E-Mail karger@karger.com
Original Paper
Med Princ Pract 2017;26:108–112
DOI: 10.1159/000454680
Natural Terpenes Influence the
Activity of Antibiotics against Isolated
Mycobacterium tuberculosis
ElwiraSieniawska
a
MartaSwatko-Ossor
b
RafałSawicki
b
KrystynaSkalicka-Woźniak
a
GrazynaGinalska
b
Departments of
a
Pharmacognosy with Medicinal Plant Unit, and
b
Biochemistry and Biotechnology,
Medical University of Lublin, Lublin, Poland
from 32 to 0.475 μg/mL for isoniazid). Combinations of myr-
cene, R-limonene, β-elemene, and sabinene with tuberculo-
static antibiotics resulted in a decreased MIC of the antibiotics
(from 3.9 to 0.475 μg/mL for ethambutol, from 15 to 0.475 μg/
mL for isoniazid, and from 0.475 to 0.237 μg/mL for rifampi-
cin) while combinations of α-pinene with ethambutol and iso-
niazid resulted in increased MIC values (from 16 to 125 μg/mL
for ethambutol, and from 32 to 125 μg/mL for isoniazid). Ri-
fampicin had a synergistic increase in activity with all the test-
ed compounds. Conclusions: Our study showed that ter-
penes enhance the activity of tuberculostatic antibiotics.
© 2016 S. Karger AG, Basel
Introduction
The World Health Organization reported that there
was a 3.3% rise in new cases of tuberculosis (TB) in 2015
[1] . It was also estimated that 20% of previously treated
Key Words
Antimycobacterial antibiotics · Minimum inhibitory
concentration · Terpenes · Synergistic activity · Tuberculosis
Abstract
Objective: In this study, we aimed to describe the influence
of natural terpenes on the antimycobacterial activity of first-
line tuberculostatic drugs against isolated Mycobacterium tu-
berculosis . Materials and Methods: The natural terpenes used
in this study were R-limonene, S-limonene, myrcene, sabi-
nene, α-pinene, and β-elemene. The values of the minimum
inhibitory concentration (MIC) for these terpenes, as well as
for combinations of terpenes with tuberculostatic antibiotics
(ethambutol, isoniazid, and rifampicin), were determined us-
ing a tube log
2
dilution method in the range of 125–0.059 μg/
mL. Results: S-limonene had a strong synergistic effect with
all tested antibiotics (MIC decreased from 16 to 0.475 μg/mL
for ethambutol, from 16 to 0.237 μg/mL for rifampicin, and
Received: May 23, 2016
Accepted: November 23, 2016
Published online: November 23, 2016
Elwira Sieniawska
Department of Pharmacognosy with Medicinal Plant Unit
Medical University of Lublin, Chodzki 1
PL–20-093 Lublin (Poland)
E-Mail elwira.sieniawska
@ gmail.com
© 2016 S. Karger AG, Basel
www.karger.com/mpp
Significance of the Study
• The study revealed the synergistic effect of combinations of R-limonene, S-limonene, myrcene, sabi-
nene, and β-elemene with first-line tuberculostatic antibiotics against isolated Mycobacterium tuber-
culosis . Hence, such combinations might be promising for treating resistant M. tuberculosis .
is is an Open Access article licensed under the terms of the
Creative Commons Attribution-NonCommercial 3.0 Un-
ported license (CC BY-NC) (www.karger.com/OA-license),
applicable to the online version of the article only. Distribu-
tion permitted for non-commercial purposes only.
Terpenes Strengthen the Activity of
Antibiotics against Mtb
Med Princ Pract 2017;26:108–112
DOI: 10.1159/000454680
109
cases involved multidrug-resistant (MDR)-TB, and that,
in this group, 9.7% were extensive drug-resistant (XDR)-
TB
[1] . Drug resistance in Mycobacterium tuberculosis
(Mtb) is caused by the sequential accumulation of muta-
tions in the genes encoding the targets of tuberculostatic
antibiotics [2] . More importantly, the active transmission
of ge notypes of several strains circulating worldwide is
the cause of increased resistance
[2] . Molecular finger-
printing methods enable the identification of MDR-TB
and XDR-TB, which constitute 0.5% of the circulating
strains in Japan and 40% in Russia, respectively
[2] . XDR-
TB is the biggest threat because it is a form of the disease
that is nearly untreatable
[1] and may develop as a multi-
system disease
[3] .
