Construction and validation of EGFP-expressing Staphylococcus aureus clinical strains for adhesion and internalization assays on epithelial cells

Background: Staphylococcus aureus is both a major pathogen and a commensal bacterium in humans. It is able to adhere at the surface of epithelial cells of the anterior nares and can trigger its internalization inside these non-professional phagocytic cells. To better understand the interactions of clinical isolates with keratinocytes in the anterior nares, we developed and validated a one-step protocol expressing enhanced green fluorescent protein (EGFP) in S. aureus clinical strains with the aim to study adhesion to and internalization into mammalian cells.Methods: Twenty S. aureus clinical isolates belonging to clonal complexes 5, 8, 30, 45, 398 were selected for one-step transformation protocol with the EGFP-encoding plasmid pBSU101. EGFP expression was analysed by flow cytometry and confocal microscopy. Wild type and isogenic EGFP-expressing strains were compared for adhesion and internalization levels by using the HaCaT cell model.Results: Transformation was achieved in all the S. aureus strains regardless of their genetic background. The flow cytometry analysis showed that the mean proportion of EGFP-expressing bacteria was 97.2% (±2.1) after 4h of incubation. Adhesion and internalization levels were similar in wild-type and isogenic EGFP-expressing S. aureus strains. Confocal laser scanning microscopy confirmed that EGFP-expressing S. aureus bacteria could be easily identified inside HaCaT keratinocytes.Conclusion: This study reports an efficient protocol for expressing EGFP in S. aureus clinical strains and demonstrates that these EGFP-expressing strains are suitable for adhesion and internalization assays using HaCaT cells, which allows to perform static and dynamic in vitro studies of S. aureus colonization.