In vitro activity of Citrus bergamia (bergamot) oil against clinical isolates of dermatophytes

Abstract

Recently, bergamot oil was shown to be a potent antifungal agent in vitro against clinically important Candida species. In this study, the activities of bergamot natural essence and its furocoumarin-free and distilled extracts on dermatophytes such as Trichophyton, Microsporum and Epidermophyton species were investigated.
In vitro susceptibility testing assays on 92 clinical isolates of dermatophytes (Trichophyton mentagrophytes n = 20, Trichophyton rubrum n = 18, Trichophyton interdigitale n = 15, Trichophyton tonsurans n = 2, Microsporum canis n = 24, Microsporum gypseum n = 1 and Epidermophyton floccosum n = 12) were performed using the CLSI M38-A broth microdilution method, except for employing an inoculum of 1-3 x 10(3) cfu/mL. MICs were determined at a visual endpoint reading of 80% inhibition compared with the growth control.
MICs (v/v) of all fungi ranged from 0.156% to 2.5% for the natural essence, from 0.02% to 2.5% for the distilled extract, and from 0.08% to 1.25% for the furocoumarin-free extract. The three isolates of T. tonsurans and M. gypseum exhibited the highest MIC values.
Data from this study indicate that bergamot oil is active in vitro against several common species of dermatophytes, suggesting its potential use for topical treatment of dermatophytoses.

