2019_SILVA_Antimicrobial activity of Mimosa caesalpiniifolia

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ORIGINAL ARTICLE
Antimicrobial activity of Mimosa caesalpiniifolia Benth and
its interaction with antibiotics against Staphylococcus
aureus strains overexpressing efflux pump genes
S.W.C. Silva1, N.B.N. Monc�~ao2, B.Q. Ara�ujo3, D.D.R. Arcanjo4, J.H.L. Ferreira1, J.S. Lima Neto3,
A.M.G.L. Cit�o3, J.P. de Siqueira J�unior5, G.W. Kaatz6 and H.M. Barreto1
1 Laboratory of Research in Microbiology, Federal University of Piau�ı, Teresina, Brazil
2 Agricultural College of Floriano, Federal University of Piau�ı (UFPI), Floriano, Brazil
3 Laboratory of Natural Products, Federal University of Piau�ı, Teresina, Brazil
4 Department of Biophysics and Physiology, Federal University of Piau�ı (UFPI), Teresina, Brazil
5 Laboratory of Genetics of Microorganisms, Federal University of Paraiba (UFPB), Jo~ao Pessoa, Brazil
6 Department of Medicine, Division of Infectious Diseases, Wayne State University School of Medicine, Detroit, MI, USA
Significance and Impact of the Study:Drug resistance is a common problem in patients with infectious
diseases. Dichloromethane fraction from the stem bark of Mimosa caesalpiniifolia showed antimicrobial
activity against Gram-positive bacterium Staphylococcus aureus and against Candida albicans, but did
not show activity against Gram-negative specie Escherichia coli. Moreover, this fraction was able to
potentiate the action of norfloxacin, ciprofloxacin and tetracycline against S. aureus strains overexpress-
ing different efflux pump genes. Thus, Mimosa caesalpiniifolia is a source of efflux pump inhibitors
which could be used in combination with fluoroquinolones or tetracycline in the treatment of infec-
tious diseases caused by S. aureus strains overexpressing efflux pump genes.
Keywords
antimicrobials, E. coli (all potentially
pathogenic types), resistance, Staphylococci,
yeasts.
Correspondence
Humberto Medeiros Barreto, Laboratory of
Research in Microbiology, Department of Par-
asitology and Microbiology, Center of Health
Sciences, Federal University of Piau�ı – UFPI,
Teresina, PI 64049-550, Brazil. E-mail: hmbar-
reto@ufpi.edu.br
2019/0122: received 20 January 2019, revised
15 April 2019 and accepted 15 April 2019
doi:10.1111/lam.13163
Abstract
This study aimed to evaluate the antimicrobial activity of the dichloromethane
fraction (DCMF) from the stem bark of Mimosa caesalpiniifolia and its effect
on the activity of conventional antibiotics against Staphylococcus aureus strains
overexpressing specific efflux pump genes. DCMF showed activity against S.
aureus, Staphylococcus epidermidis and Candida albicans. Addition of DCMF at
subinhibitory concentrations to the growth media enhanced the activity of
norfloxacin, ciprofloxacin and ethidium bromide against S. aureus strains
overexpressing norA suggesting the presence of efflux pump inhibitors in its
composition. Similar results were verified for tetracycline against S. aureus
overexpressing tetK, as well as, for ethidium bromide against S. aureus
overexpressing qacC. These results indicate that M. caesalpiniifolia is a source
of molecules able to modulate the fluoroquinolone- and tetracycline-resistance
in S. aureus probably by inhibition of NorA, TetK and QacC respectively.
Introduction
Resistance to antimicrobial agents has become wide-
spread leading to high prevalence of infectious diseases
through the world (Tong et al. 2015). Treatment of
infections caused by multidrug-resistant micro-organ-
isms is difficult because it frequently requires prolonged
use of different antibiotic combinations (Nguyen and
Graber 2010) resulting in higher toxicity for patients
and higher costs for the health care system (Heppner
et al. 2013).
Some strategies have been devised to face antimicrobial
resistance, such as discovery of new antimicrobial com-
pounds (Svietnicki 2018) and the development of new
derivatives from conventional antibiotics (Silverman et al.
2017). New synthetic or natural compounds also have
Letters in Applied Microbiology 69, 57--63 © 2019 The Society for Applied Microbiology 57
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been investigated for their ability to inhibit mechanisms
of resistance (Bharate et al. 2015; Coêlho et al. 2016). In
this approach, an antibiotic is combined with a com-
pound that inhibits a known mechanism of resistance
recovering the effectiveness of the antibiotic against resis-
tant micro-organisms (Beytur et al. 2014).
