Abstract

Background: Bacillus clausii SKB/BCL21 (MCC 0569) is a novel strain that shows promise as a probiotic for both human and animal healthcare. Objectives: The objective is to evaluate the safety profile of B. clausii SKB/BCL21 through genomic and toxicity assessments in Wistar rats, as well as to assess its efficacy as a probiotic in broiler chickens challenged with Clostridium perfringens. Methods: The identification of genus and species was performed using 16S rRNA and whole genome sequencing (WGS). A genomic analysis was conducted through bioinformatic screening of the B. clausii SKB/BCL21 genome to identify virulence factors, genes encoding toxins, mobile genetic elements, and antibiotic resistance genes. In vitro biosafety assays were conducted to evaluate mucin degradation, gelatinase, hemolytic activity, and DNase activity. The in vivo safety evaluation was performed by acute and subacute oral toxicity studies as per the OECD 423 guidelines. In efficacy testing broilers challenged with C. perfringens were administered with low dose (1 × 108 cfu/kg of feed) and high dose (1 × 109 cfu/kg of feed) of B. clausii SKB/BCL21. Performance metrics, such as average weight gain, feed conversion ratio (FCR), and mortality rates, were evaluated in comparison to a positive control group that received Virginiamycin 50% (15 ppm). Results: The isolate SKB/BCL21 was identified as Bacillus clausii based on 16S rRNA and whole genome sequencing (WGS). The bioinformatic analysis of the B. clausii SKB/BCL21 genome reveals that it lacks genes associated with toxins, mobile genetic elements, and virulence factors. However, it does contain intrinsic and non-transferable antibiotic resistance genes within its chromosomal DNA. In the acute toxicity study, an oral dose of 2000 mg/kg (400 billion cfu/kg) body weight was found to be nontoxic. The No Observed Adverse Effect Level (NOAEL) for B. clausii SKB/BCL21 was found to be 1000 mg/kg (200 billion cfu) body weight/day by oral route in the subacute toxicity study. The findings of in vivo toxicity studies indicate that there were no treatment-related changes in any of the endpoints assessed. The effects of low (1 × 108 cfu/kg of feed) and high (1 × 109 cfu/kg of feed) doses of B. clausii SKB/BCL21 on the growth performance metrics, including average weight gain, feed conversion ratio, and mortality rates in broiler chickens infected with C. perfringens, showed results similar to those of the positive control (Virginiamycin 50%, 15 ppm). Conclusions: Based on these preliminary studies, B. clausii SKB/BCL21 can serve as a potential alternative to antibiotic growth promotors in broiler production. These results suggest that the B. clausii SKB/BCL21 is safe and could be a potential probiotic for animal feed supplements.

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