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Journal of Antimicrobial Chemotherapy Advance Access published March 18, 2011
J Antimicrob Chemotherdoi:10.1093/jac/dkr108
In vitro synergistic effects of baicalin with oxytetracycline
and tetracycline against Staphylococcus aureus
Pavel Novy1, Jan Urban2, Olga Leuner1, Jaroslav Vadlejch3 and Ladislav Kokoska1,3*
1Department of Crop Sciences and Agroforestry, Institute of Tropics and Subtropics, Czech University of Life Sciences Prague,
Kamycka 129, 165 21 Prague 6—Suchdol, Czech Republic; 2National Reference Laboratory for Disinfection and Sterilization, National
Institute of Public Health, Srobarova 48, 100 42 Prague 10, Czech Republic; 3Department of Zoology and Fisheries, Faculty of Agrobiology,
Food and Natural Resources, Czech University of Life Sciences Prague, Kamycka 129, 165 21 Prague 6—Suchdol, Czech Republic
*Corresponding author. Tel: +420-224382180; Fax: +420-234381829; E-mail: email@example.com.
Received 29 November 2010; returned 6 January 2011; revised 4 February 2011; accepted 18 February 2011
Objectives: In this study we examined the in vitro synergistic effect of baicalin, the flavone constituent ofScutellaria spp., in combinations with oxytetracycline and tetracycline on the growth of Staphylococcus aureus.
Methods: The MICs were determined by the broth microdilution method and the effect of combinations wasevaluated according to the sum of fractional inhibitory concentration indices (FICIs).
Results: Synergistic activity (FICI≤0.5) was observed for combinations of baicalin with oxytetracycline or tetra-cycline against 4 of 10 strains tested, whereas the baicalin/oxytetracycline combination possessed the stron-gest synergistic effect (FICI¼ 0.418).
Conclusions: Baicalin acts synergistically with oxytetracycline and tetracycline, enhancing its antimicrobialactivity against S. aureus, including methicillin- and tetracycline-resistant strains.
Keywords: antimicrobial agents, flavonoid, staphylococci, synergy
with penicillin binding proteinsDespite the fact that the synergyof baicalin with b-lactams against S. aureus has already been
Staphylococcus aureus has become one of the most serious human
reported, its ability to potentiate antistaphylococcal activity in
pathogens during recent decades owing to its acquisition of anti-
other antibiotics has not previously been scrutinized. Therefore,
biotic resistance, whereas methicillin-resistant S. aureus (MRSA) is
we decided to perform a screening test focused on determining
at the present time one of the most important causes of
its combined effect with representatives of the main antibiotic
antibiotic-resistant healthcare-associated infections worldwide,
groups, namely b-lactams (ampicillin, cefamandole, oxacillin, peni-
resulting in prolonged hospital stays and increased mortality.
cillin), tetracyclines (doxycycline, minocycline, oxytetracycline,
Because of its exceptional ability to also acquire resistance to
tetracycline), aminoglycosides (streptomycin), quinolones (cipro-
other commonly used classes of antibiotics, such as aminoglyco-
floxacin), macrolides (erythromycin), lincosamides (lincomycin),
sides, fluoroquinolones, macrolides and tetracyclines, the contin-
glycopeptides (vancomycin) and sulphonamides (sulfanilamide),
ual development of new antibacterial compounds and, more
against three standard strains of S. aureus. Together with
importantly, new templates with novel modes of action,
b-lactams, tetracyclines produced results worth further investi-
represented for example by synergistically acting plant-derived
gation (P. Novy and L. Kokoska, unpublished data). As a continuation
compounds, is urgently needed to eradicate the resistance of
of this work, the present study reports on a detailed examination of
S. aureus and to slow down its spread and development.
the synergistic effect of oxytetracycline and tetracycline with baica-
Among a number of potential candidates of this type of agent is
lin against four standard strains and six clinical isolates of S. aureus.
baicalin (Figure ), a flavonoid present in Scutellaria baicalensisGeorgi. It exerts, along with other medicinal properties,remark-
able synergistic antistaphylococcal activity with antibiotics. Ithas been reported to restore the effectiveness of b-lactam anti-
biotics against MRSA and other strains of b-lactam-resistant
Oxacillin, oxytetracycline and tetracycline were purchased from
S. aureus, probably via its direct antibacterial action on cell
Sigma-Aldrich (Prague, CZ). DMSO, ethanol (Lach-Ner, Neratovice, CZ)
growth or due to the inhibition of b-lactamase, or by interactions
and deionised water were used as solvents.
The Author 2011. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
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oxytetracycline and tetracycline. The combination of baicalinand oxytetracycline showed the strongest synergistic effect,
whereas the addition of baicalin in concentrations of 75 and
50 mg/L resulted in an 8- and 4-fold reduction (FICI ¼0.418
and 0.445), respectively, in oxytetracycline MIC against S.
aureus ATCC 29213. The baicalin/oxytetracycline combination
showed synergistic activity against 4 of 10 S. aureus strains
tested, of which 1 was a MRSA strain and 1 was a TRSA strain.