The natural terpenes are known for their antimicrobial
properties, and their detrimental effects on the structure
and function of the microbial membranes and cell walls
are thought to be evidence of antimicrobial action
[4] .
Combinations of essential oils and their constituents have
been reported to have synergistic, additive, or inhibiting
activity with antimicrobial interactions against several mi-
croorganisms
[5] . A high antimycobacterial activity of
thymol and carvacrol has been described against Mtb and
Mycobacterium bovis
[4] . Synergistic in vitro interactions
between oleanolic acid and isoniazid, rifampicin, or eth-
ambutol against Mtb have also been described
[6] .
In this study, we aimed to describe the influence of R-
limonene, S-limonene, myrcene, sabinene, α-pinene, and
β-elemene on the antimycobacterial activity of first-line
tuberculostatic drugs, i.e. isoniazid (an inhibitor of fatty
acid synthesis), rifampicin (an inhibitor of RNA poly-
merase), and ethambutol (an inhibitor of arabinose trans-
ferases involved in cell wall biosynthesis).
Materials and Methods
Standards and Media
The standard antibiotics, rifampicin, isoniazid, and ethambutol,
and also the terpenes (with the exception of sabinene) were pur-
chased from Sigma-Aldrich (Munich, Germany). Gas chromatog-
raphy-mass spectrometry (GC-MS) was used to verify the purity of
the terpenes: 98% for α-pinene, 97% for R-limonene, 98% for S-
limonene, 97% for β-elemene, and 95% for myrcene. Middlebrook
medium 7H9 supplemented with Middlebrook oleic albumin dex-
trose catalase growth supplement (OADC enrichment) was ob-
tained from BD Difco
TM
(Franklin Lakes, NJ, USA). Analytical
grade n -hexane and acetonitrile were obtained from Polish Re-
agents (Gliwice, Poland), tert- butyl-methyl ether was purchased
from Acros Organics (part of Thermo Fisher Scientific, Belgium).
The helium 5.0 used for the GC-MS analysis was 99.999% pure,
supplied by PGNiG (Warsaw, Poland). The commercially available
carrot-seed essential oil was purchased from Primavera (Germany).
Isolation of Sabinene
Sabinene used in the susceptibility testing was isolated from
commercially available Daucus carota- seed essential oil, as de-
scribed previously
[7] . Separation was carried out with Spectrum
high-performance counter-current chromatography (HPCCC)
equipment (Dynamic Extraction Co., Ltd. Slough, UK). The sol-
vent system used for the separation was a mixture of n -hexane,
acetonitrile, and tert -butyl-methyl ether in the ratio 2:
1:0.1 (v/v),
in the reverse-phase mode. A sample of 280 mg of essential oil was
used for semipreparative separation, and continuously monitored
with a UV detector at 210 nm. The 1-min fractions collected from
the beginning of the run were monitored by GC-MS performed
with a Shimadzu gas chromatograph, GC-2010 Plus, coupled with
a Shimadzu QP2010 Ultra mass spectrometer. A fused-silica capil-
lary column ZB-5 MS (length 30 m, internal diameter 0.25 mm)
with a film thickness of 0.25 mm (Phenomenex) was used. The
retention indices were determined in relation to a homologous se-
ries of n -alkanes (C8–C24) under the same operating conditions.
Compounds were identified using a computer-supported spectral
library (http://www.massfinder.com), mass spectra of reference
compounds, and MS data from the literature
[8] .
