Microbiol. Res.
(2003) 158, 353–357
http://www.urbanfischer.de/journals/microbiolres
In vitro antimicrobial activity of propolis and synergism
between propolis and antimicrobial drugs
Srdjan Stepanović, Nataša Antić, Ivana Dakić, Milena Švabić-Vlahović
Department of Bacteriology, Institute of Microbiology and Immunology, School of Medicine, University of Belgrade,
Dr Subotića 1, 11000 Belgrade, Serbia
Accepted: November 5, 2003
Abstract
The aim of this study was to investigate antimicrobial proper-
ties of ethanolic extract of 13 propolis (EEP) samples from
different regions of Serbia against 39 microorganisms (14 re-
sistant or multiresistant to antibiotics), and to determine syn-
ergistic activity between antimicrobials and propolis. Anti-
microbial activity of propolis samples was evaluated by agar
diffusion and agar dilution method. The synergistic action of
propolis with antimicrobial drugs was assayed by the disc
diffusion method on agar containing subinhibitory concentra-
tions of propolis. Obtained results indicate that EEP, irrespec-
tively of microbial resistance to antibiotics, showed significant
antimicrobial activities against Gram-positive bacteria (MIC
0.078%–1.25% of EEP) and yeasts (0.16%–1.25%), while
Gram-negative bacteria were less susceptible (1.25%–>5%).
Enterococcus faecalis was the most resistant Gram-positive
bacterium, Salmonella spp. the most resistant Gram-negative
bacteria, and Candida albicans the most resistant yeast. EEP
showed synergism with selected antibiotics, and displayed
ability to enhance the activities of antifungals. The shown
antimicrobial potential of propolis alone or in combination
with certain antibiotics and antifungals is of potential medical
interest.
Keywords: Antimicrobial activity – Propolis – Resistance –
Synergism
Introduction
There is a steady increase in the incidence of antimicro-
bial resistance worldwide. Resistance has particularly
spread in pathogens causing nosocomial infections, but
Corresponding author: Srdjan Stepanović
e-mail: stepan@afrodita.rcub.bg.ac.yu
0944-5013/03/158/04-353 $15.00/0
also in organisms causing community acquired infec-
tions (Levy 2002). Beside the well known pathogens,
resistance has appeared in opportunistic microorgan-
isms (Levy 2002). Antimicrobial resistance results in
increased illness, deaths, and health-care costs, high-
lighting the need for novel antimicrobial agents.
Propolis (bee glue) is a resinous product that honey-
bees collect from living plants and use in construction
and adaptation of their hives (Bankova et al. 2000). Pro-
polis is extensively used in folk medicine, and a number
of investigations have shown that propolis posses anti-
bacterial, antiviral and antifungal properties (Mirzoeva
et al. 1997; Park et al. 1998; Kujumgiev et al. 1999;
Bosio et al. 2000; Drago et al. 2000; Hegazi et al. 2000;
Sforcin et al. 2000; Hegazi and El Hady 2001; Ota et al.
2001). However, most studies were done only with a
limited number of strains and/or with strains of
unknown susceptibility to antibiotics. Moreover, it has
been shown that there were variations in the antimicro-
bial activity according to the propolis origin (Hegazi
et al. 2000; Hegazi and El Hady 2001).
The objective of this work was to investigate anti-
microbial properties of 13 propolis samples obtained
from different regions of Serbia against 39 microorgan-
isms, and to explore synergistic activity between anti-
microbials and propolis.
Materials and methods
Propolis. Thirteen propolis samples were obtained from
the beehives situated in different areas of Serbia. The
propolis samples were ground into a fine powder, and
thereafter 2 g of the each propolis powder was mixed
Microbiol. Res. 158 (2003) 4 353
with 10 ml of 95% ethanol to obtain 20% (w/v) propolis
extract. Extraction was carried out at room temperature
in the dark for 7 days, with periodical strong hand
shaking. After extraction, the mixture was centrifuged,
and supernatants were designated as an ethanolic extract
of propolis (EEP).
Microorganisms. A total of 39 microorganisms, refer-
ence strains and clinical isolates, were used in this study,
21 Gram-positive bacteria, 12 Gram-negative bacteria
and six yeast strains. Among them, four Staphylococcus
aureus, and one strain of S. sciuri, S. epidermidis, S.
xylosus, Enterococcus faecalis, Klebsiella pneumoniae,
Serratia marscescens, Providencia stuartii, P. rettgeri,
Morganella morganii, Salmonella enteritidis were re-
sistant or multiresistant to antibiotics commonly used
for treatment of infections caused by these bacteria.
Stock cultures of bacteria were maintained on Tryptose
agar (Difco laboratories, Detroit, Michigan, USA) at
4 °C. Prior to inoculation, all strains were transferred
from the stock cultures to Tryptose agar and incubated
overnight at 35 °C. All strains were subsequently sub-
cultured one more time under the same conditions. The
grown cultures were used for preparation of suspensions
in sterile phosphate buffered saline (PBS; pH 7.2) with
densities adjusted to 0.5 McFarland standard. As need-
ed, the obtained suspensions were further diluted in PBS
to achieve appropriate number of cells per ml. Yeasts
were cultured on Sabouraud dextrose agar (bioMérieux,
Marcy-l’Etoile, France).
Antimicrobial activity. Antimicrobial activity of propo-
lis extracts was determined by two techniques.
For agar well diffusion method 13 microbial strains
were used. Suspensions of microorganisms containing
106 cells/ml were inoculated onto plate surfaces with a
sterile cotton swab. Test plates (diameter 10 cm) were
prepared with 20 ml of Mueller-Hinton agar (Oxoid,
Unipath Ltd., Basingstoke, Hampshire, England), and
holes of 6 mm in diameter were punched in the agar
plates. Each hole was filled with 50 µl of EEP or as the
control 50 µl of 95% ethanol. The diameters of the
growth inhibition zones around the holes were measured
after incubation for 48 h at 35°C.
Agar dilution method was performed with 39 micro-
organisms. Test plates were prepared with 19 ml of
Mueller-Hinton agar, and 1 ml of 2-fold dilutions of
each of the thirteen EEP made in 95% ethanol as
the diluent. Control plate was made with 1 ml of 95%
ethanol. After cooling and drying, the plates were spot
inoculated with 10 µl of the 10 6 cells/ml suspension.
Plates were incubated for 48 h at 35°C, and the minimal
inhibitory concentrations (MIC) were defined as the
lowest concentration of propolis that inhibited visible
growth of microorganisms spots.
Synergy assay. Synergy assay was based on previously
described procedure (Mirzoeva et al. 1997). Three
microorganisms were used for synergy assay: oxacillin
resistant and multiresistant S. aureus, multiresistant
K. pneumoniae and C. albicans. The synergistic action
of EEP with antibiotics/antifungals was assayed by the

