Staphylococcus aureus is an opportunistic pathogenic bacterium that poses a major threat to food safety and public health. Due to its remarkable adaptability, it can persist and survive in complex environments, causing a wide range of diseases in both humans and animals. The emergence of multidrug-resistant strains, combined with their ability to develop in diverse biological matrices, complicates traditional surveillance methods and highlights the need for rapid and reliable detection tools. Among these innovative approaches, aptamer-based biosensors represent a promising alternative. 

The first part of this thesis investigates the adaptation of S. aureus in complex, lipid-rich environments such as milk and serum. These environments alter the composition and morphology of the bacterial membrane, influence oxidative stress resistance, and modulate antibiotic resistance. Comparative proteomic analysis reveals significant changes in the expression of proteins involved in central carbon metabolism, biosynthetic pathways, and fermentation processes. Furthermore, adaptation to milk induces an upregulation of virulence factors and stress-response proteins, as confirmed by pathogen lethality studies in an insect infection model. These findings highlight a strong correlation between bacterial virulence and its ability to adapt to complex environments. 

The second part of this thesis focuses on the optimization and structural analysis of an aptamer named SA61, specifically selected for its ability to bind S. aureus cells. This DNA oligonucleotide targets an as-yet unidentified epitope present on the bacterial surface. The second part of this thesis focuses on the optimization and structural analysis of an aptamer named SA61, specifically selected for its ability to bind S. aureus cells. This DNA oligonucleotide targets an as-yet unidentified epitope present on the bacterial surface. Using biophysical techniques such as circular dichroism, UV spectroscopy, and nuclear magnetic resonance (NMR), the structure and stability of SA61 were characterized. The results indicate that the aptamer adopts an intercalated motif (motif-I) in vitro, consisting of C:C+ base pairs, which are characteristic of cytosine-rich sequences under slightly acidic conditions. This unique architecture could enhance the aptamer’s specificity for S. aureus. Based on these findings, nucleotide modifications were introduced to improve its binding affinity and stability. To date, no motif-I specific to a pathogenic biomarker has been clearly identified or studied, opening the door to further innovative research in this field. 

The results obtained suggest that the development of a robust, selective, and sensitive aptasensor relies on a deep understanding of S. aureus adaptations to different environments, as well as on the optimization and thorough structural characterization of aptamers. This knowledge is crucial for designing targeted chemical modifications that are essential for the development of new detection tools. The integration of these parameters is fundamental to creating reliable diagnostic devices capable of addressing the challenges posed by this pathogenic bacterium.