Introduction. Karri M. Beck*, Stephen E. Waisglass*, Hani L. N. Dick and J. Scott Weese

Vet Dermatol 2012; 23: 369 e67 Prevalence of meticillin-resistant Staphylococcus pseudintermedius (MRSP) from skin and carriage sites of dogs after treatment of their meticillin-resistant or meticillin-sensitive staphylococcal pyoderma Karri M. Beck*, Stephen E. Waisglass*, Hani L. N. Dick and J. Scott Weese DOI: 10.1111/j.1365-3164.2012.01035.x *Veterinary Emergency Clinic and Referral Centre, 920 Yonge Street Suite 117, Toronto, Ontario M4W 3C7, Canada Department of Microbiology, IDEXX Reference Laboratories, 1345 Denison Street, Markham, Ontario L3R 5V2, Canada Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, Canada Correspondence: Karri M. Beck, Veterinary Emergency Clinic and Referral Centre, 920 Yonge Street Suite 117, Toronto, Ontario M4W 3C7, Canada. E-mail: kbeckvet@gmail.com Background Meticillin-resistant staphylococci are significant pathogens in veterinary dermatology, yet longitudinal studies of the impact of routine antimicrobial therapy on emergence or resolution of resistance are lacking. Objectives To determine the prevalence of meticillin-resistant staphylococci on skin and carriage sites in dogs with bacterial pyoderma and evaluate the prevalence of meticillin-resistant Staphylococcus pseudintermedius (MRSP) colonization after successful treatment of pyoderma. Animals One hundred and seventy-three dogs that presented to a dermatology referral service with pyoderma and 41 healthy control dogs. Methods Skin, nasal and rectal swabs for bacterial culture were collected at the time of referral and after clinical resolution of the pyoderma. Meticillin resistance was confirmed by demonstration of penicillin binding protein 2a antigen. Results Initially, skin cultures yielded MRSP in 70 (40.5%) dogs, meticillin-resistant Staphylococcus aureus (MRSA) in three (1.7%) and meticillin-resistant Staphylococcus schleiferi ssp. coagulans (MRSScoag) in five (2.9%). Samples collected from the nose and rectum (carriage sites) yielded MRSP in 59 (34.1%) dogs, MRSA in 11 (6.4%) and MRSScoag in seven (4.0%). One hundred and two dogs were available for follow-up cultures after clinical cure. Of 42 dogs initially diagnosed with MRSP pyoderma, MRSP was isolated at follow-up from skin in 19 (45.2%) and carriage sites in 20 (47.6%). Of 60 dogs that did not have MRSP pyoderma initially, MRSP was isolated post-treatment from the skin in 17 (28.3%), and MRSP from carriage sites increased from 7.8% (initially) to 26.7% (P = 0.0022). Conclusions and clinical importance Colonization by MRSP often persists after resolution of MRSP pyoderma. Acquisition of MRSP during treatment appears to be common. Introduction Accepted 14 January 2012 Sources of Funding: Ontario Veterinary College Pet Trust Fund. Conflict of Interest: No conflicts of interest have been declared. Preliminary findings were presented at the North American Veterinary Dermatology Forum in 2011 and published as an abstract in Veterinary Dermatology 2011; 22: 293 and at the second ASM ESCMID conference on antimicrobial resistance in staphylococci in 2011. Staphylococci are facultatively anaerobic, Gram-positive cocci, whose primary habitats are the skin and mucous membranes of mammals and birds. 1 Dogs acquire Staphylococcus spp. from their dam in the neonatal period, 2,3 and Staphylococcus pseudintermedius colonization rates are typically high. 4 In dogs, S. pseudintermedius and, to a lesser extent, Staphylococcus aureus and Staphylococcus schleiferi ssp. coagulans, are important causes of skin and ear infections. 5 11 As opportunistic pathogens, these species can also be found in healthy animals, particularly in the nasal passages and intestinal tract and on the skin. 12,13 In recent years, meticillin resistance has emerged as a considerable problem in staphylococci. Meticillin-resistance in staphylococci is conferred by the meca gene encoding the penicillin binding protein 2a (PBP2a), which has reduced affinity for all b-lactam antimicrobials. 14 Meticillin-resistant S. aureus (MRSA) is a critically important pathogen in human medicine and is now a significant concern in veterinary medicine as well. 14,15 Meticillin resistance can also be found in other staphylococci. Of particular note in dogs is the dramatic emergence of meticillin-resistant S. pseudintermedius (MRSP) internationally. 14,16,17 In addition to b-lactam resistance, MRSP is often resistant to multiple other classes of antibiotics, thus few treatment options may be available. 16,18 21 Meticillin-resistant staphylococci pose major clinical challenges in the treatment of canine bacterial pyoderma. Longitudinal study or evaluation of the impact of routine antimicrobial therapy on emergence or resolution of resis- ª 2012 ESVD and ACVD, Veterinary Dermatology, 23, 369 e67. 369