Resistance to antibiotics can occur by several mecha-
nisms, including lower antibiotic influx, enzymatic inac-
tivation of the antibiotic or its target, mutation
resulting in lower affinity of the antibiotic by its target,
and active efflux pumps able to extrude antibiotics to
the extracellular medium (Redgrave et al. 2014). Efflux
pumps are transmembrane proteins able to efflux one
(non-multidrug efflux pump) or more than one (mul-
tidrug efflux pump) antibiotic using ATP hydrolysis
(primary transporters) or the electrochemical ion gradi-
ent across a membrane (secondary transporters) as
energy sources (Hernando-Amado et al. 2016). Resis-
tance mediated by efflux pumps can be modulated by
efflux pump inhibitors which could be an interesting
strategy for increasing the effectiveness of conventional
antibiotics against resistant micro-organisms (Costa
et al. 2016).
Mimosa caesalpiniifolia Benth (Fabaceae) is a native
plant from Caatinga and Cerrado vegetation, found in
several states of Northeast Brazil, including in Piau�ı state,
where it is commonly known as sabi�a (Ribaski et al.
2003). Ethnobotanical studies provided evidence for use
of M. caesalpiniifolia in the treatment of inflammatory
processes, respiratory tract diseases and hypertension
(Albuquerque et al. 2007; Agra et al. 2008). In folk medi-
cine, stem bark decoction is used to stop bleeding and
wound washing, whereas ingestion of stem bark infusion
has been used to treat cough and gastritis (Aguiar and
Barros 2012).
The ethanoic extract obtained from the stem bark of
M. caesalpiniifolia and their hexane and dichloromethane
fractions, as well as, betulinic acid alone showed moderate
to high inhibitory activity against colon, ovarian and
glioblastoma tumour cells (Monc�~ao et al. 2015). Ethanoic
extract from inflorescences of M. caesalpiniifolia showed
hypotensive and vasorelaxant effects in normotensive rats
(Santos et al. 2015). Ethanoic extract from the leaves of
M. caesalpiniifolia and its ethyl acetate fractions exhibited
antioxidant activity and prevented oxidative DNA damage
in Wistar rats (Silva et al. 2014).
The objective of the present study was to evaluate the
antimicrobial activity of the dichloromethane fraction
obtained from stem bark of M. caesalpiniifolia and its
main compound, betulinic acid (Fig. 1), as well as, to
investigate its effect on the antimicrobial activity of differ-
ent antibiotics against Staphylococcus aureus strains that
overexpress genes encoding efflux proteins.
Results and discussion
Results obtained from assays to evaluate antimicrobial
activity showed that DCMF was active against Gram-posi-
tive bacterial strains with MIC values ranging from 32 to
512 lg ml�1 (Table 1). Taking into account the criteria
to evaluate antimicrobial activity of plant extracts previ-
ously proposed (Holetz et al. 2002), DCMF showed a
weak inhibitory effect for the drug-sensitive standard
strain S. aureus ATCC 25923. However, it showed a good
inhibitory effect for drug-resistant strains S. aureus IS-58
and S. aureus K2068 (64 lg ml�1). For the other S. aur-
eus strains, as well as, for Staphylococcus epidermidis
ATCC 12228, DCMF showed a moderate inhibitory effect.
Similar results were previously found for ethyl acetate
extract from the stem bark of M. caesalpiniifolia that
showed antimicrobial activity for S. aureus at 128 lg
ml�1 (Callou et al. 2012). However,
DCMF did not show
activity against Escherichia coli. Gram-negative bacteria as
E. coli have an outer membrane which could be hindering
the uptake of hydrophobic compounds from DCMF
(Nikaido 2003).
Dichloromethane fraction exhibited a weak antimicro-
bial activity tested against Candida albicans (Table 1),
corroborating previous data obtained for cyclohexane and
ethyl acetate extracts from stem bark of M. caesalpiniifolia
(Callou et al. 2012). Thus, results found in the present
study confirmed that stem bark of M. caeasalpiniifolia is a
source of bioactive phytoconstituents which could be
additionally investigated as potential antimicrobial agents
for prevention or treatment of infectious diseases caused
by tested micro-organisms.
A previous study showed that BA is the main com-
pound (70�3%) present in the DCMF (Monc�~ao et al.
2015). Results showed that BA was inactive against all
strains tested, indicating that isolated betulinic acid is not
involved with the antimicrobial activity presented by
DCMF, although we cannot rule out a possible synergism
H
H
H
H
HO
CO2H
Figure 1 Chemical structure of the betulinic acid.