Table 1. Antimicrobial activity of ethanolic extracts of 13 propolis samples obtained from different regions of Serbia
Region where Zone of inhibition of microbial growth (mm)
propolis was without the size of the hole
collected
Gram-positive bacteria Gram-negative bacteria Yeast
1 2 3 4 5 6 7 8 9 10 11 12 13

Čačak 9 9 6 6 9 10 / / / 6 7 5 4
Zaklopača 10 13 8 7 11 12 / / 1 6 10 7 6
Raška 9 12 8 6 11 12 / 1 1 6 10 5 5
Vlasotince 9 12 7 6 10 13 / / / 4 10 6 5
Surčin 9 12 8 7 10 13 / / / 4 12 5 5
Pančevo 9 12 8 7 12 13 / / / 4 11 5 5
Kragujevac 10 12 7 6 13 12 / / / 5 11 6 5
East Serbia 1 9 12 8 6 11 12 / / / 2 10 5 5
East Serbia 2 10 11 7 6 13 12 / 1 / 5 10 5 5
East Serbia 3 10 12 8 7 10 12 / / / 4 11 6 5
East Serbia 4 10 12 8 7 10 12 1 1 1 2 10 6 6
Plandište 9 10 7 6 10 10 / / / 2 10 6 5
Aleksinac 11 12 7 6 10 11 1 2 1 6 11 6 5

1 = S. epidermidis ATCC 14990; 2 = S. aureus ATCC 25923; 3 = S. sciuri ATCC 29062; 4 = E. faecalis ATCC 29212; 5 =
B. subtilis; 6 = L. monocytogenes SLCC 2375; 7 = E. coli ATCC 25922; 8 = P. aeruginosa ATCC 27853; 9 = S. marscenscens;
10 = P. stuartii; 11 = C. guilliermondii; 12 = C. parapsilosis; 13 = C. albicans