Beck et al. tance is lacking. The objectives of this study were to determine the prevalence of meticillin-resistant staphylococcal pyoderma in dogs presented to a dermatology referral practice, to determine the prevalence of meticillin-resistant staphylococcal colonization of carriage sites in dogs with pyoderma and to evaluate the prevalence of MRSP colonization after successful treatment of their bacterial pyoderma. Materials and methods Population All dogs that were presented to a private dermatology referral service in Ontario, Canada and diagnosed with bacterial pyoderma from November 2009 until December 2010 were eligible for inclusion. The diagnosis of bacterial pyoderma was based on clinical and cytological findings. Clinical findings included the presence of typical pyoderma lesions, such as papules, pustules, collarettes, scale, crust or lichenification. 22,23 Cytological criteria included the presence of bacteria (cocci or rods) at more than five per oil immersion field, associated with degenerate neutrophils or engulfed within neutrophils. A control population of healthy dogs was randomly recruited from households in the same region on a voluntary basis. Control dogs were excluded if they had any physical evidence or history of dermatological disease or if they were being treated with systemic or topical antimicrobials. In addition, dogs were excluded if their owner worked in a veterinary hospital. This study was approved by the University of Guelph Animal Care Committee. Sampling procedure With owner consent, three sterile saline-moistened swabs were used to sample the distal nares, rectum and affected skin. Swabs were inserted approximately 0.5 cm into the distal nares and approximately 1 cm into the rectum. The skin was sampled from the pyoderma lesions (e.g. pustules, collarettes, beneath crusts). When the pyoderma had clinically resolved, follow-up samples were collected from the distal nares and rectum in the same manner as at initial presentation. Skin samples were collected by rubbing the swabs vigorously for approximately 10 15 s in the axillary area as well as onto the location where the original skin culture was collected. All swabs were placed in Amies gel transport media (Starswab II â ; Starplex Scientific Inc., Etobicoke, Ontario, Canada) and stored at 4 C until processing. The healthy control dogs were sampled on two occasions in their homes, 6 weeks apart. The sampling procedure was identical to that used for the affected dogs, except that the skin samples were collected only from the axillary region. Laboratory methods Swabs were inoculated into 2 ml of enrichment broth containing 10 g L Tryptone T, 75 g L sodium chloride, 10 g L mannitol and 2.5 g L yeast extract, and incubated for 24 h at 35 C. Aliquots of 100 ll were inoculated onto both MRSA Chromogenic agar (BBL CHROMagar; Becton, Dickinson and Co., Sparks, MD, USA) and mannitol salt agar with 2 lg ml oxacillin (MSA-OX) and incubated at 35 C for 48 h. Suspect staphylococcal isolates were subcultured onto Columbia blood agar and incubated at 35 C for 24 h. Isolates were identified as staphylococci based on colony morphology, Gram stain appearance and a positive catalase reaction. Meticillin resistance was confirmed by demonstration of PBP2a antigen with a latex agglutination test (Oxoid Ltd, Basingstoke, UK). A tube coagulase test (BBL TM Coagulase Plasma with EDTA; Becton, Dickinson and Co.) was performed on all meticillin-resistant staphylococci. Staphylococcus aureus was identified by a latex agglutination test (Pastorex TM Staph-Plus; Bio-Rad Laboratories, Redmond, WA, USA). Speciation of the remaining coagulase-positive meticillin-resistant isolates was performed using a multiplex-pcr assay. 24 Meticillinresistant coagulase-negative isolates were speciated by partial soda sequence analysis. 25 Statistical analysis Categorical comparisons were performed using Fisher s exact test or chi-squared test. A value of P < 0.05 was considered significant. Results One hundred and seventy-three affected dogs and 41 healthy control dogs were included in the study. The underlying aetiology of the bacterial pyoderma in affected dogs is listed in Table 1. There was a history of antibiotic usage in 154 (89%) of the affected dogs, 36 (20.8%) of which had received a fluoroquinolone antibiotic, at any point prior to presentation. Glucocorticoids and or ciclosporin had been used in 93 (53.8%) affected dogs within 6 months prior to presentation. Initial cultures The prevalence of meticillin-resistant coagulase-positive staphylococci from skin and carriage sites of affected dogs on initial presentation is summarized in Table 2. In total, meticillin-resistant coagulase-positive staphylococci were isolated from the skin in 78 of 173 (45.1%) dogs. Meticillin-resistant S. pseudintermedius was isolated from the nose and or rectum of 51 of 70 (73%) dogs with MRSP pyoderma and eight of 103 (7.8%) without MRSP pyoderma (P < 0.0001). Meticillin-resistant S. aureus was detected in nasal and or rectal swabs of one of three (33%) dogs with MRSA pyoderma and 10 of 170 (5.9%) others (P = 0.18). Meticillin-resistant S. schleiferi ssp. coagulans was isolated from nasal and or rectal swabs of three of five (60%) dogs with MRSScoag pyoderma and four of 168 (2.4%) others (P = 0.0004). There was no significant difference in the nasal versus rectal carriage of MRSP or MRSScoag (P = 0.53 and P = 1.0, respectively). Table 1. Confirmed or presumptive underlying aetiologies in 173 dogs with pyoderma Underlying aetiology n Atopic dermatitis* 109 Hypothyroidism 4 Pemphigus foliaceus 2 Erythema multiforme 1 Sebaceous adenitis 1 Vitamin A-responsive dermatosis 1 Hyperadrenocorticism 1 Intertrigo 1 Juvenile-onset demodicosis 1 Unknown 52 *Includes food induced, nonfood induced and partly food induced. 46,47 Table 2. Prevalence of meticillin-resistant coagulase-positive staphylococci on initial presentation in 173 dogs with pyoderma Carriage site* Skin [n (%)] [n (%)] MRSP 70 (40.5) 59 (34.1) MRSA 3 (1.7) 11 (6.4) MRSScoag 5 (2.9) 7 (4.0) Abbreviations: MRSP, meticillin-resistant S. pseudintermedius; MRSA, meticillin-resistant S. aureus; and MRSScoag, meticillinresistant S. schleiferi ssp. coagulans. *Carriage site refers to nose and or rectum. 370 ª 2012 ESVD and ACVD, Veterinary Dermatology, 23, 369 e67.