Letters in Applied Microbiology 69, 57--63 © 2019 The Society for Applied Microbiology58
Antimicrobial activity of Mimosa caesalpiniifolia S.W.C. Silva et al.
between BA and other components. These results are in
agreement with previous studies which also verified that
BA was inactive against S. aureus and E. coli (Fontanay
et al. 2008; Silva et al. 2012; Duric et al. 2013). However,
another study verified that BA was weakly active against
E. coli (MIC 512 lg ml�1) and moderately active against
S. aureus (MIC 256 lg ml�1), with its inhibitory effect
caused by an enhanced electron transport chain activity
leading to an increased oxidative stress in the bacterial
cells (Oloyede et al. 2017).
A previous study demonstrated that acetyl acetate
extract from stem bark of M. caesalpiniifolia potentiated
the activity of polymyxin B against P. aeruginosa, increas-
ing its inhibition zones from 12 to 16 mm (Callou et al.
2012). Results obtained in the present study with SA1199-
B strain showed that addition of DCMF to the growth
media at subinhibitory concentrations caused a decrease
in the MIC for norfloxacin and ciprofloxacin in a concen-
tration-dependent way (Fig. 2). This modulating effect
was similar to that verified for chlorpromazine (CPZ), a
known efflux pump inhibitor (EPI) (Neyfakh et al. 1993),
suggesting that DCMF contain phytochemicals able to
modulate the fluoroquinolone-resistance in S. aureus
overexpressing norA.
The modulatory effect was also verified when antibi-
otics were replaced by ethidium bromide (EtBr) (Fig. 2),
a well-known NorA substrate, as resistance mediated by
efflux pumps is the only known mechanism of resistance
Table 1 Minimum inhibitory concentrations (MICs) of the dichloro-
methane fraction from the stem bark of Mimosa caesalpiniifolia
(DCMF) and betulinic acid (BA) against different micro-organisms
Microbial strains
MIC (lg mL�1)
DCMF BA
Staphylococcus aureus IS-58 32 ≥1024
Staphylococcus aureus RN-4220 128 ≥1024
Staphylococcus aureus SA1199-B 128 ≥1024
Staphylococcus aureus K2068 64 ≥1024
Staphylococcus aureus K4100 256 ≥1024
Staphylococcus aureus ATCC 25923 512 ≥1024
Staphylococcus epidermidis ATCC 12228 256 ≥1024
Escherichia coli ATCC 25922 ≥1024 ≥1024
Candida albicans ATCC 10231 512 ≥1024
72
64
56
48
40
32
24
16
8
0
ns ns
****
**** ****
****
+ 
MlC1/8
+ 
Nor Nor Nor Nor Nor Nor Nor
DCMF DCMF 
MlC1/4 MlC1/8 MlC1/4 MlC1/8 MlC1/4
+ 
BA
+ 
BA
+ +
CPZ CPZ 
+ 
MlC1/8
+ 
Cip Cip Cip Cip Cip Cip Cip 
DCMF 
M
in
im
al
 in
hi
bi
to
ry
 c
on
ce
nt
ra
tio
n 
(µ
g 
m
l–
1 )
 
M
in
im
al
 in
hi
bi
to
ry
 c
on
ce
nt
ra
tio
n 
(µ
g 
m
l–
1 )
 
M
in
im
al
 in
hi
bi
to
ry
 c
on
ce
nt
ra
tio
n 
(µ
g 
m
l–
1 )
 
DCMF 
MlC1/4 MlC1/8 MlC1/4 MlC1/8 MlC1/4
+ 
BA
+ 
BA
+ +
CPZ CPZ 
+ 
MlC1/8
+ 
DCMF DCMF 
MlC1/4 MlC1/8 MlC1/4 MlC1/8 MlC1/4
+ 
BA
+ 
BA
+ +
CPZ CPZ 
18
ns ns
16
14
12
10
8 ****
6
4 **** ****
2 ****
0
72
64 ns ns
56
48
40
32 **** ****
24
16 ****
8 ****
0
EtBr EtBr EtBr EtBr EtBr EtBr EtBr
(a)
(b)
(c)
Figure 2 MIC of the norfloxacin (Nor) (a), ciprofloxacin (Cip) (b) and
ethidium bromide (EtBr) (c) against Staphylococcus aureus SA1199-B
(norA) in the absence or presence of the dichloromethane fraction
from the stem bark of Mimosa caesalpiniifolia (DCMF), betulinic acid
(BA) or chlorpromazine (CPZ). Each result represents the geometric
mean of three simultaneous experiments. ***Statistically significant
values (P < 0�0001).
Letters in Applied Microbiology 69, 57--63 © 2019 The Society for Applied Microbiology 59
S.W.C. Silva et al. Antimicrobial activity of Mimosa caesalpiniifolia
to this DNA-intercalating dye (Markham et al. 1999).