Antimycobacterial Assay
The isolated strain of Mtb (attenuated) was donated by the Na-
tional Institute of Tuberculosis and Lung Diseases, Warsaw, Po-
land. The minimum inhibitory concentration (MIC) for the tested
terpenes, antibiotics, and combinations of terpenes and antibiotics
were determined in a liquid medium, in a tube log
2
dilution test
[9] . The antibiotics: rifampicin, isoniazid, and ethambutol, with
known tuberculostatic activity, were used as a reference. Mtb was
grown in roller bottles at 37
° C in Middlebrook 7H9 liquid medium
supplemented with oleic acid, bovine albumin fraction V, dex-
trose, and catalase (OADC enrichment). The stock solutions of
terpenes and antibiotics were prepared in dimethyl sulfoxide, and
then diluted in Middlebrook 7H9 broth supplemented with OADC
enrichment. The final concentrations of tested compounds were
in the range of 0.059–125 g/mL.
The MIC values for terpenes and antibiotics alone were deter-
mined after 24 h of exposure to the tested substances in a bacterial
suspension (equivalent to a McFarland No. 1 standard). For the
determination of MICs of the combinations of antibiotics and ter-
penes, the antibiotics were added to the medium. Next, all samples
were inoculated in 12 L of Mtb cultures kept in the incubator for
12 h to equilibrate, and dissolved with broth to obtain the turbid-
ity of the suspensions comparable to McFarland No. 1 standard.
Turbidity of the suspensions was measured using a nefelometer
(BD Phoenix Spec., USA). The tested terpenes were then added to
each vial containing antibiotic and Mtb cultures, grown, and col-
lected after 24 h of exposure at 37
° C. The concentration of ter-
penes in the samples was as follows: α-pinene, 16 µg/mL; S-limo-
nene and R-limonene, 64 µg/mL; β-elemene, 2 µg/mL; sabinene,
32 µg/mL; and myrcene, 32 µg/mL. Because the bacteria inoculum
could cause traces of turbidity, the sample inoculated with Mtb and
kept at 4
° C for 12 h was used as a negative control. The second
control contained the highest concentration of DMSO used in the
samples, in order to eliminate the bactericidal effect of the solvent.
The third, positive, control for bacteria growth contained only me-
dium inoculated with Mtb.
The assessment of the activity of antibiotic-terpene combina-
tions was done according to x / y methodology
[10] . Briefly, x rep-
Sieniawska/Swatko-Ossor/Sawicki/
Skalicka-Woźniak/Ginalska
Med Princ Pract 2017;26:108–112
DOI: 10.1159/000454680
110
resents the MIC value of a drug-terpene combination, while y rep-
resents the lowest MIC value obtained with any of the single com-
pounds used within the combination tested. An x / y quotient of
<0.5 in the case of a 2-compound combination indicates enhanced
drug action. Respective x / y quotients were interpreted as follows:
no synergistic effect (–), x / y ≥ 0.5; moderate synergistic effect (+),
x / y ≤ 0.5; significant synergistic effect (++), x / y ≤ 0.1; and high syn-
ergistic effect (+++), x / y ≤ 0.05 ( Table1 ).
Results
The HPCCC technique used in this work enabled the
separation of sabinene from carrot-seed essential oil. Sa-
binene was present in fractions collected between 58 and
61 min. GC-MS evaluation confirmed the 99% purity of
the target compound.
The MIC values obtained for terpenes and antibiotics
tested alone as well as in combination are presented in
Table 1 . The cyclic monoterpene limonene showed the
lowest antimycobacterial activity, regardless of the ste-
reoisomers (MIC 64 µg/mL). The acyclic monoterpene
myrcene and the bicyclic monoterpenes sabinene and
α-pinene had higher activity than the limonenes (MIC
range 16–32 µg/mL). The lowest MIC value was obtained
for β-elemene (2 µg/mL). The investigated strain had a
similar sensitivity to terpenes and antibiotics. Ethambu-
tol and rifampicin inhibited bacterial growth at a 16-µg/
mL concentration, and isoniazid at a 32-µg/mL concen-
tration. Of the combinations of terpenes with first-line
tuberculostatic antibiotics, S-limonene had a high syner-
gistic effect with all antibiotics (MIC range 0.237–0.475
µg/mL). Rifampicin showed a high synergistic increase in
activity with every compound tested, except for β-elemene
(MIC range 0.237–0.475 µg/mL). Combinations of myr-
cene, R-limonene, and sabinene with antibiotics de-
creased the MIC for the antibiotics, while combinations
of α-pinene with ethambutol and isoniazid had no syner-
gistic effect ( Table1 ). Except for α-pinene, all the studied
compounds enhanced the susceptibility of Mtb to the ef-
fects of the antimycobacterial drugs, but differences in
sensitivity to the drugs used were observed.
