Veterinary Dermatology 2005, 16, 330 333

Blackwell Publishing, Ltd.

Brief communication

Inhibition of the growth in vitro of Trichophyton mentagrophytes,

Trichophyton erinacei and Microsporum persicolor by
miconazole and chlorhexidine
NATALIE PERRINS*, SUSAN A. HOWELL, MARY MOORE and ROSS BOND*
*Department of Veterinary Clinical Sciences, Royal Veterinary College, Hawkshead Lane, North Mymms,
Hatfield, Herts AL9 7TA, Department of Medical Mycology, St Johns Institute of Dermatology,
St Thomas Hospital, Lambeth Palace Road, London SE1 7EH, UK
(Received 24 May 2005; accepted 16 August 2005)

Abstract An agar dilution technique was used to assess the minimum inhibitory concentrations (MICs) of
miconazole, chlorhexidine and a 1:1 combination of both agents for 9 isolates of Trichophyton mentagrophytes,
9 isolates of Trichophyton erinacei and 5 isolates of Microsporum persicolor. MICs of chlorhexidine did not vary
significantly between the three dermatophyte species tested, but the MICs of miconazole alone and in combination
with chlorhexidine for T. erinacei were significantly greater that those for T. mentagrophytes and M. persicolor. A
synergistic drug interaction was noted with one isolate of T. erinacei and one isolate of M. persicolor. An additive
effect was demonstrated for 13 isolates (5 T. mentagrophytes, 6 T. erinacei, 2 M. persicolor), and indifference was
noted in 8 isolates (4 T. mentagrophytes, 2 T. erinacei, 2 M. persicolor). Although synergy was less often seen when
compared with a previous study of Microsporum canis, the synergistic or additive effects seen with the majority
(15 out of 23) of isolates studied in vitro provides a rationale for the combined use of miconazole and chlorhexidine
in the adjunctive topical therapy of dermatophytosis caused by T. mentagrophytes, T. erinacei and M. persicolor.

Systemic therapy is advocated for the therapy of dermatophytosis of haired skin, reflecting the anatomical
location of fungal hyphae and spores deep within the
hair shafts and follicles.1 However, adjunctive topical
therapy may reduce environmental contamination and
the risk of transmission to other in-contact humans and
animals. Miconazole 2% and 2% chlorhexidine shampoo
have been shown to be of value when used in combination
with griseofulvin for the treatment of feline dermatophytosis caused by Microsporum canis.2,3 Furthermore,
we have previously demonstrated that miconazole and
chlorhexidine had a synergistic effect in vitro against 5
out of 10 strains of M. canis, and an additive effect against
four strains.4 Although M. canis was the most common
cause of dermatophytosis in dogs and cats in the UK, in
a survey spanning 35 years, the sylvatic dermatophytes
Trichophyton mentagrophytes, Trichophyton erinacei and
Microsporum persicolor, usually obtained from wildlife,
accounted for approximately 30% of 474 isolates obtained
from dogs.5 In order to evaluate the antifungal efficacy
of miconazole and chlorhexidine against these dermatophyte species, the minimum inhibitory concentrations
(MIC) of both drugs alone and in combination were
assessed in vitro.

Correspondence: Ross Bond, Department of Veterinary Clinical

Sciences, Royal Veterinary College, Hawkshead Lane, North
Mymms, Hatfield, Herts AL9 7TA, UK. E-mail: rbond@rvc.ac.uk
330

