Fingerprinting PCR Reveals Potential Dissemination of Multidrug Efflux System Genes and Antimicrobial Resistance in Staphylococcus aureus Across Primary Healthcare Units in Brazil

Multidrug efflux systems (MESs) are major contributors to antimicrobial resistance (AMR) in Staphylococcus aureus , yet their role in primary healthcare settings is poorly understood. Under a One Health framework, we investigated MES‐mediated resistance in 38 S. aureus isolates (27 from humans, 11 from dogs) from three Basic Health Units (BHUs) in Viçosa, Brazil. Isolates were characterized by antimicrobial susceptibility testing, PCR for six key efflux genes, and (GTG) 5 ‐PCR fingerprinting. Phenotypic efflux activity was evaluated using ethidium bromide fluorescence assays. Thirty‐seven isolates were resistant to at least one antimicrobial, most commonly penicillin (57.9%) and erythromycin (55.3%), while all remained susceptible to chloramphenicol, trimethoprim, and linezolid. While the msrA gene was rare (10.5%), other efflux genes like norA/B/C , lmrS , and tet38 were nearly ubiquitous (> 94%). This high genetic prevalence contrasted with low phenotypic resistance, indicating that most MES genes were not expressed. Fingerprinting revealed seven genetic clusters, demonstrating the circulation of closely related strains between human and animal hosts across different health units. Eight isolates showed clear genotype–phenotype concordance, with MES activity confirmed phenotypically. The four msrA ‐positive, erythromycin‐intermediate isolates formed two clonal groups (100% similarity): one shared between two users from different BHUs and another shared between a healthcare worker and a dog from different BHUs, providing direct evidence of interhost and cross‐geographic AMR dissemination. Moreover, co‐colonization of a single individual with two genetically distinct tetracycline‐resistant strains (60% similarity) suggests possible horizontal gene transfer. Although phenotypic MES‐mediated resistance was limited (21%), we demonstrate the potential AMR spread across hosts and geographic boundaries, as primary healthcare settings harbor a significant reservoir of MES genes in S. aureus even if they are silent. These results highlight the critical need for integrated One Health surveillance in community settings to mitigate AMR dissemination.