Discussion
In this study, we tested the influence of natural terpenes
on an isolated Mtb strain. The results revealed a similar
sensitivity to the terpenes and antibiotics tested. What is
more, a significant enhancement of antibiotic activity in
the presence of terpenes was obtained. The MIC values
obtained for R-limonene, S-limonene, myrcene, sabinene,
α-pinene, and β-elemene tested alone suggest that acyclic,
monocyclic, and/or bicyclic structure and the number of
double bonds have no significant influence on the antimy-
cobacterial activity of monoterpenes. Similar findings
were reported in a previous study
[11] but a higher activ-
ity for aromatic and/or oxygenated monoterpenes was ob-
tained than for the corresponding carbonyl compounds
[12] . Equally important, quantitative structure-activity re-
lationship (QSAR) studies revealed that the number of
conjugated carbons, the number of phenolic and hydrox-
yl groups, and the number of acceptor atoms of hydrogen
bonds are the most important structural descriptors in the
antimycobacterial activity of terpenes
[4] .
Despite having the highest MIC, limonene showed tu-
berculostatic activity. Several studies have proved that lim-
Table 1. The influence of terpenes on antibiotics activity
Compounds MIC,
µg/mL
Enhancement of drug
activity (and respective
x/y quotients)
a
Ethambutol 16
Rifampicin 16
Isoniazid 32
α-Pinene alone 16
α-Pinene + ethambutol 125 – (7.81)
α-Pinene + isoniazid 125 – (7.81)
α-Pinene + rifampicin 0.475 +++ (0.03)
β-Elemene alone 2
β-Elemene + ethambutol 0.475 + (0.24)
β-Elemene + isoniazid 0.475 + (0.24)
β-Elemene + rifampicin 0.237 + (0.12)
Myrcene alone 32
Myrcene + ethambutol 3.9 + (0.24)
Myrcene + isoniazid 0.95 +++ (0.03)
Myrcene + rifampicin 0.475 +++ (0.03)
S-limonene alone 64
S-limonene + ethambutol 0.475 +++ (0.03)
S-limonene + isoniazid 0.475 +++ (0.01)
S-limonene + rifampicin 0.237 +++ (0.01)
R-limonene alone 64
R-limonene + ethambutol 0.95 ++ 0.06
R-limonene + isoniazid 15 + (0.47)
R-limonene + rifampicin 0.475 +++ (0.03)
Sabinene alone 32
Sabinene + ethambutol 3.9 + (0.24)
Sabinene + isoniazid 1.95 ++ (0.06)
Sabinene + rifampicin 0.475 +++ (0.03)
a
An x/y quotient of <0.5 in the case of a 2-drug combination
indicates enhanced drug action. –, ≥0.5; +, ≥0.5; ++, ≥0.1; +++,
≥0.05.
Terpenes Strengthen the Activity of
Antibiotics against Mtb
Med Princ Pract 2017;26:108–112
DOI: 10.1159/000454680
111
onene interacts with the cytoplasmic membranes of bacte-
ria, resulting in a loss of membrane integrity, the dissipa-
tion of the proton-motive forces, and the inhibition of the
respiratory enzymes [13] . What is more, a D-limonene,
organogel-based nanoemulsion was found to cause irre-
versible damage to the cytoplasmic membranes of bacteria
other than Mycobacterium
[14] . In our studies, myrcene
was more active than limonene, but Gallucci et al.
