◦C) and lower . Before enterotoxins are produced from the bacteria, spores can be eliminated by heating to 145◦F (63◦C) or higher. Upon formation of the enterotoxins, food needs to be heated or reheated to at least 249◦F (121◦C) for over 80 minutes. In terms of refrigeration, foods such as leftovers should be kept at 41◦F (5◦C) or lower to prevent spores from forming . Refrigerated or frozen foods should not be taken out of the freezer or refrigerator until they're needed; furthermore, cooked foods should not sit in a room temperature environment for more than 2 hours . Electron beam irradiation has been shown to decontaminate rice husks from the bacterium using a 7.5 kGy dosage of radiation. Interestingly, B. cereus has also been found possessing some degree of resistance against radiation. In a Korean study, it was found that certain sanitizer concentrations, such as 500 ppm of hydrogen peroxide, 100 ppm of chlorine, and 650 ppm of calcium hydroxide, were ideal disinfectants to eliminate B. cereus spores on rice as well. Thus, these two solutions are processing methods that can be implemented to eliminate B. cereus spread. B. cereus has also been located in processed foods, such as heat-treated milk. Some researchers advocate that in such situations, it is best to keep milk at a low temperature in all stages of processing in order to limit bacterial spore spread and development.

Common Locations of the Pathogen

B. cereus is found in soil and in the gut of different animal species. It is especially common in several food production facilities because its endospores easily stick to food and its surrounding production area. B. cereus found in soil has been noted with having an efficient conjugation system and high recombination properties.

Impact of B. cereus as a Pathogen

B. cereus is one of the most prevalent food-borne illness-causing pathogens in the world, the third most common bacteria to cause food-related illnesses in Europe specifically. Through an ecological perspective, B. cereus strain CITVM-11.1 has been known to possess certain antifungal properties, acting against the rot fungus Macrophomina phaseolina. Genetic sequencing has also revealed that the strain may act to destroy mycotoxins in plants as well as inhibit negative impacts of heavy metal pollutants. B. cereus also exhibits a high degree of genetic diversity and is widely studied for that reason. In one study of mobile genetics elements in B. cereus, it was found that 16 insertion sequence families, 30 known group II introns, 18 different Bacillus cereus repeats, and the Tn3 family were all found to be present in the genome. As a widely spread human pathogen, its toxicity has been an area of interest as well. Cereulide, tripartite hemolysin BL, and nonhemolytic enterotoxins have been identified as emetic and/or diarrheal toxins present in the species. It has also been suggested that more virulence characteristics exist within the species that have not been identified.

Antibiotic Treatments

B. cereus produces a strong beta lactamase, making it resistant to beta-lactam antibiotics. Some antibiotic resistances of B. cereus include amoxicillin-clavulanic acid, cefotaxime, penicillin, and vancomycin. It has also been found with possessing resistance to rifampin and tetracycline. In contrast, ciprofloxacin and vancomycin both work against B. cereus. Furthermore, azithromycin, ciprofloxacin, erythromycin, and gentamicin have all been studied as effective antibiotics against B. cereus. Another study has also cited clindamycin and chloramphenicol as effective treatments and noted that vancomycin generally works as a good antibiotic as well.

References

1. Franklin, G. and P. 1887. Studies on some New Mirco-organisms obtained from Air. Philos Trans Soc Lond B Biol Sci 178: 281-284.

2. Ford, W. W. 1916. Studies on Aerobic Spore-Bearing Non-Pathogenic Bacteria. Journal of Bacteriology 1(3): 281-288. DOI: 10.1128/jb.1.3.273-320.51.1916

3. Bottone EJ. 2010. Bacillus cereus, a Volatile Human Pathogen. Clinical Microbiology Reviews 23:382–398.

4. Sunkar S, Nachiyar CV. 2012. Biogenesis of antibacterial silver nanoparticles using theendophytic bacterium Bacillus cereus isolated from Garcinia xanthochymus. Asian Pacific Journal of Tropical Biomedicine 2:953–959.

5. Binnerup SJ, Højberg O, Sørensen J. 1998. Gram characteristics determined on single cells and at the microcolony level of bacteria immobilized on polycarbonate membrane filters. Journal of Microbiological Methods 31:185–192.

6. Okamoto A, Otsuji S, Kamako M, Inoue I, Tasaka K, Kato J. 2020. Bacillus cereus Group Exhibits More Resistant to Chlorhexidine Rather Than Bacillus subtilis Group. Open Journal of Medical Microbiology 10:139–152.

7. https://www.rki.de/EN/Content/infections/Diagnostics/NatRefCentresConsultantLab/CONSULAB/EM-images/EM_Tab_Bacillus_cereus_en.html

8. https://atlas.sund.ku.dk/microatlas/veterinary/bacteria/Bacillus_subtilis/

9. https://learn.chm.msu.edu/vibl/content/differential/images/macconkey_n.gonorrhoeae.jpg

10. Rabha M, Sharma S, Acharjee S, Sarmah BK. 2017. Isolation and characterization of Bacillus thuringiensis strains native to Assam soil of North East India. 3 Biotech 7.

11. Jasuja, N.D., Subhash C, Suresh J, Richa S. 2013. Isolation and Identification of microorganism from polyhouse agriculture soil of Rajasthan. African Journal of Microbiology Research 7(41):4886-4891.

12. Asha, S. and M. Krishnaveni. 2012. Isolation and Molecular Level Identification of DNase Producing Halophilic Bacillus cereus family isolates from Marine Sediment Sample. J. Pure and App Microbiol. 14(1): 423-435.

