Usefulness of cefovecin disk-diffusion test for predicting mecA gene-containing strains of Staphylococcus pseudintermedius and clinical efficacy of cefovecin in dogs with superficial pyoderma
Background – Cefovecin has been widely used to treat skin infections in dogs. The relationship of the cefovecin disk-diffusion test results to the presence of the mecA gene and the clinical efficacy of cefovecin have not been fully evaluated.
Hypothesis/Objectives – To determine the usefulness of an in vitro cefovecin disk-diffusion test in predicting the presence of the mecA gene in Staphylococcus pseudintermedius, as well as the in vivo efficacy of cefovecin therapy in dogs with superficial pyoderma.
Methods – Twenty-six S. pseudintermedius strains isolated from 22 dogs with pyoderma were used. In vitro disk-diffusion test results of cefovecin were compared with agar-dilution test results, the presence of the mecA gene, and the improvement in clinical scores of dogs with superficial pyoderma at 14 days post treatment.
Results – There was a significant linear correlation (r = -0.83) between the diameter of the obvious zone of inhibition by disk diffusion and the minimal inhibitory concentration for cefovecin (P < 0.0001). Receiver operating characteristic analysis revealed that zone diameters between 25 and 27 mm exhibited better sensitivity (92.9%) and specificity (100.0%) for detection of strains carrying the mecA gene. The mean improvement in clinical scores in dogs carrying cefovecin-resistant strains was significantly lower than in dogs carrying cefovecin-susceptible strains (P < 0.01).
Conclusions and clinical importance – The cefovecin disk-diffusion test with a cut-off value estimated in this study was valuable for predicting mecA gene carriage in S. pseudintermedius, as well as the in vivo efficacy of cefovecin therapy in dogs with superficial pyoderma caused by S. pseudintermedius.
Superficial pyoderma is a common bacterial disease in dogs.1 Staphylococcus pseudintermedius is the major staphylococcal species isolated from lesions of canine superficial pyoderma.2–4 Cephalosporins are commonly recommended as the first choice for the treatment of canine superficial pyoderma.1 Cefovecin (Convenia®; Pfizer Animal Health, New York, NY, USA), a broad-spectrum and long-acting cephalosporin injectable antibiotic, has recently been approved for the treatment of skin infections in dogs. The pharmacokinetic and pharmacodynamic properties of cefovecin, which are characterized as having slow elimination and long-lasting antibacterial activity,5 allow veterinarians to prescribe the drug at 14 day intervals for the treatment of cutaneous infections in dogs. Two studies demonstrated that cefovecin is as effective as oral cefadroxil or amoxicillin-clavulanate for improving the clinical signs in dogs with pyoderma.6,7
Meticillin-resistant or multidrug-resistant staphylococci are increasingly being isolated from dogs with superficial pyoderma and are a therapeutic problem.3,8,9 Meticillin-resistant staphylococci express the penicillin-binding protein 2a (PBP2a), which is encoded by the mecA gene; thus, organisms possessing this gene show low affinity for all β-lactam antibiotics currently marketed for clinical use.1,10-12 It is therefore important to determine the presence of the mecA gene in clinical isolates of staphylococci prior to the selection of an antibiotic for the treatment of canine pyoderma.
The disk-diffusion and agar-dilution tests are used to determine susceptibility of bacteria to antimicrobial drugs. Disk-diffusion susceptibility tests are used in clinical laboratories because of their rapid and uncomplicated procedures. Oxacillin-susceptibility tests have been used to determine whether isolates are sensitive or resistant to meticillin. Meanwhile, detection of the mecA gene or PBP2a is considered an alternative method to detect meticillin-resistant staphylococcal strains.13,14 There is limited information on the correlation between disk-diffusion results and the presence of the mecA gene using clinical isolates from veterinary patients. The objectives of the present study were to determine the usefulness of an in vitro cefovecin disk-diffusion test for predicting the presence of the mecA gene in S. pseudintermedius and as a predictor of in vivo efficacy of cefovecin therapy in dogs with superficial pyoderma caused by S. pseudintermedius.
Materials and methods
Twenty-two client-owned dogs diagnosed with superficial pyoderma between September 2010 and July 2011 at Tokyo University of Agriculture and Technology Animal Medical Center, Japan, were enrolled in this study. The diagnosis of superficial pyoderma was made on the basis of compatible clinical signs (papules, erythema, scales, epidermal collarettes or a combination thereof) and cytological examination of skin lesions (infiltration of neutrophils with presence of extra- and intracellular cocci). After obtaining informed consent from owners, samples were obtained and cefovecin (8 mg/kg) was injected subcutaneously for the treatment of the superficial pyoderma. Dogs were excluded from the study if they had ectoparasitic infestations or fungal skin infections or were being treated with antiseptic shampoos during the cefovecin trial. Previous history of antibiotic use, underlying diseases and concurrent nonantibiotic therapy were also recorded for all study dogs. All studies were given ethical approval by Tokyo University of Agriculture and Technology as a sponsored research project with Pfizer Japan Inc.
Clinical evaluation of canine superficial pyoderma
Clinical signs of superficial pyoderma were evaluated before (day 0) and after the administration of cefovecin (day 14) in accordance with the guidelines of the Japanese Society of Antimicrobials for Animals.15 Briefly, the severity and extent of erythema, papules or pustules, crusts and/or alopecia were scored using a scale from 0 (no visible lesions) to 4 (severe). The clinical score for each lesion type (e.g. erythema) was calculated by multiplying the lesion severity score by the lesion extent score. The total clinical score for each case was calculated as the sum of the clinical score for the four types of skin lesions. The improvement rate of the clinical score was calculated by the following formula: improvement score (%) = [(total clinical score at day 0 – total clinical score at day 14)/total clinical score at day 0] × 100. Improvement scores were standardized as 0% in cases in which total clinical scores worsened after cefovecin therapy. Dogs with total clinical scores <10 at day 0 were excluded from this study, because in such cases the clinical efficacy of cefovecin treatment was difficult to evaluate.
