Susceptibility in vitro of canine methicillin-resistant and -susceptible staphylococcal isolates to fusidic acid, chlorhexidine and miconazole: opportunities for topical therapy of canine superficial pyoderma

Objectives Increasing multidrug resistance amongst canine pathogenic staphylococci has renewed interest in topical antibacterial therapy for skin infections in the context of responsible veterinary prescribing. We therefore determined the activity in vitro of three clinically relevant topical agents and synergism between two of them against Staphylococcus pseudintermedius and Staphylococcus aureus. Methods The MICs of fusidic acid (n = 199), chlorhexidine (n = 198), miconazole (n = 198) and a 1:1 combination of miconazole/chlorhexidine (n = 198) were determined for canine isolates [50 MRSA and 49 methicillin-resistant S. pseudintermedius (MRSP), 50 MSSA and 50 methicillin-susceptible S. pseudintermedius (MSSP)] collected from the UK and Germany using an agar dilution method (CLSI VET01-A4). Fractional inhibitory concentration (FIC) indices were calculated to assess the interaction of miconazole with chlorhexidine. Results MICs of each drug/combination were significantly (P < 0.0005) higher for S. aureus when compared with S. pseudintermedius. Most strains (n = 172) had an MIC of fusidic acid of ≤0.03 mg/L (MIC ≥64 mg/L, n = 5 MRSA). All strains had MICs of chlorhexidine of 0.5–4 mg/L, except for one MRSA (MIC = 8 mg/L). All but four strains had MICs of miconazole of 1–4 mg/L (MIC = 16 mg/L, n = 3; MIC = 256 mg/L, n = 1). Miconazole/chlorhexidine (1:1 ratio) had a synergistic effect against 49/50 MRSA, 31/50 MSSA, 12/49 MRSP and 23/49 MSSP. Conclusions Since the majority of these staphylococci, including methicillin-resistant isolates, had MICs that should be readily exceeded by topical skin application of these agents, their therapeutic efficacy for canine superficial pyoderma should be assessed. The synergistic interaction shown in vitro supports further clinical evaluation of miconazole/chlorhexidine combination therapy for staphylococcal infection.


Introduction
The alarming increase in canine skin infections caused by methicillin-resistant Staphylococcus pseudintermedius (MRSP) and MRSA 1 paralleled by recognition of zoonotic (and reverse-zoonotic) infections, 2 highlights the urgent need to develop strategies to limit further emergence of MDR strains. Topical antibacterial therapy can provide an alternative treatment option for many dogs with bacterial skin infections and thus limit the need for oral antibiotics. 3 Licensed drugs include fusidic acid, chlorhexidine and a 1:1 shampoo combination of miconazole and chlorhexidine.
Although there is concern in human medicine over reduced efficacy of agents such as chlorhexidine and fusidic acid, 4,5 clinical evidence for treatment failure remains inconsistent. Assessment of resistance is hampered by lack of breakpoint standards for agents used topically. In dog-derived staphylococci, MICs of fusidic acid, chlorhexidine and miconazole have been low, 6 -8 but geographical differences can be expected. 9

Bacterial isolates
Coagulase-positive staphylococci [50 MRSA and 50 MSSA obtained in 2005-07 and 49 MRSP and 50 methicillin-susceptible S. pseudintermedius (MSSP) obtained in 2010-13] isolated from dogs were randomly selected from our collection. Staphylococcus aureus isolates were from canine infections in a UK-wide study. 11 Examples of S. pseudintermedius comprised clinical isolates (Germany, 25 MRSP and 25 MSSP; UK, 24 MRSP) and carriage isolates (UK, 25 MSSP). Species identification and methicillin resistance were confirmed using both phenotypic and genotypic (nuc, mecA) methods. 12,13 MIC determination MICs were determined by agar dilution (CLSI VET01-A4). 14 Prior to MIC determination, strains were subcultured twice on blood agar base (CM0271, Oxoid) containing 5% sheep blood (TCS Biosciences, Buckingham, UK) at 358C for 24 h. Stock solutions were prepared at 10× final concentration in distilled water (fusidic acid sodium salt F0881, chlorhexidine digluconate C9394; Sigma-Aldrich Inc.) or 1% DMSO (miconazole nitrate PHR1163; Sigma), adjusted for drug potency. 14 Final concentrations of the active fraction ranged from 0.015 to 2048 mg/L for fusidic acid and from 0.03 to 256 mg/L for chlorhexidine, miconazole and a 1:1 combination of chlorhexidine and miconazole. 14 Discrepancy between duplicate MICs was accepted provided they varied by only one dilution; in such cases, the higher value was identified as the final MIC. S. aureus ATCC 25923, S. aureus ATCC 29663 and S. pseudintermedius LMG 22219 were included for quality control purposes. Three MRSA strains previously reported with low, medium and high MICs of fusidic acid 6 were also included for comparative purposes.