MRSP in canine pyoderma Meticillin-resistant S. aureus more frequently colonized the nose versus the rectum (P = 0.03; Table 3). There were significant differences in isolation rates with MRSA Chromogenic agar versus MSA-OX (Table 4). Meticillin-resistant coagulase-negative staphylococci (MR-CoNS) were isolated from the skin of 25 of 173 (14.4%) affected dogs and from at least one carriage site in 28 (16.2%) dogs. A subset of the isolates were available for speciation (Table 5). Twenty-two of the 25 MR-CoNS isolated from the skin were found concurrently with a coagulase-positive Staphylococcus spp. The MR-CoNS isolated alone were two meticillin-resistant Staphylococcus schleiferi ssp. schleiferi and one meticillinresistant Staphylococcus epidermidis. There was no association between prior antimicrobial treatment and meticillin-resistant staphylococcal infection or colonization (P = 0.44 and 0.62, respectively). There was also no association between administration of any specific antimicrobial class and meticillin-resistant infection (all P > 0.70). There was a trend, although not statistically significant, towards prior glucocorticoid or ciclosporin use and decreased likelihood of MRSP infection (P = 0.087). Follow-up cultures Follow-up samples were collected from 102 of 173 (59%) dogs upon clinical resolution of their bacterial pyoderma. Table 3. Comparison of recovery of meticillin-resistant coagulasepositive staphylococci from the nose and rectum from 173 dogs with bacterial pyoderma Nose Rectum P-value MRSP 46 42 0.53 MRSA 9 3 0.03 MRSScoag 5 4 1.0 Of the 71 dogs that did not have follow-up cultures, 58 were lost to follow-up, three died of unrelated causes and 10 (seven MRSP pyoderma, three meticillin-sensitive Staphylococcus pseudintermedius pyoderma) did not reach a clinical cure by the end of the study. Samples were collected between 3 and 15 weeks (mean 6.6 weeks) after initial presentation. The prevalence of meticillin-resistant coagulase-positive staphylococci from skin and carriage sites on followup is summarized in Table 6. Forty-two of the 102 dogs available for follow-up were diagnosed with an MRSP pyoderma upon initial presentation. The remaining 60 dogs had a non-mrsp pyoderma, defined as an infection with either a susceptible organism or a different meticillin-resistant Staphylococcus spp. Table 7 summarizes the skin and carriage site prevalence of MRSP on follow-up, comparing the colonization of dogs that initially had an MRSP pyoderma with those that did not. Of the 42 dogs with an MRSP pyoderma initially, 26 (61.9%) still harboured MRSP at one or more body sites at follow-up. The prevalence of MRSP colonization was significantly lower than at initial presentation for both skin (P < 0.0001) and carriage sites (P = 0.0089). Of the 60 dogs with a non-mrsp pyoderma on initial presentation, 23 (38.3%) were colonized with MRSP at one or more sites at follow-up. The acquisition of MRSP on the skin in 28.3% and the increase of MRSP at carriage sites from 7.8% (at initial presentation) to 26.7% were both statistically significant (P = <0.0001 and P = 0.0022, respectively). There was no difference in the follow-up skin prevalence of MRSP in dogs that initially had an MRSP pyoderma versus those that did not (P = 0.0944). There was, however, a significant difference in carriage site prevalence of MRSP between dogs that originally had an MRSP pyoderma and those that did not (P = 0.0364). Table 4. Comparison of recovery of MRSP, MRSA and MRSScoag from 173 dogs with bacterial pyoderma with two different culture media Organism MSA-OX MRSA chromogenic agar P-value MRSP 207 of 224 (92%) 67 of 224 (30%) <0.0001 MRSA 9 of 17 (53%) 12 of 17 (71%) 0.029 MRSScoag 19 of 20 (95%) 2 of 20 (10%) <0.0001 Table 6. Prevalence of meticillin-resistant coagulase-positive staphylococci on follow-up cultures in 102 dogs once a clinical cure was attained Carriage site* Skin [n (%)] [n (%)] MRSP 36 (35.3) 36 (35.3) MRSA 1 (1.0) 1 (1.0) MRSScoag 2 (2.0) 4 (3.9) *Carriage site refers to nose and or rectum. Table 5. Prevalence of meticillin-resistant coagulase-negative staphylococci at initial presentation Skin (n = 25) S. epidermidis 7 8 S. schleiferi ssp. schleiferi 2 4 S. lugdunensis 2 4 S. haemolyticus 2 2 S. warneri 2 0 S. sciuri 0 2 S. vitulus 1 2 S. cohnii 1 1 S. hominis 1 0 Not speciated 7 8 Carriage site (n = 31) Table 7. Prevalence of MRSP on follow-up cultures from 102 dogs once a clinical cure was attained MRSP prevalence [n (%)] Dogs with MRSP pyoderma on presentation (n = 42) Dogs with a non-mrsp pyoderma on presentation (n = 60) Skin Carriage site* 19 (45.2) 20 (47.6) 17 (28.3) 16 (26.7) *Carriage site refers to nose and or rectum. Refers to the total number of dogs that were available for follow-up and were diagnosed with MRSP pyoderma on presentation. Refers to the total number of dogs that were available for follow-up and were diagnosed with a non-mrsp pyoderma on presentation. ª 2012 ESVD and ACVD, Veterinary Dermatology, 23, 369 e67. 371

