Veterinary Microbiology 142 (2010) 432434

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Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic

Short communication

Antimicrobial activity of propolis extract against Staphylococcus

coagulase positive and Malassezia pachydermatis of canine otitis
Rosemari Laura Cardoso a,1, Franciele Maboni b,1,*, Gustavo Machado a, Sydney Hartz Alves c,
Agueda Castagna de Vargas a
a

Departamento de Medicina Veterinaria Preventiva, Universidade Federal de Santa Maria, Santa Maria, Rio Grande do Sul, Brazil
Programa de Pos-Graduacao em Biologia Celular e Molecular, Departamento de Biotecnologia, Universidade Federal do Rio Grande do Sul, Av. Bento Goncalves
9500, Porto Alegre, Rio Grande do Sul, Brazil
c
Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Maria, Santa Maria, Rio Grande do Sul, Brazil
b

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 24 June 2009
Received in revised form 25 September 2009
Accepted 29 September 2009

The aims of this study were to evaluate the antimicrobial potential of propolis extract by
determining the minimum bactericidal concentration (MBC) for coagulase-positive
Staphylococcus isolates (Staphylococcus aureus and Staphylococcus intermedius) and the
minimum fungicidal concentration (MFC) for Malassezia pachydermatis isolates. The
microorganisms were assayed using broth microdilution techniques. The MBC90 was
21 mg mL 1, and the MFC90 was 5.3 mg mL 1. The propolis extract was found to exhibit
antimicrobial activity against both pathogens.
2009 Elsevier B.V. All rights reserved.

Keywords:
Staphylococcus aureus
Staphylococcus intermedius
Yeasts
Fitotherapics

1. Introduction
Propolis is a natural compound well known since
ancient times for its therapeutic effects; the substance can
be used in the development of alternative therapies for the
treatment of many diseases (Bankova, 2005). It is
produced by bees from the resins of sprouts, exudates
and other parts of plant tissues (Kusumoto et al., 2001).
The biological and pharmacological activities, in particular the antimicrobial, anti-virus, anti-cancer, antiinammatory, and anti-oxidant effects (Bankova, 2005),
depend on the chemical composition, which in turn
depends on the geographic localization of the plants that
the bees feed on. The most common compounds in
propolis are avonoids, aromatic acids, and phenolic
esters (Krol et al., 1996).

* Corresponding author. Tel.: +55 51 3308 6077; fax: +55 51 3308 7009.
E-mail address: franmaboni@gmail.com (F. Maboni).
1
These authors contributed equally to this work.
0378-1135/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetmic.2009.09.070

In veterinary medicine, fungal and bacterial diseases can

exhibit a high level of antibiotic resistance (Pedersen et al.,
2007) and thus require alternative therapies. Included in
this group of diseases is canine otitis, which is highly
prevalent; 21.742% of dogs have been reported to be
infected (Oliveira et al., 2006; Ramalho et al., 2007). Canine
otitis develops due to predisposing factors and the action of
bacteria and fungi. This infection, normally presents with a
high level of microbial resistance (Lyskova et al., 2007). The
majority of dogs with otitis have a simultaneous infection
with two etiological agents (Staphylococcus intermedius and
Malassezia pachydermatis). Infection by only one of these
pathogens is not common (Ramalho et al., 2007). Both
microorganisms belong to the normal microbial ora of the
auricular area; they only become pathogenic when they
multiply very fast and overgrowth occurs.
The aims of this study were to evaluate the antimicrobial activity of propolis extract in vitro against
coagulase-positive Staphylococcus and M. pachydermatis
isolates from dogs with otitis, with the aim of determining
the minimum bactericidal concentration (MBC) and the
minimum fungicidal concentration (MFC).

R.L. Cardoso et al. / Veterinary Microbiology 142 (2010) 432434

433

Fig. 1. The susceptibility of nine strains of Staphylococcus aureus, 25 strains of Staphylococcus intermedius, and 33 strains of Malassezia pachydermatis to
different propolis extract concentration using the broth, microdilution techniques. The numbers of strains are indicated at the bottom.

2. Materials and methods

2.1. Preparation of the propolis extract
The propolis used in this work was obtained from Santa
Maria, Rio Grande do Sul, Brazil. The propolis extract was
prepared according Szewczak and Godoy (1984). Propolis
was cold-macerated to make an extract with ethanol 70%
(brute propolis 300 g/ethyl alcohol 700 mL), resulting in a
300 mcg 1 mL concentration. The solution was stored at
room temperature and protected from light for 45 days,
after which the supernatant was extracted from a dark
bottle using a siphon.
2.2. Microorganisms
Sixty-seven microbial isolates were used: 34 Staphylococcus coagulase-positive (S. intermedius, n = 25, Staphylococcus aureus, n = 8, and, for control, one bacterial reference
strain S. aureus ATCC 25923) and 33 M. pachydermatis
isolates. All of them originated from canine otitis, which
was phenotypically identied according to Quinn et al.
(1994).
To obtain inocula, bacterial isolates were grown on
ovine blood agar 5% and incubated at 35 8C for 24 h. The
fungal isolates were grown on sabouraud agar under the
same conditions.
2.3. Determination of the antimicrobial activityminimum
bactericidal concentration (MBC) and minimum fungicidal
concentration (MFC)
To evaluate the minimum bactericidal concentration
(MBC) and minimum fungicidal concentration (MFC),
propolis extract concentrations in solution with 70%

ethanol ranging from 0.69 to 42.8 mg mL 1 were tested.

