Bacillus licheniformis

Bacillus licheniformis: A Comprehensive Reference

1. Identity

1.1 Taxonomic and Microbiological Classification

Bacillus licheniformis is a Gram-positive, rod-shaped bacterium belonging to the genus Bacillus, family Bacillaceae, order Bacillales. It is a Gram-positive, rod-shaped bacterium that belongs to the Bacillus genus, commonly found in soils. A taxonomical approach shows that it is closely related to Bacillus subtilis. Historically, B. licheniformis and two closely related species, B. subtilis and B. pumilus, were grouped taxonomically into what was known as the subtilis-group.

It is a Gram-positive, mesophilic bacterium. Its optimal growth temperature is around 50 °C, though it can survive at much higher temperatures. The optimal temperature for enzyme secretion is 37 °C. It can exist in a dormant spore form to resist harsh environments, or in a vegetative state when conditions are good. Generally, most bacilli are predominantly aerobic; however, B. licheniformis is a facultative anaerobe compared to other bacilli in ecological niches.

A key taxonomic complication has emerged in recent years. Bacillus paralicheniformis, a species known to produce the antimicrobial bacitracin, could be misidentified as Bacillus licheniformis, depending on the identification method used. For this reason, the European Commission requested EFSA to review the taxonomic identification of formerly assessed B. licheniformis production strains. EFSA retrieved the raw data from 27 technical dossiers submitted and found that the taxonomic identification was established by 16S rRNA gene analyses for 15 strains and by whole genome sequence analysis for 12 strains. As a conclusion, only these 12 strains could be unambiguously identified as B. licheniformis.

1.2 Natural Habitat and Sources

The natural habitat for B. licheniformis is soil. Therefore, long-term survival in soil may be expected to occur. Bacillus licheniformis is a ubiquitous bacterium thought to be of importance in the environment as a contributor to nutrient cycling due to the production of protease and amylase enzymes.

B. licheniformis is found in a wide variety of environments, but especially in soil and in the feathers of birds, where B. licheniformis degrades β-keratin. There is evidence that red feathers, with psittacofulvin, are more resistant to degradation. It is found on bird feathers, especially chest and back plumage, and most often in ground-dwelling birds (like sparrows) and aquatic species (like ducks).

It is one of the dominant populations of the soil and plant microbiota but is not an inherent bacterium in the human intestinal tract. B. licheniformis produces a variety of extracellular enzymes that are associated with the cycling of nutrients in nature. It is an apathogenic soil organism that is mostly associated with plant and plant materials in nature.

Bacillus licheniformis has been widely detected in fermented soybean foods. B. licheniformis has been isolated as a predominant species in fermented soybean foods from Korea and exhibits the highest salt tolerance among isolated Bacillus spp.

1.3 Sporulation

Bacillus licheniformis forms spores in soil. A pathway that leads to endospore formation is initiated when the bacterium is starved. Endospore formation is actually desired and serves as a great example of prokaryotic development and differentiation. These spores are quite tolerant of heat, cold, radiation, and other environmental stresses. Under good conditions, the spores will germinate and produce vegetative cells. The spore-forming capacity of B. licheniformis is of particular significance for its use as a probiotic supplement: the benefits of B. licheniformis as a probiotic when compared to other sources of natural and encapsulated probiotics mainly lie in sporulation, since this can contribute to greater survival in the adverse conditions found in the stomach and allow its arrival to the small intestine.

1.4 Commercial Forms and Preparations

High capacity of secretion of the alkaline serine protease has made B. licheniformis one of the most important bacteria in industrial enzyme production. B. licheniformis is used industrially for manufacturing biochemicals, enzymes, antibiotics, and aminopeptidase. Several proteases such as α-amylase, penicillinase, pentosanase, cycloglucosyltransferase, β-mannanase, and certain pectinolytic enzymes are synthesized industrially using B. licheniformis.

As a dietary supplement and probiotic, B. licheniformis is available in several forms. A B. licheniformis live bacterial preparation (Zhengchangsheng®, Northern Pharmaceutical Group Shenyang No.1 Pharmaceutical Co., Ltd., Shenyang, China) is a microecological preparation made from the B. licheniformis BL20386 strain. Commercial preparations such as B-Act® contain viable spores of a Bacillus licheniformis strain intended for use in animal feed and poultry. Human supplement products typically contain lyophilized or dried spore preparations in capsule or powder form, often combined with other spore-forming Bacillus species. The commercially employed species include B. subtilis, B. clausii, B. coagulans, B. licheniformis, B. polyfermenticus, and B. pumilus.

