Staphylococcus aureus, an opportunistic pathogen, is responsible for a spectrum of severe chronic diseases such as chronic osteomyelitis and chronic mastitis in humans and in cows, respectively. Although traditionally regarded as an extracellular pathogen, S. aureus has been identified within non-immune cells such as osteoblasts (associated with osteomyelitis) or mammary gland epithelial cells (linked to cow mastitis), potentially influencing the infection outcome. The detection of signals specific from infected bacteria-bearing cells is complicated by dilution and confounding signals by bystander effects in uninfected cells. To address this, we developed a novel long-term infection model for non-immune cells using a flow cytometric approach, enabling the isolation of cells containing internalized S. aureus from mixed populations. This model facilitates a detailed analysis of the impact of prolonged S. aureus infection on the transcriptional program of human non-immune (osteoblast-like) cells. RNA-seq, KEGG, and Reactome pathway enrichment analyses revealed a remodeled transcriptomic profile marked by heightened immune and inflammatory responses, metabolic dysregulations influencing bacterial intracellular life, and downregulation of genes encoding epigenetic regulators, including those involved in chromatin-repressive complexes (e.g., NuRD, BAHD1, and PRC1) and epifactors associated with DNA methylation. Additionally, sets of genes related to cell adhesion or neurotransmission were deregulated. Our findings suggest a long-term impact of intracellular S. aureus infection through epigenetic modifications in host cells, potentially leading to pathophysiological dysfunctions alongside the defense response during infection. These results not only enhance our understanding of biological processes in long-term S. aureus infections of non-immune cells but also provide an atlas of deregulated host genes and biological pathways, identifying potential markers and candidates for prophylactic and therapeutic approaches. Moreover, building on this in vitro work, we have initiated a collaboration with the NuMeCan Institute to validate these observations in vivo in a mini-pig model of S. aureus infection. This animal study will be complemented by an exploration of the serum metabolome and the intestinal microbiota in the chronicity of S. aureus infection. Furthermore, these investigations will be extended to the context of S. aureus mastitis in cows through collaboration with Canadian colleagues.