The sub-inhibitory effect of curcumin and norfloxacin on gene expression of Staphylococcus aureus isolates

Background: Staphylococcus aureus is a major cause of hospital- and community-acquired infections, with increasing resistance to antibiotics, biofilm formation, efflux pump activity, and quorum sensing regulation contributing to its pathogenicity. This study aimed to identify S. aureus isolates from clinical samples, assess their antibiotic resistance and biofilm-forming ability, detect key virulence and resistance genes (norA, norC and agrA), evaluate the sub- MIC effects of Norfloxacin, Curcumin, and their combination on gene expression. Methods: A total of 250 clinical specimens were collected from Baqubah Teaching Hospital and Al-Batoul Hospital (Diyala, Iraq) between December 10, 2024, and January 30, 2025. Bacteriological identification was performed using standard methods. Antibiotic susceptibility was tested using disk diffusion. Biofilm formation was assessed before and after sub-MIC treatment with Curcumin. Molecular detection of (norA, norC and agrA genes), and their expression changes after treatment with Curcumin, Norfloxacin, and their combination were analyzed using PCR-based methods. Results: 25/250 (10%) of S. aureus was isolated from specimens including: wounds (24%), burns (24%), nasal swabs (16%), urine (20%), and ear swabs (16%). Among these, ten isolates (40%) were methicillin-resistant (MRSA) and 15 (60%) were methicillin-sensitive (MSSA). Antibiotic susceptibility tests revealed that MRSA isolates exhibited 100% resistance to Azithromycin, Levofloxacin, and Norfloxacin, while MSSA isolates showed lower resistance rates (20% to Norfloxacin and 40% to Tetracycline). Biofilm assays demonstrated that all MRSA isolates (100%) were strong biofilm formers; after treatment with sub-MIC Curcumin, biofilm intensity decreased to 40% strong and 60% moderate. For MSSA isolates, biofilm formation decreased from strong (93%) and moderate (7%) to strong (27%), moderate (33%), and weak (40%). Gene expression analysis revealed significant downregulation of agrA, norA, and norC after treatment with sub-MIC Curcumin, Norfloxacin, and their combination, with mean fold-change values decreasing to (0.402, 0.199, 0.228), (0.104, 0.053, 0.308), and (0.052, 2.527, 0.413), respectively. Conclusion: This study demonstrates that MRSA and MSSA isolates are prevalent and characterized by multidrug resistance, strong biofilm-forming ability, and the presence of efflux-associated genes. Treatment with sub-MIC levels of Curcumin, Norfloxacin, and particularly their combination markedly reduced biofilm formation and suppressed the expression of resistance and virulence genes. These findings suggest that Curcumin may serve as a valuable adjuvant to antibiotics in combating S. aureus infections.