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2. Traditional and Historical Use

2.1 Fermented Foods of Asia and Africa

While Bacillus licheniformis was not historically identified or isolated as a named microorganism before the era of modern microbiology, it has been an active constituent of traditional fermented foods across multiple cultures for centuries. Its role in these preparations was not consciously recognized as a discrete species, but rather was an inherent feature of open-vessel, ambient-temperature fermentation processes.

Bacillus is present in alkaline-fermented foods of Asia and Africa. Species of Bacillus that are present, mostly in legume-based fermented foods, are Bacillus amyloliquefaciens, Bacillus circulans, Bacillus coagulans, Bacillus firmus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus subtilis, Bacillus subtilis variety natto, and Bacillus thuringiensis.

In Korea, B. licheniformis has historically been part of two major fermented soybean preparations. Cheonggukjang, a traditional fermented soybean food from Korea, is produced by fermenting steamed soybeans with natural microbial cultures. The main microbes in Cheonggukjang are B. thermoamylovorans and B. licheniformis. Doenjang, another ancient fermented soybean dish from Korea, has been taken for millennia as both a protein source and a flavor, comparable to miso in Japan and tempeh in Indonesia. Traditionally, Doenjang is made by mixing and fermenting brine with moldy cooked soybeans, in which naturally transported bacteria destroy soy proteins and produce a variety of nutritious chemicals. Bacillus is believed to be dominant in soybean paste, and the probiotic Bacillus spp. involved in its fermentation are B. licheniformis and B. subtilis.

Doenjang is a classic Korean condiment produced through the fermentation of soybeans and brine. It has garnered significant recognition as a nutritious source and flavor enhancer, offering a range of essential nutrients, including amino acids, flavonoids, vitamins, and minerals. A variety of Bacillus species have been found in Doenjang samples during fermentation, with B. subtilis, B. amyloliquefaciens, B. siamensis, B. methylotrophicus, and B. licheniformis having significant roles in the fermentation process.

The traditional Korean fermented food 'Chungkookjang' (soup prepared with fermented soybeans) has long been in the center of attention in Korea as a functional food having intestinal-protecting effect and blood circulation improving effect. Chungkookjang is also called Tempeh, Dushi, Kinema, and Natto in different countries. Chungkookjang is soybean fermented soup. During fermentation, microorganisms, enzymes and diverse bioactive materials are newly generated.

Integrating the Bacillus species into fermented food is a longstanding culinary tradition that reflects deeply rooted cultural customs and regional expertise. In traditional cuisines, these species contribute beneficial metabolites and distinctive flavors, adding a unique cultural dimension to the food.

2.2 Industrial and Pharmaceutical History

Bacillus licheniformis is a ubiquitous, saprophytic, soil bacterium which is thought to contribute to nutrient cycling due to its ability to produce a wide variety of enzymes. This latter feature of the microorganism has been commercially exploited for over a decade. B. licheniformis has been used for industrial production of proteases, amylases, antibiotics, and specialty chemicals. Its use in the production of bacitracin — the antibiotic derived from it — dates to the mid-20th century, and the bacterium has since been an important industrial workhorse in enzyme biotechnology. B. licheniformis has been used in industry for production of proteases, amylases, antibiotics, and specialty chemicals for over a decade with no known reports of adverse effects to human health or the environment.

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3. Key Constituents and Active Compounds

3.1 Antimicrobial Peptides and Lipopeptides

B. licheniformis produces a notable repertoire of bioactive secondary metabolites. Chief among these is bacitracin. Bacitracin is a dodecapeptide antibiotic produced by some strains of Bacillus licheniformis and Bacillus subtilis. The synthesis is nonribosomally catalyzed by a multienzyme complex composed of three subunits, BacA, BacB, and BacC, whose genes have been cloned and sequenced. Bacitracin has potent antibiotic activity against gram-positive bacteria. The inhibition of peptidoglycan biosynthesis is the best-characterized bactericidal effect of bacitracin. It forms a complex mediated by a metal ion (Zn2+) with the lipid C55-isoprenyl pyrophosphate (IPP), which is a carrier of a peptidoglycan unit or a disaccharide with pentapeptide across the membrane. Bacitracin, by binding to IPP, inhibits the conversion of IPP to C55-isoprenyl phosphate, which is catalyzed by a membrane-associated pyrophosphatase.

