Beyond Diarrhea Control: Emerging Neuroimmune and Psychobiotic Roles of Bacillus clausii

Shravani A. Mete*, Pratiksha M. Tarale, Dr. Sagar N. Ande,     

       Dr. Pramod V. Burakle

Dept. of Pharmacology, Dr. Rajendra Gode Institute Of Pharmacy, Ghatkheda, Amravati – 444602

*Correspondence: shravanimete@gmail.com

DOI: https://doi.org/10.71431/IJRPAS.2026.5306       

INTRODUCTION   

The word “Probiotics” is derived from a Greek word in which “Pro” means favor and “Bios” means life. Probiotics are nonpathogenic microorganism and are beneficial for its host as they improve microbial load in gastrointestinal tract. Probiotics are defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”[1].

The characteristics of an ideal probiotic preparation includes:[2]

·         High cell viability in adverse conditions.

·         Survivability in the intestinal environment.

·         Adhesion to intestinal epithelium.

·         Interaction with host immune system.

·         Non-pathogenic and safe.

·         Stability during processing and storage.

·         Ability to influence local metabolic activity.

Growing research supports the positive health effects of probiotics, such as promoting digestive health, strengthening the immune system, lowering blood cholesterol levels, and potentially reducing the risk of cancer. These benefits depend on the specific probiotic strains used and are influenced by different underlying biological mechanisms[3]. The GIT influences the brain function and vice versa. There are various ways through which the gut microbiota can interact with the brain. These include complex and adaptable neural circuits, as well as subtle signaling mechanism involving small molecules that act both within the gut and at distant sites such as the brain[4].

Lactic acid bacteria are the most well researched probiotics to date. However, until recently, the processes underlying the beneficial effects of other probiotics particularly those involving Bacillus species had not been thoroughly investigated. The following Bacillus strains are used in medicinal preparations for human consumption and as animal feed: B. subtilis, B. licheniformis, B. coagulans, B. toyoi (cereus), B. natto (subtilis), B. clausii, B. polyfermentans, and B. cereus[5]. In contrast to lactic acid bacteria, Bacillus when exposed to hard environmental conditions, spore forming bacteria go through a complicated developmental process that transforms the bacterial cell into a spore that can last eternally without water, nutrition, extremes in pH, temperature, UV light, or harmful chemicals.  The spores develop into vegetative cells that are capable of growth and reproduction once the right environmental conditions are restored[6].Clinically, Bacillus clausii has established a strong record of safety and effectiveness in treating antibiotic associated diarrhea and gut imbalances across both adult and pediatric populations[7].

In addition to its gastrointestinal benefits, Bacillus clausii may also have immunomodulatory and neuromodulatory effects, according to recent research. In rodent models of stress, for example, oral Bacillus clausii administration has been shown to elevate central monoamine neurotransmitter levels and reduce behavioral markers of stress, indicating impact on CNS circuits linked to mood and stress response[8]. Furthermore, probiotic administration has shown promise in preventing the development of seizures in other preclinical models by modifying oxidative stress and neurotransmitter balance, supporting neuroprotective effects mediated via gut derived signaling pathways[9]. The depicts modulation of neuroimmune signaling, cortisol regulation, vagal pathways, cytokine balance, and microbial metabolite production highlighting the unique spore-forming probiotic properties of Bacillus clausii relative to conventional psychobiotic strains represents in figure 1.

In vitro and in vivo, Bacillus clausii has been shown to interact with host immune cells and alter immunological responses, including attenuating the production of proinflammatory cytokine genes and interacting with macrophage pathways[10].When Bacillus clausii spores are administered to allergic patients, the Th2 cytokine IL-4 is downregulated while anti-inflammatory cytokines like IL-10, TGF-β, and IFN-γ are upregulated. This suggests a change toward a more balanced immune phenotype and improved regulatory signaling[11]. As seen by decreased reactive oxygen species and decreased expression of Toll like receptor pathway genes in cellular infection models, probiotic strains of Bacillus clausii also suppress proinflammatory pathways, underscoring their potential to attenuate excessive inflammatory signaling[12].Since immune mediators like cytokines can affect CNS function and neuroinflammation through both peripheral and central pathways, modulation of immune signaling is a key mechanism of gut–brain communication[13]. Pretreatment with Bacillus clausii and another Bacillus species delayed the onset of seizures and lessened their severity in a pentylenetetrazole (PTZ) induced epileptogenesis model in mice. This effect was linked to decreased oxidative stress and the normalization of genes related to tight junctions and neurotransmitter systems, suggesting protective actions at the level of both the blood–brain barrier and neuronal signaling pathways[9].

