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Brazilian Journal of Microbiology

Print version ISSN 1517-8382On-line version ISSN 1678-4405

Braz. J. Microbiol. vol.33 no.4 São Paulo Oct./Dec. 2002

http://dx.doi.org/10.1590/S1517-83822002000400018 

Propolis antimicrobial activity against periodontopathic bacteria

 

Atividade antimicrobiana da própolis contra bactérias periodontopatogênicas

 

 

Elaine C.E. GebaraI,II; Luiz A. LimaI; Marcia P.A. MayerI

IDepartamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brasil
II
Departamento de Estomatologia, Divisão Periodontia, Faculdade de Odontologia, Universidade de São Paulo, São Paulo, SP, Brasil

Correspondence

 

 


ABSTRACT

Propolis extract antimicrobial activity against periodontopathic (ATCC) bacteria was investigated "in vitro". Bacterial strains tested were: Prevotella intermedia, Prevotella melaninogenica, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Capnocytophaga gingivalis and Fusobacterium nucleatum. Minimal inhibitory concentration (MIC) for the strains tested was determined using the method of broth dilution with the propolis extract in serial concentrations. Results showed MIC of 1 µg/ml for Actinobacillus actinomycetemcomitans and Capnocytophaga gingivalis; and 0.25 µg/ml for Prevotella intermedia, Prevotella melaninogenica, Porphyromonas gingivalis and Fusobacterium nucleatum. Some superinfectant organisms were also tested: Candida albicans susceptibility to propolis ethanolic extract was demonstrated at a concentration of 12 µg/ml. The MIC for Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus (wild types) was 14 µg/ml. All periodontal pathogens and superinfectants tested were susceptible to the propolis extract. The positive results suggest that the propolis extract should be further tested as an adjuvant to periodontal therapy.

Key words: bacteria, periodontal pathogens, periodontal diseases, minimal inhibitory concentration, propolis


RESUMO

A atividade antimicrobiana da própolis contra bactérias periodontopatogênicas (ATCC) foi investigada através de testes "in vitro". As cepas bacterianas testadas foram: Prevotella intermedia, Prevotella melaninogenica, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Capnocytophaga gingivalis e Fusobacterium nucleatum. A concentração inibitória mínima (CIM) foi determinada usando-se o método de diluição do extrato de própolis no meio de cultura em diferentes concentrações. Os resultados demonstraram CIM de 1 µg/ml para Actinobacillus actinomycetemcomitans e Capnocytophaga gingivalis; e 0,25 µg/ml para Prevotella intermedia, Prevotella melaninogenica, Porphyromonas gingivalis e Fusobacterium nucleatum. Alguns microrganismos que desempenham "in vivo" papel de superinfectantes também foram testados: a susceptibilidade de Candida albicans ao extrato etanólico de própolis foi observada na concentração de 12 µg/ml. A CIM para Pseudomonas aeruginosa, Escherichia coli e Staphylococcus aureus (tipo selvagem) foi de 14 µg/ml. Todos os patógenos periodontais e microrganismos superinfectantes testados foram sensíveis ao extrato de própolis testado. Os resultados obtidos encorajam a realização de novos estudos com esse extrato de própolis, para avaliar sua utilização como coadjuvante ao tratamento periodontal.

Palavras-chave: bactéria, patógeno periodontal, doença periodontal, concentração inibitória mínima, própolis


 

 

INTRODUCTION

Human periodontal disease has been associated with a complex microbiota. The development of destructive periodontitis seems to be the result of a specific infection (46). Gram positive coccoid bacteria have been related to periodontal health, while periodontal disease was associated with Gram negative rods and spirochetes (28). Many authors (5,10,13,46,47,51) suggest that the presence of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis (Porphyromonas gingivalis) and Bacteroides intermedius (Prevotella intermedia) is related to active periodontal disease. Other species like Fusobacterium nucleatum (13,49,51) and Capnocytophaga sp. (45,51) were also associated with disease. According to the Consensus report of the World Workshop on Clinical Periodontics (1996), human periodontitis is initiated and perpetuated by a small group of bacteria that colonize the subgingival region, mainly Gram-negative, anaerobic or microaerophilic bacteria. Furthermore, most cases of human periodontitis are caused by Porphyromonas gingivalis, Bacteroides forsythus and Actinobacillus actinomycetemcomitans (52).

Because periodontitis is an infectious disease, and taking into consideration that some patients do not respond to conventional mechanical therapy, sometimes antimicrobial agents have been prescribed as adjuvants to periodontal treatment (9). However, the emergence of pathogenic bacteria that are resistant to antibiotics, due to inappropriate systemic usage, has become a serious clinical problem. Loesche (29) pointed out that in order not to contribute to a "coming plague", dentists should add the knowledge of an infectious disease specialist to their surgical skills. Gillette (17) suggested that antibiotics should be used only when there is a reasonable specific goal, so that the expected benefits will outweigh the risks to the patient and society.