Beck et al. Control population Meticillin-resistant coagulase-positive staphylococci were not found at any of the sites on the healthy control dogs at either time point. Treatments Topical therapy alone was used to treat two of 60 (3.3%) dogs with a non-mrsp pyoderma that were available for follow-up and nine of 42 (21%) dogs with an MRSP pyoderma that were available for follow-up. The most common topical therapy consisted of 2 or 4% chlorhexidine shampoo, with or without topical mupirocin. Concurrent systemic antimicrobial therapy was used to treat the remainder of the dogs. The systemic antimicrobials used in the dogs that initially presented with a non-mrsp pyoderma and no MRSP carriage are listed in Table 8. Clindamycin exposure was associated with the acquisition of MRSP on follow-up (P = 0.011). Further study would be needed to corroborate this finding and investigate possible reasons. There was unlikely to be any bias involved in the patients with sensitive infections for whom clindamycin was prescribed versus another antibiotic, because the main factor that determined the use of cefalexin versus clindamycin was drug availability. Cefalexin was unavailable for periods of time during the study. Clindamycin was also chosen for two dogs that had a sensitivity to cefalexin, one dog with a deep bacterial pyoderma, three dogs with MRSA and two dogs with MRSScoag. Discussion The prevalence of meticillin-resistant staphylococcal pyoderma (particularly MRSP) in this study was high, a rate that is both striking and concerning. Yet, it is important to consider the study population. This study was performed at a dermatology referral practice, and it is reasonable to Table 8. Treatment used in 58 dogs without MRSP, at any location, on initial presentation* Treatment Dogs with no MRSP on follow-up (n = 37) Dogs with MRSP on follow-up (n = 21) Cefalexin 22 7 Clindamycin 4 7 Cefovecin 5 2 Amoxicillin clavulanic acid 1 1 Marbofloxacin 1 0 Enrofloxacin 0 0 Cefalexin and enrofloxacin 1 0 Clindamycin and enrofloxacin 1 1 Cefalexin, changed to cefovecin 1 0 Clindamycin, changed to cefovecin 0 1 Cefalexin, changed to clindamycin 0 1 Topical treatment only 1 1 *Refers only to dogs that had follow-up cultures performed. Refers to dogs colonized with MRSP at any of the three locations (skin, nose or rectum). Antibiotic was changed part way through treatment due to adverse effects. Antibiotic was changed part way through treatment due to cefalexin becoming unavailable. suspect that the prevalence of resistance could be higher amongst a referral population. In this population, there may be a bias towards animals that have failed empirical therapy and or have recurrent disease and accompanying recurrent antimicrobial exposure, even though this study could not find an association between prior antimicrobial administration and MRSP colonization or infection. With such a high rate of resistance, empirical antimicrobial usage with many of the common antimicrobials would have been ineffective in a significant percentage of our cases, potentially resulting in prolonged treatment times and high failure rates. This highlights the importance of an accurate understanding of the prevalence of meticillinresistant staphylococcal infections in individual veterinary practices and the usefulness of routine culture and susceptibility testing, particularly in practices where meticillin resistance rates are high or increasing. As studies of MRSP and MRSA increase, it is important to consider microbiological differences between these two organisms that might impact screening methods. This was highlighted by the finding in this study that MRSA Chromogenic agar was poorly sensitive for detection of MRSP and MRSS compared with MSA-OX, while it was more effective for recovery of MRSA. This is presumably accounted for by the antimicrobial present in the selective media. MSA-OX contains oxacillin, which has been shown to be a better indicator of meticillin resistance in MRSP. 21 In contrast, oxacillin is a poor indicator of meticillin resistance in MRSA, for which cefoxitin is the recommended indicator. 26 Therefore, the discrepancy between recovery rates with these two media is not surprising, but it highlights the potential for underestimation of the prevalence of MRSP or MRSA if only one type of selective medium is used. Meticillin-resistant S. aureus was isolated infrequently from dogs in this study. This is in agreement with previous studies that report low incidence rates of S. aureus in dogs, which may be due to the fact that S. aureus is not a predominant commensal of dogs. 7,14,27 Although MRSA is still of concern because treatment options may be limited and there is potential for zoonotic transmission, 4,28 31 MRSP is clearly the more important canine health concern. Meticillin-resistant S. schleiferi ssp. coagulans was also isolated infrequently, similar to previous studies. 