Full text

Journal of Antimicrobial Chemotherapy (2007) 59, 305–308
doi:10.1093/jac/dkl473
Advance Access publication 20 November 2006
In vitro activity of Citrus bergamia (bergamot) oil against
clinical isolates of dermatophytes
M. Sanguinetti
1
*, B. Posteraro
1
, L. Romano
2
, F. Battaglia
3
, T. Lopizzo
1
, E. De Carolis
1
and G. Fadda
1
1
Institute of Microbiology, Catholic University of the Sacred Heart, Rome, Italy;
2
Laboratory of
Clinical Pathology and Microbiology, Center for High Technology Research and Education in
Biomedical Sciences, Catholic University of the Sacred Heart, Campobasso, Italy;
3
Unit of
Gynaecology and Obstetrics, Hospital of San Filippo Neri, Rome, Italy
Received 4 May 2006; returned 5 July 2006; revised 26 October 2006; accepted 29 October 2006
Objectives: Recently, bergamot oil was shown to be a potent antifungal agent in vitro against clinically
important Candida species. In this study, the activities of bergamot natural essence and its
furocoumarin-free and distilled extracts on dermatophytes such as Trichophyton, Microsporum and
Epidermophyton species were investigated.
Methods: In vitro susceptibility testing assays on 92 clinical isolates of dermatophytes (Trichophyton
mentagrophytes n = 20, Trichophyton rubrum n = 18, Trichophyton interdigitale n = 15, Trichophyton
tonsurans n = 2, Microsporum canis n = 24, Microsporum gypseum n = 1 and Epidermophyton floccosum
n = 12) were performed using the CLSI M38-A broth microdilution method, except for employing an
inoculum of 1–3 · 10
3
cfu/mL. MICs were determined at a visual endpoint reading of 80% inhibition
compared with the growth control.
Results: MICs (v/v) of all fungi ranged from 0.156% to 2.5% for the natural essence, from 0.02% to 2.5%
for the distilled extract, and from 0.08% to 1.25% for the furocoumarin-free extract. The three isolates of
T. tonsurans and M. gypseum exhibited the highest MIC values.
Conclusions: Data from this study indicate that bergamot oil is active in vitro against several common
species of dermatophytes, suggesting its potential use for topical treatment of dermatophytoses.
Keywords: MIC, broth microdilution, antifungal susceptibility
Introduction
Unlike other superficial fungal infections, the incidence of
dermatophytoses, commonly known as ringworm or tinea, has
increased considerably,
1,2
and this trend has paralleled the
increased number of individuals with impaired immunity follow-
ing treatment with cytotoxic drugs, broad-spectrum antimicro-
bials, or immunosuppressive agents.
3
Some of these infections are
still difficult to resolve completely, and remissions and relapses
are often observed.
1
Clinical and mycological cure of dermato-
phytoses may be prevented by the inability of the antifungal drug
to penetrate the site of infection or by the intrinsic resistance of
the fungus. Cases of infections due to griseofulvin-resistant
isolates have been described,
4
as well as a high-level primary
resistance to terbinafine displayed by Trichophyton rubrum
isolates obtained sequentially from a single onychomycosis
patient who failed oral terbinafine therapy.
5,6
The poor availability of antifungals and increasing number of
treatment failures have motivated current searches for therapeutic
alternatives to include the testing of essential oils (e.g. from
Thymus vulgaris and Melaleuca alternifolia) as potential
antimicrobial agents.
7–9
Most of them contain large amounts of
phenolic monoterpenes, which are responsible for activity against
viruses, bacteria and fungi.
9–11
The essential oil of Citrus bergamia, also called bergamot oil,
is primarily produced in Calabria, in southern Italy, and from this
country came the first information on the antimicrobial properties
of this compound.
12
This oil, termed by us as ‘natural essence’, is
a yellow-green liquid directly obtained from the cold-pressed
peels of the fruit, and consists of c. 80 volatile (e.g. limonene,
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*Corresponding author. Tel: +39-06-30154964; Fax: +39-06-3051152; E-mail: msanguinetti@rm.unicatt.it
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305
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linalool and linalyl acetate) and non-volatile (e.g. bergamottin,
citroptene and bergaptene) components.
13
As a consequence of
the phototoxic action of furocoumarins (i.e. bergaptene) present in
bergamot oil, furocoumarin-free and distilled extracts are often
used, instead of the natural essence, in pharmaceutical products
(Bergamon S.r.l., Rome, Italy). Of note, some of these products
are empirically used for prevention and treatment of mycoses.
In our previous work, we investigated the antifungal properties
of the bergamot natural essence and its furocoumarin-free and
distilled extracts against vaginal isolates of several Candida
species in vitro.
14
We established that these preparations were
effective agents, mainly when tested in association with boric
acid, which suggested that they are potentially active against
filamentous fungi as well. Accordingly, the aim of this study was
to assess the effects of the three compounds on dermatophytes by
the use of in vitro susceptibility assays.
Materials and methods
Fungal isolates
Ninety-two isolates belonging to seven species of dermatophytes
were tested. They were chosen from the culture collection of clinical
isolates maintained at the Mycology Section of the Catholic
University Medical Centre, and included 20 Trichophyton menta-
grophytes isolates, 18 T. rubrum,15Trichophyton interdigitale,
2 Trichophyton tonsurans,24Microsporum canis,1Microsporum
gypseum and 12 Epidermophyton floccosum. Isolates had originally
been identified to the species level by standard procedures
15
and
stored as water suspensions at room temperature. Prior to testing,
each isolate was subcultured on a potato dextrose agar (PDA) slant
and incubated at 30

C for 4–5 days or until good conidiation was
produced. T. rubrum isolates were subcultured on rice agar plates
to induce conidium sporulation.
2
Candida parapsilosis ATCC 22019
and Candida krusei ATCC 6258 were used as quality control
strains.
16
Fungal inoculum preparation
For each dermatophyte isolate, a suspension of conidia was prepared
in 0.85% saline by swabbing the colony surface with a sterile swab, as
reported recently.
17
After the settling of the larger particles, conidia
were counted with a haemocytometer and diluted in RPMI 1640
medium (Sigma, Milan, Italy) to correspond to a final inoculum
concentration of 1 · 10
3
–3 · 10
3
cfu/mL, as described previously.
17
Yeast control strains were subcultured on PDA and incubated at 35

