Differential Expression Analysis of a Regulatory and a Virulence Gene in Biofilm and Planktonic Staphylococcus Aureus Under Dual Antibiotic Stress

Background: Staphylococcus aureus is a major human pathogen whose clinical persistence is driven by biofilm formation, antibiotic resistance, and sophisticated virulence regulatory networks. Objective: The current investigation investigated the difference in their production of virulence gene hla (alpha-hemolysin) and stress-regulatory gene sigB (sigma factor B) in biofilm and planktonic S. aureus clinical isolates when compared at sub-lethal values of vancomycin and ciprofloxacin. Methods: Forty non-duplicate isolates were obtained from diabetic foot infection (DFI) patients from Baghdad, Iraq, verified by VITEK-2 and phenotypically characterized in biofilm formation and antibiotic susceptibility. Minimum inhibitory concentration profiling also showed high-level resistance of both antibiotics, in great proportion meeting VRSA criteria. Gene expression was assessed with the 2⁻ΔΔCT RT-qPCR method with 16S rRNA as standard after 3-hour sub-MIC treatments. Results: Isolates from biofilms showed a strong upregulation of hla under both antibiotics (fold-change: 1.04–7.41), while planktonic isolates showed much weaker responses (0.52–1.34-fold), indicating that virulence activation is dependent on lifestyle. Regarding sigB, biofilm isolate BF-4 showed a remarkable upregulation under ciprofloxacin and vancomycin (11.63× and 23.43×, respectively), whereas planktonic isolate PL-1 exhibited strong induction for vancomycin (14.93×) with the other planktonic isolates down-regulating. These opposite trends highlight some mechanistic differences between the two drugs: vancomycin preferentially activates the SigB stress-response system, while ciprofloxacin induces the SOS system hla expression in an antagonistic manner. Conclusions: These results imply that low doses of antibiotics modulate S. aureus virulence regulation circuitries differentially in a manner associated with lifestyle and the type of antibiotics, which may hamper the treatment of biofilm-associated DFIs.