7,8 The role of coagulase-negative staphylococci in bacterial pyoderma is poorly understood, probably in part because speciation of coagulase-negative staphylococci is uncommonly performed. Historically, coagulasenegative staphylococci have been considered minimally pathogenic. 32 Broad characterization of coagulase-negative species may be misleading. In particular, there is recent evidence that S. schleiferi ssp. schleiferi is an opportunistic pathogen in dogs, something that is of concern given the high rate of meticillin resistance in this species. 8,33,34 In this study, the majority of MR-CoNS were isolated concurrently with a coagulase-positive Staphylococcus spp. However, meticillin-resistant S. schleiferi ssp. schleiferi was isolated alone from the skin in two dogs, and meticillin-resistant S. epidermidis was the sole isolate in one dog. These isolates were considered pathogenic and of significance in these three dogs. 372 ª 2012 ESVD and ACVD, Veterinary Dermatology, 23, 369 e67.

MRSP in canine pyoderma Meticillin-resistant staphylococci were frequently recovered from carriage sites in the affected dogs. This is not unexpected, because the nares, oral cavity and anal mucosa are postulated to be the source of the staphylococcal population that colonizes the skin. 12,13,35 However, a study such as this cannot determine whether the presence of the organism on the skin is the result or cause of colonization of other body sites. The more frequent isolation of MRSP or MRSScoag from the nose or rectum of dogs with pyoderma caused by those pathogens is logical. There was not an association between MRSA pyoderma and MRSA colonization, but this may simply be the result of lack of statistical power with the small number of MRSA pyoderma cases. Nasal carriage was more frequent than rectal carriage in dogs colonized with MRSA, whereas dogs with MRSP and MRSScoag were colonized equally in both locations. This may reflect the frequent close proximity of dogs noses and their human caregivers. It has been suggested that MRSA in dogs is more likely to be transmitted from humans to dogs, although the direction of transmission has not yet been elucidated. 4,14,30,36 It may also represent a true biological predisposition of S. aureus for the nasal passages of dogs compared with other sites. A predominance of nasal over rectal colonization has been previously reported, 37 although other studies have reported no significant difference 38 or higher yield from rectal swabs. 39 Clearly, more information about the ecology of MRSA in dogs is required. Screening results after clinical resolution were striking and of significant concern from the standpoint of infection control. Of dogs that initially had an MRSP pyoderma, 26 of 42 (61.9%) were colonized at one or more sites at follow-up, even though the pyoderma had resolved. This is perhaps not surprising, because the goal of treatment is clinical cure, not microbiological cure, and S. pseudintermedius is a well-adapted canine commensal. These data indicate that a dog cannot be assumed to be free of MRSP after clinical resolution, something that may be of relevance for infection control in veterinary hospitals. The duration of MRSP colonization has not been adequately studied, although the data here indicate that colonization may persist for weeks. These data also suggest that systemic treatment with an antimicrobial effective in vitro against MRSP is unlikely to be effective for decolonization therapy. The number of dogs that had a sensitive staphylococcal infection at presentation which became carriers of MRSP on their skin or carriage sites at follow-up was astounding. This study could not establish what factors led to the acquisition of MRSP after treatment of the pyoderma, or the source of exposure. It is possible that these dogs had a heterogeneous staphylococcal population initially, with small subpopulations of resistant organisms which were not detected by initial culture. This, however, is unlikely to account for the large difference in pre- and posttreatment results, given the selective culture media used. Emergence of resistance among endogenous meticillinsensitive S. pseudintermedius is unlikely because of the relatively poor transmissibility of SCCmec, the large genetic element that contains meca. While this can occur, it is exceedingly unlikely that it occurs this often. The highly clonal nature of MRSP in dogs, against a background of meticillin-sensitive S. pseudintermedius with much genetic diversity, is indicative of successful dissemination of a few clones that acquired meticillin-resistance rather than frequent de novo emergence of resistance amongst endogenous meticillin-sensitive S. pseudintermedius. 