Interaction of chlorhexidine with trisEDTA or miconazole in vitro against canine meticillin-resistant and -susceptible Staphylococcus pseudintermedius isolates from two UK regions

Introduction

In the light of the emergence of multidrug-resistant (MDR) staphylococci, especially meticillin-resistant Staphylococcus pseudintermedius (MRSP) in veterinary medicine,1 the role of antiseptics is becoming increasingly important. Improved treatment strategies for staphylococcal skin disease are needed to limit the spread of MRSP and to reduce selective pressure from repeated systemic antimicrobial therapy. Topical therapy, in particular with chlorhexidine, has been recommended as an option for treatment of canine superficial pyoderma.2

Previous studies have shown that chlorhexidine shampoo is an effective monotherapy in canine superficial pyoderma,3, 4 and in vitro data indicate that chlorhexidine is effective against MRSP as well as its meticillin-susceptible counterpart (MSSP), and the predominant human commensal (S. aureus; both meticillin-resistant and susceptible strains).5, 6 Genes that encode drug efflux proteins in S. aureus, including qacA/B and smr which confer low level and high level chlorhexidine resistance, respectively,7, 8 have rarely been described in S. pseudintermedius.9, 10 However, the clinical relevance of this has not been assessed. The development of biocide resistance in S. pseudintermedius might well be minimized by combining chlorhexidine with other agents with additive or synergistic effects. Current products used for canine superficial skin infections include shampoos containing chlorhexidine alone, or in combination with miconazole or tromethamine + ethylenediaminetetraacetic acid (trisEDTA). Minimum inhibitory concentrations (MICs) have been established for miconazole and chlorhexidine in combination, with synergistic and additive interactions being shown.5 A shampoo formulation of chlorhexidine and miconazole was shown to significantly reduce coagulase-positive staphylococcal counts on seborrhoeic canine skin when determined by cup-scrub in a blinded study design.11 It was reported that chlorhexidine and trisEDTA was more active than chlorhexidine alone against S. aureus,12 although fractional inhibitory concentration (FIC) indices were not used to assess the interaction between the drugs. TrisEDTA has been shown to enhance the efficacy of marbofloxacin and gentamicin13 against Pseudomonas aeruginosa, and has been shown to act synergistically with cefalexin, oxytetracycline, ampicillin, streptomycin and sulfadimethoxine against S. aureus.14 This is thought to be due to EDTA damaging the outer cell wall, causing the bacteria to become more permeable to the antimicrobial agents that act intracellularly.13

There has been limited investigation into the potential synergistic activity of different ratios of trisEDTA amongst chlorhexidine products; furthermore MICs for chlorhexidine/miconazole have not yet been compared to chlorhexidine/trisEDTA. We therefore determined MICs of chlorhexidine, miconazole, trisEDTA and the combination of chlorhexidine with either miconazole or trisEDTA in two ratios, in a large collection of MRSP and MSSP isolates obtained from the northern and southeastern regions of the United Kingdom.

Materials and methods

Results

TrisEDTA (16:1 ratio) alone did not inhibit growth (MIC ≥ 512 mg/L, n = 196) and thus was omitted from analysis in the linear mixed effect model. MICs of the 1:1 combination of chlorhexidine and miconazole in the initial MIC determination ranged from 0.25 to 2 mg/L (Table 1), with 90.3% (177 of 196) of isolates having an MIC of 0.25 or 0.5 mg/L. Values were very closely similar in the repeat analysis of 185 isolates with a range of 0.25–1 mg/L and 75% of isolates (139 of 185) having an MIC of 0.25 or 0.5 mg/L (Table 3). For the whole collection of 196 isolates, chlorhexidine/miconazole MICs (MIC₉₀ = 0.5 mg/L) were significantly lower than chlorhexidine alone (P < 0.0005; MIC₉₀ chlorhexidine = 2 mg/L), chlorhexidine/trisEDTA 80:16:1 combination (P < 0.0005; MIC₉₀ 80:16:1 2 mg/L) and miconazole alone (P < 0.005; MIC₉₀ miconazole = 1 mg/L; except in NUK MSSP). Miconazole MICs were significantly lower than chlorhexidine alone or in 80:16:1 combination with trisEDTA (P < 0.005, Table 1).

Combination with miconazole reduced the chlorhexidine MICs by zero (n = 1), one (n = 100), two (n = 83) or three dilutions (n = 12). In 79 of 196 isolates, an additive interaction was observed between miconazole and chlorhexidine (0.5 < FICi ≤ 1; Table 2).20 No antagonistic interactions were observed; all other isolates fell into the ‘no interaction’ group.