Inocula were prepared with bacterial and fungal saline
suspension, with a cell concentration between 0.40 and
0.49 nm of absorbance for bacterial inoculum, and
between 0.60 and 0.69 nm for the yeast inoculum. The
spectrophotometer readings were taken at a wavelength of
600 nm; these procedures created suspensions with a
concentration of approximately 1.0  106 CFU/mL. The
M07-A8 (Clinical and Laboratory Standards Institute
CLSI, 2008) and the CLSI M27-A3 (2008) protocols were
used to obtain the MBC and MFC, respectively. The time of
contact among the microorganisms and propolis extract
was 24 h. The innocuity of the 70% ethanol was evaluated
by diluting it to 3% in MullerHinton broth. The controls for
inoculum viability and growth medium sterility were
performed according to the above protocols.
3. Results and discussion
The results obtained in tests of antimicrobial activity
demonstrated the bacterial and fungal action of propolis
extract; both coagulase-positive Staphylococcus and M.
pachydermatis isolates were found to be sensitive to
propolis extract. The alcohol control did not inhibit
bacterial or fungal isolate growth.
3.1. Action of propolis extract against coagulase-positive
Staphylococcus
Three of the seven tested concentrations were found to
be more effective against coagulase-positive Staphylococcus (see Fig. 1).
The in vitro antibacterial activity of propolis has
previously been well demonstrated against a great variety
of bacteria, particularly gram-positive species. Its action

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R.L. Cardoso et al. / Veterinary Microbiology 142 (2010) 432434

against Staphylococcus sp. has also been reported (Kujumgiev et al., 1999; Miorin et al., 2003; Uzel et al., 2005).
Fernandes et al. (2003) described minimum inhibitory
concentrations (MICs) ranging from 126.23 to
185.7 mg mL 1 for S. aureus, whereas Kujumgiev et al.
(1999) and Bankova et al. (1999) demonstrated propolis
activity at a concentration of 0.4 mg mL 1 against S. aureus.
Studies with propolis produced by three different bee
species showed variations at MIC ranging from 117 to
468 mg mL 1 against S. aureus isolates (Longhini et al.,
2007). The majority of research addressing propolis
antimicrobial activity has been performed in human
medicine, using a wide range of techniques.
3.2. Action of propolis extract against M. pachydermatis
The results of our tests in M. pachydermatis isolates are
described in Fig. 1, which conrms the antifungal activity
of propolis. Fig. 1 shows that extract concentration
2.6 mg mL 1 was required to inhibit 50% of strains under
in vitro growth conditions.
Studies performed with Malassezia species are less
common, especially in animal samples. The fungistatic and
fungicidal activities of propolis extract against M. pachydermatis were previously demonstrated by Lilenbaum and
Barbosa (1994) using the serial dilution method; these
authors demonstrated activity over a range of 0.8
2.4 mg mL 1, which represents concentrations similar to
those tested in this study.
In human medicine, the antifungal activity of propolis
has been previously reported in numerous yeast species.
Longhini et al. (2007) for example, demonstrated the
antifungal potential of propolis extract at 0.4 mg mL 1.
Kujumgiev et al. (1999) showed the best inhibitory zone
using 0.5 mg mL 1 of alcoholic propolis extract against
yeasts. Tests performed by Silici and Kutluca (2005) with
Candida albicans demonstrated a MIC of 3.7 mg mL 1.
However, the lowest published concentration of propolis
extract with demonstrable activity against lamentous
fungi and yeast is 16 mg mL 1, which was obtained by Uzel
et al. (2005) against a reference strain Candida tropicalis.
Another study, using the clinically important fungus,
Cryptococcus neoformans, yielded a MFC of 1.6 mg mL 1
(Fernandes et al., 2007). The differences observed between
the results described in the literature and those in the
current study can be attributed to the different types of
yeasts originating from human samples, due to geographical variations or because of variations in the propolis
extract preparations themselves. Thus, a number of
variables must be considered in every propolis study.
MBC and MFC results using our propolis extract against
coagulase-positive Staphylococcus and M. pachydermatis
demonstrated efcacy at concentrations less than
42.8 mg mL 1, demonstrating the antimicrobial potential
of the propolis extract tested against both of these agents
and the lower bactericidal and fungicidal concentration for
inhibit their growth.

Conict of interest statement

All authors disclose there are no nancial and personal
relationships with other people or organizations that could
inappropriately inuence their work.
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