An Open Labeled, Placebo Controlled Trial to Evaluate the Role of Probiotics- Bacillus subtilis HU58 and Bacillus coagulans SC208 0n Antibiotic Associated Diarrhoea In Humans

Objective: To evaluate the activity and tolerability of a probiotic mix of Bacillus subtilis HU58 plus Bacillus coagulans SC208 and Bacillus coagulans SC208 alone compared with placebo in a group of 75 patients suffering from Antibiotic Associated Diarrhea (AAD). Materials and Methods: An open labeled placebo-controlled trial was conducted for


Introduction
Antibiotics are one of the most over-prescribed drugs in modern medicine. The side effects of antibiotics are nausea, indigestion, vomiting, diarrhea, bloating and feeling of fullness, loss of appetite, stomach cramping and pain. Among these, diarrhea is the most common one and can range from mild to watery stools.
' Antibiotic-associated diarrhea is defined as otherwise unexplained diarrhea that occurs in association with the administration of antibiotics. The frequency and severity of AAD varies with different antibacterial agents and occurs in about 5-25% in patients and can occur with oral as well as parental antibiotics like ampicillin, amoxicillin, fluoroquinolones, cephalosporins, clarithromycin, erythromycin, and tetracycline [1]. About 9.6% of the hospitalized patients consuming antibiotics suffered from AAD [2]. Incidence of diarrhea in pediatrics in outpatient wards was found to be 6-75%.
In adults, the AAD occurs in 7-33 patients out of 100 patients taking antibiotics and even after the treatment for diarrhea, a relapse is seen in almost 20% of cases [1]. Probiotics reduce and shorten the duration of AAD which is shown in many studies [3][4][5][6]. AAD occurs due to the antimicrobial action which kills the normal gut microflora and enables the pathogenic organisms that induce diarrhea to overgrow.
One of the most notorious organisms which cause AAD in around 10-25% of cases is the spore forming anaerobe Clostridium difficile. Other species associated but to a lesser extent are C. perfringens, S. aureus, Candida spp, Klebsiella spp, and Salmonella spp [1]. Probiotics are often prescribed with the belief that the intake of healthy bacteria would correct the dysbiosis and recolonize the gut and thereby reduce AAD. There have been a wide range of studies with different strains of organisms and with patchy results [7]. Some of the commonly used organisms include Bacillus spp., Bifidobacterium spp., Clostridium butyricum, Lactobacilli spp., Lactococcus spp., Leuconostoccremoris, Saccharomyces spp., Streptococcus spp., Enterococcus spp., Pediococcus spp., Escherichia coli alone or in combination. In a probiotic preparation these organisms may be used as a single species or a mixture of multi species with variable spore counts [8]. The present study was undertaken to evaluate the efficacy of two probiotic preparations in patients with AAD.

Study Design
We conducted an open labeled, placebo-controlled trial in patients with AAD to compare the efficacy, safety and tolerability of the probiotic combination of Bacillus subtilis HU58 + Bacillus coagulans SC208, Bacillus coagulans SC208 alone and placebo. The study was conducted at Konkan Hospital, Mumbai with the approval of an Independent Ethics Committee and registered at Clinical Trial Registry of India (CTRI) CTRI/2018/06/014439.

Subjects
The study was conducted at Department of Medicine of Kokan Hospital, Mumbai, India. Eighty patients (aged 18-65 years) who were on antibiotics and suffered from AAD were screened and selected after taking an informed consent. Out of 80 screened patients, 75 patients completed the study. All patients were randomized in group A, group B and group C as shown in Table 1. Values are expressed in Mean (SD); Group A: Bacillus subtilis HU58 + Bacillus coagulans SC208, group B: Bacillus coagulans SC208 and group C: Placebo. SD: Standard Deviation.