17,40 It is more likely that the selection pressure exerted by antimicrobial therapy facilitated colonization of MRSP from other sources. Potential sources of exposure include dogs in the general population, environments frequented by dogs and the veterinary hospital environment, but no assessment of possible sources was performed here. The potential for routine use of antibacterials to have such a dramatic effect on MRSP colonization is quite concerning and could be an important factor accounting for the rapid dissemination of this pathogen. This emphasizes the importance of repeating culture and sensitivity in dogs with bacterial pyoderma upon relapse or in those dogs that fail to respond to appropriate treatment. The lack of acquisition of MRSP in our healthy control population suggests that affected dogs acquire MRSP with increased frequency, although further specific study of risk factors for MRSP acquisition is needed. In addition to the population bias discussed above, other limitations must be considered. There is currently no gold standard for MRSP and MRSA screening in dogs, either for sites to be tested or laboratory procedures. 21,33,41,42 This study was performed in only one region, and extrapolation to other geographic areas should be done with caution. Follow-up samples were collected at a single point in time; therefore, the duration of colonization cannot be established, nor can persistent colonization be differentiated from re-exposure. It would be beneficial to investigate how long dogs remain colonized with MRSP, with more frequent testing over an extended period of time, and whether persistent colonization with MRSP increases the likelihood of future infections as has been demonstrated in humans with MRSA colonization. 43 There was no association in this study between prior antimicrobial exposure and meticillin-resistant staphylococcal infection or colonization. Previous studies report fluoroquinolone treatment as a risk factor for meticillinresistant infections in both dogs and humans, 44,45 but neither fluoroquinolones nor any other antimicrobial class were identified as risk factors in our patients. Interestingly, there was an association between clindamycin treatment in dogs without MRSP infection or colonization and subsequent identification of MRSP at recheck following clinical cure. This has not been reported, and it is unclear why clindamycin would pose an increased risk compared with cefalexin. Further study of antimicrobial influences on MRSP acquisition is needed. The trend towards a decreased likelihood of MRSP infection in dogs treated with glucocorticoids or ciclosporin is not surprising. At least 63% of the subjects had atopic dermatitis as an underlying aetiology of their bacterial pyoderma. Controlling the underlying inflammation and pruritus in these patients may result in fewer secondary infections and less antibacterial usage. This study identified a high prevalence of MRSP in dogs with bacterial pyoderma and frequent colonization of carriage sites. This raises the question of whether empirical ª 2012 ESVD and ACVD, Veterinary Dermatology, 23, 369 e67. 373

Beck et al. antibiotic usage for canine bacterial pyoderma should still be a standard practice or whether more targeted approaches, such as topical biocides, should be used more widely. Study on the efficacy of topical therapy, along with impact of topical therapy on MRSP emergence and persistence, is needed. The international epidemic of antimicrobial resistance in companion animal patients is perhaps most clearly demonstrated by MRSP in canine pyoderma. Further study of the reasons for the emergence and rapid dissemination of MRSP (and other meticillin-resistant staphylococci) and investigation of measures to reduce the prevalence and impact of these pathogens are required. Acknowledgements The authors would like to thank Joyce Rousseau for assistance with specimen processing and data entry. References 1. Cox HU. Staphylococcal infections. In: Greene CE, ed. Infectious Diseases of the Dog and Cat, 3rd edition. St Louis, MO: Saunders Elsevier, 2006: 316 320. 2. 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Saunders Co., 2001: 86 99. 23. Scott DW, Miller WH, Griffen CE. Bacterial Skin Diseases. In: Muller and Kirk s Small Animal Dermatology. 6th edition. Philadelphia, PA: W.B. Saunders Co., 2001: 288 296. 