This result indicates that modulation of the drug resis-
tance by DCMF could be due to the presence of NorA
inhibitors, leading to drug accumulation in bacterial cells.
Modulation of the drug resistance by DCMF was also
verified for EtBr in the strain K4100 (qacC), as well as for
tetracycline in the strain IS-58 (tetK) (Table 2), indicating
that potential EPIs present in DCMF could inhibit the
activity of other efflux proteins besides NorA. However,
DCMF was not able to modulate the drug resistance in
the strains K2068 (mepA) or RN4220 (msrA).
Plants have been known as a source of EPIs able to
inhibit efflux pumps from Gram-positive or Gram-nega-
tive bacteria (Rao et al. 2018). EPIs could inhibit the
efflux of antibiotics in different ways, such as dissipation
of the proton gradient across the cell membrane, inhibi-
tion of the expression of genes encoding efflux pumps,
blocking the binding site of the substrate in the efflux
pump, causing disruption of the transcription of genes
that regulate the expression of genes encoding efflux
pumps, or causing conformational changes in efflux pro-
teins (Pag�es and Amaral 2009; Marino et al. 2014).
NorA, TetK and QacC are efflux pumps dependent on
the proton motive force (Schindler and Kaatz 2016).
Thus, the modulating effect on norfloxacin-, tetracycline-
and ethidium bromide-resistance against SA1199-B, IS-58
and K4100 may be caused by hydrophobic phytochemi-
cals within the DCMF that were able to cause destabiliza-
tion of the bacterial plasma membrane leading to
dissipation on the proton motive force, as well as,
increasing the cell permeability to the antibiotics (Burt
2004). However, the damage in the cell membrane seems
to have been insufficient to cause inhibition of MepA
which is also dependent on proton motive force for its
activity (Schindler and Kaatz 2016).
Modulatory effect was not verified for BA indicating that
the modulatory effect showed by DCMF could be attribu-
ted to their minor components or that it results of a syn-
ergy between BA and their minor constituents. The marked
action of DCMF when compared with BA is possibly due
to its lipophilic characteristics, where other compounds
might act as a coadjuvant probably forming BA-conjugates
in order to better interact with the cell membrane (Costa
et al. 2017). The advantage of BA-conjugated compounds
in inducing a higher cytotoxic activity involving apoptosis
associated with loss of mitochondrial membrane potential
and increase in intracellular free Ca2+ have been reported
in eukaryotic cells (Xu et al. 2017). Thus, these minor com-
pounds could be acting as a BA carrier into the plasma
membrane, and then potentiating this effect.
The present study is the first report showing that a natu-
ral product extracted from the stem bark of M. caesalpini-
ifolia is able to increase the activity of norfloxacin,
ciprofloxacin, tetracycline and ethidium bromide against S.
aureus strains overexpressing efflux pumps NorA, TetK and
QacC respectively. Although it had the limitation of not
being able to identify specific
compounds from DCMF as
modulators of drug resistance, our results showed that its
major component alone is not related to this activity.
In conclusion, DCMF showed intrinsic antimicrobial
activity against S. aureus, S epidermidis and C. albicans,
suggesting that M. caesalpiniifolia is a source of secondary
metabolites that could be used as an antiseptic to prevent
microbial growth. Moreover, it was able to potentiate the
antibiotic activity of norfloxacin, and ciprofloxacin
against SA1199-B and was able to modulate tetracycline-
resistance against S. aureus IS-58. These results indicate
the occurrence of phytochemicals which can modulate
fluoroquinolone- and tetracycline-resistance, probably by
inhibition of NorA and TetK efflux pumps. Such com-
pounds could be used as adjuvants of norfloxacin, cipro-
floxacin or tetracycline for treatment of infections caused
by S. aureus strains overexpressing efflux pumps.
Materials and methods
Plant material and extraction
Stem bark of M. caesalpiniifolia was collected in Teresina
(latitude 5°03025,24″ south and longitude 42°47042,48″
west), Piau�ı, Brazil in May 2010. Plant material was identi-
fied and a voucher specimen was deposited at the Herbar-
ium Graziela Barroso, with the number TEPB 26�824. Stem
bark of M. caesalpiniifolia was dried at room temperature
and pulverized in a knife mill (MA680, Marconi Equipa-
mentos Laborat�orio, Piracicaba, SP, Brazil). Stem bark
Table 2 Minimum inhibitory concentrations (MICs) of antibiotics and
ethidium bromide in the absence or presence of the dichloromethane
fraction from the stem bark of Mimosa caesalpiniifolia (DCMF) against
effluxing strains of Staphylococcus aureus. Each result represents the
geometric mean of three simultaneous experiments
Strain (drug)
Efflux
pump
(family)
MIC (lg ml�1)
Drug
alone
+DCMF
(MIC 1/8)
+DCMF
(MIC 1/4)
IS-58
(tetracycline)
TetK (MFS) 64 32 (2�0)* 32 (2�0)
IS-58 (EtBr) TetK (MFS) 8 4 (2�0) 3 (2�7)
K4100 (EtBr) QacC (SMR) 32 16 (2�0) 16 (2�0)
RN4220
(erythromycin)
MsrA (ABC) 32 32 (0�0) 32 (0�0)
K2068 (EtBr) MepA (MATE) 64 64 (0�0) 64 (0�0)
ABC, ATP-binding cassette; MFS, Major facilitator superfamily; MATE,
Multidrug and toxin extrusion; SMR, Small multidrug resistance.