In addition to bacitracin, the production of several bacteriocin-like substances with different characteristics and a wide spectrum of activity against pathogenic bacteria was recorded in the strains of B. licheniformis. The genome annotation of relevant strains has detected gene clusters responsible for antimicrobial component production (lichenysin, fengycin, lichenicidin and bacillibactin biosynthetic gene clusters).

Lichenysin is a particularly characteristic lipopeptide of B. licheniformis. B. licheniformis additionally produces antibiotics such as lichenysin, surfactin, and iturin. B. licheniformis has been shown to inhibit the growth of various fungi, and its potential utility as a biological control agent against several fungal plant pathogens has recently been investigated. Surfactins were first isolated from a culture broth of B. subtilis and include compounds surfactin A, B, C, and lichenysin. Surfactins exert their antibacterial activities by acting on the plasma membrane through the pore-forming mechanism.

The strain also produces several classes of non-ribosomal peptides with various modes of action. Non-ribosomal peptides are synthesized by special non-ribosomal multimodular peptide synthetases and contain unnatural amino acids or fatty acids. Their harmful effect is due to their ability to form pores in biological membranes, destabilize lipid packaging, and disrupt the peptidoglycan layer. Lipopeptides, as biosurfactants, are able to destroy bacterial biofilms. Special attention has been paid to the antimycobacterials produced by B. licheniformis as a possible approach to combat multidrug-resistant and latent tuberculosis. In particular, licheniformins and bacitracins have shown strong antimycobacterial activity.

3.2 Extracellular Enzymes

The commercial utility of the extracellular products of B. licheniformis makes this microorganism an economically interesting species. The bacterium secretes a wide array of enzymes including proteases, amylases, and other carbohydrolases. Among these, subtilisin Carlsberg (also known as subtilisin A or alcalase) is a serine protease widely used in industrial applications ranging from detergents to food processing. High capacity of secretion of the alkaline serine protease has made B. licheniformis one of the most important bacteria in industrial enzyme production. These digestive enzymes contribute to nutrient breakdown in the gastrointestinal tract when the bacterium is administered as a probiotic.

3.3 Exopolysaccharides

Secreted polysaccharides are high molecular weight compounds, composed of repeated units of sugar moieties attached to a carrier lipid. Their antagonistic action was revealed in relation to bacteria, viruses, and fungi. Exopolysaccharides also inhibit the formation of biofilms by pathogenic bacteria and prevent their colonization on various surfaces.

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4. Mechanisms of Action

4.1 Sporulation and Gastrointestinal Survival

The primary functional advantage of B. licheniformis as a probiotic, compared to non-spore-forming organisms such as Lactobacillus species, is its capacity to form heat- and acid-resistant endospores. The cortex-lytic enzymes involved in the depolymerization of cortical peptidoglycan in B. licheniformis spores are close to those found in other Bacillus species; however, some investigations indicate that the primary enzymatic activity found during germination is about a lytic transglycosylase, probably SleB, and this protein seems to play a more significant role in B. licheniformis spore germination than in other Bacillus species. This allows the probiotic to survive passage through the acidic gastric environment and the bile salt-rich duodenum, germinating and becoming metabolically active in the small intestine.

4.2 Modulation of the Gut Microbiota

The intake of B. licheniformis has been related to the effects of modulation of the intestinal microbiota, antimicrobial activity, growth promotion, anti-inflammatory and immunostimulatory effects, and promotion of the regulation of the lipid profile, increase of neurotransmitters, and stress reduction, among others.

In animal models, the supplementation of Bacillus licheniformis could increase the growth of Firmicutes and Lactobacillus and encourage the construction of a more mature and stable microbiome. Studies in antibiotic-disrupted microbiota also showed that Bacillus licheniformis supplementation and treatment groups significantly increased the relative abundance of Firmicutes and Lactobacillus and decreased the relative abundance of Proteobacteria and Acinetobacter compared to the control group.

4.3 Intestinal Barrier Function

Mechanistic studies at the cellular level have documented that B. licheniformis can strengthen the intestinal epithelial barrier. Immunofluorescence and western blotting analysis revealed that B. licheniformis PF9 increased the expression levels of zona occludens 1 (ZO-1) and occludin (OCLN) in pathogen-infected intestinal epithelial cells. In animal studies, supplementation with probiotics improved intestinal mucosal integrity as evidenced by higher villus heights and a higher ratio of villus heights to crypt depths (duodenum and jejunum) and higher mRNA and protein levels of occludin and ZO-1 in jejunum mucosa. ZO-1 and occludin are key tight junction proteins whose upregulation indicates a more impermeable intestinal barrier.