C
for 24 h, and the corresponding inocula were prepared to final
concentrations of 0.5 · 10
3
–2.5 · 10
3
cfu/mL.
18
Test compounds and susceptibility testing assays
Natural essence of bergamot (NE) and its distilled (DE) and
furocoumarin-free (FF) extracts, produced by the Consorzio del
Bergamotto of Reggio Calabria, Italy, were supplied by Bergamon
S.r.l. (Rome, Italy). The chemical composition of the three
preparations, as determined by gas and gas chromatography/mass
spectroscopy analyses, has been reported previously.
14
In particular,
the furocoumarin-free extract is bergaptene-free, whereas the distilled
extract is absolutely devoid of non-volatile residues.
14
Standard
powders of antifungal drugs, such as itraconazole (Janssen, Beerse,
Belgium) and griseofulvin (Sigma), were used to prepare stock
solutions.
17,18
In vitro susceptibility testing of dermatophytes to all
compounds was based on a modification of the CLSI M38-A broth
microdilution method.
16
Each oil preparation was diluted (v/v) in
RPMI 1640 (Sigma), and Tween 80 (Sigma, final concentration
0.001% v/v) was included to enhance oil solubility. At this
concentration, the detergent did not show any inhibitory effect on
fungal growth (data not shown). Serial 2-fold dilutions of each test
compound, prepared in RPMI 1640, were placed in 96-well microtitre
plates.
18
The individual ranges of each substance used were as
follows: bergamot oil preparations, 0.02–10% (v/v); itraconazole,
0.03–16 mg/L; and griseofulvin, 0.125–64 mg/L. Growth and sterility
control wells were included in each plate. After the addition of
inocula (prepared as described earlier), plates were incubated for
96 h at 35