Nine isolates of T. mentagrophytes, nine of T. erinacei

and five isolates of M. persicolor obtained from animals
and humans were studied. The isolates were identified
according to gross colonial and microscopical morphology.6,7 Although some sources refer to T. erinacei
as T. mentagrophytes var. erinacei ,7 recent molecular
studies indicate that T. erinacei is a valid taxon.8 Previous
exposure to antifungal drugs was unknown. The isolates
were maintained on Sabourauds dextrose agar (SDA)
plates (Oxoid CM41 65 g L1, Oxoid, Basingstoke, Hants,
UK) and freshly cultured on SDA plates in 90-mm Petri
dishes at 26 C for 10 days prior to use.
An agar dilution assay was used to establish the
minimum inhibitory concentrations (MIC) of chlorhexidine, miconazole and a combination of both drugs at a
ratio of 1:1 against the dermatophytes using methods
described previously.4 Stock solutions (2.5 mg mL1) of
chlorhexidine diacetate (C-6143, Sigma, St. Louis, MO,
USA), miconazole nitrate (M-3512, Sigma) and both
drugs in combination in a 1:1 ratio were prepared by
solubilizing them in dimethylsulfoxide (DMSO, D-5879,
Sigma). Aliquots were added to bijou bottles and serial
twofold dilutions were made in DMSO. SDA medium
was autoclaved at 121 C for 15 min, cooled to 48 C in a
water bath and 24 mL of medium was then pipetted into
90 mm Petri dishes. Aliquots (1 mL) of the dissolved drug
were added to paired plates to give final concentration
ranging from 100 to 3.12 g mL1 for chlorhexidine, 3.12
0.024 g mL1 for miconazole, and 3.120.012 g mL1
2005 European Society of Veterinary Dermatology

Dermatophyte susceptibility testing

Table 1. Minimum inhibitory
concentrations (g mL1) of chlorhexidine,
miconazole, and 1:1 combination of
miconazole and chlorhexidine, and
fractional inhibitory concentration indices
for 9 isolates of Trichophyton
mentagrophytes, 9 isolates of Trichophyton
erinacei and 5 isolates of Microsporum
persicolor

331

Isolate

CH

MI

CH + MI

FIC index

Outcome

TM 1
TM 2
TM 3
TM 4
TM 5
TM 6
TM 7
TM 8
TM 9
Mean SE
TE 1
TE 2
TE 3
TE 4
TE 5
TE 6
TE 7
TE 8
TE 9
Mean SE
MP 1
MP 2
MP 3
MP 4
MP 5
Mean SE

37.5
25
25
50
25
25
37.5
18.75
18.75
29.17 3.45*
50
50
50
18.75
18.75
75
25
12.5
50
38.89 6.97*
50
37.5
50
18.75
37.5
38.75 5.73*

0.585
0.39
0.39
0.196
0.78
0.78
0.439
0.244
0.585
0.49 0.07
1.17
0.78
0.78
1.17
2.34
1.56
1.56
1.56
1.56
1.39 0.16
0.59
0.24
0.29
0.24
0.39
0.35 0.06

1.17
0.293
0.39
0.39
0.488
0.585
0.39
0.488
0.293
0.50 0.09
0.80
0.41
1.56
0.78
1.66
1.66
0.78
0.11
1.17
0.99 0.19
0.29
0.29
0.59
0.10
0.24
0.30 0.08

2.03
0.76
1.02
2.00
0.65
0.77
0.90
2.03
0.52

Indifference
Additivity
Indifference
Indifference
Additivity
Additivity
Additivity
Indifference
Additivity

0.70
0.54
2.03
0.71
0.80
1.08
0.53
0.08
0.77

Additivity
Additivity
Indifference
Additivity
Additivity
Indifference
Additivity
Synergy
Additivity

0.51
1.21
2.01
0.41
0.63

Additivity
Indifference
Indifference
Synergy
Additivity

TM, Trichophyton mentagrophytes; TE, Trichophyton erinacei; MP, Microsporum persicolor;

CH, chlorhexidine; MI, miconazole; CH + MI, chlorhexidine plus miconazole in a 1:1 ratio;
FIC, fractional inhibitory concentration.
Comparison with MI and MI + CH within species, *P < 0.01.
Comparison with TM and MP within treatment, P < 0.05, P < 0.01.

for the combination of miconazole and chlorhexidine.

Control plates consisted of SDA containing 4% DMSO.
Preliminary studies indicated that all the T. mentagrophytes, T. erinacei and M. persicolor isolates tested
grew readily on plates containing 4% DMSO.
Paired SDA plates were stab inoculated using a
mycological probe as described previously4 and incubated at 26 C for 7 days. The MIC was determined as
the lowest concentration at which no mycelial growth
was evident, providing growth was seen on the control
plate. In order to evaluate the interaction of the two
antifungal drugs, fractional inhibitory concentration
(FIC) indices were calculated for each isolate using the
formula [M]/[Ma] + [C]/[Ca], where [M] is the lowest
inhibitory concentration obtained in combination plates
containing miconazole, [Ma] is the MIC of miconazole
alone, [C] is the lowest inhibitory concentration obtained
in combination plates containing chlorhexidine, and
[Ca] is the MIC of chlorhexidine alone.9 An FIC index
of 0.5 was considered indicative of synergy, 0.5 to
1.0 as additivity, 1.0 to 4.0 as indifference and > 4.0
as antagonism.9 Analyses of variance were used to
compare the MICs for each drug or drug combination
between the three dermatophyte species (Unistat
statistical software package version 3.0, Unistat Ltd,
London, UK).
For all three species of dermatophyte tested, the
mean MIC of chlorhexidine exceeded (P < 0.01) that
of miconazole and of the combination of chlorhexidine
and miconazole (Table 1). The mean MICs of chlorhex-