Restoration of tight junction proteins after antibiotic disruption was also demonstrated: antibiotic treatment suppressed the mRNA expression of tight junction proteins ZO-1, claudin, occludin, and Ki67. However, B. licheniformis supplementation after antibiotic treatment restored the expression of the above genes, and there was no statistically significant difference compared to the control group.

4.4 Anti-Inflammatory and Immunomodulatory Mechanisms

Multiple cellular pathway investigations have characterized how B. licheniformis exerts anti-inflammatory effects. B. licheniformis PF9 treatment was capable of remarkably attenuating the expression levels of inflammation cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-8, and IL-6. Furthermore, the gene expression of Toll-like receptor 4 (TLR4)-mediated upstream related genes of NF-κB signaling pathway has been significantly inhibited. These changes were accompanied by significantly decreased phosphorylation of p65 NF-κB during infection.

B. licheniformis B410 was found to possess significant anti-inflammatory effect by suppressing the NF-κB and IRF signaling pathways. In cytokine analyses, B. licheniformis B410 could significantly inhibit the expression of pro-inflammatory cytokines IL-1β and TNF-α induced by LPS and promote the expression of the anti-inflammatory cytokine IL-10.

In animal studies with Clostridium perfringens-challenged mice, serum cytokine analysis revealed that B. licheniformis increased anti-inflammatory IL-4 and IL-10 levels while reducing pro-inflammatory IL-1β, IL-6, and TNF-α levels.

Immunoglobulin modulation has also been reported. The intake of B. licheniformis is related to a reduction of pro-inflammatory cytokine IL-8 and an increase in IgM and IgG, while IgA antibodies, and higher concentrations of total serum proteins and globulins were also found.

4.5 Short-Chain Fatty Acid Production

A few studies focused on neurological and psychological disorders were obtained, in which an improvement in the modulation of serum γ-aminobutyric acid, glutamic acid, 5-HT, and higher colonic concentrations of butyrate and valerate was observed. Butyrate, in particular, is a key short-chain fatty acid that serves as an energy source for colonocytes and has potent anti-inflammatory properties within the gut.

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5. Scientific Evidence by Area of Use

5.1 Gastrointestinal Health

5.1.1 Diarrhea and Intestinal Microbiota Balance (Human Evidence)

The B. licheniformis BL20386 strain can regulate gut microbiota balance and is mainly used for acute and chronic diarrhea, ulcerative colitis, spontaneous bacterial peritonitis, and other intestinal diseases caused by the gut microbiota imbalance and other reasons in clinical practice. With the expansion of the application range of B. licheniformis in recent years, it is used to treat not only diarrhea and intestinal inflammation but also other diseases caused by gut microbiota disorder. Many clinical studies have found that it affects patients' inflammatory response and neurotransmitter levels.

5.1.2 Clostridioides difficile Infection (Human Clinical Trial)

A randomized, placebo-controlled human clinical trial assessed B. licheniformis as adjuvant therapy in patients with Clostridioides difficile infection (CDI). B. licheniformis 250 mg or placebo three times per day was administered for five days in the study and placebo groups, respectively. The placebo, which contained starch as its main component, was designed to match the study drug in color and shape. CDI treatment was performed using 125 mg of vancomycin four times per day for both groups. Enrollment was performed between July 2021 and December 2022 at six academic hospitals in Korea. This represents one of the few rigorously designed human clinical trials specifically involving B. licheniformis in an identified disease context.

5.1.3 Post-Antibiotic Microbiota Restoration (Preclinical)

In a preclinical mouse study, the results showed that treatment with ceftriaxone sodium over 7 days suppressed the expression of NF-κB pathway mRNA levels, which caused cytoplasmic vacuolization in intestinal tissues; afterward, the administration of Bacillus licheniformis could effectively restore intestinal morphology and inflammation levels. Moreover, the ceftriaxone sodium treatment entirely affected the intestinal microbial ecology, leading to a decrease in microbial abundance. Firmicutes, Proteobacteria, and Epsilonbacteraeota were the most predominant phyla in each of the four groups. The antibiotic treatment resulted in a significant decrease in the relative abundance of 2 bacterial phyla and 20 bacterial genera compared to the administration of Bacillus licheniformis after ceftriaxone sodium treatment. The supplementation of Bacillus licheniformis could increase the growth of Firmicutes and Lactobacillus and encourage the construction of a more mature and stable microbiome. Evidence level: Preclinical (animal/in-vitro).