The gut microbiota plays a crucial role in the regulation of both central nervous system (CNS) function and immune responses through a complex bidirectional network known as the microbiota–gut–brain axis. This axis enables gut microbes to influence brain development, behavior, and neuroimmune interactions by producing metabolites and neurotransmitters that communicate with neural and immune cells, while alterations in microbial composition have been linked to immune related neurological disorders. Furthermore, modulation of immune cells by gut microbiota can affect CNS resident immune cells such as microglia and impact blood–brain barrier integrity, demonstrating how gut microbes are integrally involved in gut–brain and gut–immune communication in both health and disease contexts[14].

With the combination effects on gut immunity and neurological processes, Bacillus clausii is a strong candidate for more research as a psychobiotic a probiotic with the potential to affect brain function and behavior. A thorough analysis of recent preclinical and clinical research is necessary because the molecular underpinnings of its activity in neurological and immunological situations are still not fully understood.

Microbiological profile of Bacillus Clausii:

Bacillus clausii is a gram positive, aerobic, and motile rod shaped bacterium within the phylum Firmicutes, distinguished by its robust ability to form resilient endospores [15,16].These spores provide high resistance to heat, desiccation, gastric acidity and bile salts, ensuring the microbe passes through the gastrointestinal tract intact to germinate into vegetative cells in the intestines[17–19]. Clinically, Bacillus clausii is widely utilized to prevent and treat gastrointestinal disorders like antibiotic associated diarrhea[17]. The most prominent commercial preparation, Enterogermina®, contains four proprietary strains (O/C, N/R, SIN, and T) specifically selected for their chromosomal resistance to antibiotics like chloramphenicol, rifampicin, and tetracycline[20]. While numerous other products like BACIPRO® and TUFPRO® exist, studies indicate that only a few maintain the rigorous purity and viable spore counts found in Enterogermina® [17,21,22]. Beyond survival, Bacillus clausii supports the host by secreting antimicrobial compounds and bacteriocins, such as gallidermin, which inhibit pathogens like Staphylococcus aureus and Clostridioides difficile [5,20,23]. Its efficacy is further bolstered by stress response proteins and adhesion mechanisms that facilitate its transient colonization of the intestinal mucosa[24].

Mechanism of action of Bacillus clausii including gut-brain axis activity:

Bacillus clausii supports its beneficial effects in the overall wellness of the human body in various complex ways to the gut-brain axis (GBA), a bidirectional communication pathway that connects the gastrointestinal tract to the central nervous system. Being a spore producing probiotic organism, Bacillus clausii is able to resist the acidity of the stomach to colonize the gastrointestinal tract for a shorter period in the process, the bacterium affects the vagal nerve to allow the nerve to communicate with the parts of the brain concerned with the regulation of stress responses, such as the hypothalamus and the limbas[17,18]. Bacillus clausii has been shown to affect the production of neuroactive substances such as GABA, precursors of serotonin, and short chain fatty acids (SCFAs), which results from fermentation of dietary fibers, which may modulate CNS function in an indirect manner and affect synaptic plasticity and mood and behavior[19,20]. Additionally, Bacillus clausii limits peripheral and neuroinflammation, a crucial pathway in gut–brain communication, by promoting regulatory T cells and lowering pro-inflammatory cytokines (TNF-α, IL-6)[22].It also affects the hypothalamic-pituitary-adrenal (HPA) axis, reducing corticosterone responses brought on by stress and fostering emotional stability and stress resilience[21]. These mechanisms show that Bacillus clausii affects the neuroendocrine, immune, and metabolic pathways of the host. This strengthens the gut-brain axis and helps protect the brain, reduce stress, and improve overall mental health.