The clinical use of antibiotics and other antimicrobial agents, as adjuvants for the treatment of periodontitis, has been extensively investigated in the past decade (15,20,22,43,44). Recently, special attention has been paid to natural medication, and propolis has been reported to possess certain medicinal properties (30).

Propolis is a natural composite balsam, produced by honey bees (Apis melifera) from the gum of various plants. Bees collect vegetal exudates and form pellets with their mandibles, mixing the exudates with wax and products of their salivary glands. The resulting material is used to strengthen the nest, provide protection from microorganisms, and as an embalming substance to cover the carcass of a hive invader (21). The medicinal properties of propolis have been widely investigated (11,14,15,21,22,23,25,30,31,38,40,41).

Antimicrobial action of propolis has already been shown. Various studies report on antibacterial (11,14,15,21,22,41), antifungic (11) and antiparasitic (21) actions.

Gebara et al. (15) in 1996 demonstrated propolis antimicrobial activity against Streptococcus mutans and Streptococcus sobrinus, as well as its action in inhibiting the production of polysaccharides. In 1998, Rosalen et al.(40), observed that the application of propolis extract on rat molars reduced the severity of carious lesions in these animals.

Once propolis is known to function as an antibacterial agent against specific organisms, the aim of this study was to investigate the antimicrobial "in vitro" action of a propolis extract (with a previously determined composition) against periodontopathic bacteria, as well as against superinfectants.

 

MATERIALS AND METHODS

The propolis ethanolic extract (70% ethanol) used in this study was provided by UNESP - Botucatu - Department of Animal Production- Brazil.

Susceptibility tests were performed using the following ATCC strains: Prevotella intermedia (33563), Prevotella melaninogenica (25845), Porphyromonas gingivalis (33277), Actinobacillus actinomycetemcomitans (29523 and 29522), Capnocytophaga gingivalis (33624), Fusobacterium nucleatum (10953) and Candida albicans (10231). Isolates of Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus from our own culture collection were also tested.

Aliquots of frozen stocks in 20% glycerol of the different strains were inoculated on agar plates. P. intermedia, P. melaninogenica, P. gingivalis, A. actinomycetemcomitans, C. gingivalis, F. nucleatum were cultured in enriched Brain Heart Infusion agar, for 5 days. C. albicans was grown for 2 days in Saboraud dextrose agar, and P. aeruginosa, E. coli and S. aureus were cultured in Brain Heart Infusion agar for 1 day. The resultant cultures were diluted in PBS (phosphate buffer solution), to reach concentrations equivalent to Mac Farland scale nº 1. P. intermedia, P. gingivalis and F. nucleatum were resuspended to a concentration of 105 cfu/ml. A. actinomycetemcomitans, C. gingivalis, P. melaninogenica, were diluted to a concentration of 107 cfu/ml. The concentration was 3 x 108 cfu/ml for C. albicans, P. aeruginosa, E. coli and S. aureus.

Minimal inhibitory concentrations (MIC) for propolis against the tested strains were determined using the propolis extract in serial concentrations: 0 (negative control), 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 14 and 16 µg/mL. Control plates with serial concentrations of ethanolic alcohol solution were also tested. The strains were inoculated by a Steer apparatus. All tests were performed in quadruplicate.

All strains were grown in Brain Heart Infusion Agar (BHI - Difco) except for Candida albicans, which was grown in Saboraud agar (Difco), and incubated at room temperature for 4 days.

Pseudomonas aeruginosa, E. coli and S. aureus inoculated in Brain Heart Infusion agar were incubated aerobically at 37ºC for 2 days. Brain Heart Infusion agar enriched with hemin (0.1% - Sigma) and menadione (0.01% - Sigma) was used to grow strains of P. intermedia, P. melaninogenica, P. gingivalis, A. actinomycetemcomitans, C. gingivalis and F. nucleatum. The plates were incubated anaerobically (Gas-Pak-BBL) at 37ºC for 7 days.

 

RESULTS

Minimum Inhibitory Concentration (MIC) was determined as the lowest concentration of the propolis extract, which inhibited the growth of the tested microorganisms.

The propolis extract showed antimicrobial activity against all tested strains. Table 1 presents the Minimal Inhibitory Concentrations obtained for each strain tested. All control plates, including those with different ethanolic alcohol concentrations and the negative controls, presented regular bacterial growth.

 

 

 

DISCUSSION

Besides showing antimicrobial activity against periodontopathic bacteria, the propolis extract did not demonstrate selection of superinfectant organisms.

The verification of the antimicrobial action of the propolis extract is not surprising. The primary function of propolis in the hive is to act as a biocide, being active against invasive bacteria, fungi and even invading larvae (16,27,32). There are a number of studies documenting the biocidal functions of propolis, its extracts and constituents. The spectrum of activity is fairly broad, with action against Gram positive and Gram negative rods and cocci, yeast and fungi (6).

The antimicrobial activity of propolis ethanolic extract has been studied by several authors, however, few studies have investigated its activity towards oral pathogens (15,22,38,50).