5.1.4 Subhealth and Gut Microbiota (Preclinical)

A study in rats used chronic stress and excessive antibiotics to induce a "subhealth" state, then evaluated the effect of B. licheniformis on microbiota and inflammatory biomarkers. TNF-α in the model group showed no significant difference from the control group after the modeling period, but TNF-α in the subgroup was significantly higher than in the control group; TNF-α in the B. licheniformis-treated group significantly decreased. Evidence level: Preclinical (rodent model).

5.2 Endotoxemia and Cardiovascular Risk Factors

5.2.1 Reduction of Dietary Endotoxemia (Human Evidence)

In humans, a study of 30-day probiotic supplementation of B. licheniformis with other oral spore-based probiotics could reduce dietary endotoxemia (McFarlin et al., 2017). This 30-day human supplementation study with spore-based probiotics including B. licheniformis could reduce dietary endotoxemia. Even though endotoxemia is the result of a translocation of LPS into the circulation, studies revealed its link to an elevated risk of many cardiovascular diseases. It should be noted that this study used a multi-species spore-based product and did not isolate the effects of B. licheniformis alone. Evidence level: One human study; multi-species formulation; cannot attribute effects solely to B. licheniformis.

5.2.2 Triglycerides and Lipid Profile

Animal studies demonstrated that regulation of disorders indirectly related to cardiocirculatory problems, such as triglyceride regulation for atherosclerosis, and its role in risk reduction of heart attacks, coronary diseases, cardiopathies, and many other heart illnesses have been reported. Although animal research has revealed that gut microorganisms may influence heart disease risk, no such relation has been observed in humans. Evidence level: Preliminary, primarily from animal models; no confirmed human cardiovascular benefit specific to B. licheniformis.

5.3 Immune Modulation and Infection Resistance

A total of 23 studies have revealed the potential effect of B. licheniformis on immune parameters. These are predominantly animal studies (poultry, fish, pigs). It was shown that this probiotic is associated with hepatoprotection and cardioprotection since it shows reduced dietary endotoxemia and modulation of liver toxicity and other molecules with a direct or indirect relation.

In animal models involving infection challenge, the probiotic administration significantly prevented infection-induced weight loss and immune organ enlargement. Serum cytokine analysis revealed that B. licheniformis increased anti-inflammatory IL-4 and IL-10 levels while reducing pro-inflammatory IL-1β, IL-6, and TNF-α levels. Histological analysis showed that B. licheniformis preserved intestinal morphology and inhibited epithelial cell apoptosis. Evidence level: Predominantly animal (poultry, porcine, fish, murine). Human immune data are limited.

5.4 Neurological and Psychological Applications

A few studies focused on neurological and psychological disorders were obtained, in which an improvement in the modulation of serum γ-aminobutyric acid, glutamic acid, 5-HT, and higher colonic concentrations of butyrate and valerate was observed. Moreover, better stress and anxiety response and reduction of norepinephrine could lead to a promising therapy aid in the treatment of this type of disorder. Evidence level: Preliminary; mostly animal or in-vitro; no confirmed human neuropsychiatric benefit.

5.5 Fermented Food and Anti-Diabetic Activity

A laboratory investigation studied whether chungkookjang fermented specifically with B. licheniformis would differ in anti-diabetic activity from preparations fermented with other Bacillus species. Traditionally fermented soybeans (chungkookjang) may have potent anti-diabetic activity, depending on the ambient microorganisms and conditions. It was hypothesized that one of the major Bacillus species in TFC contributes to the anti-diabetic activity and could be used to standardize a highly functional fermented food. Cell-based studies were used to evaluate insulin sensitizing and insulinotropic action of chungkookjangs fermented with various Bacillus spp. and fermentation periods. Evidence level: Preliminary (cell-based); results apply to the fermented food product, not oral B. licheniformis supplementation directly.