Bacillus clausii exerts probiotic effects by modulating gut microbiota, enhancing immune responses (↑IgA, ↑IL-10, ↓IL-6), and strengthening intestinal barrier integrity through tight-junction regulation. These actions influence the gut–brain axis via microbial metabolites and vagus nerve signaling, ultimately contributing to neurotransmitter balance and neuroimmune homeostasis. Figure 2. represents mechanistic overview of Bacillus clausii in the gut–immune–brain axis.

Neurological activity of Bacillus clausii:

A number of recent experimental studies have begun investigating the effects of Bacillus clausii on the nervous system using animal models that have already been established to be useful in examining the gut-brain axis. One of the most pertinent studies performed oral gavage of Bacillus clausii spores to mice at a daily dose of 1010 CFU per animal during acute and subacute restraint stress experimental paradigms which are commonly accepted as models of psychological stress and how they affect behaviour and neurobiological function. Using behavioural tests (Elevated Plus Maze, Light Dark Box, and Open Field), this study quantifies the amount spent in the open arms of the Elevated Plus Maze, in the light compartment of the Light Dark Box, and in the centre of the Open Field test in Bacillus clausii administered restraint stressed mice versus restraint stress treated mice and indicates an improvement in stress management behaviour and a significant reduction in anxiety like behaviours in Bacillus clausii treated restraint stressed mice, as well as lower concentrations of serum cortisol and ACTH. This indicates that Bacillus clausii has an attenuating effect on the neuroendocrine stress response[8].

Aside from the stress models, evidence also points to the neuroprotective effect of Bacillus clausii in pathological conditions like epileptogenesis. Using the pentylenetetrazole (PTZ)-induced seizure model, the pretreatment with Bacillus clausii together with other probiotic species reduced the incidence of seizures, the intensity and duration of seizures, as well as the mortality rate compared to the control group. These effects were related to the reduction of oxidative stress in the brain, the restoration of blood–brain barrier associated gene expression of tight junction proteins such as ZO-1, occludin, and claudin, and the normalization of the expression of gene proteins of glutamatergic and GABA receptor functions inhibited by PTZ. These results indicate the neuroprotective function of Bacillus clausii in modulating the excitability of the brain via the preservation of the blood–brain barrier function[9].

Mechanistically, these neurological impacts of Bacillus clausii are postulated to be attributed to its action on the microbiota-gut-brain axis, revolving around the interface between microbial products, the immune system, and central neurotransmitter mechanisms. Interaction with the microbiota-gut-brain axis involves a multitude of communication processes mediated via the vagal nerve, immune system modifications, and neuromodulatory mechanisms, allowing the host microbiota to impact the central nervous system. Elevated production and/or modulation of central monoamines and reduced stress response mechanisms are thought to be mediators of its psychobiotic properties, improving stress and excitability related neurological disorders[23].

Immunological activity of Bacillus clausii:

In vitro immunological models of murine macrophages and intestinal epithelial cells have revealed the capacity of Bacillus clausii to trigger the activation of immune responses and manipulate the mechanism of inflammation. The vegetative form of Bacillus clausii has been identified to stimulate nitric oxide synthase II (NOS II) activity, produce interferon-gamma (IFN-γ), and increase CD4+ T-cell proliferation in murine cells, which suggests the activation of both innate and adaptive components of the immune system[5].