The Study Procedure and Assessments
After proper history, examination and investigations seventyfive patients were selected as per inclusion criteria in the protocol. was used. One capsule of either Bacillus subtilis HU58 + Bacillus coagulans SC208 in combination or Bacillus coagulans SC208 alone or placebo was prescribed to the patients daily for 7 days. The stool frequency was assessed with Bristol Stool Scale ( Figure 1).
BSC is a common tool and has been used in extensively to study the consistency of stool [9,10]. As per the Bristol stool scales, 1-2 is constipated type of stool, 3-5 is normal stool and 6-7 is loose stool i.e., diarrhea [10]. The safety of probiotic Bacillus subtilis HU58 + Bacillus coagulans SC208 in combination and Bacillus coagulans SC208 was assessed by clinical tolerability, side effects and adverse events and by any change in the organ function tests. Improvement in stool consistency compared to baseline was noted to assess the therapeutic efficacy. Other beneficial and adverse effects which were observed during therapy were recorded.

Results
According to BSC, the stool consistency measured at baseline was same for all the three groups (scale 6-7 The other symptoms like pain in abdomen, sensation of bloating and flatulence also decreased. In group C, the scale remained the same as baseline which was at 6-7. Improvement in stool consistency was not seen. Patients were not feeling better with the therapy. Table 2 shows the details about average stool consistency. The pain in abdomen was continuous with no relief. Also, the symptoms of flatulence and bloating sensation did not reduce.

Statistical Analysis
Analysis of data was done for the two sets using student's unpaired t test. In Group A the mean, standard deviation (SD), Standard Error of Mean (SEM) at baseline was found to be 6.63, 0.49, 0.10 respectively whereas on the15th day the mean, SD and SEM was found to be 3.17, 0.38, 0.08 respectively. The unpaired t test for this group was found to be statistically significant (p value< 0.0001). Mean, SD, SEM ingroup B for baseline was found to be 6.60, 0.50, 0.10 respectively and by the end of 15 th day the mean, SD, SEM was found to be 3.16, 0.37, 0.07 respectively. The unpaired t test for this group was also found to be statistically significant (p value< 0.0001). Mean, SD, SEM in group C for baseline was found to be 6.48, 0.51, 0.10 respectively and by the end of 15 th day the mean, SD, SEM was found to be 6.24, 0.44, 0.09 respectively. The data for group C was not statistically significant (p value 0.0799) as compared to group A and group B. Table 3 shows the statistical analysis of group A, group B and group C.  Figure   2). TNF-α by 55% [14].

Further, in an open labeled study conducted by
Mehta DS et al in 60 patients suffering from AAD, Bacillus subtilis HU58 (2 X 10 9 CFU) daily for 7 days showed a reduced incidence of AAD in comparison with placebo [15]. The gut microbiota controls essential human functions like digestion, energy metabolism, and inflammation by modifying multiple endocrines, neural, and immune pathways of the host [16]. The interaction of microbes in intestinal tissue where the growth of microbes takes place in the lumen contributes to digestion of food and chemical milieu of the gut [17]. The non-digestible carbohydrates produce primary endproduct such as short chain fatty acids there-by providing health benefits [18]. Administration of antibiotics even for short periods, disturbs the normal microbiota of the gut leading to a dysbiosis state which aggravates the disease [16]. Antibiotic administration triggers changes in enteric bacterial flora and a reduction in flora of fecal anaerobes due to which leads to alteration in carbohydrate metabolism and digestion followed by a concurrent decrease in absorption of short chain fatty acids causing osmotic diarrhea [19].
Probiotics administration helps to improve the microbial Patients also gained weight in varying degrees [22].

Conclusion
Improvement in stool frequency and consistency along with pain in abdomen, sensation of bloating and flatulence were observed in both the probiotic groups. Data of patients in group A and group B was found to be statistically significant (p value< 0.0001) compared to group C. It was also found that patients receiving Bacillus subtilis HU58 + Bacillus coagulans SC208 in combination experienced better relief in terms of stool frequency and consistency along with pain in abdomen, sensation of bloating and flatulence than the patients receiving Bacillus coagulans SC208 alone. It has been reported that the symptoms of pain in abdomen, diarrhea, sensation of bloating and flatulence have also been noted in the IBS-D cases. Hence, application of Bacillus coagulans SC208 in IBS-D can also be considered. Further these claims can be supported by the use of other strains of Bacillus coagulans in the IBS-D trails (vide supra). This comparison however needs to be studied in detail by conducting double blind randomized study in a larger sample size.