13. https://www.mgc.co.jp/eng/products/sc/anaeropack/img/kenki_ph_06.jpg

14. https://microbiologyinfo.com/wp-content/uploads/2019/04/featured-Phenol-Red-Fermentation-Test.jpg

15. Trick, Iris, Olga Salcher, Franz Lingens. 1984. Characterization of filament forming Bacillus strains isolated from bulking sludge. App Microbiol and Biotechnol 19: 120-124.

16. https://microbiologyinfo.com/wp-content/uploads/2019/04/featured-Motility-Test.jpg

17. Chandra, Ramesh. 2015. Cloning and Expression of Invertase Gene from Invertase producing bacteria to Nonproducing bacteria. Intl Journal of Adv Research in Biotech 4(5).

18. Valero, J.A. and M.C. Salmeron. 2002. Enumeraion, isolation and characterization of Bacillus cereus strains from Spanish wild rice. Food Microbiology 19(6): 589-595.

19. https://microbiologyinfo.com/oxidase-test-principle-uses-procedure-types-result-interpretation-examples-and-limitations/

20. Tongeren SP van, Roest HIJ, Degener JE, Harmsen HJM. 2014. Bacillus anthracis-Like Bacteria and Other B. cereus Group Members in a Microbial Community Within the International Space Station: A Challenge for Rapid and Easy Molecular Detection of Virulent B. anthracis. PLOS ONE 9:e98871.

21. Messelhäußer U, Ehling-Schulz M. 2018. Bacillus cereus—a Multifaceted Opportunistic Pathogen. Curr Clin Micro Rpt 5:120–125.

22. Stenfors Arnesen LP, Fagerlund A, Granum PE. 2008. From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiol Rev 32:579–606.

23. Sarrı́as JA, Valero M, Salmerón MC. 2003. Elimination of Bacillus cereus contamination in raw rice by electron beam irradiation. Food Microbiology 20:327–332.

24 .Min-Jeong L, Dong-Ho B, Dong-Ha L, Ki-Hyo J, Deog-Hwan O, Sang-Do H. 2006. Reduction of Bacillus cereus in Cooked Rice Treated with Sanitizers and Disinfectants. Journal of Microbiology and Biotechnology 16:639–642.

25. Bartoszewicz M, Hansen BM, Swiecicka I. 2008. The members of the Bacillus cereus group are commonly present contaminants of fresh and heat-treated milk. Food Microbiology 25:588–596.

26. Helgason, Caugant, Lecadet, Chen, Mahillon, Lovgren, Hegna, Kvaloy, Kolsto. 1998. Genetic Diversity of Bacillus cereus/B. thuringiensis Isolates from Natural Sources. Current Microbiology 37:80-87.

27. Glasset B, Herbin S, Granier SA, Cavalié L, Lafeuille E, Guérin C, Ruimy R, Casagrande-Magne F, Levast M, Chautemps N, Decousser J-W, Belotti L, Pelloux I, Robert J, Brisabois A, Ramarao N. 2018. Bacillus cereus, a serious cause of nosocomial infections: Epidemiologic and genetic survey. PLOS ONE 13:e0194346.

28. Cabellero J., Peralta, Molla, Del Valle, Caballero P., Berry, Felipe, Yaryura. 2018. Draft Genome Sequencing of Bacillus cereus CITVM-11.1, a Strain Exhibiting Interesting Antifungal Properties. J Mol Microbiol Biotechnol 28:47-51.

29. Fayad N, Kallassy Awad M, Mahillon J. 2019. Diversity of Bacillus cereus sensu lato mobilome. BMC Genomics 20:436.

30. Schoeni JL, Lee Wong AC. 2005. Bacillus cereus Food Poisoning and Its Toxins. J Food Prot 68:636–648.

31. Turnbull PCB, Sirianni NM, LeBron CI, Samaan MN, Sutton FN, Reyes AE, Peruski LF. 2004. MICs of Selected Antibiotics for Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides from a Range of Clinical and Environmental Sources as Determined by the Etest. Journal of Clinical Microbiology 42:3626–3634.

32. Park KM, Jeong M, Park KJ, Koo M. 2018. Prevalence, Enterotoxin Genes, and Antibiotic Resistance of Bacillus cereus Isolated from Raw Vegetables in Korea. J Food Prot 81:1590–1597.

33. Turnbull PCB, Sirianni NM, LeBron CI, Samaan MN, Sutton FN, Reyes AE, Peruski LF. 2004. MICs of Selected Antibiotics for Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides from a Range of Clinical and Environmental Sources as Determined by the Etest. Journal of Clinical Microbiology 42:3626–3634.

34. Sacchi CT, Whitney AM, Mayer LW, Morey R, Steigerwalt A, Boras A, Weyant RS, Popovic T. Sequencing of 16S rRNA Gene: A Rapid Tool for Identification of Bacillus anthracis -Volume 8, Number 10—October 2002 - Emerging Infectious Diseases journal - CDC https://doi.org/10.3201/eid0810.020391.

About the Researcher

Hi everyone! My name is Dominika Dzurny, and I am a second-year Microbiology major from St. Petersburg, FL hoping to pursue double minors in Bioinformatics and Nutritional Sciences. I intend to continue Microbiology or a related subject in graduate school with the dream of later working in research or medicine (or both!). During my free time, you can find me baking sourdough bread, hanging out with friends, or starting a new hobby to keep me busy.

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