Studies conducted using human intestinal epithelial cell systems also demonstrate the anti-inflammatory potential of Bacillus clausii. Efficacies were observed with vegetative forms of Bacillus clausii strain CSI08, which showed strong suppression of lipopolysaccharide (LPS) and Poly I:C-induced expression of pro-inflammatory genes such as IL-8, TNF-α, IL-17C, and CXCL10 in HT-29 intestinal epithelial cells, which were attributed to suppressed activation of the NF-κB signaling pathway, a central regulator of inflammation. In addition, Bacillus clausii CSI08-activated immune responses of U937-derived macrophages, which produced a strong signature pattern of released cytokines and chemokines, including TNF-α, IL-1β, and IL-18, and regulatory factors such as IL-10 and GM-CSF, suggesting immune regulation of macrophage responses and innate immune pathways in these immune effector cells[10].The effects of administering Bacillus clausii in disease models related to the immune system have been studied using experimental animal models (i.e., murine models). In a mouse model of allergic airway inflammation (i.e., asthma induced by ovalbumin), treatment with Bacillus clausii resulted in a reduction of eosinophil, neutrophil and lymphocyte infiltration into lung tissues, an improvement in airway epithelial thickness and decreased levels of Th2 associated cytokines (i.e., IL-4 and IL-5). Therefore, it appears that Bacillus clausii may modulate the immune response to allergies by promoting an anti-inflammatory environment and a healthy immune status in lung tissue[6].

Clinical evidence is also supportive of the immunomodulatory effects on the human immune system. In a pilot clinical study on allergic patients with allergic rhinitis, oral ingestion of the spores of Bacillus clausii for four weeks showed a significant change in the pattern of cytokines present in the nasal lavage fluid: the level of the Th2 cytokine IL-4 was significantly lowered, while anti-inflammatory and Th1-type cytokines like IL-10, transforming growth factor beta (TGF-β), and IFN-γ were elevated, suggesting a relative balance in the expression of Th1 and Th2 immunity. Such modulation in the pattern of the immune response in allergic patients suggests the potential modulatory effect of Bacillus clausii on the mucosal and systemic immune response[11].

Bacillus clausii is known to stimulate nitric oxide synthase II (NOS II) and the production of interferon gamma (IFN-γ), indicating the stimulation of macrophage function and Th1 immune responses. It is also known to stimulate the proliferation of CD4+T cells. This suggests the action of the adaptive immune component[25]. Lipoteichoic acids of Bacillus clausii strains have been found to induce the production of nitric oxide in macrophages, which is a means of pathogen killing and activation of the immune system[6]. Studies conducted with Bacillus  clausii strains (O/C, N/R, SIN, T) in mice and humans revealed a boosted systemic IgG response and increased secretion of sIgA by oral treatment in healthy individuals, emphasizing an augmented humoral immune system function. Bacillus clausii strains increased the expression of innate AMPs (like HBD-2 and LL-37), as well as proinflammatory cytokine secretion by enterocytes in rotavirus infected models, which implies an augmentation of natural gut immune mechanisms[12,25].

Role of Bacillus clausii:

New preclinical findings also show that Bacillus clausii has been able to affect the activity of the hypothalamic-pituitary-adrenal (HPA) axis, which is a key part of the gut-brain axis and plays a vital role in controlling stress responses by secreting corticotropin releasing factors, adrenocorticotropic hormone (ACTH), and glucocorticoid (cortisol/corticosterone) hormones. In restraint stressed mice, oral treatment of mice with spores of Bacillus clausii was found to decrease serum ACTH and cortisol concentrations as indicators of HPA axis activation compared to controls. Improvements in behavioral symptoms of stress in affected mice administered Bacillus clausii resulted in a reduction of HPA axis markers together with elevated levels of monoaminergic neurotransmitters in the brain as well as higher expression of dopamine receptor and synaptophysin in the hippocampus and prefrontal cortical regions[8].These observations indicate that Bacillus clausii has a potential ability to impact stress response by decreasing HPA axis hyperactivation, which may mechanistically underlie improvements in gut barrier function, microbiota derived metabolites, immune regulation, and vagal nerve stimulation, which have been established as pathways in microbiota-gut-brain interaction[26]. Moreover, other research within broader probiotic studies has found that the normalization of gut microbiota composition is effective in alleviating hyperactivity of the HPA axis, caused by stress, and, consequently, decreasing glucocorticoids secretion[27].