idine did not vary significantly among the three species

of dermatophyte tested. In contrast, the mean MIC of
miconazole for the T. erinacei isolates exceeded (P <
0.01) those for T. mentagrophytes and M. persicolor
(Table 1). Similarly, the MIC of the combination of
chlorhexidine and miconazole was significantly higher
(P < 0.05) for T. erinacei when compared with the other
two dermatophyte species. A synergistic effect of miconazole and chlorhexidine was noted for only one isolate
of T. erinacei and one isolate of M. persicolor (Table 1).
An additive effect was demonstrated for 13 isolates
(5 T. mentagrophytes, 6 T. erinacei, 2 M. persicolor),
and indifference was noted in 8 isolates (4 T. mentagrophytes, 2 T. erinacei, 2 M. persicolor). An antagonistic
effect was not seen in any of the isolates tested.
The lack of a synergistic antifungal effect of chlorhexidine and miconazole against T. mentagrophytes, and its
infrequent occurrence among the isolates of T. erinacei
and M. persicolor tested, is in marked contrast against
the results of our previous study of M. canis where
synergy was found in 5 out of 10 strains.4 The MICs
of chlorhexidine obtained for the three species tested
in the present study were comparable with those of M.
canis.4 The MICs of miconazole for T. mentagrophytes
and M. persicolor were also comparable with those
of M. canis, whereas the mean MIC for T. erinacei was
of the order of one to two dilutions higher. Based on
the present and our previous study of a relatively small
number of isolates, synergistic or additive antifungal
effects of miconazole and chlorhexidine seem to occur

2005 European Society of Veterinary Dermatology, Veterinary Dermatology, 16, 330333

332

N Perrins et al.

more frequently against M. canis and T. erinacei than

with T. mentagrophytes and M. persicolor. The molecular
mechanisms responsible for these differences are unknown but these observations highlight the important
species differences in dermatophyte susceptibility to
antifungal drugs in vitro, both alone and in combination.
The clinical relevance of species and strain differences
in antifungal susceptibility in vitro in the therapy of
sylvatic dermatophyte infections requires further assessment; in vitro testing might not correlate fully with clinical
efficacy. In humans with scalp dermatophytosis, it has
been suggested that different therapeutic regimens should
be adopted, depending on the species of dermatophyte
responsible for the infection; terbinafine may be a poor
choice in scalp infections caused by M. canis.10 Advances
in the methods for in vitro testing of dermatophyte
susceptibility to antifungal drugs11 are likely to be of
fundamental importance in designing clinical trials to
establish optimal therapies for dermatophytosis in dogs
and other veterinary species, especially against the
Trichophyton spp. that often cause severe and progressive
disease in dogs that can be difficult to treat.12 The
possibilities for combination antifungal therapy has
received more attention in medical mycology recently,
particularly in serious invasive infections associated with
Candida albicans and Aspergillus spp.13 Such approaches
might improve the outcome of the treatment of severe
dermatophytosis in dogs, and may delay the emergence
of drug-resistant dermatophyte strains.

