Very limited data on the nasal carriage of S. aureus in donkeys are available. In this sense, a study conducted in Ethiopia described the detection of S. aureus in the upper respiratory tract of 13% of tested donkeys . Other study performed in Italy detected S. aureus in 6% of donkey milk samples . The high recovery rate of S. aureus detected among nasal samples of healthy donkeys in the present report (50% of tested samples) is relevant, and represents the first study of this type in Tunisia. The high diversity of genetic lineages among the S. aureus recovered is noteworthy. However, it should be noted that all our strains were methicillin susceptible. Elevated clonal variety has been already detected in other studies among MSSA strains and it seems that MSSA of human show a higher genetic diversity than MRSA .
The 16 different STs identified by MLST among S. aureus isolates, with seven of them new, were distributed in eight clonal complexes (CC133, CC1, CC6, CC522, CC72, CC7, CC15 and CC22), and five singletons. Among these, CC133, CC1 and CC6 were predominant and grouped 68% of typed isolates.
Twenty two of our isolates (44%) corresponded to the major ruminant lineage CC133 [6, 22], being the major detected lineage. Several previous studies have reported the existence of CC133 in clinical S. aureus isolates of cattle, goats and sheep [26–28]; nevertheless, this clone was not detected in an earlier study carried out in Tunisia on healthy sheep . It is interesting to remark that the clonal complex CC133 was associated with different spa-types in donkey isolates in our study (t1166, t1403, t2420, t3583, t7718, t8836) or in sheep and goat isolates (t544, t2678, t3495, t4560, t5592, t7294, t7296, t7297, t7298, t7300) in previous studies . It has been reported that S. aureus isolates of the clonal complex CC133 have been also responsible for most cases of mastitis in dairy farms , and it seems that this clone may have a broad geographic distribution. The agr-type I detected among the CC133 isolates in our study was coincident with former reports . Some authors propose that strains of lineage CC133 could have evolved and adapted to small ruminants derived from humans due to an adaptive genome diversification resulted from allelic variation, gene loss, and horizontal acquisition of mobile genetic elements containing virulence genes with attenuated or enhanced activity in ruminants. The capacity to coagulate bovine plasma detected in our isolates belonging to this CC is a characteristic previously reported for isolates adapted to small ruminants and bovine ; and the acquisition of a novel staphylococcal pathogenicity island (SaPIov2) carrying a novel von Willebrand factor-binding protein (vWBP) with ruminant-specific coagulase activity has been described among isolates of this lineage . On the other hand, a very recent study undergone in a Danish Zoo  has revealed that ST133 may have a broader host distribution since it was detected in S. aureus of a wide variety of animal species. However, to the best of our knowledge, this is the first description of this genetic lineage in donkeys .
Seven MSSA isolates were ascribed to the clonal complex CC1 (those of ST1 and ST1738) which is a common lineage among human isolates . This data is in accordance with earlier reports  where S. aureus isolated from equines were more likely to cluster into human associated lineages. In the referred study performed in United Kingdom, equine-associated S. aureus isolates were assigned to the major human lineage CC1. Although this CC seems to be related to human isolates, a possible animal origin has also been suggested in other studies, given that its presence in other animal species is not exceptional .
The lineage CC6, less frequently detected among our isolates (5 isolates), has been previously detected in both human and animal S. aureus isolates [35, 36]. Our CC6 MSSA isolates were classified into the agr group I, which is in harmony with previous studies .
Three MSSA isolates were ascribed to the clonal complex CC72; this clone has been detected in humans in different countries [38, 39]. One isolate belonged to the clonal complex CC15, which has been before detected in humans at both community and hospital settings [40, 41]. Another study conducted in five African countries  showed that 12% of S. aureus obtained from humans in the community corresponded to the CC15 clone. In our study, the characteristics of our CC15 isolate were different from other previous reports in relation to the spa-types, agr-types and virulence determinants [40, 42].
One MSSA isolate was ascribed to lineage CC22, associated to the United Kingdom EMRSA-15, which is commonly isolated from pet animals, especially dogs [35, 43]. This result is consistent with that of  where the MRSA isolates of equines belonged to the same clone EMRSA-15 (CC22) and harbored the egc cluster-like comprising the enterotoxin genes: seg, sei, sem, sen, seo, and seu.
Several virulence genes were detected in our MSSA isolates. The detection of the tst gene in 12% of isolates is interesting, although this percentage was lower than the one detected among S. aureus isolates from healthy sheep (78%) or healthy humans (20%) in Tunisia [29, 44]. Nevertheless, the tst gene was not detected among S. aureus isolates of donkey milk in a previous report . Most of our MSSA isolates harboured haemolysin genes, where the hla and hld genes were present in all our isolates. Similarly, a significant number of strains harboured the hlb gene, while the hlg and hlg
genes were only present in one isolate. High occurrence of the hld gene in nasal S. aureus isolates of healthy sheep in Tunisia has also been reported . Remarkably, most of our isolates (90%) carried the lukED genes, which are commonly present in cattle causing bovine mastitis infections . In addition, a high number of enterotoxin genes, with 3 distinct egc-cluster-like variants, were observed. With this regard, some strains presented some but not all of the genes comprising the egc-cluster or egc-cluster-like. The absence of one or more genes in the egc-cluster has been previously reported [45, 46]. Even though S. aureus associated food poisoning outbreaks are normally due to human isolates , the presence of a wide variety of enterotoxin genes in S. aureus from donkey reflects the adaptation of enterotoxigenic strains to different mammalian species. The presence of the exfoliatin etb gene in one isolate, which also revealed novel genetic characteristics (t8842-ST2152), is remarkable.
Most of our MSSA showed susceptibility to the antimicrobials tested (60%) with several exceptions. The low frequency of penicillin resistance detected among our isolates contrasts with the high frequency of this type of resistance reported for human isolates, even in commensal strains [44, 45]; however, it is in agreement with isolates of sheep origin . With regard to fusidic acid resistance, the rate found in different countries is very variable, with percentages ranging from <1% to >50% . No mutations in elongation factor G have been detected in the fusA sequences of the analyzed fusidic acid resistant S. aureus isolates and the fusC gene was only detected in one strain. The resistance mechanism of the remaining fusidic acid resistant MSSA strains of this study remains unknown. In addition, 2 streptomycin- and one chloramphenicol-resistant isolates did not present any of the tested resistance genes.