Bacillus clausii, being a spore forming probiotic, has gained increasing interest in the antioxidant and cytoprotective properties that can be associated with neuroprotection through various pathways involved in the reduction of oxidative stress, modulation of inflammatory pathways, and preservation of cellular integrity. While direct studies of Bacillus clausii in CNS oxidative stress models are scanty, evidence from preclinical and in vitro research accounts for its antioxidative potential, which may have an indirect influence on neuroprotective outcomes as applied via gut–brain axis interactions. One line of evidence comes from models of epileptogenesis where combined probiotic administration including Bacillus clausii resulted in a significant attenuation of oxidative stress in the brain and was associated with reduced seizure severity, delay of seizure onset, and protection of neuronal integrity and blood–brain barrier integrity. In PTZ-kindled mice, pretreatment with probiotics lowered oxidative stress markers and restored the expression of genes related to glutamatergic and GABAergic neurotransmission, evoking impressive neuroprotective effects with probable mediation through antioxidative pathways[9]. Novel strain studies constitute further evidence for the antioxidant capacity of Bacillus clausii. The Bacillus clausii CSI08 strain exerted marked antioxidant activity in vitro and in vivo, where pretreatment of epithelial cells with the probiotic partially recovered the viability of cells after hydrogen peroxide induced oxidative injury, while C. elegans nematodes fed with Bacillus clausii exhibited significantly higher survival under acute oxidative stress compared with controls, indicating dose dependent protection against reactive oxygen species(ROS)[10]. Endotoxemia and acute renal injury models showed the effect of Bacillus clausii, which decreased the oxidative stress markers of lipid peroxidation and myeloperoxidase (MPO) activity, and the inflammatory cytokines (IL-6 and TNF-α), indicating the potential of Bacillus clausii as an antioxidant and anti-inflammatory agent protecting the tissues systemically[28].

Although studies directly focusing on Bacillus clausii’s synthesis of neurotransmitters are only now beginning, evidence supports its impact upon the central neurotransmitter pathways via the guts-brain axis, specifically concerning dopamine, serotonin (5-HT), and gamma aminobutyric acid (GABA) levels, all integral in modulating mood, stress, and cognition.

The oral coadministration of Bacillus clausii and Lactobacillus fermentum significantly elevated levels of brain monoamines such as serotonin and dopamine and boosted expression levels of dopamine D1 and D2 receptors in the hippocampus and prefrontal cortical regions of restraint-stressed mice compared to controls. The findings indicate Bacillus clausii has potential roles and actions in modulating levels and functions of fundamental transmitters and factors participating in stress and mood homeostasis through possible vegal nerve signaling and interaction mechanisms. The positive results were also associated with alleviated behavioral symptoms and decreased stress hormones (ACTH and cortisol) regarding its pronounced neuromodulatory properties[8].Although not all microbes produce these compounds, an implied inference from studies of the gut and brain axis is that gut microbes have an ability to modulate the production and levels of the aforementioned compounds such as serotonin, dopamine, and GABA. Another way by which gut microbes can modulate host neurotransmitter metabolism is by altering the synthesis of their precursors (like serotonin from tryptophan) and/or influencing the activities of the enterochromaffin cells of the gut, which are responsible for the secretion of approximately 90% of the body’s serotonin[29]. Evidence from neurological models, such as epilepsy studies using Bacillus clausii containing probiotic interventions, suggests that pretreatment with probiotics restores expression of GABAergic and glutamatergic genes, indicating a balancing effect on inhibitory neurotransmission associated with GABA and excitatory signaling associated with glutamate. Such an action is necessary for maintaining neural network stability and preventing excitotoxicity. The PTZ epileptogenesis study supports this idea, although Bacillus clausii itself was part of a probiotic mix in this model, pointing out its contribution to the potential GABAergic modulation and neuroprotective signaling in the brain by Bacillus safensis + B. clausii[9].