Bacterial samples were collected using sterile swabs from skin lesions on days 0 and 14. If the skin lesions were absent on day 14, bacterial samples were collected from the skin sites sampled on day 0. Samples were inoculated onto sheep blood agar plates (Nissui Pharmaceutical, Tokyo, Japan) and incubated at 37°C for 24 h. Catalase-positive colonies were selected for additional diagnostic testing. Genomic DNA was isolated from bacterial colonies using the Ultra Clean Microbial lsolation kit (MO BIO, Carlsbad, CA, USA). Identification of S. pseudintermedius was determined by PCR after amplification of the thermonuclease gene (nuc) using a previously reported primer pair.16 The PCR amplification was performed using AmpliTaq Gold 360 Master Mix (Applied Biosystems, Carlsbad, CA, USA) for 30 cycles of 30 s at 95°C, 30 s at 55°C and 60 s at 72°C. Polymerase chain reaction products were resolved by electrophoresis through 1.2% (w/v) agarose gels, and visualized using ultraviolet light after staining with ethidium bromide.
Disk-diffusion susceptibility test
Staphylococcus pseudintermedius strains were cultured in Luria-Bertani (LB) broth (Invitrogen, San Diego, CA, USA) and incubated at 37°C for 24 h. The culture was diluted in sterile saline, adjusted to 0.5 McFarland (McFarland Turbidity Standard No. 0.5; Becton, Dickinson and Co., Franklin Lakes, NJ, USA), and inoculated on Mueller–Hinton agar (Becton, Dickinson and Co.). An antimicrobial susceptibility disk containing 30 μg of cefovecin (Sensi-Disc®; Becton, Dickinson and Co.) was placed on culture plates, which were then incubated at 35°C for 16-18 h. Zone diameters with obvious growth inhibition were measured after culture.
The minimal inhibitory concentrations (MICs) of cefovecin were determined by the agar-dilution test with Mueller–Hinton agar (Oxoid, Cambridge, UK) in accordance with the guidelines of the Clinical Laboratory Standards Institute: CLSI Ninteenth Informational Supplement, M100-S19.17 The concentration range tested for cefovecin was 0.125-512 μg/mL.
Detection of mecA
The presence of the mecA gene in S. pseudintermedius isolates was detected by PCR analysis using a primer pair as previously reported.18 DNA amplification was performed as described in the section of “bacterial strains” to amplify the nuc gene.
Pearson’s correlation coefficient (two-tailed) test was used to confirm the correlation between inhibition zone diameters and MICs for cefovecin. Receiver operating characteristic (ROC) analysis was performed to estimate a breakpoint of the inhibition zone diameters for detection of the mecA gene. The Mann-Whitney U-test was used to compare the zone of inhibition diameters among mecA-positive and mecA-negative S. pseudintermedius isolates. Among dogs with pyoderma, Student’s paired t-test and Fisher’s exact test were used to compare the clinical efficacies of cefovecin therapy in dogs carrying S. pseudintermedius isolates with a zone of inhibition diameter above the estimated breakpoint and those below the breakpoint. All statistical analyses were carried out with STATVIEW software (version 5.0; Hulinks, Tokyo, Japan). A P-value of <0.05 was considered statistically significant.
Dogs and clinical data
There were 17 male dogs (five neutered) and five females (four spayed) in this study (Table 1). The most common breed was the miniature dachshund (n = 5). Possible underlying diseases for pyoderma were identified in five of 22 cases (18.2%), as follows: atopic dermatitis (n = 3), hypothyroidism (n = 1) and concurrent atopic dermatitis with hypothyroidism (n = 1). Prior therapies were noted in 13 dogs and included cephalosporin [nine of 22 dogs (40.9%)], recombinant canine interferon-γ (dog no. 2), prednisolone (dog no. 12), ciclosporin (dog no. 16), and allergen-specific immunotherapy (dog no. 21). Concurrent medical therapies were unchanged in dogs during the study (see Table 1).
Correlation between inhibition zone diameters and MICs
Disk-diffusion susceptibility and agar-dilution tests for cefovecin were performed on 36 S. pseudintermedius isolates cultured from the skin of the dogs (22 from day 0 and 14 from day 14; Table 2). The results of cefovecin disk-diffusion tests and MICs of 10 strains isolated on day 14 (SP nos. 23, 25, 27, 28, 29, 31, 32, 33, 34 and 36) were similar to 10 strains isolated on day 0 (SP nos 3, 5, 7, 8, 11, 15, 16, 19, 20 and 22) and were excluded from further statistical analysis; therefore, a total of 26 strains were characterized further. There was a significant linear correlation (r = -0.83) between the diameter of the zone of inhibition by disk diffusion and the MICs for cefovecin (Pearson’s correlation coefficient, two-tailed, P < 0.0001; Figure 1). The range of zone diameters of inhibition for cefovecin was 6-39 mm (mean, 26.5 mm). The MICs of seven strains with zones of inhibition ≤6 mm were ≥512 μg/mL (n = 5), 256 μg/mL (n =1) and 1 μg/mL (n = 1). The MICs of 13 strains whose zones of inhibition ≥30 mm were ≤0.25 μg/mL (Table 2).