[15] re-
port that it was not active against slime-producing/non-
slime-producing staphylococci, and that this is associated
with its low aqueous solubility. This low aqueous solubility
could limit the dose required to inhibit the growth of staph-
ylococci, but in the case of Mtb, the amount of myrcene
dissolved in the aqueous medium was sufficient to pene-
trate the cell wall barrier. Our previous study showed that
monoterpenes caused morphological changes in mycobac-
terial cells, suggesting that they may affect the cell wall syn-
thesis/maintenance pathways, with the consequences be-
ing changes in cell permeability and also microbial death
[16] . In this study, the best antimycobacterial activity was
observed for sesquiterpene, β-elemene. This several-times
lower MIC value may be explained by higher lipophilicity
(log
P
= 6.1) compared to the other studied substances, and
a greater affinity to lipophilic structures in the mycobacte-
rial cell wall and phospholipid bilayer
[4] . Vik et al. [17]
observed very good antimycobacterial activity for geranyl-
geraniol, which has a similar lipophilicity (log
P
= 6.6) to
β-elemene. Furthermore, in the QSAR model, the lipophi-
licity descriptor octanol/water (log
P
) showed a high contri-
bution to the activity of terpenes against Mtb
[4] .
One would expect that the MIC values obtained for the
control antibiotics would have been the lowest, but antibi-
otics tested alone showed activity comparable with that of
α-pinene, sabinene, and myrcene. The low bacteria sensi-
tivity to antibiotics is characteristic for isolated strains of
Mtb, which usually acquires resistance to ≥ 1 tuberculo-
static drugs
[18–20] . What is more, Mtb strains possess
natural resistance due to the rich composition of mycolic
acids in the mycobacterial cell wall, which is highly lipo-
philic and responsible for a low permeability to most an-
tibiotics
[4, 10] . The lipophilic properties of natural ter-
penes are responsible for their affinity to the phospholipid
bilayer. The study performed on isolated bacterial mem-
branes suggests that cell membranes may be a site of action
of terpenes, and that antibacterial activity is a result of
their lipophilic properties, the potency of their functional
groups, and their aqueous solubility
[21] . Terpenes change
the permeability of the outer membrane of bacteria, but
their absorption is also determined by the permeability of
the cell wall
[4] . The changed permeability of the cell wall
alters the absorption and activity of antibiotics. In our
study, the best results were obtained with combinations of
terpenes with rifampicin, which is a lipophilic compound
that inhibits DNA-dependent RNA polymerase, by form-
ing a stable steric block with the enzyme and subsequently
inhibiting a transcription elongation
[22] . Rifampicin
works on a genetic material level. A less significant in-
crease in activity was observed for isoniazid and ethambu-
tol under the influence of terpenes. These antibiotics act at
the cell wall level. Isoniazid, when activated in vivo, can
oxidize or acylate protein groups that are part of the syn-
thesis of mycolic acids. It leads to the inhibition of the
elongation of fatty acids during the synthesis of mycolic
acids, by interacting with the NADH-dependent enoyl-
ACP reductase, while ethambutol inhibits arabinosiltrans-
ferase and interferes with the biosynthesis of the arabino-
galactan
[23] . These differences in drug susceptibility
caused by terpenes confirm that the site of action of ter-
penes may be the cell membrane
[24] . The drugs interfer-
ing with cell envelope formation are less efficient in com-
bination with terpenes because the terpene disrupts the
cell wall/membrane
[25] . In the case of rifampicin, ter-
penes enable better penetration into the cell. The signifi-
cant increase in the activity of rifampicin in combination
with terpenes indicates that the resistance acquired by the
examined strain was not associated with gene mutation.
Also studies on Mtb clinical isolates resistant to tubercu-
lostatic drugs demonstrated that not only classical gene
mutations play role in resistance. Additional mechanisms
that contribute to drug resistance in mycobacteria are:
drug-modifying and -inactivating enzymes and efflux-re-
lated mechanisms
[25] . Because the combinations of anti-
biotics with terpenes increase the bacterial susceptibility to
antibiotics the drug modifications probably is not a reason
of resistance. Decreased membrane permeability alone is
also unlikely to contribute in significant antibiotic resis-
tance
[25] and it was shown that Mycobacteria extrude
many drugs via active efflux systems. The active efflux is
an immediate stress response to the presence of noxious
agents
[26] . No specific gene was associated with extrusion
of isoniazid via an efflux pump
[26] , however in case of
rifampicin several efflux systems (Rv2333, DrrB, DrrC,
Rv0842, BacA, and EfpA) were indicated
[27] . Terpenes
can modulate the membrane proteins and receptors in a
nonspecific manner
[28] , but some naturally occurring li-
pophilic alkaloids, terpenes, and flavonoids have been de-
scribed as efflux pump inhibitors
[29] . The terpene car-
nosic acid from Rosmarinus officinalis and the diterpene
totarol from Cupressus nootkatensis inhibit the efflux
pump NorA in Staphylococcus aureus
[30] . This kind of
Sieniawska/Swatko-Ossor/Sawicki/
Skalicka-Woźniak/Ginalska
Med Princ Pract 2017;26:108–112
DOI: 10.1159/000454680
112
interaction of terpenes with efflux pumps would make
mycobacteria more susceptible to antimycobacterial drugs
and may contribute to the increased activity of tuberculo-
static antibiotics tested in combination with terpenes.