5.6 Growth Performance and Intestinal Integrity (Veterinary Models)

For the specific application of B. licheniformis in animals and growth, a total of 55 studies were found, being one of the most relevant uses for this probiotic. Most animal publications reported positive results on growth, mass gain, and feed conversion. Weaned piglets receiving B. licheniformis had improved growth, reduced post-weaning diarrhea, and improved intestinal epithelium and gut microbiota. Supplementation with probiotics improved intestinal mucosal integrity as evidenced by higher villus heights and a higher ratio of villus heights to crypt depths (duodenum and jejunum) and higher mRNA and protein levels of occludin and ZO-1 in jejunum mucosa. The intestinal sIgA levels were elevated in the probiotic group. Furthermore, weaning piglets who were given probiotics had a better balance of the cecum microbiota, with Lactobacillus abundance increased and Clostridium_sensu_stricto_1 abundance decreased. Evidence level: Strong in veterinary/animal models; cannot be directly extrapolated to human benefit.

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6. Body Systems and Health Areas

Based on the available scientific literature, B. licheniformis has been associated with effects on the following body systems and health areas:

• Gastrointestinal system: Intestinal barrier function, microbiota modulation, diarrhea (acute and chronic), ulcerative colitis, antibiotic-associated dysbiosis, and Clostridioides difficile infection adjuvant therapy.
• Immune system: Innate and adaptive immune responses; modulation of pro- and anti-inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8, IL-10); upregulation of IgA, IgM, IgG.
• Cardiovascular/metabolic system: Reduction of dietary endotoxemia (a cardiovascular risk factor); preliminary evidence of lipid profile modulation (triglycerides) in animal studies.
• Neurological system: Preliminary animal evidence of influence on neurotransmitter levels (GABA, serotonin, norepinephrine) via the gut-brain axis.
• Liver: Hepatoprotective associations reported in animal models through reduction of dietary endotoxemia.
• Metabolic (anti-diabetic): Preliminary cell-based evidence from fermented food studies.

The intake of B. licheniformis has been related to the effects of modulation of the intestinal microbiota, antimicrobial activity, growth promotion, anti-inflammatory and immunostimulatory effects, promotion of the regulation of the lipid profile, increase of neurotransmitters, and stress reduction, among others.

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7. Dosage Forms and Dosages Reported in Studies

Dosages used in studies vary considerably by formulation type, species studied, and clinical objective. The following are reported directly from source publications:

• Human CDI adjuvant trial (Korea): B. licheniformis 250 mg three times per day was administered for five days.
• Animal (fish) supplementation study: Oreochromis mossambicus were fed dietary Bacillus licheniformis Dahb1 at 10⁷ CFU g⁻¹ for 8 weeks.
• Animal (weaned piglets) study: 108 weaned piglets were fed a basal diet supplemented with probiotic at 1000 g/t of B. licheniformis for a 14-day trial.
• Rat subhealth study: B. licheniformis was prepared into a 1 × 10⁸ CFU/mL bacterial suspension for oral administration in rats.
• 90-day rodent toxicity safety study: A 90-day subchronic oral toxicity study used 2 doses (1.1 × 10¹⁰ and 1.1 × 10¹¹ colony-forming unit [CFU]/kg body weight, respectively).

The use of Bacillus licheniformis as a probiotic has increased significantly in recent years. Published reports demonstrate that it provides multiple benefits for health. Although there are already studies in humans and it is marketed, it is mostly used in the veterinary industry still. No consensus human dosage has been established by a regulatory authority.

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8. Safety Considerations

8.1 Regulatory Classification (EFSA QPS)

This species is considered by EFSA to be suitable for the qualified presumption of safety (QPS) approach to safety assessment, which requires the identity of the strain to be established and evidence that it is not toxigenic and does not show acquired resistance to relevant antibiotics. The species B. licheniformis is included in the list of organisms for which the qualified presumption of safety (QPS) may be applied, provided that the absence of acquired antimicrobial resistance (AMR) genes and toxigenic activity are verified for the specific strain used. The QPS qualification is strain-level and conditional — it is not a blanket approval for all B. licheniformis strains without verification.

B. licheniformis can be intentionally added to foods or feeds in the European Union based on EFSA's qualification of the species as a safe biological agent, and the US Food and Drug Administration allows genetically modified strains of this species to be used for enzyme production.

