Probiotic potential of Streptomyces sp. Md12 strain for poultry: in vitro and in silico characterization
Abstract
Regulatory restrictions on in-feed antimicrobial growth promoters have renewed interest in actinobacterial probiotics, particularly Streptomyces, as promising candidates for poultry feed-additive development following preliminary in vitro and genomic safety assessment. This study characterizes Streptomyces sp. Md12, a soil-derived strain, to evaluate its probiotic potential for poultry through combined in vitro assessment and in silico whole-genome analysis. Thermotolerance, gastrointestinal survival, antibacterial activity, antimicrobial susceptibility, and hemolytic activity were assessed in vitro, while the genome, sequenced on an Illumina platform and assembled and annotated using standard bioinformatics pipelines, was screened for biosynthetic gene clusters, digestive enzymes, vitamin biosynthesis pathways, antimicrobial-resistance genes, virulence factors, and mobile genetic elements. Strain Md12 grew at 41 °C, indicating thermotolerance compatible with poultry body temperature and showed post-exposure growth recovery after 180 min of exposure to gastric pH (2 to 4), bile salts (0.1 to 0.5%), pepsin (3 mg mL-1), and after 120 min of exposure to lysozyme (100 mg L-1), indicating tolerance to simulated gastrointestinal conditions in qualitative assays. It inhibited Listeria monocytogenes, Escherichia coli, and Salmonella typhi, remained susceptible to all antimicrobials tested, and showed no hemolytic activity. The 8.33 Mb genome (7,553 coding sequences) encodes candidate determinants consistent with observed phenotypes, including 46 biosynthetic gene clusters predicted to contribute to antimicrobial activity, alongside a broad enzymatic repertoire and four vitamin B biosynthesis pathways suggesting feed-digestive and nutritional potential. No acquired antimicrobial-resistance (AMR) gene or genuine virulence factor was detected, and none of the flagged homologs were located on plasmid or provirus contigs, indicating no mobile-element association in this short-read assembly. Digital DNA-DNA hybridization indicated that Md12 represents a novel Streptomyces species. Together, these findings support Md12 as a safe and functionally promising candidate warranting in vivo evaluation for development as a poultry probiotic feed additive.