*Fold reduction.
Letters in Applied Microbiology 69, 57--63 © 2019 The Society for Applied Microbiology60
Antimicrobial activity of Mimosa caesalpiniifolia S.W.C. Silva et al.
powder (883�5 g) was extracted exhaustively with ethanol
at 1 : 4 (w/v) plant material/solvent 10 consecutive times.
The filtered and combined ethanolic extracts were concen-
trated under reduced pressure on a rotary evaporator
(Laborota 4000; Heidolph Instruments, Schwabach, BY,
Germany) and lyophilized (Edwards Micro Modulyo freeze
dryer/Valpump VLP80 Savant, West Sussex, UK), yielding
36�7 g (4�2%) of dried EtOH extract. The stem bark etha-
nol extract (30�0 g) was suspended in MeOH-H2O (2 : 1,
v/v) and subjected to successive partitioning, resulting in
the following fractions: n-hexane (2�7 g, 7�4%), dichloro-
methane (3�9 g, 10�6%), EtOAc (3�7 g, 10�1%) and aque-
ous (13�8 g, 37�7%). In this study, we investigated only the
dichloromethane fraction (DCMF). Chemical composition
of the DCMF has already been determined by gas chro-
matography coupled to mass spectrometer and results
showed the presence of betulinic acid (70�30%), lupeol
(3�26%), methyl octacosanoate (3�19%), methyl hexa-
cosanoate (3�08%), methyl tetracosanoate (2�16%) as main
compounds (Monc�~ao et al. 2015).
Strains and chemicals
Evaluation of the intrinsic antimicrobial activity of the
DCMF was performed against standard microbial strains,
including S. aureus ATCC 25923, S. epidermidis ATCC
12228, Escherichia coli ATCC 25922 and C. albicans ATCC
10231. Antimicrobial activity of the DCMF was also evalu-
ated against drug-resistant S. aureus strains that overex-
pressed efflux pumps (Table 3). Assays for evaluation of
the modulating effect on drug resistance were performed
only with drug-resistant S. aureus strains overexpressing
specific efflux pumps. Bacterial strains were maintained on
Brain Heart Infusion Agar (BHIA, Himedia, India) slants at
4°C, and prior to assay the cells were grown overnight at
37°C in Brain Heart Infusion (BHI, Himedia, India). The
yeast strain was maintained on Sabouraud Dextrose Agar
(SDA, Himedia, India) slants at 4°C and prior to assay the
cells were grown for 24 h at 37°C in Sabouraud Dextrose
Broth (SDB, Himedia, India). Ciprofloxacin (Cip), nor-
floxacin (Nor), erythromycin (Eri), tetracycline (Tet),
ethidium bromide (EtBr), chlorpromazine (CPZ) and betu-
linic acid (BA) (Fig. 1) were obtained from Sigma Chemi-
cal Corp., St. Louis. With the exception of Eri which was
dissolved in absolute ethanol, Cip, Nor, Tet, EtBr, CPZ and
BA were dissolved in sterile water.
Assays for evaluation of the intrinsic antimicrobial
activity
Stock solutions of DCMF or CPZ were prepared in
DMSO followed by dilution in sterile distilled water to a
final concentration of 1024 lg ml�1. A stock solution of
BA was prepared in sterile distilled water to a final con-
centration of 1024 lg ml�1. Minimal inhibitory concen-
trations (MICs) were determined by microdilution assay
in BHI broth with bacterial suspensions of 105 CFU per
ml and partition fraction concentrations ranging from 8
to 512 lg ml�1. Microtitre plates were incubated at 37°C
for 24 h, then 20 ll of resazurin (0�01% w/v in sterile
distilled water) was added to each well to detect bacterial
growth by visual colour change from blue to pink.
