Bacillus subtilis: A Comprehensive Guide

Bacillus subtilis also known as hay bacillus or grass bacillus is one of the best characterized bacteria and is used as a model organism for Gram positive bacteria to study bacterial chromosome replication and cell differentiation. It is a rod-shaped bacterium which is found in soil and gastrointestinal tract of ruminants, humans and marine sponge. B. subtilis produce endospores which help the bacteria to survive in adverse environmental conditions like heat and desiccation.

Taxonomy and Classifications

Kingdom: Bacteria

Phylum: Firmicutes

Class: Bacilli

Order: Bacillales

Family: Bacillaceae

Genus: Bacillus

Species: Bacillus subtilis

Morphology and Microscopy

• Gram positive, Rod shaped, 0.7-0.8 µm*2-3µm appear either singly or in chains
• Sporing
• Motile with peritrichous flagella
• Non-capsulated
• Historically classified as obligate aerobe but evidence shows that it is facultative anaerobes.
• Commonly found on plants.
• Is model organism of Gram positive bacteria.
• Valuable for producing enzymes like amylase, proteases, antibiotics like bacitracin
• Used as probiotics in agriculture and livestock feed.
• Under microscope, B. subtilis appear as red either single or in chains with green endospore.

Cultural and Growth Characteristics

• Grow on media in presence or absence of oxygen.
• Optimum temperature: 28-30˚C
• Optimum pH: 5.5-8.5
•  Most strain tolerate 7% Nacl but some can tolerate up to 10%
• Facultative anaerobes
• On Nutrient Agar: round to irregular in shape, 2-3mm, undulate to fimbriate margin, opaque with dull or wrinkled surface, mostly white sometimes creamy, brown , yellow, orange, pink, red or black in color.
• On Blood Agar: round, 3-4mm, flat, opaque, dry with white colored colonies, β-hemolysis.
• On Tryptic Soy Agar: circular or irregular, flat, irregular margin, opaque with mucoid surface, white to creamy colored colonies.

Biochemical and Identification Tests

Pathogenesis and Virulence Factors

B. subtilis is not considered to be pathogenic to human however it can contaminate food but rarely causes food poisoning and is considered opportunistic pathogen in immunecompromised patients. As they are capable of producing antibiotics, they are mainly used on seeds, vegetables and plants. These strains are mainly used by farmers as they have capacity to produce various toxins for insects which inhabits the root of plants and compete with disease causing organisms. Proteolytic enzymes like subtilisin are produced by B. subtilis and it also have spores which help them survive extreme environmental condition like heat and desiccation. It produces sticky, stringy consistency ropiness in spoiled bread dough. B. subtilis have low degree of virulence to humans. It has been found that it does not produce significant amount of virulence factors or other extracellular enzyme that cause infection in human. Only the possible virulence factor is toxin production as these bacteria produces enzyme lecithinase which have been involved in food poisoning. Similarly, an extracellular enzyme called subtilisin is produced which is responsible for allergic reactions in some individuals.

Reproduction

It divide symmetrically to make two daughter cells or asymmetrically to produce single endospore which is resistant to adverse environmental conditions like heat, acid and desiccation. During the time of nutritional stress, endospore is formed permitting organism to survive in the environment till the conditions become suitable. Before the production of spore, bacteria become motile by the formation of flagella also utilize DNA from the environment.

Epidemiology and Transmission

The bacterium was originally known as Vibrio subtilis in 1835 by Christian Gottfried Ehrenberg. But later it was renamed by Ferdinand Cohn as Bacillus subtilis in 1872. Throughout 1950s it was used as alternative medication because of its immunostimulatory effects of cell matter, which after digestion have ability to stimulate broad spectrum immune activity together with activation of antibody IgM, IgG and IgA secretion and release of CpG dinucleotide along with INF A/Y generating activity of leukocytes and cytokines which is crucial in evolution of cytotoxicity against tumor cells. Since 1946, it was used as an immunostimulatory support in gut and urinary tract diseases treatment such as Shigella and Rotavirus. But later on, after establishment of low-cost antibiotics, its popularity decreased in spite of low toxicity and reduced chance of allergic reaction.

B. subtilis is transmitted mainly through following:

• Consumption of contaminated cooked foods which is subjected to inadequate post-cooking temperature.
• Consumption of contaminated poultry products.
• Consumption of meat or vegetables with pastry products.
• Consumption of seafood dishes.
• Consumption of inappropriately stored bakery products such as bread, crumpets, sandwiches.

Clinical Manifestations

The incubation period is 10 minutes to 14 hours. The most common clinical features include:

• Nausea
• Vomiting
• Diarrhea

B. subtilis is associated with following clinical features in lesser extent:

• Bacteremia
• Septicemia
• Endocarditis
• Meningitis
• Infections of wounds, ear, eyes, respiratory tract, urinary tract and gastrointestinal tract

Laboratory Diagnosis

 Microscopy (Gram’s Staining)

• Appears as purple in color with spores.

Culture

• Specimens are inoculated in suitable media.
• Then the culture was inoculated into liquid LB culture medium incubated at 37˚C for 18-24hr with vigorous shaking.
• On Nutrient Agar: round, grey to white in color, flat, opaque, medium sized colonies.
• On Rabbit Blood Agar: round, grayish white in color, opaque, flat, drying, medium sized, shows complete hemolysis.
• On TSA agar containing 5% fetal calf serum: round, grayish white in color, smooth, thick ridges, moist, medium sized colonies.
• On LB Agar: round, gray-white in color, flat, opaque, medium sized colonies.

Biochemical Tests

After culture, colonies from incubated plates are performed for biochemical tests and identified as B. subtilis based on following results:

Treatments

Antibiotics like Vancomycin, Clindamycin, Ciprofloxacion, Imipenem are used for infection caused by B. subtilis.

Application

• Industrial Enzyme Production: Used in industry to produce various enzymes like amylases, proteases etc. for food processing, detergents and biofuels. Used in fermentation.
• Environment: Used in treatment of sewage and helps in sewage disposal. Useful in biodegradation of waste materials.
• Medicine: Commonly used in vaccine and in probiotic supplements for gut health.
• Agriculture: Used in agriculture as biocontrol as it have natural fungicide properties and enhance plant growth. Used in animal feed and insect resistance.

Antibiotic Resistance

B. subtilis secrets toxins known as penicillinase, which is responsible for the inactivation of penicillin, interfering with penicillin based antibiotic treatment. This organism has capacity to secret antibiotic compounds like ribosomal antibiotics (e.g subtilosin and sublancin) that play crucial role in preliminary step of infection via the process of niche establishment and microbial monopolization. This secretion removes the competing bacteria and permits the bacteria to survive as this pathogen has acquired immunity against the self-generated antibiotics.

Conclusion

B. subtilis is a ubiquitous bacterium commonly isolated from water, air, soil and decomposing plant residue, that is responsible for production of endospores which can resist adverse environment conditions making them useful as biological indicators in sterilization processes. This bacterium produces various enzymes which help them in the development of antibiotic and enable them to degrade various natural substrate contributing to nutrient cycle.

References

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Biochemical tests for identification of Bacillus subtilis and Bacillus pumilus [Table]. (n.d.). ResearchGate.
https://www.researchgate.net/figure/Biochemical-tests-for-identification-of-Bacillus-subtilis-and-Bacillus-pumilus_tbl2_228484931

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smoubarak. (n.d.). Bacillus subtilis [Document]. Scribd.
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Turnbull, P. C. B. (1996). Bacillus. In S. Baron (Ed.), Medical microbiology (4th ed.). University of Texas Medical Branch at Galveston. NCBI Bookshelf.
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