354 Microbiol. Res. 158 (2003) 4

disc diffusion method on Mueller-Hinton agar for bac-
teria, and glucose enriched Mueller-Hinton agar (Lee
et al. 2001) for C. albicans. One ml of appropriate EEP
dilution and 19 ml of the cooled medium were mixed
to obtain concentration of EEP which did not inhibit
the growth of tested microorganism. Final concentra-
tions of EEP in the media were 0.63% and 0.31% for
K. pneumoniae, 0.16% and 0.078% for C. albicans and
0.078% and 0.039% for S. aureus. Plates containing
sub-inhibitory concentrations of EEP, as well as control
plates containing 95% ethanol instead of EPP, were
seeded with the tested bacterial strains as described for
agar well diffusion method, and as it was described by
Lee et al. (2001) for C. albicans. The following discs
(Torlak, Belgade, Serbia) were placed on the seeded
plates: ampicillin (10 µg), ceftriaxone (30 µg), doxycy-
cline (30 µg), amikacin (30 µg), nalidixic acid (30 µg)
and trimethoprim/sulfamethoxazole (1.25 + 23.75 µg)
for bacteria, and nystatin (100 UI) (Sanofi Diagnostics
Pasteur, Marnes la Coquette, France) for C. albicans.
Plates were incubated 48 h at 35°C, before the effect of
EEP was measured by the relation of diameters of the
growth inhibition zones around the discs in the presence
of EEP to the diameters of the growth inhibition zones
around the discs without the presence of EEP (Mirzo-
eva et al. 1997).
Results
Results of the evaluation of antimicrobial activities of
13 propolis samples obtained by agar well diffusion
method, showed significant antimicrobial activities
against Gram-positive bacteria and yeast, but far less
against Gram-negative bacteria (Table 1).
More precise information on antimicrobial activities
of 13 propolis samples was obtained by agar dilution
method (Table 2). MICs for Gram-positive bacteria

Table 2. Minimum inhibitory concentrations for ethanolic extracts of 13 different propolis

Microorganism Number of propolis samples displaying various MICs against different microbial strains*
(number of strains)
MIC: >5 5 2.5 1.25 0.63 0.31 0.16 0.078

Gram-positive bacteria
S. epidermidis (2) 2 17 6 1
S. xylosus (1) 1 11 1
S. lentus (1) 9 4
S. sciuri (2) 7 17 2
S. intermedius (1) 8 5
S. aureus (6) 12 59 7
M. luteus (2) 10 16
L. monocytogenes (1) 1 12
E. faecalis (2) 1 5 20
B. subtilis (2) 2 16 8
B. cereus (1) 10 3
Gram-negative bacteria
S. flexneri (1) 7 6
Y. enterocolitica (1) 4 9
S. typhimurium (1) 3 10
S. enteritidis (1) 4 9
E. coli (1) 3 5 5
K. pneumoniae (1) 10 2 1
S. marscescens (1) 2 2 9
P. stuartii (2) 11 7 8
M. morganii (1) 1 4 8
P. rettgeri (1) 3 6 4
P. aeruginosa (1) 6 4 3
Yeasts
C. guilliermondii (2) 1 17 8
C. parapsilosis (1) 7 6
C. albicans (3) 2 4 30 3

* The numbers presented were calculated as: number of propolis samles displaying indicated
MIC value × number of tested microbial strains