Assays for evaluation of the drug-resistance modulation
To evaluate if DCMF or BA were able to modulate antibi-
otic resistance in S. aureus strains overexpressing specific
efflux proteins, MICs of antibiotics were determined in the
presence or absence of subinhibitory concentrations of each
natural product (1/8 or 1/4 MIC). Antibiotic concentra-
tions ranged from 0�125 to 128 lg ml�1. Microtitre plates
were incubated at 37°C for 24 h and readings were per-
formed with resazurin as described above. To verify if the
drug-resistance modulation occurred due to efflux pump
inhibition, the modulation assay was performed by replac-
ing antibiotics with EtBr, which is a known substrate of
efflux pumps (Markham et al. 1999), here used as an indi-
cator of efflux pump inhibition. Control assays were also
performed replacing DCMF or BA by CPZ which is a
known efflux pump inhibitor (Neyfakh et al. 1993).
Statistical analysis
Experiments were performed in triplicate and results were
normalized by calculation of geometric mean values.
Error deviation and standard deviation of the geometric
mean were revealed. Statistical analyses were performed
using GraphPad Prism, ver. 5.02. Differences between
Table 3 Characteristics of the efflux pumps expressed by the Staphy-
lococcus aureus strains tested
Microbial
strains
Efflux
pump
Protein
family
Main
substrates References
RN-4220 MsrA ABC MC and SR Ross et al. (1989)
IS-58 TetK MFS TT Gibbons and
Udo (2000)
SA1199-B NorA MFS FQ, EtBr, QAC Kaatz and Seo
(1993)
K2068 MepA MATE FQ, EtBr, QAC Kaatz et al. (2005)
K4100 QacC SMR EtBr and QAC Littlejohn et al.
(1991)
ABC, ATP-biding cassette; MFS, Major facilitator superfamily; MATE,
Multidrug and toxin extrusion; SMR, Small multidrug resistance; QAC,
quaternary ammonium compounds; EtBr, ethidium bromide; FQ, fluo-
roquinolones; MC, macrolides; SR, streptogramins; TT, tetracycline.
Letters in Applied Microbiology 69, 57--63 © 2019 The Society for Applied Microbiology 61
S.W.C. Silva et al. Antimicrobial activity of Mimosa caesalpiniifolia
treatment with antibiotics (or EtBr) alone or associated
with DCMF, betulinic acid or CPZ were examined using
one-way analysis of variance (ANOVA). The differences
mentioned above were analysed by Bonferroni post-test
and P < 0�05 were considered statistically significant.
Acknowledgements
This study was funded by
the Fundac�~ao de Amparo �a
Pesquisa do Estado do Piau�ı, Conselho Nacional de
Desenvolvimento Cient�ıfico e Tecnol�ogico and Universi-
dade Federal do Piau�ı.
Conflict of Interest
The authors declare that they have no conflict of interest.
References
Agra, M.F., Silva, K.N., Bas�ılio, I.J.L.D., Franc�a, P.F. and
Barbosa-Filho, J.M. (2008) Survey of medicinal plants used
in the region Northeast of Brazil. Rev Bras Farmacogn 18,
472–508.
Aguiar, L.C.G.G. and Barros, R.F.M. (2012) Plantas medicinais
cultivadas em quintais de comunidades rurais no dom�ınio
do cerrado piauiense (Munic�ıpio de Demerval Lob~ao,
Piau�ı, Brasil). Rev Bras Plantas Med 14, 419–434.
Albuquerque, U.P., Medeiros, P.M., Almeida, A.L.S., Monteiro,
J.M., Lins-Neto, E.M.F., Melo, J.G. and Dos Santos, J.P.
(2007) Medicinal plants of the caatinga (semi-arid)
vegetation of NE Brazil: a quantitative approach. J
Ethnopharmacol 114, 325–354.
Beytur, A., Yakupogullari, Y., Oguz, F., Otlu, B. and Kaysadu,
H. (2014) Oral amoxicillin-clavulanic acid treatment in
urinary tract infections caused by extended-spectrum beta-
lactamase–producing organisms. Jundishapur J Microbiol 8,
1–5.
Bharate, J.B., Singh, S., Wani, A., Sharma, S., Joshi, P., Khan,
I.A., Kumar, A., Vishwakarma, R.A. et al. (2015) Discovery
of 4-acetyl-3-(4-fluorophenyl)-1-(p-tolyl)-5-methylpyrrole
as a dual inhibitor of human P-glycoprotein and
Staphylococcus aureus NorA efflux pump. Org Biomol
Chem 13, 5424–5431.
Burt, S. (2004) Essential oils: their antibacterial properties and
potential applications in foods. Int J Food Microbiol 94,
223–253.
Callou, M.J.A., Miranda, R.C.M., Feitosa, T.R., Arruda, F.V.F.,
Nascimento, M.S. and Gusm~ao, N.B. (2012) Avaliac�~ao da
atividade antimicrobiana da casca da Mimosa
caesalpiniifolia Benth (Sabi�a). Sci Plena 8, 1–7.