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Walsh TJ, Peter J, McGough DA et al. Activities of
amphotericin B and antifungal azoles alone and in combination against Pseudallescheria boydii. Antimicrobial
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Rsum Une technique de dilution en glose a t utilsi pour dterminer les concentrations minmales inhibitrices (MICs) du miconazole, de la chlorhexidine et dune combinaison de ces deux agents vis vis de 9 souches
de Trichophyton mentagrophytes, 9 souches de Trichophyton erinacei et 5 souches de Microsporum persicolor. Les
MICs de la chlorhexidine nont pas vari selon lespce fongique, mais les MICs du miconazole ou de lassociation
taient plus leves pour T. erinacei que pour T. mentagrophytes et M. persicolor. Une interaction synergique a
t note avec une souche de T. erinacei et une souche de M. persicolor. Un effet additif a t dmontr pour 13
isolats (5 T. mentagrophytes, 6 T. erinacei, 2 M. persicolor), et une indiffrence pour 8 isolats (4 T. mentagrophytes,
2 T. erinacei, 2 M. persicolor). Bien quune synergie soit moins frquemment note que dans une tude prcdente
avec Microsporum canis, leffet synergique ou additif observ ici pour la majorit (15 sur 23) des souches tudies
in vitro suggre dutiliser lassociation de miconazole et de chlorhexidine pour le traitement topique des dermatophytoses T. mentagrophytes, T. erinacei et M. persicolor.
Resumen Se utiliz una tcnica de dilucin en agar para evaluar las concentraciones inhibidoras minimas
(MICs) de miconazol, clorhexidina y de la combinacin de ambos a partes iguales, frente a 9 aislados de Tricophyton mentagrophytes, 9 aislados de Tricophyton erinacei y cinco aislados de Microsporum persicolor. Las concentraciones inhibidoras mnimas (MICs) de clorhexidina no variaron de forma significativa entre los distintos
aislados de dermatofitos, pero las MICs de miconazol slo y en combinacin con clorhexidina para T. erinacei
fueron significativamente mayores que aquellas para T. mentagrophytes and M. persicolor. En el uso combinado,
apreciamos una interaccin sinergstica slo frente a un aislado de T. erinaci y frente a otro de M. persicolor.
Asmismo, demostramos un efecto aditivo para 13 aislados (5 de T. mentagrophytes, 6 de T. erinacei, 2 de M. persicolor), y un resultado indiferente para 8 aislados (4 de T. mentagrophytes, 2 de T. erinacei, 2 de M. persicolor).
Aunque una reaccin sinergstica fue menos frecuente que en un estudio previo con M. canis, los efectos sinergsticos
2005 European Society of Veterinary Dermatology, Veterinary Dermatology, 16, 330 333

Dermatophyte susceptibility testing

333

o aditivos observados con la mayora (15 de 23) de los aislados estudiados in vitro es razn suficiente para recomendar el uso combinado de miconazol y clorhexidina en la terapia tpica de dermatofitosis producida por T.
mentagrophytes, T. erinacei y M. persicolor.
Zusammenfassung Eine Agarverdnnungstechnik wurde verwendet, um die minimalen inhibitorischen
Konzentrationen (MICs) von Miconazol, Chlorhexidin und einer 1:1 Kombination beider Reagentien fr 9 Isolate von Trichophyton mentagrophytes, 9 Isolate von Trichophyton erinacei und 5 Isolate von Microsporum persicolor zu bestimmen. Die minimalen inhibitorischen Konzentrationen (MICs) von Chlorhexidin variierten nicht
signifikant zwischen den drei getesteten Dermatophytenspezies, die MICs von Miconazol alleine und in Kombination mit Chlorhexidin fr T. erinacei waren signifkant hher als die fr T. mentagrophytes und M. persicolor.
Eine synergistische Wechselwirkung wurde bei einem Isolat von T. erinacei und einem Isolat von M. persicolor
festgestellt. Eine additive Wirkung wurde fr 13 Isolate (5 T. mentagrophytes, 6 T. erinacei, 2 M. persicolor)
demonstriert, und eine Indifferenz wurde bei 8 Isolaten (4 T. mentagrophytes, 2 T. erinacei, 2 M. persicolor) gesehen. Obwohl eine Synergie im Vergleich mit einer vorhergehenden Studie ber Microsporum canis seltener gesehen wurde, liefert der synergistische oder additive Effekt, der bei einer Mehrheit (15 von 23) der in vitro
untersuchen Isolate gefunden wurde, eine Begrndung fr die Kombination von Miconazol und Chlorhexidin
als begleitende topische Therapie von Dermatophytosen, welche durch T. mentagrophytes, T. erinacei und M. persicolor verursacht wurden.

2005 European Society of Veterinary Dermatology, Veterinary Dermatology, 16, 330333