Probiotics are live microbes, which when administered in adequate amounts confer a health benefit to the host [1]. Bacillus species which are spore forming bacteria have been used as probiotics for the last five decades. The advantage of spore forming probiotics over nonspore formers such as Lactobacillus spp. is that they are heat stable and can be stored at room temperature without any loss of viability. Spore forming bacteria are also resistant to acidic conditions of the stomach (low pH) and hence can survive the transit to reach the intestine [2,3]. Experimental data suggest that both Bacillus clausii (B. clausii) spores and cells can adhere to the bowel wall and colonize the mucosa [4]. As B. clausii is extremely stable to acidic conditions, the entire dose of ingested bacteria reach the small intestine intact. Three principal reviews have covered the field of Bacillus spp. as probiotics [5-7]. The most extensively studies Bacillus probiotics are B. subtilis, B. clausii, B. cereus, B. coagulans and B. licheniformis [8].

Clinical studies with Bacillus clausii

B. clausii is a probiotic widely used in Italy since the1960s for viral diarrhoea in children and for antibiotic related side-effects [9]. We had earlier conducted a study to evaluate the anti-diarrhoeal activity of our B. clausii strain UBBC-07 in patients suffering from acute diarrhoea [10].

Diarrhoea is one of the principal causes of morbidity and mortality among adults and children globally [11]. In industrialised countries, the incidence of acute diarrhoea was observed to be an average of 0.5 to 2 episodes per year per person. The incidence is higher in developing and underdeveloped countries than in industrialised countries [12]. While viruses are the major causative agent in children, both bacterial and viral pathogens are implicated in adults [13]. Other causes for acute diarrhoea include irritable bowel syndrome, intake of some types of drugs, and ileal bile salt malabsorption. The treatment of acute diarrhoea mainly involves the prevention of dehydration, shortening the length of the illness, and reducing the period that a person is infectious [14]. Treatment with oral rehydration solutions (ORS) have significantly reduced the incidence of mortality and morbidity caused by diarrhoea, however, ORS neither shortens the duration of diarrhoea nor stool consistency. Other treatment options include antibiotics, gut motility suppressing agents (e.g., loperamide, codeine), and probiotics [13].

Normal gut microbiota plays an important role in the protection of the host against gastrointestinal tract diseases [15,16]. During acute diarrhoea, the normal gastrointestinal microbiota is found to undergo radical changes that facilitate the overgrowth of unwanted microorganisms, including pathogenic strains. Several authors have reported that the administration of probiotics can restore the gut microbiota and also control the severity of diarrhoea [17-19].

In our study, we evaluated the anti-diarrhoeal activity of B. clausii strain UBBC 07 in patients suffering from acute diarrhoea. A total of 27 patients (average age of 35.44 ± 8.08 years) with acute diarrhoea were included in a prospective, Phase II clinical study. The criteria included for all subjects were ≥ 3 loose stool motions within 24 hours and for more than 7 days. All patients were assigned to receive one capsule of B.clausii strain UBBC-07 (containing 2 × 10⁹ cfu) two times a day for a period of 10 days. Efficacy assessment of duration of diarrhoea, frequency of defecation, abdominal pain and stool consistency were tested on days 1, 3, 6 and 10. Safety was evaluated by assessing the incidence and type of adverse effects such as increase in blood pressure and pulse rate, physical examination and clinical laboratory tests, i.e., complete blood count, serum glutamic pyruvic transaminase, serum creatinine, and stool examination and microscopy, on day 1 and day 10. The results of this study clearly showed that the mean duration of diarrhoea decreased from 34.81 ± 4.69 to 9.26 ± 3.05 (P<0.0001) min per day, the frequency of defecation also decreased from 6.96 ± 1.05 to 1.78 ± 0.50 (P<0.0001) times per day, abdominal pain decreased from 3.22 ± 0.93 (severe) to 0.74 ± 0.71 (absent) (P<0.0001), and stool consistency improved from 3.93 ± 0.38 (watery) to 1.22 ± 0.42 (soft) (P<0.0001). No significant change in safety parameters were observed during treatment. Our study indicated that B. clausii strain UBBC-07 was effective in alleviating the symptoms of diarrhoea without causing any adverse effects.

Studies conducted by other researchers reaffirm the efficacy and safety of B. clausii in reducing the incidence of diarrhoea. We performed and assessed a comprehensive range of relevant literature from PubMed, Medline and other online sources including Google Scholar.

For the PubMed search, the following search terms were initially used (MeSH/All fields) "bacillus" AND "clausii" AND ("diarrhoea OR "diarrhea"). Subsequently, filters like clinical trials and human species were used. The search was restricted only to diarrhoea and other conditions like upper respiratory tract infections, small intestinal bacterial overgrowth (SIBO) wherein B. clausii was used as treatment, were excluded.

B. clausii therapy was found to reduce side-effects of anti- Helicobacter pylori treatment in a randomized, double-blind, placebo controlled trial [20]. The effect of B. clausii on incidence (primary variable) and severity of antibiotic-associated side-effects during anti-H. pylori therapy was studied. One hundred and twenty H. pylori-positive patients were randomly screened to receive: (i) a standard 7 days triple therapy with rabeprazole 20 mg b.d., clarithromycin 500 mg b.d. and amoxicillin 1 g b.d. and B. clausii t.d.s. (each preparation containing 2 x 10⁹cfu) for 14 days starting from the first day of treatment. (ii) The same 7 days triple therapy and placebo t.d.s. for 14 days starting from the first day of treatment. Side-effects were assessed using a validated questionnaire and were recorded for 4 weeks from the start of therapy. It was found that the incidences of nausea, diarrhoea and epigastric pain in patients treated with B. clausii were significantly lower than in placebo group.

