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Veterinary Microbiology 142 (2010) 432–434

Veterinary Microbiology 142 (2010) 432–434 Contents lists available at ScienceDirect Veterinary Microbiology

Contents lists available at ScienceDirect

Veterinary Microbiology

journal homepage: www.else

journal homepage: www.else Short communication Antimicrobial activity of propolis

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 Veterina´ ria Preventiva, Universidade Federal de Santa Maria, Santa Maria, Rio Grande do Sul, Brazil b Programa de Po´ s-Graduac¸ a˜ o em Biologia Celular e Molecular, Departamento de Biotecnologia, Universidade Federal do Rio Grande do Sul, Av. Bento Gonc¸ alves 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


Article history:

Received 24 June 2009 Received in revised form 25 September 2009 Accepted 29 September 2009


Staphylococcus aureus Staphylococcus intermedius Yeasts Fitotherapics


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 MBC 90 was 21 mg mL 1 , and the MFC 90 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.

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 parti- cular the antimicrobial, anti-virus, anti-cancer, anti- inflammatory, 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 flavonoids, aromatic acids, and phenolic esters ( Krol et al., 1996 ).

* Corresponding author. Tel.: +55 51 3308 6077; fax: +55 51 3308 7009. E-mail address: (F. Maboni). 1 These authors contributed equally to this work.

0378-1135/$ – see front matter 2009 Elsevier B.V. All rights reserved.

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; 21.7–42% 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 flora 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 anti- microbial 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) 432–434


et al. / Veterinary Microbiology 142 (2010) 432–434 433 Fig. 1. The susceptibility of nine strains

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 Staphylo- coccus coagulase-positive ( S. intermedius , n = 25, Staphylo- coccus 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 identified according to Quinn et al. (1994) . To obtain inocula, bacterial isolates were grown on ovine blood agar 5% and incubated at 35 8 C for 24 h. The fungal isolates were grown on sabouraud agar under the same conditions.

2.3. Determination of the antimicrobial activity—minimum

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 10 6 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 Mu¨ ller–Hinton 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 Staphylococ- cus (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


R.L. Cardoso et al. / Veterinary Microbiology 142 (2010) 432–434

against Staphylococcus sp. has also been reported ( Kujum- giev 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 m g 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 confirms 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. pachy- dermatis 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 filamentous fungi and yeast is 16 m g 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 geogra- phical 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 efficacy 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.

Conflict of interest statement

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


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