Conclusions
This study revealed that natural terpenes with high li-
pophilicity inhibited the growth of mycobacteria to a
greater extent. Equally important, R-limonene, S-limo-
nene, myrcene, sabinene, and β-elemene increased the
antimycobacterial activity of tuberculostatic antibiotics
due to inhibition of the natural mechanisms of mycobac-
teria resistance.
Acknowledgements
This work was financially supported by grant No. 2013/11/D/
NZ7/01613 from the Polish National Science Centre.
References
1 WHO: Global Tuberculosis Report, 2015.
2 Sougakoff W: Molecular epidemiology of
multidrug-resistant strains of Mycobacterium
tuberculosis . Clin Microbiol Infec 2011;
17:
800–805.
3 Guler SA, Bozkus F, Inci MF, et al: Evaluation
of pulmonary and extrapulmonary tubercu-
losis in immunocompetent adults: a retro-
spective case series analysis. Med Princ Pract
2015;
24: 75–79.
4 Andrade-Ochoa S, Nevárez-Moorillón GV,
Sánchez-Torres LE, et al: Quantitative struc-
ture-activity relationship of molecules con-
stituent of different essential oils with antimy-
cobacterial activity against Mycobacterium
tuberculosis and Mycobacterium bovis . BMC
Complem Altern Med 2015;
15: 333.
5 Bassolé IHN, Juliani HR: Essential oils in
combination and their antimicrobial proper-
ties. Molecules 2012;
17: 3989–4006.
6 Ge F, Zeng F, Liu S, et al: In vitro synergistic
interactions of oleanolic acid in combination
with isoniazid, rifampicin or ethambutanol
against Mycobacterium tuberculosis . J Med
Microbiol 2010;
59: 567–572.
7 Sieniawska E, Swiatek L, Rajtar B, et al: Carrot
seed essential oil – source of carotol and cyto-
toxicity study. Ind Crop Prod 2016;
92: 109–115.
8 Stein SE: IR and mass spectra; in Mallard WG,
Linstrom PJ (eds): NIST Standard Reference
Database No. 69. Gaithersburg, National In-
stitute of Standards and Technology, 2000.
http://webbook.nist.gov (accessed March 15,
2016).
9 Turnidge JD, Jorgensen JH: Antimicrobial
susceptibility testing: general considerations;
in Murray PR, Baron EJ, Pfaller MA, et al
(eds): Manual of Clinical Microbiology, ed 7.
Washington, American Society for Microbi-
ology, 1999, pp 1469–1473.
10 Rastogi N, Goh KS, Horgen L, et al: Synergis-
tic activities of antituberculous drugs with ce-
rulenin and trans-cinnamic acid against My-
cobacterium tuberculosis . FEMS Immunol
Med Microbiol 1998;
21: 149–157.
11 Zygadlo JA, Maestri DM, Lamarque AL, et al:
Essential oil variability of Minthostachys ver-
ticillata . Biochem Sys Ecol 1996;
24: 319–323.
12 Zygadlo JA, Juliáni HR: Bioactivity of essen-
tial oil components . Curr Topic Phytochem
2000;
3: 204–214.
13 Sun J: D-limonene: safety and clinical applica-
tions. Altern Med Rev 2007;
12: 259–264.