The baicalin/tetracycline combination had a synergistic effectagainst one S. aureus strain, where the addition of 50 mg/L bai-
Figure 1. Chemical structure of baicalin.
calin resulted in a 4-fold reduction in the MIC of tetracycline(FICI¼ 0.445). In contrast to a report by Fujita et al.,describing
that baicalin at concentrations below its MIC does not signifi-cantly affect the growth-inhibitory activity of tetracycline
The standard strains [ATCC 25923, ATCC 29213, ATCC 43300 (MRSA) and
against MRSA, our results showed that 50, 75 and 100 mg/L bai-
ATCC BAA-976] were purchased from Oxoid (Basingstoke, UK), whereas
calin (,1/2 MIC) caused a 2- to 8-fold reduction in the MIC of
clinical isolates (M02, M05), including MRSA (M238) and tetracycline-
tetracycline against TRSA and MRSA strains (FICI¼ 0.516 –
resistant S. aureus (TRSA) strains (M003, M079, M926), were obtained
from the Motol University Hospital in Prague, Czech Republic. For antibac-
0.793). Despite the differences in results obtained from both
terial assays, the strains were grown in cation-adjusted Mueller– Hinton
studies, which may be influenced by variations in experimental
broth (MHB; Oxoid, Basingstoke, UK), which was equilibrated with Tris-
design (e.g. only two strains were tested in the above mentioned
buffered saline (Sigma-Aldrich, Prague, CZ).
report), our findings suggest a certain degree of combined anti-staphylococcal effect of baicalin and tetracycline against resist-ant strains. Combinations of baicalin with doxycycline and
minocycline exerted no interaction when tested against three
A broth microdilution method was used to determine the MICs of test
standard S. aureus ATCC strains (FICI¼ 0.73–1.5), while baicalin
antimicrobial agents following the guidelines described by the CLSI
at concentrations below its MIC caused a 2-fold reduction in
using 96-well microtitre plates. Briefly, the samples were 2-fold diluted
in MHB (100 mL), inoculated with bacterial suspension to obtain the
Our positive results on the synergy of baicalin with oxytetracy-
final concentration of 5×105 cfu/mL and then incubated at 378
cline or tetracycline can also be supported by the fact that the
24 h. The bacterial growth was measured as turbidity by a Multiscan
FICI of both combinations reached values of ,0.6 for 75% of
Ascent Microplate Photometer (Thermo Fisher Scientific, Waltham, MA,
S. aureus strains tested, which would be assessed as a strong
USA) at 405 nm. The MIC was defined as the lowest concentration ofthe compound that inhibited the growth of the test bacteria by ≥80%.
additive effect if evaluated according to the European Commit-
Oxacillin and tetracycline were used as markers of methicillin and tetra-
tee for Antimicrobial Susceptibility Testingcriteria for synergy.
cycline resistance, respectively, and S. aureus ATCC 29213 was used as a
Thus, although synergistic interactions of baicalin with oxytetra-
reference strain for the standardization of antibiotic susceptibility testing.
cycline and baicalin with tetracycline were observed against 4
All tests were performed as three independent experiments, each carried
and 1 of 10 strains, respectively, we can conclude that baicalin
significantly potentiated the antimicrobial effect of oxytetracy-cline and tetracycline against S. aureus. These results suggest
baicalin is a promising compound for the development of newsynergistically acting drugs with the potential to extend the
Initially, 2-fold serial dilutions of an antibiotic were tested in combination
pharmacological action of tetracyclines, which, as antistaphylo-
with 2-fold serial dilutions of baicalin, where individual MICs of each com-
coccal agents, are clinically applied in limited cases only (e.g. in
pound were used as starting concentrations. The most promising combi-
the treatment of skin or skin structure infections due to
nations were then selected for detailed testing, where 2-fold serial
S. aureus susceptible strIn addition, since the effectiveness
dilutions of antibiotics starting at quadruple concentration of their par-
of oxytetracycline and tetracycline (FICI ¼0.42 –0.7) was signifi-
ticular MICs were combined with constant concentrations of baicalin
cantly more improved than the action of doxycycline and mino-
(100, 75 and 50 mg/L). The fractional inhibitory concentration (FIC) wascalculated as the ratio of the MIC of agents A and B in combination
cycline (FICI ¼0.75 –1.25) when tested against S. aureus ATCC
with the MIC of agent A (or B) alone. The FIC index (FICI) was then calcu-
29213, 25923 and 43300 (P. Novy and L. Kokoska, unpublished
lated using the combined FICs of agents A and The obtained results
data), we estimate that baicalin has the ability to augment the
were interpreted in accordance with the BSAC recommendation as
antistaphylococcal effect of the short-acting tetracyclines to a
follows: synergy (FICI≤0.5); no interaction (FICI.0.5–4); antagonism
much greater extent than the representatives of the long-acting
Previously it has been suggested that baicalein, the aglycone
of baicalin, may produce a similar effect on the cell wall as epi-gallocatechin which interferes with cell wall integrity
The susceptibilities of S. aureus strains to the antibiotics tested,
through direct binding to peptidoglycanand exhibits the charac-
either alone or in the presence of baicalin, are summarized in
teristics of a cell wall active agent against enterococci.Since
Table . The results show that baicalin possesses only a weak
both baicalin and baicalein possess a flavone structure and
antistaphylococcal effect (MICs ranging from 128 –512 mg/L),
similar pharmacological activity, both have a weak antimicrobial
but its addition resulted in significant MIC reduction in
effect against S. aureus and both exert synergistic antimicrobial
Antistaphylococcal synergistic effect of baicalin with tetracyclines
Table 1. In vitro inhibitory activity of baicalin in combination with oxytetracycline and tetracycline against S. aureus
OXY with B at following concentrations (mg/L):
TET with B at following concentrations (mg/L):
B, baicalin; OXA, oxacillin; OXY, oxytetracycline; TET, tetracycline.