24. Sasaki T, Tsubakishita S, Tanaka Y et al. Multiplex-PCR method for species identification of coagulase-positive staphylococci. J Clin Microbiol 2010; 48: 765 769. 25. Sasaki T, Kikuchi K, Tanaka Y et al. Reclassification of phenotypically identified Staphylococcus intermedius strains. J Clin Microbiol 2007; 45: 2770 2778. 26. Smyth RW, Kahlmeter G. Mannitol salt agar-cefoxitin combination as a screening medium for methicillin-resistant Staphylococcus aureus. J Clin Microbiol 2005; 43: 3797 3799. 27. Fulham KS, Lemarie SL, Hosgood G et al. In vitro susceptibility testing of meticillin-resistant and meticillin-susceptible staphylococci to mupirocin and novobiocin. Vet Dermatol 2010; 22: 88 94. 28. Coughlan K, Olsen KE, Boxrud D et al. 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MRSP in canine pyoderma 36. Baptiste KE, Williams K, Williams NJ et al. Methicillin-resistant staphylococci in companion animals. Emerg Infect Dis 2005; 11: 1942 1944. 37. Floras A, Lawn K, Slavic D et al. Sequence type 398 meticillinresistant Staphylococcus aureus infection and colonisation in dogs. Vet Rec 2010; 166: 826 827. 38. Kottler S, Middleton JR, Perry J et al. Prevalence of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus carriage in three populations. J Vet Intern Med 2010; 24: 132 139. 39. Lefebvre SL, Reid-Smith RJ, Waltner-Toews D et al. Incidence of acquisition of methicillin-resistant Staphylococcus aureus, Clostridium difficile, and other health-care-associated pathogens by dogs that participate in animal-assisted interventions. JAm Vet Med Assoc 2009; 234: 1404 1417. 40. Bannoehr J, Ben Zakour NL, Waller AS et al. Population genetic structure of the Staphylococcus intermedius Group: insights into agr diversification and the emergence of methicillin-resistant strains. J Bacteriol 2007; 189: 8685 8692. 41. Bemis DA, Jones RD, Hiatt LE et al. Comparison of tests to detect oxacillin resistance in Staphylococcus intermedius, Staphylococcus schleiferi, and Staphylococcus aureus isolates from canine hosts. J Clin Microbiol 2006; 44: 3374 3376. 42. Schissler JR, Hillier A, Daniels JB et al. Evaluation of Clinical Laboratory Standards Institute interpretive criteria for methicillinresistant Staphylococcus pseudintermedius isolated from dogs. J Vet Diagn Invest 2009; 21: 684 688. 43. Safdar N, Bradley EA. The risk of infection after nasal colonization with Staphylococcus aureus. Am J Med 2008; 121: 310 315. 44. Faires MC, Traverse M, Tater KC et al. Methicillin-resistant and - susceptible Staphylococcus aureus infection in dogs. Emerg Infect Dis 2010; 16: 69 75. 45. Weber SG, Gold HS, Hooper DC et al. Fluoroquinolones and the risk for methicillin-resistant Staphylococcus aureus in hospitalized patients. Emerg Infect Dis 2003; 9: 1415 1422. 46. Olivry T, DeBoer DJ, Favrot C et al. Treatment of canine atopic dermatitis: 2010 clinical practice guidelines from the International Task Force of Canine Atopic Dermatitis. Vet Dermatol 2010; 21: 233 248. 47. Olivry T, DeBoer DJ, Prélaud P et al. Food for thought: pondering the relationship between canine atopic dermatitis and cutaneous adverse food reactions. Vet Dermatol 2007; 18: 390 391. Résumé Contexte Les staphylocoques résistants à la méticilline sont des pathogènes importants en dermatologie vétérinaire. Cependant, nous manquons d études longitudinales de l impact des traitements antimicrobiens de routine sur l émergence ou la résolution des résistances. Objectifs Déterminer la prévalence des staphylocoques résistants à la méticilline sur la peau et les sites de portage chez les chiens atteints de pyodermite bactérienne et évaluer la prévalence de colonisation des MRSP après le succès du traitement d une pyodermite. Animaux 173 chiens présentés à un service de dermatologie référée atteints de pyodermite et 41 chiens contrôles sains. Méthode Des écouvillons cutanés, nasaux et rectaux ont été prélevés pour culture bactérienne au moment du référé et aprèsrésolution clinique de la pyodermite. La résistance à la méticilline a été confirmée par la mise en évidence de l antigène de la protéine se liant à la pénicilline (PBP2a). Résultats Initialement, les cultures cutanées ont révélé la présence de MRSP chez 70 chiens (40.5%), de Staphylococcus aureus résistant à la méticilline (MRSA) dans 3 cas (1.7%) et de Staphylococcus schleiferi ssp. coagulans résistant à la méticilline (MRSScoag) dans 5 cas (2.9%). Les échantillons collectés à partir du nez et du rectum (sites porteurs) ont révélé des MRSP dans 59 cas (34.1%), des MRSA dans 11 cas (6.4%) et des MRSScoag dans sept cas (4.0%). Des cultures de suivis ont pu être réalisées chez 102 chiens après la guérison clinique. Sur les 42 chiens atteints initialement de pyodermite à MRSP, les MRSP ont été isolés lors du suivi sur la peau dans 19 cas (45.2%), et sur les sites de portage dans 20 cas (47.6%). Sur les 60 chiens qui n étaient pas initialement atteints de pyodermite à MRSP, les MRSP ont été isolés après le traitement à partir de la peau dans 17 cas (28.3%) et la présence de MRSP au niveau des sites de portage a augmenté de 7.8% initialement à 26.7% (P = 0.0022). Conclusions et importance clinique La colonisation des MRSP persiste souvent après la résolution de la pyodermite. L acquisition de MRSP en cours de traitement semble être fréquente. Resumen Introducción los estafilococos resistentes a meticilina son agentes patógenos significativos en dermatología veterinaria, pero aun faltan estudios longitudinales acerca del impacto de la terapia antimicrobiana rutinaria en la aparición o en la resolución de la resistencia. Objetivos determinar el predominio de estafilococos resistentes a meticilina en piel y en otros órganos que portan bacterias en perros con pioderma bacteriana y evaluar el predominio de la colonización de MRSP después del tratamiento adecuado de la pioderma. Animales 173 perros que se presentaron a un servicio de referencia en dermatología con pioderma, 41 perros sanos como control. Métodos se obtuvieron muestras con hisopos de la piel, nasales y rectales para cultivo bacteriano y fueron recogidas durante la primera visita y después de la resolución clínica de la pioderma. La presencia de resistencia a meticilina fue confirmada por la demostración del antígeno 2a de la proteína de unión a la penicilina. Resultados inicialmente, los cultivos de la piel resultaron con MRSP en 70 (40,5%) perros, Staphylococcus aureus resistente a meticilina (MRSA) en tres (1,7%) y S. scheliferi ssp. coagulans (MRSScoag) en cinco (2,9%). Las muestras obtenidas de la nariz y el recto (lugares de transporte asintomático) dieron MRSP en 59 (34,1%) perros, MRSA en 11 (6,4%) y MRSScoag en siete (4,0%). 102 perros estuvieron disponibles para seguimiento y cultivo bacteriano tras la curación clínica. De 42 perros diagnosticados ini- ª 2012 ESVD and ACVD, Veterinary Dermatology, 23, 369 e67. 375

Beck et al. cialmente con pioderma por MRSP, MRSP fue aislado en el seguimiento en 19 (45,2%), y en 20 sitios portadores (47,6%). De 60 perros que no tenían pioderma por MRSP inicialmente, MRSP fue aislado post-tratamiento en la piel en 17 (28,3%) y MRSP de los portadores aumentó de un 7,8% (inicialmente) a un 26,7% (P = 0,0022). Conclusiones e importancia clínica La colonización por MRSP persiste a menudo después de la resolución de la pioderma por MRSP. La adquisición de MRSP durante el tratamiento parece ser un fenómeno común. Zusammenfassung Hintergrund Meticillin resistente Staphylokokken stellen signifikante Pathogene in der Veterinärdermatologie dar, obwohl es zurzeit noch keine Langzeitstudien, die den Einfluss routinemäßiger antimikrobieller Therapie auf die Entstehung oder das Verschwinden von Resistenzen, gibt. Ziele Das Vorkommen von Meticillin-resistenten Staphylokokken auf der Haut und auf den typischen Trägerstellen von Hunden mit bakterieller Pyodermie zu bestimmen und das Vorkommen einer Kolonisierung mit MRSP nach erfolgreicher Behandlung einer Pyodermie zu evaluieren. Tiere 173 Hunde, die in einem Überweisungszentrum für Dermatologie mit Pyodermie vorgestellt wurden, 41 gesunden Kontrollhunde. Methoden Haut, Nasen- und Rektaltupfer für eine Bakterienkultur wurden zum Zeitpunkt der Überweisung und nach der klinischen Abheilung der Pyodermie entnommen. Eine Meticillin Resistenz wurde durch den Nachweis des Penicillin bindenden Proteins 2a Antigen nachgewiesen. Ergebnisse Zu Beginn ergaben die Hautkulturen bei 70 Hunden (40,5%) MRSP, bei drei Hunden (1,7%) Meticillin-resistenten Staphylococcus aureus (MRSA) und bei fünf Hunden (2,9%) Meticillin-resistenten Staphylococcus schleiferi ssp. coagulans (MRSScoag). Proben, die aus der Nase und dem Rektum (Trägerstellen) genommen wurden, ergaben bei 59 Hunden (34,1%) MRSP, bei 11 Hunden (6,4%) MRSA und bei sieben Hunden (4,0%) MRSScoag. Von 102 Hunden gab es nach der klinischen Heilung so genannte,,follow-up Kulturen. Von 42 Hunden, bei denen ursprünglich eine MRSP Pyodermie diagnostiziert worden war, wurde durch die Folgekultur bei 19 (45,2%) Hunden MRSP von der Haut und bei 20 Hunden (47,6%) von den Trägerstellen isoliert. Von 60 Hunden, die zu Beginn keine MRSP Pyodermie hatten, wurde bei 17 Hunden (28,3%) MRSP nach der Behandlung der Haut isoliert und von den Trägerstellen isolierter MRSP nahm von 7,8% (zu Beginn) auf 26,7% (P = 0,0022) zu. Zusammenfassung und klinische Bedeutung Die Kolonisierung mit MRSP bleibt häufig nach Abheilung einer MRSP Pyodermie bestehen. Das Auftreten von MRSP während der Behandlung scheint häufig vorzukommen. e66 ª 2012 ESVD and ACVD, Veterinary Dermatology, 23, 369 e67.

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