Microbiol. Res. 158 (2003) 4 355

Table 3. Synergy between antibiotics/antifungals and 13 different ethanolic extracts of propolis (EEP)
Microorganism/antibitoic (susceptibility to antibiotics)
S. aureus
ampicillin – resistant
ceftriaxone – resistant
doxycycline – resistant
amikacin – susceptible
nalidixic acid – resistant
trimethoprim/sulfamethoxazole – resistant
K. pneumoniae
ampicillin – resistant
ceftriaxone – susceptible
doxycycline – susceptible
amikacin – intermediate susceptible
nalidixic acid – resistant
trimethoprim/sulfamethoxazole – resistant
C. albicans
nystatin – 16 mm***
* Data presents the minimal and maximal values of 13 tested ethanolic extracts of propolis
** 1, no synergism; >1, synergism present
*** Because of known problems with interpretation of yeast susceptibility to antifungals, the diameter of the growth inhibition
zone around the nystatin is given.
were in the range of 0.078%–1.25% EEP, Gram-nega-
tive bacteria 1.25%–>5% EEP, and yeasts 0.16–1.25%
EEP. E. faecalis was the most resistant Gram-positive
bacterium, Salmonella spp. was the most resistant
Gram-negative bacteria, and C. albicans was the most
resistant yeast.
The results of synergy between antibiotics/antfungals
and 13 different EEP on multiresistant S. aureus, multi-
resistant K. pneumoniae and C. albicans are presented
in Table 3.
Discussion
The chemical composition of propolis is very complex
and depends on the flora in the areas where it was col-
lected (Bankova et al. 2000). Therefore variations
shown in the antimicrobial activity according to the pro-
polis origin (Hegazi et al. 2000; Hegazi and El Hady
2001) are not surprising. However, results of the evalu-
ation of antimicrobial activities of 13 propolis samples
from different parts of Serbia, obtained by agar well dif-
fusion method revealed only minor variations in the
antimicrobial activity according to the propolis origin.
Bosio et al. (2000) found that diffusion method is not
suitable for comparison of the specimens since the mini-
mal concentration of propolis that permitted measure-
ment of the diameter of the inhibition zone is relatively
high, and propolis solutions display irregular diffusion.
However, MIC of the EEP obtained by agar dilution
356 Microbiol. Res. 158 (2003) 4
Synergism with indicated concentration of EEP*
0.078% EEP 0.039% EEP
1–1.3** 1–1.23
1–1.33 1–1.25
1.08–1.19 1–1.08
1 1
1–1.1 1–1.1
1 1
0.63% EEP 0.31% EEP
1 1
1.5–2.0 1.45–1.6
1.04–1.15 1.11–1.41
1 1
1 1
1 1
0.16% EEP 0.078% EEP
1.56–1.88 1.38–1.63
method confirmed only small variations in the antimi-
crobial activity according to the propolis origin. Similar
antimicrobial activity does not necessarily mean that
there is no difference in the composition of tested pro-
polis samples, since Kujumgiev et al. (1999) observed
that in different samples, different substance combina-
tions are essential for the biological activity of the pro-
polis.
Although the antimicrobial properties of propolis
have been the subject of many investigations, it is diffi-
cult to compare the results of different studies, due to the
different compositions of propolis and/or different
methods used for the evaluation of propolis antibacteri-
al activities (Drago et al. 2000). However, it is gener-
ally recognized that Gram-positive bacteria are more
susceptible to antibacterial action of propolis than
Gram-negative bacteria (Mirzoeva et al. 1997; Drago
et al. 2000; Sforcin et al. 2000), which is confirmed in
this study on various microorganisms. The result of
particular interest of this study is that resistance of tested
bacteria to antibiotics has no influence on the suscep-
tibility to EEP.
The results of synergistic action of EEP with antibio-
tics demonstrated potential of propolis to enhance anti-
biotic action and, thus, support previous findings on
synergistic action between antibiotics and propolis
(Krol et al. 1993; Mirzoeva et al. 1997). However, dif-
ferent propolis samples showed different potential to
enhance antibiotics actions. The inhibition zones around
the discs of antibiotics, on which EEP had the influence,
were larger in the presence of higher concentrations of
EEP. The only exception from this was doxycycline
when used against K. pneumoniae. This antibiotic has
the larger inhibition zones in the presence of lower
quantities of EEP. There is no apparent explanation for
this finding. The important finding of this study is
displayed ability of propolis extracts to enhance anti-
fungal activities. Interestingly little attention has been
given previously to the synergistic effects of propolis
and antifungals. To the best of our knowledge this is the
first time that this has been shown. Combination of pro-
polis with antimicrobial agents could allow the reduc-
tion in the dose of selected antimicrobials and potentia-
tion of the antimicrobial therapy (Mirzoeva et al. 1997).
This study has shown that propolis has significant
antimicrobial potential against bacteria and yeasts, but
the effect is species dependent. The resistance of tested
bacteria to antibiotics has no influence on the suscept-
ibility to EEP. The shown potential of propolis to en-
hance antibiotics and especially antifungal action is of
potential medical interest especially for topical applica-
tion.
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