Participant flow:
Participants were randomly assigned to either the A. clausii AO1125 group or the pla- group, with 50 individuals allocated to each group. The sample size calculation was based on an expected adverse event rate of 20.0% in the AO1125 group and 0.5% in the placebo group, with a Type I error rate of 5% (alpha = 0.05) and a Type II error rate of 20% (80% power). A 10% anticipated dropout rate was also incorporated into the calculation, resulting in a minimum requirement of 41 participants per group. However, it was determined to recruit 50 participants per group, providing a buffer to accommo-date any unanticipated withdrawals. Randomization was conducted using the EpiData 3.1 software , which ensured unbiased allocation to treatment and placebo groups. Blinding was strictly maintained by dispensing the capsules in identical packaging, preventing both participants and study personnel from distinguishing between the active treatment and the placebo.
Following randomization and baseline screening, the final cohort comprised 50 par-ticipants in the AO1125 group and 49 in the placebo group, reflecting one exclusion post-randomization
Baseline characteristics:
Demographic Variables A. clausii AO1125 Cohort (n = 50) Placebo Cohort (n = 49 ) p Value
Number % Number %
Sex Female 29 58 31 63.3 0.896 a
Male 21 42 18 36.7
Age (years) Median ± SD 50.06 ± 12.87 44.43 ± 14.74 0.185 b
BMI Median ± SD 24.01 ± 3.65 23.63 ± 4.17 0.44 b
Outcome measures:
Primary Outcome:The study involved a total of 99 individuals, 49 in the placebo group and 50 in the A. clausii group. Most individuals, 83.8% (n = 83), did not show any adverse effects (AE).
Secondary Outcome:
To determine the averages of each chemical variable, the Student’s t-test for inde-pendent samples was employed. On the baseline day, the following variables were sta-tistically significant in both study groups: Total protein (p < 0.01), albumin (p = 0.008), Cholesterol (p = 0.042), Bilirubin D (p = 0.01), MHC (p = 0.03), and eosinophils (Eo) (p = 0.036). Additionally, MPV showed statistical significance only on day 60 (p = 0.034). Sig-nificant differences between the Alkalihalobacillus clausii group and the placebo group between baseline and day 60 were confirmed solely for GGT, both at baseline (p = 0.02) and on day 60 (p = 0.03).
Student’s t-test for paired samples was applied to the A. clausii group to evaluate changes between baseline and day 60. The following parameters decreased significantly: Glycemia (p < 0.01), Creatinine (p < 0.01), and Urea (p = 0.016). However, there were sig-nificant increases in ALAT (p = 0.02) and ASAT (p < 0.01).
A similar analysis in the placebo group revealed significant increases in Urea (p = 0.012), GGT (p = 0.026), Hematocrit (HTC) (p = 0.001), and Mean Corpuscular Volume (MCV) (p < 0.01). Significant reductions were observed in ALAT (p = 0.002), ASAT (p < 0.01), Cholesterol (p < 0.01), Total Bilirubin (p = 0.002), and Direct Bilirubin (p = 0.001).
Bioimpedance was measured using weight and Body Mass Index (BMI). The t-test for both independent and paired samples did not reveal significant differences between the study groups in these measures
The survey questions related to the physical and emotional health status of the study subjects were analyzed (SF-36, Questions 1, 6, 7, and 11). In both groups, the be-havior of the answers to these questions was very similar. Both the placebo group and the treatment group maintained similar responses at both times (Kappa~1, p = 0.000). The significant p values represent that the agreement between the responses of the two moments is high in both evaluated groups
Adverse events:
The study involved a total of 99 individuals, 49 in the placebo group and 50 in the A. clausii group. Most individuals, 83.8% (n = 83), did not show any adverse effects (AE) .Of these, 74% belonged to the group treated with the probiotic (n = 37) and 93.9% to the placebo group (n = 46). Significant differences were observed between the treated group and placebo according to the development of AE related to participants in the study (p = 0.007).Among the participants who received the A. clausii probiotic capsule, 13 individuals (26%) experienced mild adverse events (AEs). The reported adverse events included gastrointestinal pain or discomfort (n = 3), gas (n = 10), headache (n = 1), constipation (n = 3), and diarrhea (n = 1).
The study cohort consisted of 60 females and 39 males. Adverse events were ob-served in 8 females (8/60) and 8 males (8/39). A moderate-intensity adverse event was reported in 1 female from the placebo group (7%), who developed edema, which coin-cided with a diagnosis of dengue infection. This moderate AE was likely associated with the arbovirus rather than the intervention. None of the participants reported limitations in their daily activities, which is consistent with the mild intensity of the events. The adverse effects diminished over the course of the treatment
Summary study:
Abstract: (1) Background: Alkalihalobacillus clausii AO1125 is a Gram-positive, motile, spore-forming bacterium with potential as a probiotic due to its broad-spectrum antimicrobial ac-tivity, inhibiting pathogens like Listeria monocytogenes, Staphylococcus aureus, and Clostridium difficile, as well as anti-rotavirus activity. Its resilience in gastrointestinal conditions suggests benefits for gut health. This study evaluates the safety and probiotic potential of A. clausii AO1125. (2) Meth-ods: Genome annotation identified genes linked to probiotic traits such as stress resistance, gut colonization, immune modulation, and antimicrobial production. The genome was screened for antibiotic resistance genes using CARD, bacteriocin clusters using BAGEL4, and virulence factors via VFDB. Cytotoxicity was assessed on Vero cells and erythrocytes, and a Phase I, double-blind, placebo-controlled clinical trial was conducted with 99 healthy volunteers (50 AO1125, 49 placebo). (3) Results: Genomic analysis confirmed minimal antibiotic resistance genes and the absence of virulence factors, supporting safety. A. clausii AO1125 showed no pathogenicity, cytotoxicity, or hemolytic activity and was well-tolerated in clinical settings, with mild, transient abdominal gas as the most common adverse event. (4) Conclusions: The safety profile and genetic basis for probiotic and antimicrobial properties support A. clausii AO1125 as a promising probiotic candidate for gastrointestinal health, warranting further clinical research.
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