Genome-wide discovery and functional insights into novel sRNAs involved in responses to bioactive compounds in the emerging pathogen <i>Staphylococcus haemolyticus</i>

AIMS: Staphylococcus haemolyticus is an emerging opportunistic pathogen and a reservoir of antimicrobial-resistance genes, yet its regulatory noncoding RNAs remain poorly defined. This study aimed to identify, validate, and functionally assess novel small RNAs (sRNAs) in S. haemolyticus, focusing on their responses to bioactive compounds and their potential roles in stress adaptation, virulence regulation, and antimicrobial tolerance. METHODS AND RESULTS: Multi-algorithmic in silico prediction, RT-PCR validation, and transcriptomic analysis were used to characterize the sRNA repertoire of S. haemolyticus. Twenty-six candidates were predicted, and 25 were confirmed by RNA-seq or RT-PCR. Seven trans-acting sRNAs were selected for further study. Exposure to Ginkgo biloba exocarp extract and a fusaric-acid derivative produced compound-specific transcriptional responses: shsr05 and shsr07 were upregulated, while shsr08 and shsr09 were strongly downregulated. Coexpression networks and target predictions linked these sRNAs to translation, DNA repair, metabolism, oxidative stress response, and virulence pathways. Structural modeling supported stable sRNA-mRNA interactions, and conservation analysis revealed homologs across several staphylococcal species, suggesting broader regulatory relevance. CONCLUSION: This study provides the first integrated framework for sRNA-mediated regulation in S. haemolyticus and shows that several newly identified sRNAs respond dynamically to biologically active compounds. These findings uncover additional regulatory layers involved in stress adaptation and virulence modulation and highlight targets for future mechanistic and translational research.