Often, the use of broad spectrum antibiotics gives rise to intestinal dysbiosis, resulting in the disruption of the microbiota-gut-brain axis, hence neuropsychic side effects, such as anxiety, depression, among others[8]. Bacillus clausii, or more specifically Alkalihalobacillus clausii, acts as an extremely useful biotherapeutic tool in countering this problem in multiple different ways. First, it resists the conditions in the gastric environment, colonizes the gastrointestinal tract, and works to protect the intestinal epithelial lining, preventing the passage of pro-inflammatory cytokines, lipopolysaccharides, or LPS, among other factors, that trigger neuroinflammations[30,31].

Additionally, Bacillus clausii significantly lowers the release of stress hormones like corticosterone and ACTH by modulating the hypothalamic-pituitary-adrenal (HPA) axis[8]. It has also been recently found to function as a psychobiotic in upregulating the expression of the brain-derived neurotrophic factor (BDNF) as well as the levels of crucial monoamine neurotransmitters serotonin, dopamine, and norepinephrine in the hippocampus and prefrontal cortex[32]. By suppressing the NLRP3 inflammasome pathway and restoring microbial diversity, B. clausii effectively reverses the behavioral deficits and memory impairments typically associated with antibiotic induced microbial depletion[8,33].

Bacillus clausii is a widely used probiotic, especially appreciated for its capacity to preserve the integrity of the intestinal epithelium barrier. Gut cell line permeability is the role of the Tight Junction (TJ) proteins, which are referred to as the intestine's gatekeepers or TJ proteins. When the integrity of these junctions is impaired, leading to a leaky gut syndrome, pathogens and toxins can easily pass through the intestinal epithelial wall and enter the bloodstream. Bacillus clausii is recognized as a reestablishing factor because it increases the expression of fundamental proteins involved in the TJ complex, including occludin, Zonula occludens-1 (ZO-1), and members of the claudin family. Available studies suggest this particular probiotic bacteria exert a protective role in toxicity (such as from Clostridium difficile) because it usually breaks down these proteins, leading to reduced permeability across the gut wall[34].

Preclinical research has confirmed that Bacillus clausii has the capacity to reduce epithelial damage along with increases in epithelial permeability under pathological conditions. Oral supplementation with Bacillus clausii has been found to reduce intestinal epithelial permeability along with improving histopathological parameters in a mouse model of intestinal mucositis induced by 5-fluorouracil (5-FU). 5-FU-induced intestinal mucositis comprises inflammation, villous atrophy, along with increases in epithelial permeability, with incorrect tight junction behavior of epithelial cells being implicated as a result of increases in proinflammatory markers like TNF-α, IL-1β, along with oxidative stress. These parameters disrupt tight junction integrity[35]. Another line of work, which provided further proof for the enhancing role of Bacillus clausii in epithelial barrier function, was the study on the role of the bacterium in the damage to the epithelial cell layer induced in an in vitro model by viruses. In human enterocyte monolayers where rotavirus infection induced an increased transepithelial electrical resistance (TEER) desirable for the integrity of the epithelial cell junction Bacillus clausii strains or metabolites inhibited the virus induced increased TEER and upregulated the epithelial cell production of the mucus glycoprotein MUC5AC and the tight junction associated proteins occludin and ZO-1. These are indicative of a strengthened epithelial barrier function[12].

Alternative model for Bacillus clausii preclinical testing

As the field of preclinical assessment for Bacillus clausii and other probiotics grows, so has the diversity of animal model approaches. The focus has shifted beyond the classical rodent model to include animal model approaches that have a greater similarity with human physiology. Animal model approaches that have been typically utilized include mice or rat model approaches that have screened the mechanisms by which probiotics function. In many instances, the use of a germ-free animal model or the use of an antibiotic treated animal model has allowed for a clearer dissection of the impact that microbiota can have on the central nervous system or the gastrointestinal tract[36,37]. Apart from vertebrate models, simple organism models Caenorhabditis elegans, Drosophila melanogaster, or Danio rerio (zebrafish) have been adopted for examining fundamental probiotic interactions with their tissues, longevity, immune responses, and metabolism at lower costs in large quantities, thereby gaining an insight into universal host microbe interaction processes applicable for probiotic screening purposes[38].

