22Background: Staphylococcus aureus is both a major pathogen and a commensal bacterium in 23 humans. It is able to adhere at the surface of epithelial cells of the anterior nares and can trigger 24 its internalization inside these non-professional phagocytic cells. To better understand the 25 interactions of clinical isolates with keratinocytes in the anterior nares, we developed and 26 validated a one-step protocol expressing enhanced green fluorescent protein (EGFP) in S. 27 aureus clinical strains with the aim to study adhesion to and internalization into mammalian 28 cells. 29Methods: Twenty S. aureus clinical isolates belonging to clonal complexes 5, 8, 30, 45, 398 30 were selected for one-step transformation protocol with the EGFP-encoding plasmid pBSU101. 31 EGFP expression was analysed by flow cytometry and confocal microscopy. Wild type and 32 isogenic EGFP-expressing strains were compared for adhesion and internalization levels by 33 using the HaCaT cell model. 34Results: Transformation was achieved in all the S. aureus strains regardless of their genetic 35 background. The flow cytometry analysis showed that the mean proportion of EGFP-expressing 36 bacteria was 97.2% (± 2.1) after 4h of incubation. Adhesion and internalization levels were 37 similar in wild-type and isogenic EGFP-expressing S. aureus strains. Confocal laser scanning 38 microscopy confirmed that EGFP-expressing S. aureus bacteria could be easily identified inside 39HaCaT keratinocytes. 40 Conclusion:This study reports an efficient protocol for expressing EGFP in S. aureus clinical 41 strains and demonstrates that these EGFP-expressing strains are suitable for adhesion and 42 internalization assays using HaCaT cells, which allows to perform static and dynamic in vitro 43 studies of S. aureus colonization. 44
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