The present study has shown propolis antimicrobial activity against the following periodontal pathogens: A. actinomycetemcomitans, P. intermedia, P. melaninogenica, P. gingivalis, C. gingivalis and F. nucleatum. Antimicrobial activity against Candida albicans, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus was also demonstrated in this study, confirming previous results (3,12,18,22,26,38,50).

Different results were achieved by Nieva et al. (35) that reported antimicrobial activity against Staphylococcus aureus, but no action against Pseudomonas aeruginosa and Escherichia coli. A possible explanation for diverse results is the fact that propolis composition is variable depending on the region and season that it is collected (1,19,24,37). Consequently, the active compounds may not be present in sufficient quantities or quality.

One of the limitations to propolis use is the variability in composition and action as a consequence of variations in the flora of the region where it is produced. However, according to Bankova et al. (2), antimicrobial action is expected to be always present because of its vital importance as an antimicrobial agent to the bees, independently of the region where the propolis is produced. In a recent study, Sforcin et al. (42) did not find a seasonal effect on the antimicrobial activity of the Brazilian propolis.

Propolis mechanism of antimicrobial action, though not completely understood, seems to be complex and may vary according to its composition. The compounds known to have antimicrobial action are mainly the flavonoids and cinamic acids (2).

An aliquot of the same propolis extract tested in this study was sent to Bulgaria, for analysis of constituents by gas chromatography. The flavonoids detected were Kaempferol; 5,6,7 Trihidroxi-3,4 dimetoxiflavone and Aromadendrine-4-metil-eter. Other constituents identified were 9-E-2, 2-dimetil-6-carboxietenil-8-prenil-2H-1-benzopirano and 9-Z-2,2-dimetil-6-carboxietenil-8-prenil-2H-1-benzopirano (4).

Osawa et al. (36) reported that Kaempferol had antimicrobial activity against Streptococcus mutans and Actinomyces viscosus. Cai and Wu (7) demonstrated that the same constituent inhibited the growth of P. intermedia and P. gingivalis.

Regarding susceptibility of the tested microorganisms to propolis it seems that they were more susceptible to propolis than to some antibiotics. Carrasco et al. (8) showed the MIC for doxycycline, tetracycline, metronidazole, ofloxaxin and amoxycillin (8, 4, 8, 6 and 4 µg/mL respectively) against P. gingivalis, P. intermedia and F. nucleatum "in vitro". When their MIC results are compared to ours, it can be observed that frequently used antibiotics had greater MIC than propolis, against P. gingivalis, P. intermedia and F. nucleatum. These periodontal pathogens may be more susceptible to propolis than to the antibiotics shown above.

Previous study on the susceptibility of A. actinomycetemcomitans to selected antimicrobial agents indicated that MIC90 to penicillin varied from 1.0 to 6.25 µg/ml, to amoxicillin from 1.0 to 2.0 µg/ml, to tetracycline from 0.5 to 8.0 µg/ml, to doxycycline from 1.0 to 3.1 µg/ml, and to metronidazole from 12.5 to 32 µg/ml (34). In the present study, the MIC of this pathogen to propolis was 1 µg/ml.

Susceptibility tests of P. gingivalis have shown that MIC90 to penicillin varied from 0.016 to 0.29 µg/ml, amoxycillin from 0.023 to < 1.0 µg/ml, metronidazole from 0.023 to 2.1 µg/ml (34). Interestingly, in present study MIC to propolis was 0.25 µg/ml.

In 1990, Rams et al. (39) noted that some strains of S. aureus isolated from the periodontal pocket were resistant to tetracycline, penicillin, metronidazole and erythromycin. Additionally, when the antimicrobial activity of 18 antibiotics was tested against Enterobacteriaceae and Pseudomonadaceae, only ciprofloxacin was able to eliminate these microorganisms from the periodontal pocket (48).

Our results showed that propolis extract presented "in vitro" antimicrobial activity, not only against some periodontophatic bacteria (F. nucleatum, P. gingivalis, P. intermedia, P. melaninogenica, A. actinomycetemcomitans and C. gingivalis) but also against some organisms able to cause superinfection (S. aureus, P. aeruginosa, E. coli and Candida albicans).

It is important to remember that "in vitro" tests do not reflect the real conditions found in periodontal pockets. They do not take into account biofilm formation. In addition, determination of MIC values depends on technical details that may vary between laboratories (33).

The antimicrobial action observed for the propolis extract suggest its usage as an adjuvant to periodontal therapy. A step further should be given to verify if a dose sufficient to kill the target microorganisms can be reached within the subgingival environment, without causing major local or systemic adverse effects.

 

ACKNOWLEDGMENTS

The authors would like to acknowledge Prof. Dr. Silvia Cunha Funari for kindly providing the propolis extract.

 

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Correspondence to
Departamento de Estomatologia, Divisão Periodontia, Faculdade de Odontologia, Universidade de São Paulo
Av. Prof. Lineu Prestes, 2227, Cidade Universitária. 05508-900, São Paulo, SP, Brasil
Tel.: (+5511) 3091 7833
E-mail: gebar@uol.com.br

Submitted: July 19, 2001; Returned to authors for corrections: February 20, 2002; Approved: September 24, 2002.

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