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Journal of Venomous Animals and Toxins

Print version ISSN 0104-7930On-line version ISSN 1678-4936

J. Venom. Anim. Toxins vol. 1 n. 2 Botucatu  1995 

Original paper





A. FERNANDES Jr.1,3, M.F. SUGIZAKI , M.L. FOGO , S.R.C. FUNARI2,3 , C.A.M. LOPES1,3 Image1812.gif (888 bytes)

1 Department of Microbiology and Immunology of the Institute of Biosciences of Botucatu - UNESP, Botucatu, State of São Paulo, Brazil, 2 Department of Production and Animal Exploration of the School of Veterinary Medicine and Animal Husbandry of Botucatu - UNESP, Botucatu, State of São Paulo, Brazil, 3 Center for the Study of Venoms and Venomous Animals - CEVAP - UNESP, Botucatu, State of São Paulo, Brazil.



ABSTRACT. The in vitro activity of propolis against 118 Staphylococcus aureus, 108 Escherichia coli, 60 Salmonella typhimurium, 50 Candida albicans, 23 Candida parapsilosis, 19 Candida tropicalis and 14 Candida guilliermondii isolated from human infections was studied by the agar dilution method. Among the bacteria, the Gram-negative were the least susceptible organisms showing minimal inhibitory concentration (MIC) for 90% of the strains tested ranging from 22.5 mg/ml - 10,2% V/V to 23.1 mg/ml - 10.5% V/V. The MIC's for Candida ranged from 0.80 mg/ml to > 11 mg/ml (0.40% V/V to > 5.0% V/V), the strains of C. parapsilosis being the least susceptible. The relative order of susceptibility among all isolates, was: S.aureus > C. tropicalis > C. albicans > C. guilliermondii > C. parapsilosis > S. typhimurium > E. coli.
KEY WORDS: propolis, antimicrobial activity, bacteria, yeast pathogens, Candida sp




Propolis is a resinous hive product collected by bees known to possess several significant pharmacological properties(2). Because of propolis diverse biological activities and increasing industrial interest, its chemical composition is of great importance. Furthermore, investigations have shown that individual compounds such as the flavonoids(1) are responsible for spasmolytic (quercitin, kaempferol and pectolinargenin), antiinflamatory (acacetin), antiulcerative (apigenin) or antimicrobial (pirocembrin and galangin) activities(2,3).

Since many reports dealing with propolis are not available to most readers, except as abstracts, and most of them are published by eastern European Journals, and a few available in Brazil(6,10), this study was undertaken to compare the sensitivity of some significant bacterial and yeast pathogens from human infections with the antimicrobial activity of propolis produced in hives, maintained and controlled in the University apiary.



PREPARATION OF PROPOLIS SOLUTION FOR TESTING: Samples of propolis were obtained from hives maintained and controlled by the technical staff of the Department of Production and Animal Exploration of the School of Veterinary Medicine and Animal Husbandry of Botucatu, UNESP. After screening and homogenization procedures, samples were carried out from the production site to the laboratories of the Department of Microbiology and Immunology of the Institute of Biosciences of Botucatu, UNESP in polyethylene buckets with tight-fitting lids and stored in the dark at 5ºC. Solutions of propolis for testing were prepared asepticaly and protected from brigh light to prevent photo-degradation. For its preparation, the product "in natura" was dissolved by stirring it in ethanol 96º GL (MERCK) and submitted to filtration. The final concentration in each main solution was calculated to yield a standard of 50% (V/V), which corresponded to an amount of 220 mg/ml of propolis.

TEST ORGANISMS: Strains of bacteria and yeast pathogens for testing were as fallows: Staphylococcus aureus (No. = 118), Escherichia coli (No. = 108), Salmonella typhimurium (No. = 60), Candida albicans (No. 50), Candida parapsilosis (No. = 23), Candida tropicalis (No. = 19) and Candida guilliermondii (No. = 14), all sequentially isolated from clinical specimens of patients treated at the University Hospital of the School of Medicine of Botucatu, UNESP. The panel of microorganisms was identified by current standard microbiological methods according to Koneman et al.(4), and Kreger-Van Rij(5).

