Comparison of MRSA skin infection models with HaCaT keratinocytes and a 3D-organotypic skin model

Abstract

Background and Aims Skin is a major site for Staphylococcus aureus colonization and invasion. A 3D-organotypic skin model was generated by co-culturing keratinocytes with fibroblasts at air-liquid interface enabling the proliferation, migration and differentiation of the keratinocyte to mimic terminal differentiation of the epidermis. Methods The adherence and internalization of MRSA strain types ST8, ST30, ST59, ST22, ST45 and ST239 were evaluated in the HaCaT keratinocytes and a 3-D organotypic skin model. Acridine orange staining and/or anti-Staphylococcus aureus antibody were used for bacterial localization. TUNEL assay was used to evaluate cell death due to apoptosis. Results Maximum adherence to HaCaT cells were exhibited by both MRSA ST59 and ST8 strain types (P=0.129, Tukey post-hoc test). The maximum percentage of internalization was exhibited by both ST59 and ST30. With ST8, ST30, ST59 and ST239 types, bacteria were present within the cytoplasm whereas localization of ST22 and ST45in the phagosomes of HaCaT keratinocytes were observed. Study of cell death in HaCaT keratinocytes was limited to 24 h due to dislodgement of the keratinocytes with all six strains. The 3-D skin model proved to be better to study MRSA transmigration and cell death which could be monitored for longer time points. ST59 exhibited maximum adhesion and internalization. Conclusion The 3-D skin model proves to be a better model to study MRSA skin infections.