14 Zahi MR, Liang H, Yuan Q: Improving the
antimicrobial activity of D-limonene using a
novel organogel-based nanoemulsion. Food
Control 2015;
50: 554–559.
15 Gallucci N, Oliva M, Carezzano E, et al: Ter-
penes antimicrobial activity against slime
producing and non-producing staphylococci.
Mol Med Chem 2010;
21: 132–136.
16 Sieniawska E, Swatko-Ossor M, Sawicki R, et
al: Morphological changes in the overall My-
cobacterium tuberculosis H37Ra cell shape
and cytoplasm homogeneity due to Mutellina
purpurea L. essential oil and its main constit-
uents. Med Prin Pract 2015;
24: 527–532.
17 Vik A, James A, Gundersen LL: Screening of
terpenes and derivatives for antimycobacte-
rial activity; identification of geranylgeraniol
and geranylgeranyl acetate as potent inhibi-
tors of Mycobacterium tuberculosis in vitro .
Planta Med 2007;
73: 1410–1412.
18 Dias Matos E, Moreira Lemos AC, Bitten-
court C, et al: Anti-tuberculosis drug resis-
tance in strains of Mycobacterium tuberculosis
isolated from patients in a tertiary hospital in
Bahia. Braz J Infect Dis 2007;
11: 331–338.
19 Almeida da Silva PE, Osório M, Reinhardt
MC, et al: Drug resistance of strains of Myco-
bacterium tuberculosis isolated in Brazil. Mi-
crobes Infect 2001;
3: 1111–1113.
20 Toungoussova S, Caugant DA, Sandven P, et
al: Drug resistance of Mycobacterium tuber-
culosis strains isolated from patients with pul-
monary tuberculosis in Archangels, Russia.
Int J Tuberc Lung Dis 2002;
6: 406–414.
21 Dorman HJD, Deans SG: Antimicrobial agents
from plants: antibacterial activity of plant vola-
tile oils. J Appl Microbiol 2000;
88: 308–316.
22 Campbell EA, Korzheva N, Mustaev A, et al:
Structural mechanism for rifampicin inhibi-
tion of bacterial RNA polymerase. Cell 2001;
104: 901–912.
23 Rivera-Chavira BE, Adame-Gallegos JR,
Nevárez-Moorillón GV: Antimicrobial resis-
tance of infectious diseases of global impor-
tance: tuberculosis and malaria; in Méndez-
Vilas A (ed): The Battle against Microbial
Pathogens: Basic Science, Technological Ad-
vances and Educational Programs. Formatex
Research Center, 2015.
24 Nazzaro F, Fratianni F, De Martino L, et al:
Effect of essential oils on pathogenic bacteria.
Pharmaceuticals 2013;
6: 1451–1474.
25 Louw GE, Warren RM, Gey van Pittius NC, et
al: A balancing act: efflux/influx in mycobac-
terial drug resistance. Antimicrob Agents
Chemother 2009;
53: 3181–3189.
26 Machado D, Couto I, Perdigao J, et al: Contri-
bution of efflux to the emergence of isoniazid
and multidrug resistance in Mycobacterium
tuberculosis . PLoS One 2012, DOI: 10.1371/
journal.pone.0034538.
27 Li G, Zhang J, Guo Q, et al: Study of efflux
pump gene expression in rifampicin-mono-
resistant Mycobacterium tuberculosis clinical
isolates . J Antibiot 2015;
68: 431–435.
28 Wink M (ed): Function of plant secondary
metabolites and their exploitation in biotech-
nology; in Annual Plant Reviews, ed 2. Shef-
field Academic Press and CRC Press, 1999, pp
214–347.
29 Nelson ML, Levy SB: Reversal of tetracycline
resistance mediated by different bacterial tet-
racycline resistance determinants by an in-
hibitor of the Tet(B) antiport protein. Anti-
microb Agents Chemother 1999;
43: 1719–
1724.
30 Holler JG, Christensen SB, Slotved HC, et al:
Novel inhibitory activity of the Staphylococ-
cus aureus NorA efflux pump by a kaempfer-
ol rhamnoside isolated from Persea lingue
Nees. J Antimicrob Chemother 2012;
67:
1138–1144.