SUSCEPTIBILITY TESTING: Determination of minimal inhibitory concentration (MIC) by the agar dilution method was performed following the National Committee for Clinical Laboratory Standard guidelines (7,8). Strains of bacteria were grown in Brain Heart infusion Agar (Difco) and yeasts in Sabouraud Dextrose Agar (Difco) at 35ºC/24 h. After incubation, five colonies of each strain were suspended in 5 ml of sterile 0.85% Phosphate Buffer Solution (PBS), and then the suspensions were diluted 1/10 and 1/100 in PBS to yield a final inoculum of approximately 5 x 105 UFC/ml for the bacteria and 5 x 104 cells/ml for yeasts. By using the standardized main solution, serial concentrations of propolis were achieved (% V/V) in plates containing Sabouraud Dextrose Agar and Mueller Hinton Agar for the yeasts and bacteria, respectively, as follows: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 3.0, 4.0, 5.0, and additional concentrations of 6.0, 7.0, 8.0, 9.0, 10.5 (%V/V) for the bacteria tested. Each antimicrobial test also included a duplicate of plates containing the culture medium plus ethanol in correspondent volumes to obtain intermediary and final concentrations of propolis as a control of the solvent antimicrobial effect. Staphylococcus aureus ATCC 29213, Escherichia coli K12 and Saccharomyces cerevisiae ATCC 2601 were used as control reference strains. After the inoculation procedures by using a multiloop replicator, plates were incubated at 35ºC/24 h and MIC endpoints were read as the lowest concentration of propolis that resulted in no visible growth or haze on the surface of the culture medium. Populational analyses of data were carried out by calculating the MIC for 50% and 90% of the strains of each group of microorganisms tested.

STATISTICAL ANALYSIS: Statistical analysis of the MIC data was carried out by using the mean and median values as measures of central tendency and standard deviation (SD) and percentages [(P25%-P75 % among which 50% of the observations are seen)] as measures of variability. Comparison of MIC data among the yeasts was calculated using the analysis of variance with the F statistics. Contrasts among pairs of means were analyzed by the method of Tukey(11).



The MIC50 and MIC90 as well as the MIC range are shown in Table 1. MIC's 90% for the bacterial species varied from 1.2 mg/ml (0.6% V/V) to 23.1 mg/ml (10.5% V/V). These data do not agree with those of Miller & Lilenbaum(6) who observed inhibitory concentrations of propolis in the range of 3.3 mg/ml for S. aureus and 5.8 mg/ml and 7.2 mg/ml for E. coli and Proteus mirabilis, respectively. These authors also reported a mean difference between the inhibitory and bactericidal concentrations with an equivalence of 8-10 times to each other. On the other hand, our data confirm those of the literature which emphazise the lower sensitiveness of the Gram-negative species compared to Gram-positive(2,6) ones.


TABLE 1. Antimicrobial susceptibility of bacteria and Candida sp to propolis.



Among the tested yeasts, C. albicans and C. tropicalis showed a higher susceptibility than C. parapsilosis and C. guillermondii. MIC's for C. albicans (3.8 mg/ml - 1.7% V/V) and C. tropicalis (2.1 mg/ml - 0.9% V/V) were partially similar to those obtained by Souza et al.(9), who observed that the susceptibilty of C. albicans ranged from 1.2 mg/ml to 2.56 mg/ml, considering both the fungistatic and fungicid effects. Data on some Candida species, such as C. parapsilosis and C. guilliermondii are not available in the literature for a comparative analysis. However, C. parapsilosis strains (10.9 mg/ml - 4.9% V/V) were only inhibited by concentrations twice as low as those for Gram-negative bacteria.

In this study, there were also significant statistical differences between susceptibility patterns of Gram negative and Gram-positive bacteria, as well as among the Candida species. MIC's for C. albicans and C. tropicalis were significantly different from those for C. parapsilosis and C. guilliermondii, with differences based on median and mean response values (F = 60.32 and p < 0.001). The relative order of susceptibility based on the cumulative percentuals of microorganisms inhibited by the respective concentrations of propolis was: S. aureus > C. tropicalis > C. albicans > C. guilliermondii > C. parapsilosis > S. typhimurium > E. coli (Figure 1).


FIGURE 1. Cumulative percentual of microorganisms inhibited by the respective concentrations of propolis.


1 S. aureus 2 C. albicans 3 C. tropicalis 4 C. guilliermondii
5 C. parapsilosis 6 S. typhimurium 7 E. coli


Since variations in the susceptibility to propolis among several microorganisms have been reported, but not their specific mechanisms of action, further studies are needed to explain whether the cell structure and permeability to such compounds or even specific targets in the cell enzymatic systems are involved in microbial susceptibility.



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C.A.M. LOPES, Departamento de Microbiologia e Imunologia, Instituto de Biociências de Botucatu, UNESP - CEP 18.618-000 - Botucatu - São Paulo - Brasil.

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