A Multidisciplinary Toolkit for Studying <em>Staphylococcus aureus</em> Biofilms

Background/Objectives: The rising prevalence of antibiotic-resistant bacteria, particularly within biofilms, presents a critical challenge in treating hospital-acquired infections. Staphylococcus aureus is a clinically significant pathogen, notorious for its ability to form biofilms that drastically reduce antimicrobial susceptibility. This study aimed to charac-terize the metabolic response of S. aureus in both planktonic and biofilm states to under-stand resistance mechanisms better. A key objective was to optimize methodologies for large-scale biofilm production and high-yield RNA extraction, providing a vital tool for subsequent transcriptomic analyses. Methods: To reach our goals, we investigated two strains with differing resistance profiles: a methicillin-sensitive reference strain (ATCC 6538) and a methicillin-resistant clinical isolate (MRSA pHUA585). Biofilm morphology was characterized, and the response to vancomycin was assessed. To facilitate RNA ex-traction, we optimized a large-scale biofilm production method using a solid membrane support on agar plates, followed by RNA purification via an enhanced phenol/chloroform protocol. Results: Our results revealed distinct biofilm architectures between the strains, with differences in thickness and the distribution of nucleic acids and polysaccharides. Vancomycin exhibited a concentration-dependent inhibitory effect on both, though the response varied, suggesting strain-specific biofilm formation mechanisms and matrix composition. Furthermore, we successfully standardized the scaled-up biofilm production and obtained high-quality RNA. However, under the conditions tested, biofilms from both strains showed low expression of the icaA gene, a key mediator of polysaccharide inter-cellular adhesion, potentially indicating growth condition dependencies or biofilm ma-turity state. Conclusions: In summary, this work establishes a methodological foundation for biofilm research and provides crucial insights into the strain-specific phenotypic and potential genetic determinants that underline antibiotic tolerance in S. aureus biofilms.