FICI: ≤0.5, synergy (bold font indicates synergistically acting combinations); .0.5–4, no interaction; .4, antagonism.
can surmise that their modes of action could possibly be
similar. In addition, since baicalein has previously been reported
to restore the effectiveness of tetracycline against MRSA due tothe inhibition of TetK-mediated tetracycline efflux and possiblyinhibits TetM or some other we can presume that also
baicalin may act as an efflux pump inhibitor. Moreover, the
1 Ruhe JJ, Monson T, Bradsher RW et al. Use of long-acting tetracyclines
synergistic effect of the baicalin/tetracycline combination
for methicillin-resistant Staphylococcus aureus infections: case series and
against one TRSA strain tested in this study may indicate its
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selectivity for particular tetracycline resistances mechanisms,
for instance, against a specific efflux pump. On the other hand,
weaknesses and opportunities. Planta Med 2008; 74: 594– 602.
the fact that we did not observe any significant difference
3 Li-Weber M. New therapeutic aspects of flavones: the anticancer
between the FICIs of effective combinations obtained against
properties of Scutellaria and its main active constituents wogonin,
resistant and susceptible S. aureus strains indicates that baicalin
baicalein and baicalin. Cancer Treat Rev 2009; 35: 57– 68.
does not affect resistance mechanisms of resistant strains but
4 Liu IX, Durham DG, Richards RME. Baicalin synergy with b-lactam
affects some other processes common to both susceptible and
antibiotics against methicillin-resistant Staphylococcus aureus and
resistant S. aureus strains. In view of this, we suggest that baica-
other b-lactam-resistant strains of S. aureus. J Pharm Pharmacol 2000;
lin may have a direct antimicrobial effect on cell growth and the
synergistic activity of baicalin with tetracyclines is probably pro-
5 Clinical and Laboratory Standards Institute. Methods for Dilution
duced by a multitarget effect of combined compounds.
Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—
In conclusion, this is the first report on the synergistic effect of
Eighth Edition: Approved Standard M07-A8. CLSI, Wayne, PA, USA, 2009.
oxytetracycline and tetracycline with baicalin against S. aureus,
including its MRSA and TRSA strains. Regarding the synergy
Terminology relating to methods for determination of susceptibility
mechanism of its antistaphylococcal effect, direct enhancement
bacteria to antimicrobial agents. Clin Microbiol Infect 2000; 6: 503– 8.
of antibiotic activity rather than a resistance reversal effect is
7 Odds FC. Synergy, antagonism, and what the chequerboard puts
probably responsible for the inhibition of resistant strains. In
between them. J Antimicrob Chemother 2003; 52: 1.
addition, because of its ability to potentiate the effect of tetra-
8 Fujita M, Shiota S, Kuroda T et al. Remarkable synergies between
cyclines against S. aureus and its low toxicity,baicalin seems
baicalein and tetracycline, and baicalein and b-lactams against
to be an attractive candidate for the development of new syner-
methicillin-resistant Staphylococcus aureus. Microbiol Immunol 2005;
gistically acting antistaphylococcal drugs.
9 Zhao WH, Hu ZQ, Okubo S et al. Mechanism of synergy betweenepigallocatechin-gallate and b-lactams against methicillin-resistantStaphylococcus aureus. Antimicrob Agents Chemother 2001; 45:1737– 42.
10 Chang P-C, Li H-Y, Tang H-J et al. In vitro synergy of baicalein and
This research was supported by projects CSF 525/08/H060 and
gentamycin against vancomycin-resistant Enterococcus. J Microbiol
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