Fractional inhibitory concentration (FIC) indices
The FIC index was calculated to analyse drug interaction of chlorhexidine and miconazole when used in the 1:1 combination, using the formula SFIC ¼FIC chlorhexidine +FIC miconazole ¼ (

Statistical analysis
SPSS version 21 (IBM UK Ltd, Portsmouth, UK) was used, with P,0.05 for significance. Since data were not normally distributed (Shapiro-Wilk test), the MIC values for different bacterial groups were compared using the Kruskal -Wallis test. Post hoc comparisons were performed using Mann-Whitney U-tests with Holm-Bonferroni adjustments.

Results
One MSSP strain failed to grow in studies of MICs of chlorhexidine, miconazole and miconazole plus chlorhexidine. MICs varied by a single dilution between replicates on only five occasions.

Miconazole
The MICs of miconazole ranged from 1 to 256 mg/L (median 2 mg/L); 175 of 198 isolates had MICs of 1 or 2 mg/L and 19 had MICs of 4 mg/L (Table 1). Four isolates had higher MICs, comprising single examples of MRSA, MSSA and MSSP with MICs of 16 mg/L and one MSSA with an MIC of 256 mg/L. S. aureus isolates (median 2 mg/L, n¼ 100) had higher MICs than S. pseudintermedius isolates (median 1 mg/L, n¼ 98) (P,0.0005). There was no difference (P ¼ 0.415) in MIC between the MRSA and MSSA groups; however, the MRSP group (median 2 mg/L, n ¼ 49) had a significantly (P, 0.0005) higher MIC than the MSSP group (median 1 mg/L, n ¼49).

Miconazole and chlorhexidine in combination
The MICs of the 1:1 combination of miconazole and chlorhexidine ranged from 0.25 to 8 mg/L (median 0.5 mg/L; Table 1); 190 of 198 isolates had an MIC of 0.25 or 0.5 mg/L and all but one MSSA isolate had an MIC of ≤2 mg/L. The MICs for the S. aureus isolates (median 0.5 mg/L) exceeded (P, 0.0005) those for the S. pseudintermedius isolates (median 0.5 mg/L), and the MICs for the MSSP group (median 0.25 mg/L) were lower (P, 0.0005) than for the other three groups. The MSSA isolate whose miconazole MIC was 256 mg/L had an MIC of 8 mg/L for the combination.
A synergistic interaction between miconazole and chlorhexidine (FIC index ≤0.5) was observed for 80 out of 100 S. aureus isolates and 35 out of 98 S. pseudintermedius isolates ( Table 2). Antagonistic interactions were not observed; the other strains fell into the 'no interaction' group as defined by Odds. 15 EUCAST guidelines 16

Discussion
The low MICs of fusidic acid, miconazole and chlorhexidine for the great majority of these canine staphylococcal isolates in vitro provide strong support for their use topically in clinical cases of surface and superficial pyoderma, including cases caused by methicillin-resistant bacteria.
The interpretation of MIC values in the context of susceptibility/ resistance is hindered by the absence of defined breakpoints; whilst EUCAST guidelines indicate a value of 2 mg/L for fusidic acid when used against S. aureus, this is of questionable value for canine skin infections treated topically rather than systemically.
The prevalence of high fusidic acid MICs for human-derived S. aureus appears related to geographical variation in prescribing practice in human medicine; 17 10.7% of human-derived European S. aureus had MICs ≥2 mg/L 9 compared with only 0.3% of isolates from North America, where fusidic acid usage is infrequent. 10 The comparable frequency of fusidic acid MICs ≥2 mg/L amongst our European canine MRSA isolates (10.0%) is perhaps not surprising since these strains likely originate from human sources. 6 The strikingly uniform MICs of chlorhexidine, almost exclusively in the narrow range of 0.5 -4 mg/L, are remarkably similar to those reported previously for MRSA isolates from human sources worldwide. 18 Similarly, the MICs of miconazole in the present study are comparable to those in previous reports indicating good efficacy against staphylococci. 8 The frequent synergistic activity of the combination of chlorhexidine and miconazole indicates potential clinical value as a combination treatment in dogs, especially as they lack a critical role in human medicine.
Monitoring for the presence, and potential for transfer, of resistance between populations of MRSA on dogs and humans, and between canine S. pseudintermedius and human S. aureus strains is warranted. Genetic studies are also required to explain the rare higher MICs that were identified in individual isolates. Previous authors have reported maximum values in the order of 16 mg/L miconazole for S. aureus and 8 mg/L fusidic acid for S. pseudintermedius; the value of 16 mg/L fusidic acid seen for a single MRSP strain might correspond to 'low-level fusidic acid resistance' 17 and explanation is needed for the single high MIC of miconazole of 256 mg/L.
This study provides further evidence to support topical therapies as an alternative to systemic antibacterial drugs in superficial pyoderma. Further work is needed to confirm the clinical efficacy of these topical treatments.