In the United States, veterinary use of mupirocin is primarily limited to the treatment of canine pyoderma caused by methicillinresistant Staphylococcus pseudintermedius (MRSP). In this study, only 1 of 581 S. pseudintermedius isolates tested was resistant to mupirocin and carried the high-level mupirocin resistance gene, ileS2, on a plasmid. Staphylococcus pseudintermedius is the primary bacterial pathogen isolated from canine pyoderma and also causes postsurgical infections in dogs (1, 2). Methicillin resistance and multidrug resistance are increasing in S. pseudintermedius, thus limiting the options for therapeutic treatment of canine skin infections (2). Mupirocin is a bacteriostatic antibiotic that reversibly binds to isoleucyl-tRNA synthetase to disrupt protein synthesis and is widely used to eliminate nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) in human MRSA carriers (3). Mupirocin has been used on only a limited basis in veterinary medicine but is approved in the United States for the treatment of bacterial skin infections and superficial pyoderma in dogs (4).In S. aureus, two levels of mupirocin resistance have been identified. Low-level mupirocin resistance occurs due to a point mutation to the chromosomal ileS gene that encodes the native isoleucyl-tRNA synthetase. The MIC for mupirocin for staphylococci carrying the low-level resistance is Ն8 g/ml but Յ256 g/ml (5). Conversely, high-level mupirocin resistance (MIC of Ն512 g/ml) is usually conferred by the plasmid-borne ileS2, although a chromosomal location of ileS2 has been reported (5). Recently, ileS2 plasmid-mediated mupirocin resistance was found in a mupirocin-resistant, methicillin-susceptible S. pseudintermedius strain isolated from a dog in Croatia (6). The goal of the present study was to determine the prevalence of mupirocin resistance in S. pseudintermedius isolated from patients presented to a veterinary hospital in Texas.In this study, 581 isolates of S. pseudintermedius were screened for phenotypic low-level mupirocin resistance. Isolates were collected from veterinary patients, predominantly dogs (n ϭ 446), but also included isolates from cats (n ϭ 9). Some patients were cultured at multiple sites and contributed more than one isolate, and of these, 21 patients contributed more than two isolates. The isolates included a historical collection of 403 isolates from clinical infections and contained both methicillin-resistant S. pseudintermedius (MRSP) isolates (n ϭ 153) and methicillin-susceptible S. pseudintermedius (MSSP) isolates (n ϭ 250). The isolates from clinical infections were collected from the following anatomic sites: skin (n ϭ 96), external ear canal (n ϭ 31), wounds (n ϭ 79), postoperative infections (n ϭ 33), urine or the urinary tract (n ϭ 87), and other sources (n ϭ 77). Additional isolates were collected during a study of MRSP prevalence in canine patients without clinical staphylococcal infection that presented for elective orthopedic procedures. The MRSP prevalence study yielded 178 S. pseudin...