Coêlho, M.L., Ferreira, J.H.L., Siqueira J�unior, J.P., Kaatz,
G.W., Barreto, H.M. and Cavalcante, A.A.C.M. (2016)
Inhibition of the NorA multi-drug transporter by
oxygenated monoterpenes. Microb Pathog 99, 173–177.
Costa, L.M., de Macedo, E.V., Oliveira, F.A., Ferreira, J.H.,
Gutierrez, S.J., Pel�aez, W.J., Lima, F.D., de Siqueira J�unior,
J.P. et al. (2016) Inhibition of the NorA efflux pump of
Staphylococcus aureus by synthetic riparins. J Appl
Microbiol 121, 1312–1322.
Costa, E.V.S., Br�ıgido, H.P.C., Coelho-Ferreira, M.R., Brand~ao,
G.C. and Dolabela, M.F. (2017) Antileishmanial activity of
Handroanthus serratifolius (Vahl) S. Grose (Bignoniaceae).
Evid Based Complement Alternat Med 2017, 1–6.
Duric, K., Kovac-Besovic, E., Niksic, H. and Sofic, E. (2013)
Antibacterial activity of methanolic extracts, decoction and
isolated triterpene products from different parts of birch,
Betula pendula, Roth. J Plant Stud 2, 61–70.
Fontanay, S., Grare, M., Mayer, J., Finance, C. and Duval, R.E.
(2008) Ursolic, oleanolic and betulinic acids: antibacterial
spectra and selectivity indexes. J Ethnopharmacol 120,
272–276.
Gibbons, S. and Udo, E.E. (2000) The effect of reserpine, a
modulator of multidrug efflux pumps, on the in vitro
activity of tetracycline against clinical isolates of
methicillin resistant Staphylococcus aureus (MRSA)
possessing the tet(K) determinant. Phytother Res 14,
139–140.
Heppner, H.J., Cornel, S., Peter, W., Philipp, B. and Katrin, S.
(2013) Infections in the elderly. Crit Care Clin 29,
757–774.
Hernando-Amado, S., Blanco, P., Alcalde-Rico, M., Corona, F.,
Reales-Calderon, J.A., Sanchez, M.B. and Martinez, J.L.
(2016) Multidrug efflux pumps as main players in intrinsic
and acquired resistance to antimicrobials. Drug Resist
Update 28, 13–27.
Holetz, F.B., Pessini, G.L., Sanches, N.R., Cortez, D.A.,
Nakamura, C.V. and Dias Filho, B.P. (2002) Screening of
some plants used in the Brazilian folk medicine for the
treatment of infectious diseases. Mem Inst Oswaldo Cruz
97, 1027–1031.
Kaatz, G.W. and Seo, S.M. (1993) Efflux-mediated
fluoroquinolone resistance in Staphylococcus aureus.
Antimicrob Agents Chemother 37, 1086–1094.
Kaatz, G.W., McAleese, F. and Seo, S.M. (2005) Multidrug
resistance in Staphylococcus aureus due to overexpression
of a novel multidrug and toxin extrusion (MATE)
transport protein. Antimicrob Agents Chemother 49,
1857–1864.
Littlejohn, T.G., DiBerardino, D., Messerotti, L.J., Spiers, S.J.
and Skurray, R.A. (1991) Structure and evolution of a
family of genes encoding antiseptic and disinfectant
resistance in Staphylococcus aureus. Gene 101, 59–66.
Marino, L.B., Miyata, M., Souza, P.C., Leite, C.Q.F. and Pavan,
F.R. (2014) Drug discovery for TB: frontiers and
perspectives. Front Anti-Infect Drug Discov Edn 29, 3–31.
Markham, P.N., Westhaus, E., Klyachko, K., Johnson, M.E.
and Neyfakh, A.A. (1999) Multiple novel inhibitors of the
NorA multidrug transporter of Staphylococcus aureus.
Antimicrob Agents Chemother 43, 2404–2408.
Letters in Applied Microbiology 69, 57--63 © 2019 The Society for Applied Microbiology62
Antimicrobial activity of Mimosa caesalpiniifolia S.W.C. Silva et al.
Monc�~ao, N., Ara�ujo, B., Silva, J., Lima, D., Ferreira, P.,
Airoldi, F., Pessoa, C. and Cit�o, A. (2015) Assessing
chemical constituents of Mimosa caesalpiniifolia stem bark:
possible bioactive components accountable for the
cytotoxic effect of M. caesalpiniifolia on human tumour
cell lines. Molecules 20, 4204–4224.