B. clausii bacteriotherapy was hence found to reduce the incidence of the most common side-effects related to anti-H. pylori antibiotic therapy compared with placebo, i.e., nausea, diarrhoea and epigastric pain.

In a randomized, double-blind, placebo-controlled trial by Maugo, B. M. [21] to determine the effectiveness of B. clausii in shortening acute diarrhoeal illness in under-five population with severe dehydration in Kenya, it was observed that when B. clausii (2 x 10⁹CFUs/5 ml vial or placebo, twice daily, for 5 days) was administered to 90 children (age range: 6 months to 5 years) with acute diarrhoea and severe dehydration, the mean duration of diarrhoea in the B. clausii group was shorter (77.59 ± 34.10 hours) than the placebo group (86.74 ± 40.16 hours).There was a significant decrease in the mean number of diarrhoeal motions on day 3 (B. clausii group 2.74 ± 1.81 motions vs. Placebo group 3.80 ± 2.7 motions) and day 4 (B. clausii group 1.45 ± 1.13 motions vs. Placebo group 2.35 ± 2.19 motions). There was no significant difference in the duration of stay in hospital between the groups. The conclusion drawn from the study was that in children admitted with acute diarrhoea and severe dehydration, there was a significant decrease in the number of stools as seen on day 3 and 4 of treatment. Although, there was no significant difference in reduction of the duration of diarrhoea and duration of hospital stay in the two groups.

A clinical study on efficacy of B. clausii in preventing antibioticassociated diarrhoea among Filipino infants and children administered B. clausii indicated a lower incidence of diarrhoea in the B. clausii treated group [22]. The multicenter randomised, open-label, clinical trial, compared two parallel groups of infants and children (323) allocated to either the B. clausii probiotic strain or a “no-intervention” control while on antibiotic therapy. The treatment group received a twice daily dose of 1 vial of 2 x 109 of B. clausii spores within 24 hours of antibiotic initiation and were continued until the last day of antibiotic therapy. The “No Intervention group” was only observed for events. The duration of treatment was 7 to 21 days. Participants were evaluated daily from day 0 of antibiotic therapy until day 45 post-intervention. Although a prevalence of 31% of children presenting antibiotic– associated diarrhoea was expected in this study, only a total of 10 diarrhoea events (3%) were observed in the included population which was low to reach any conclusion. Seven (7) cases were reported in the “No intervention” group and three (3) cases in the B. clausii group but the difference did not reach statistical significance (p=0.22). The trend, though not statistically different was towards that of B. clausii reducing the incidence of antibiotic associated diarrhoea.

In a separate study, B. clausii as an adjuvant therapy in acute childhood diarrhoea was studied in a multicentric trial in India [23,24]. The prospective study included children suffering from acute diarrhoea, at a private tertiary care hospital India. Children (131) were divided into 2 groups: Group 1 included children on oral rehydration therapy (ORT)+Zinc+B. clausii (2 x 10⁹cfu) and Group 2 comprised of children on ORT+Zinc.

The duration of diarrhoea in B. clausii treated group (Group 1) was 22.64 hours as compared to the ORT and Zinc treated group (Group 2) which was 47.05 hours (p<0.01).

The frequency of diarrhoea showed improvement within 24 and 60 hours in Group 1 and Group 2 respectively (p<0.01).Similarly, the mean duration of hospital stay was 2.78 days in Group 1 and 4.30 days in Group 2. The treatment cost was also significantly reduced. The conclusion from the study was that B. clausii reduced the duration; frequency and hospital stay of diarrhoea thereby reducing the treatment and social costs.

Results of all the studies have been summarized in (Table 1).

Table 1: Summary of clinical studies on effect of Bacillus clausii treatment on diarrhoea.

The clinical benefits observed with probiotic use are mainly attributed to the antimicrobial substances produced by probiotic strains and to their immunomodulatory effects.

B. clausii can inhibit the growth of pathogens in the gastrointestinal tract via three distinct mechanisms [21]: colonization of free ecological niches, which are no longer available for the growth of other microorganisms; competition for epithelial cell adhesion, which is particularly relevant for spores in the initial or intermediate germination phase; production of antibiotics and/or enzymes secreted into the intestinal environment, especially peptide antibiotics, which are mainly active on Gram-positive bacteria, but also enzymes that exhibit lytic activity against Pseudomonas aeruginosa. Urdaci Maria et al. [25] found B. clausii to possess antimicrobial and immunomodulatory activities. B. clausii strains were found to release antimicrobial substances in the medium. The release of the antimicrobial substances was observed during stationary growth phase and coincided with sporulation. These substances were active against Gram-positive bacteria, in particular against Staphylococcus aureus, Enterococcus faecium, and Clostridium difficile. The antimicrobial activity was resistant to subtilisin, proteinase K, and chymotrypsin treatment, whereas it was sensitive to pronase treatment. The evaluation of the immunomodulatory properties of B. clausii strains was performed in vitro on Swiss and C57 Bl/6j murine cells. The authors [25] demonstrated that B. clausii strains, in their vegetative forms, were able to induce NOS II synthetase activity, IFN-γ production, and CD4⁺T-cell proliferation.

The conclusions one can draw from the aforementioned studies is that B. clausii is an effective probiotic and the probiotic of choice in the treatment of diarrhoea, prevention of antibiotic associated diarrhoea and in the prevention of side effects associated with Helicobacter pylori treatment. It has the added advantage of being a spore forming probiotic making it a very resilient, stable probiotic which can be stored at room temperature.

The authors belong to Unique Biotech Ltd., India which is involved in the manufacturing of probiotics. In this paper, they have merely presented a review of published literature.