C (yeast controls were incubated for 24 h).
17
MIC
was determined visually and recorded as the lowest concentration
of substance that reduced growth to 80% of that of the control.
17
The
minimum concentration of substance that inhibited 90% of the
isolates was defined as MIC
90
. Isolates were tested twice. For the
two isolates tested with itraconazole as quality controls, MICs
were within expected ranges (for C. parapsilosis, 0.25 mg/L; for
C. krusei, 0.5 mg/L).
18
Results and discussion
Table 1 shows the MIC values for all the dermatophyte isolates
tested against three bergamot oil preparations (NE, DE and FF).
MICs ranged from 0.156% to 2.5% for NE, from 0.02% to 2.5%
for DE and from 0.08% to 1.25% for FF. Generally, MIC
90
s were
lower for DE and FF compared with NE. Among the species with
fewer than 10 isolates, T. tonsurans (two isolates) and M. gypseum
(one isolate) exhibited the highest MICs to NE, DE and FF, but
again the MICs of DE or FF were lower than those of NE for both
species. Consistent with our previous results,
14
the MICs of NE,
FF and DE for C. parapsilosis ATCC 22019 were 1.25%, 1.25%
and 0.64%, respectively; for C. krusei ATCC 6258 were 2.5%,
2.5% and 0.64%, respectively. Griseofulvin and itraconazole
gave MICs in the ranges of 0.125–64 and 0.03–0.25 mg/L,
respectively.
In recent years, proliferation of new classes of drugs, such
as the allylamines (e.g. terbinafine) and orally active triazoles
(e.g. itraconazole), has represented the most noteworthy trend in
dermatophytosis therapy.
1
Many azoles, in particular itraconazole,
have been used effectively, often resulting in complete clearance
of the lesions.
19
However, treatment with both itraconazole and
terbinafine for prolonged times requires periodic laboratory
monitoring of liver function.
20
Moreover, these antifungal agents
may have drug interactions with other medications.
21
Griseoful-
vin, which had been for many years the only antifungal available
for the treatment of dermatophytoses, is still the long-standing
drug of choice for tinea capitis, but there are concerns with
resistance and toxicities with this agent.
21
Even though systemic antifungal therapy is often indicated—
especially for tinea unguium, the most resistant of
dermatophytoses—topical agents are still frequently used to
cure or speed the resolution of uncomplicated lesions.
1
This is in
line with the current opinion that systemic therapy should be
given consideration when lesions involving a large infected area
fail to clear with repeated topical treatment using different
drugs.
22
The topical agents, applied to the surface of the skin
in the form of creams, lotions or sprays, are known to readily
penetrate into the stratum corneum to kill the fungi or render
them unable to grow or divide. Fungicidal drugs such as
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terbinafine are often preferred over fungistatic azoles (micona-
zole, clotrimazole and ketoconazole) for treatment of dermato-
phytic fungal infections, since short-term treatments (i.e. one
application daily for 1 week) are associated with high cure rates.
23
Taken together, the improved cure rates, reduced adverse
effects, decreased drug interactions and lower cost of topical
agents make therapy with these drugs a favourable choice in the
management of superficial fungal infections including dermato-
phytoses.
21
In this context, new antifungal plant derivatives could
be useful alternatives for the treatment of dermatophytoses where
a topical therapy is required. The advantage of using these natural
compounds may be a reduced risk of side-effects and lower cost.
It is thus not surprising that, in recent years, there has been
growing interest in the use of medicinal plants to cure skin
diseases.
In this study, we demonstrated the high in vitro activity of
bergamot oil against a wide number of clinical isolates of various
pathogenic dermatophytes. In general, the three preparations
tested had low MICs. However, the two extracts, DE and FF, were
more active than NE against all of the species tested. This is of
great importance in the light of the fact that the two derivatives
are devoid (in part or completely) of non-volatile residues, in
particular of the phototoxic bergaptene. Although we found the
activities of these compounds against dermatophytes to be
superior to the anticandidal effect we observed previously,
14
our
data all indicate that bergamot oil can be used as an efficacious
antifungal agent against dermatophytes and yeast pathogens.
These results give substantial support to popular or anecdotal
beliefs in the effectiveness of treating skin and mucosal infections
with bergamot oils. The only other data in the literature on the
antimycotic action of bergamot oil are those of Hammer et al.,
24
who investigated the susceptibility of a single isolate of Candida
albicans to NE. Otherwise, in vivo and in vitro studies conducted
on M. alternifolia (tea tree) oil
25
have established that some of the
anecdotal claims made about natural oils have a scientific basis.
In an interesting review, Martin and Ernst
26
critically assessed the
evidence, from controlled clinical trials, of the efficacy of
antifungal plant oils and extracts. As reported in that systematic
review, in some of these studies, plant preparations were
compared with conventional antifungal treatments, and in all
cases encouraging results were reported. Four trials described the
promising use of tea tree oil preparations for treatment of tinea
pedis and onychomycosis.
26
Thus, this emphasizes the need for
extensive studies to understand the ways in which bergamot oils
inhibit fungi, and for clinical trials to prove their effectiveness in
the cure of dermatophytoses.
Acknowledgements
We would like to thank Dr Paul Kretchmer at San Francisco Edit
for his assistance in editing this manuscript.
Transparency declarations
None to declare.
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Table 1. In vitro activity of bergamot oils, itraconazole and griseofulvin against 92 isolates of dermatophytes, determined by the
microdilution broth method
NE
a
(% v/v) DE
a
(% v/v) FF
a
(% v/v) Itraconazole (mg/L) Griseofulvin (mg/L)
Species (no. of isolates) MIC range MIC
90
MIC range MIC
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MIC range MIC
90
MIC range MIC
90
MIC range MIC
90
T. mentagrophytes (20) 0.156–1.25 0.625 0.156–0.625 0.312 0.08–0.156 0.156 0.03–0.125 0.125 0.125–64 0.5
T. rubrum (18) 0.156–0.625 0.312 0.08–0.312 0.156 0.156–0.625 0.156 0.03–0.25 0.125 0.125–64 0.5
T. interdigitale (15) 0.312–1.25 0.625 0.156–0.625 0.156 0.08–0.312 0.312 0.03–0.125 0.125 0.125–64 0.5
T. tonsurans (2) 2.5 ND
b
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M. gypseum (1) 2.5 ND 2.5 ND 1.25 ND 0.125 ND 0.5 ND
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a
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