Neyfakh, A.A., Borsch, C.M. and Kaatz, G.W. (1993)
Fluoroquinolone resistance protein NorA of Staphylococcus
aureus is a multidrug efflux transporter. Antimicrob Agents
Chemother 37, 128–129.
Nguyen, H.M. and Graber, C.J. (2010) Limitations of
antibiotic options for invasive infections caused by
methicillin-resistant Staphylococcus aureus: is combination
therapy the answer? J Antimicrob Chemother 65, 24–36.
Nikaido, H. (2003) Molecular basis of bacterial outer
membrane permeability revisited. Microbiol Mol Biol Rev
67, 593–656.
Oloyede, H.O.B., Ajiboye, H.O., Salawu, M.O. and Ajiboye,
T.O. (2017) Influence of oxidative stress on the
antibacterial activity of betulin, betulinic acid and ursolic
acid. Microb Pathog 111, 338–344.
Pag�es, J.M. and Amaral, L. (2009) Mechanisms of drug efflux
and strategies to combat them: challenging the efflux
pump of Gram-negative bacteria. Biochim Biophys Acta
1794, 826–833.
Rao, M., Padyana, S., Dipin, K.M., Kumar, S., Nayak, B.B. and
Varela, M.F. (2018) Antimicrobial compounds of plant
origin as efflux pump inhibitors: new avenues for
controlling multidrug resistant pathogens. J Antimicrob
Agents 4, 1–6.
Redgrave, L.S., Sutton, S.B., Webber, M.A. and Piddock, L.J.V.
(2014) Fluoroquinolone resistance: mechanisms, impact on
bacteria, and role in evolutionary success. Trends Microbiol
22, 438–445.
Ribaski, J., Lima, P.C.F., Oliveira, V.R. and Drumond, M.A.
(2003) Sabi�a (Mimosa caesalpiniaefolia) �Arvore de m�ultiplo
uso no Brasil. Comunicado T�ecnico 104 da Embrapa 1-3.
Ross, J.I., Farrell, A.M., Eady, E.A., Cove, J.H. and Cunliffe,
W.J. (1989) Characterization and molecular cloning of the
novel macrolide-streptogramin B resistance determinant
from Staphylococcus epidermidis. J Antimicrob Chemother
24, 851–862.
Santos, M.E., Moura, L.H., Mendes, M.B., Arcanjo, D.D.,
Monc�~ao, N.B., Ara�ujo, B.Q., Lopes, J.A., Silva-Filho, J.C.
et al. (2015) Hypotensive and vasorelaxant effects induced
by ethanolic extract of the Mimosa caesalpiniifolia Benth.
(Mimosaceae) inflorescences in normotensive rats. J
Ethnopharmacol 164, 120–128.
Schindler, B.D. and Kaatz, G.W. (2016) Multidrug efflux pumps
of Gram-positive bacteria. Drug Resist Updat 27, 1–13.
Silva, M., David, J.P., Silva, L.C., Santos, R.A., David, J.M.,
Lima, L.S., Reis, P.S. and Fontana, R. (2012) Bioactive
oleanane, lupane and ursane triterpene acid derivatives.
Molecules 17, 12197–12205.
Silva, M.J.D., Vilegas, W., Silva, M.A., Moura, C.F.G., Ribeiro,
F.A.P., Silva, V.H.P. and Ribeiro, D.A. (2014) Mimosa
(Mimosa caesalpiniifolia) prevents oxidative DNA damage
induced by c�admium exposure in wistar rats. Toxicol
Mech
Methods 24, 567–574.
Silverman, S.M., Moses, J.E. and Sharpless, K.B. (2017)
Reengineering antibiotics to combat bacterial resistance:
click chemistry [1,2,3]-triazole vancomycin dimers with
potent activity against MRSA and VRE. Chem Eur J 23,
79–83.
Svietnicki, W. (2018) Recent advances in antibacterial drug
development. IJRSR 9, 26501–26505.
Tong, S.Y.C., Davis, J.S., Eichenberger, E., Holland, T.L. and
Fowler, V.G. (2015) Staphylococcus aureus infections:
epidemiology, pathophysiology, clinical manifestations,
and management. Clin Microbiol Rev 28, 603–661.
Xu, B., Yan, W.Q., Xu, X., Wu, G.R., Zhang, C.Z., Han, Y.T.,
Chu, F.H., Zhao, R. et al. (2017) Combination of amino
acid/dipeptide with ligustrazine-betulinic acid as antitumor
agents. Eur J Med Chem 130, 26–38.
Letters in Applied Microbiology 69, 57--63 © 2019 The Society for Applied Microbiology 63
S.W.C. Silva et al. Antimicrobial activity of Mimosa caesalpiniifolia