| Summary: | Donkeys (<i>Equus asinus</i>) are in decline in Europe. Occupational exposure to farm animals has been associated with increased staphylococci carriage. We aimed to isolate <i>S. aureus</i> and coagulase-negative staphylococci (CoNS) from donkeys and handlers and characterize the antimicrobial resistance profiles and genetic lineages of <i>S. aureus</i> strains. Oral and nasal swab samples were collected from 49 Miranda donkeys and 23 handlers from 15 different farms. Staphylococci species were identified by MALDI-TOF MS. The presence of antimicrobial resistance genes and virulence factors was investigated by PCR. Molecular typing was performed in <i>S. aureus</i> isolates. From the 49 donkey samples, 4 <i>S. aureus</i> (8.2%) and 21 CoNS (42.9%) were isolated. Ten handlers (43.5%) were carriers of <i>S. aureus</i> and 4 (17.4%) carried CoNS. The CoNS isolates showed resistance to several classes of antimicrobials encoded by the <i>mec</i>A, <i>aph</i> (3′)-IIIa, <i>ant</i> (4′)-Ia, <i>tet</i>M, <i>tet</i>K, <i>lnu</i>A, <i>erm</i>B, <i>erm</i>C, <i>dfr</i>A and <i>dfr</i>G genes. <i>S. aureus</i> isolates were resistant to penicillin, aminoglicosides and tetracycline harboring the <i>bla</i>Z, <i>aph</i> (3′)-IIIa, <i>tet</i>L, <i>tet</i>M and <i>tet</i>K genes. All <i>S. aureus</i> isolates from donkeys belonged to ST49 and <i>spa</i>-type t208 while the strains isolated from the handlers were ascribed to 3 STs and 7 <i>spa</i>-types. However, human isolates were from different STs than the donkey isolates. Donkeys are mainly colonized by methicillin-resistant <i>S. sciuri</i>. <i>S. aureus</i> transmission between donkeys and their handlers appears not to have occurred since the isolates belonged to different genetic lineages.
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