8.2 Preclinical Toxicology

An in vivo toxicological safety assessment of Bacillus licheniformis Me1, a native isolate from milk, was performed. An acute toxicity study in male albino Wistar rats demonstrated no treatment-related illness or mortality. A 90-day subchronic oral toxicity study using 2 doses (1.1 × 10¹⁰ and 1.1 × 10¹¹ CFU/kg body weight, respectively) failed to show dose-dependent illness or mortality. Moreover, neither significant differences in serum biochemical and hematological analyses nor histopathological changes in organs or tissues were found when compared to the control groups. The no-observed-adverse-effect level (NOAEL) was found to be greater than 1.1 × 10¹¹ CFU/kg body weight. The in vivo micronucleus assay in mice did not reveal any signs of genotoxic effect at any of the doses tested. Furthermore, dermal and acute eye irritation tests conducted in rabbits showed no edema or erythema and ocular lesions.

8.3 Opportunistic Infections in Susceptible Individuals

One concern about the application of Bacillus in food products is that certain strains of Bacillus can cause opportunistic infections. There are several reports in the literature of human infections with B. licheniformis; however, these occurred in immunosuppressed individuals or following trauma.

Bloodstream infection cases have been documented. Bacillus licheniformis is a facultative anaerobe, gram-positive, endogenous, spore-forming bacillus. It is included in a probiotic preparation commonly used in clinical practice and is usually safe for oral administration. Two cases of bloodstream infection resulting from using B. licheniformis probiotic preparations for gastrointestinal bleeding were reported. The results suggest that B. licheniformis should be used with caution in people who are immunocompromised and suffering from severe damage to the intestinal mucosal barrier.

The presence of B. licheniformis at high levels (>5 log CFU/mL) in foods may pose a health risk to humans.

8.4 Intrinsic Antibiotic Resistance

The antibiotic susceptibilities and characteristics of 94 Bacillus licheniformis strains isolated from traditional Korean fermented soybean foods were assessed. The minimum inhibitory concentration tests revealed that all strains were susceptible to gentamicin, kanamycin, tetracycline, and vancomycin, and that antibiotic resistances were expressed in a strain-specific manner. The resistances of B. licheniformis to chloramphenicol and streptomycin were established as intrinsic characteristics.

The risk of transferable antibiotic resistance genes is a recognized concern for all probiotic bacteria. The probability for the presence of transmissible antibiotic resistance genes in the probiotic candidates should be considered in any probiotic applications. Therefore, the use of these spore-forming bacteria as dietary supplements, functional foods, and for incorporation in pharmaceutical products requires careful safety assessment using suitable models.

8.5 Confounding Due to Taxonomic Misidentification

A specific safety concern particular to B. licheniformis involves its relationship with B. paralicheniformis, a closely related species that produces bacitracin and was formally separated as a distinct species in 2015. B. paralicheniformis, classified in 2015 as a new species, is closely related to B. licheniformis within the Bacillus genus. Among the few differences between the two species, it is notable the capacity of B. paralicheniformis to produce bacitracin, fengycin and a lantipeptide. Bacitracin is considered a medically important antimicrobial and it has been previously shown to induce cross-resistance to colistin. This raises concerns that historical B. licheniformis studies and commercial products may have inadvertently involved B. paralicheniformis, with different safety and resistance profiles.

8.6 Foodborne Illness Risk

Bacillus licheniformis is a Gram-positive bacterium associated with foodborne illnesses and opportunistic infections in immunocompromised individuals, resulting in significant economic and health burdens. Its presence in foods at high concentrations, particularly in contaminated dairy or processed food products, is monitored by food safety authorities as part of broader spore-forming bacteria surveillance.

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9. Overall Evidence Assessment

Most of the 70% of published studies about the health benefits of B. licheniformis have been published from 2016 until now. The bulk of the research base is thus recent, and the field is still developing a clinical evidence base. The use of Bacillus licheniformis as a probiotic has increased significantly in recent years. Published reports demonstrate that it provides multiple benefits for health. Although there are already studies in humans and it is marketed, it is mostly used in the veterinary industry still.

Human clinical evidence is currently limited. The most relevant human data include a randomized controlled trial in CDI patients (multi-centre, Korea) and a 30-day open or controlled study on dietary endotoxemia reduction. The overwhelming majority of published evidence pertains to agricultural animal species (poultry, pigs, fish), where the evidence base is considerably more extensive. Its benefits could be extrapolated to humans in the future, but rigorous, powered randomized controlled trials in human populations remain sparse. Caution is warranted in interpreting animal or in-vitro findings as evidence of human clinical benefit.

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