One of the best accepted alternatives for probiotic evaluation in vivo is Caenorhabditis elegans. Being a bacterivorous nematode, C. elegans provides for the direct administration of Bacillus clausii by oral means, thus enabling the assessment of the survival of bacteria, spore germination, and host microbe interaction within the intestinal lumen[39]. Innate immune pathways conserved and known targets for the modulation by probiotics include p38 MAPK (PMK-1), DAF-2/DAF-16 insulin signaling, and TGF-β pathways, which are highly pertinent to the immunoprotective effect attributed to Bacillus clausii[40]. Additionally, C. elegans shows dose response effects for stress resistance, lifespan, and neurobehavioral parameters, making it potentially responsive to probiotic therapy. Previous studies have successfully demonstrated that gut microbiota metabolite cues modulated neuronal function and behavior; thus, it shows relevance to early stage screening for the gut-brain axis. On that basis, it acts as a predictive platform for preclinical screening, owing to its suitability as it connects in vitro work to studies involving rodents[41].

Drosophila melanogaster is another nonvertebrate potent alternative model available for research on probiotics. The gut of this fly exerts functional similarities to the mammalian intestine, including epithelial renewal, innate immune signaling (IMD and JAK-STAT pathways), and microbial sensing[42]. Probiotic bacteria have also been reported to control gut homeostasis, the immune response, and lifespan in the Drosophila model, which makes the model suitable to determine the host protective effects of the Bacillus clausii[43].

Zebrafish, or Danio rerio, have emerged as the ideal alternative model to the mammalian model to experimentally prove the probiotic potential of probiotic candidates like Bacillus clausii, given their genetic, physiological, and immunological make up, which is akin to higher vertebrates. In addition to their innate and adaptive immunity, the enteric microbial flora of the Zebrafish also allows the researcher to understand the interaction of microbes with their host while assessing the probiotic effects of the probiotic compound of interest. The transparency of the Zebrafish larva allows the researcher to view the intestinal colonization of the probiotic compound of interest through noninvasive procedures[44].

In related studies on probiotics using zebrafish as a model organism, several studies proved that exogenous beneficial bacteria could affect zebrafish gut health and even influence immune related gene expressions. It has been proved that probiotics could affect the gut microbiota of zebrafish, mucosal membrane functions, and inflammation related markers in zebrafish intestinal disease models[45].To date, while our own studies have focused on the Lactobacillus and Bifidobacterium species, these mechanisms also appear to apply to spore forming species such as Bacillus, and indeed there have been studies conducted in the context of a model organism, such as a zebrafish model for Bacillus species and these species in aquatic livestock[46]. Significantly for the use of Bacillus clausii, one of the more interesting aspects of the study, conducted to determine the ability of human gut flora to colonize the zebrafish larva gut, highlighted the presence of spore forming bacteria within the gut of the zebrafish larva. The relevance of this finding for the ability of Bacillus clausii to colonize the gut of the human host cannot be underestimated, therefore[47] Zebrafish offer a platform for the quantification of immunomodulatory effects of probiotics. Administration of probiotics in zebrafish has been associated with changes in immune gene expression and improved resistance of the host against pathogenic challenge a proof of how the host immune system responds to beneficial microbes[48].

DISCUSSION:

Unlike traditional probiotics like Lactobacillus and Bifidobacterium, which are delicate, non spore forming bacteria, Bacillus clausii is a robust, spore forming organism designed to survive extreme heat and stomach acid without refrigeration[15,49,50]. Its unique biology allows it to remain stable during antibiotic treatment a time when other probiotics are often killed off making it a primary clinical choice for preventing antibiotic-associated diarrhea and maintaining gut homeostasis[7]. Beyond simple digestion, Bacillus clausii secretes specialized antimicrobial peptides like clausii and has shown a remarkable ability to modulate the gut-brain axis in fact, preclinical studies suggest it may even outperform certain Lactobacillus strains in suppressing stress hormones like cortisol and boosting neurotransmitters such as serotonin and dopamine[6,23]. While animal models use high doses to see these neuroimmune changes, human therapeutic benefits are typically seen at doses of 109CFU/day, with symptoms and inflammatory markers often improving within 3 to 7 days as the spores rapidly germinate in the small intestine[15,17,51,52].