MICs for chlorhexidine/trisEDTA in 80:16:1 combination (MIC₉₀ 2 mg/L, range 0.5–16 mg/L, Table 1) were significantly lower than that of chlorhexidine alone in all bacterial groups (P < 0.05) except SEUK MRSP. A 80:16:1 combination with trisEDTA reduced the chlorhexidine MICs by one dilution in 43 cases, and had no effect in 150. There was an additive interaction between chlorhexidine and trisEDTA in a 80:16:1 combination in 21.9% isolates (43/196) whereas three isolates (1.5%) showed an antagonistic interaction where the MIC was increased by one dilution (Table 2).

As with the chlorhexidine/miconazole combination, MICs for the 80:5:1 ratio of chlorhexidine/TrisEDTA were closely comparable to those seen with the 80:16:1 ratio; the MIC₅₀ and MIC₉₀ for both combinations were 1 and 2 mg/L, respectively, and the lowest MIC was 0.5 mg/L in both studies (Table 3). As before, the chlorhexidine/miconazole combination (MIC₅₀ = 0.5 mg/L; MIC₉₀ = 1.0 mg/L) had greater in vitro activity (P < 0.0005) than the combination of chlorhexidine and TrisEDTA.

Amongst the MSSP, NUK isolates were significantly more susceptible to all drugs (except 80:5:1 chlorhexidine/trisEDTA) than SEUK isolates (P < 0.05; Table 4). In the MRSP groups, SEUK isolates were significantly less susceptible to miconazole than NUK isolates (P > 0.0005); however, no significance was found in differences between NUK and SEUK MRSP for the other drugs.

MRSP had significantly higher MICs of miconazole (NUK MRSP MIC₉₀ = 0.5 mg/L, SEUK MRSP MIC₉₀ = 1 mg/L) than MSSP (NUK MSSP MIC₉₀ = 0.5 mg/L, SEUK MSSP MIC₉₀ = 0.5 mg/L; P < 0.05; Table 4). NUK MRSP had significantly higher MICs of chlorhexidine and chlorhexidine/trisEDTA 80:16:1 combination when compared with MSSP; however, no differences were seen in other MRSP/MSSP comparisons (Table 4). Reference strains had MICs equal to or within one dilution of previously established values;5 the quality control MSSA isolate (B122) showed greater susceptibility to miconazole than previously determined, reflecting the overall increased susceptibility to miconazole seen in these results. MICs varied by a single dilution between replicates on only six occasions.

Discussion

Continued spread of MRSP and the potential for the emergence of tolerance to chlorhexidine8, 9 warrants the monitoring of the efficacy of these combination products. Further investigation into the interaction of these combinations may indicate whether additive or synergistic combinations can be used to delay development of resistance, as well as to improve the efficacy of chlorhexidine. The efficacy of miconazole in this study against staphylococci is not unexpected considering previous research documenting its anti-staphylococcal activity.21 The good activity of chlorhexidine and miconazole in combination against MRSP and MSSP is in accordance with and supports the results of a study of isolates originating from both the UK and Germany.5 Previously, both synergistic and additive effects have been observed with miconazole and chlorhexidine, whereas only additive effects were noted in the present study; these differences appear to reflect a somewhat higher activity of miconazole alone. A degree of variation is well recognized in MIC determinations even when standardized protocols are followed.22 Otherwise, MICs of field and reference strains were similar to values reported previously and there was good repeatability in the data within this study. The lower MICs and increased rates of additivity in this study indicated that the combination of miconazole/chlorhexidine is more efficacious in vitro than both tested combinations of trisEDTA/chlorhexidine.

The combination of trisEDTA with chlorhexidine resulted in lower MICs than with chlorhexidine alone (in most cases); however, this disparity appears modest in comparison to the enhancement seen when trisEDTA is combined with marbofloxacin or gentamicin against Pseudomonas aeruginosa.13 This may reflect a difference between the effect of TrisEDTA on Gram-positive and -negative bacteria, or could indicate that the mechanism of activity of chlorhexidine is not improved by the increased permeability afforded to the bacteria by the action of TrisEDTA. The mechanisms of interaction of these combinations warrants further investigation. The emergence of MRSP is attributed to the expansion of a few dominant lineages, rather than the acquisition of resistance genes by multiple lineages;1 this is similar to the clonal expansion of MRSA in human medicine. This may account for the lack of regional differences in MIC amongst this study's MRSP isolates. Greater regional variability in MSSP MICS is likely to reflect greater genetic diversity in this group,9 but the overall low MICs for both NUK and SEUK regions indicates that there should be no difference in response to treatment with these combinations according to region.

The low MICs in this study showed that chlorhexidine shampoos are rational choices for treatment of canine superficial pyoderma associated with S. pseudintermedius, including MRSP. The combination of chlorhexidine with miconazole showed apparent superiority over combination with trisEDTA in vitro, although product formulation, ability of the antiseptic to penetrate into the infected area of skin and client compliance may have confounding effects on treatment that must be investigated through in vivo studies. The very limited differences in susceptibility amongst MSSP and the apparent absence of regional differences amongst MRSP indicates that topical therapy remains an important option for veterinarians in the treatment of canine superficial pyoderma.