CONCLUSION:

Bacillus clausii has several clinical advantages over other non spore forming probiotics in that it has exceptional survivability in the gastrointestinal tract. Its spore forming ability gives it high tolerance to acid, bile, and heat, hence a longer shelf life and high survivability in the gastrointestinal tract[15]. Moreover, its compatibility in co administration with antibiotics gives it the advantage of effectively preventing and treating antibiotic associated and acute infectious diarrhea, a feature that has rarely been achieved by strains such as Lactobacillus and Bifidobacterium[6,53]. In addition to its effects on the digestive system, Bacillus clausii has immunomodulatory effects that balance the cytokines and improve the integrity of the epithelium[12]. It has preclinical potential to support the health of the neuroimmune system by normalizing stress hormones and neurotransmitters via the gut-brain axis[8].

However, there remain several limitations: the routine use of Bacillus clausii in combination with multiple strains prevents the precise determination of species specific effects, which is crucial to understand the mechanisms of action[8,9,52]. Moreover, despite the encouraging results from pre-clinical CNS studies, there is a notable absence of good quality RCTs conducted on humans to explore the direct neuroimmune effects of probiotics, creating a chasm between the results obtained from animal studies and the evidence based practice of medicine[8,15]. Last but not least, the effectiveness of probiotics is strain and species dependent  the absence of deep sequencing or functional metagenomics from human studies on Bacillus clausii means that the effects of Bacillus clausii on the microbial ecosystems remain to be fully explored[54].

The modulation of brain derived neurotrophic factors (BDNF) through short chain fatty acid (SCFA) mediated and vagal pathways is another area which can be explored in the future for Bacillus clausii. Though there is no direct evidence available currently regarding the association between Bacillus clausii and the modulation of BDNF, the knowledge based understanding gained through the study of the gut-brain axis and the mechanisms through which SCFAs potentiate the modulation of BDNF and other neurotrophic factors can provide the much needed platform to explore this area. Moreover, the meta analytical results have also established the fact that probiotic supplementation can increase the levels of BDNF in the circulation of human subjects. Considering the robustness and the ability of Bacillus clausii to survive in the gastrointestinal tract and to modulate the gut microbiota, it is highly recommended to explore the potential of this probiotic to modulate SCFAs and the vagal pathways to regulate BDNF-mediated neuroplasticity in neuroimmune and stress related disorders[23,55,56].

The new strain of the novel species Bacillus clausii CSI08 (Munispore(R)) has been previously characterized in vitro and in vivo, showing its remarkable immunomodulatory, antioxidant, and cytoprotective activities, such as its ability to strongly adhere to mucus producing cells of the intestine, suppress pro-inflammatory responses, and enhance the capacity to counteract oxidative stress, which could suggest its potential use in the regulation of neuroimmune homeostasis[10]. Considering that there are established links between chronic inflammation, oxidative stress, and neuropsychiatric disorders such as anxiety disorders, these functional characteristics offer further rationale for exploring CSI08 in clinical trials of anxiety and stress related disorders. There is a significant body of evidence regarding probiotics and their effect on inflammation and oxidative stress and how it may impact the gut-brain axis, including cytokines, vagal afferent activity, and stress hormones in anxiety disorders[4]. Although little evidence exists of its effect on anxiety and central neurotransmitters in humans, its immunomodulatory, antioxidant, and barrier enhancing properties, combined with its systemic efficacy, make CSI08 an interesting candidate for future randomized, placebo controlled trials targeting anxiety and stress responses and their related neuroimmune endpoints. Future studies should include assessment of dose effect relationships using standardized psychological or physiological anxiety measures such as anxiety scales and HPA axis and neuroinflammation endpoints.

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