Comparative Immunology, Microbiology and Infectious Diseases 57 (2018) 22–28

Contents lists available at ScienceDirect

Comparative Immunology, Microbiology

and Infectious Diseases
journal homepage: www.elsevier.com/locate/cimid

In vitro e in silico evaluation of the inhibition of Staphylococcus aureus efflux T

pumps by caffeic and gallic acid
Joycy F.S. dos Santosa, Saulo R. Tintinob, Thiago S. de Freitasb, Fábia F. Campinab,
⁎
Irwin R. de A. Menezesc, José P. Siqueira-Júniord, Henrique D.M. Coutinhob, ,
Francisco A.B. Cunhaa
a
Laboratory of Semi-Arid Bioprospecting (Lab-Bioprospec), Department of Biological Chemistry/CCBS/URCA, Ceará, Brazil
b
Laboratory of Microbiology and Molecular Biology (LMBM), Department of BiologicalChemistry/CCBS/URCA, Ceará, Brazil
c
Laboratory of Pharmacology and Molecular Chemistry (LFQM), Department of Biological Chemistry/CCBS/URCA, Ceará, Brazil
d
Laboratory of Microrganisms Genetics (LGM), Department of Molecular Biology/CCEN/UFPB, Paraíba, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: Staphylococcus aureus has been reported as one of the most difficult to treat. In the search for new treatment
Antibacterial drugs alternatives, isolated plant substances such as phenolic compounds, have demonstrated the ability to reverse
Active efflux bacterial resistance. The present study aims to evaluate the inhibitory action of caffeic acid and gallic acid on
Gram-positive efflux pumps from S. aureus resistant strains. The broth microdilution assay was carried out to obtain the MICs of
caffeic acid and gallic acid while the efflux pump inhibition test was assessed through the reduction of the
minimum inhibitory concentration of the antibiotic and ethidium bromide. In addition, in silico theoretical
parameters were analyzed to determine the theoretical efficacy of the compound and its free energy of inter-
action. In the results, the inhibition concentration of the two compounds did not certify clinical relevance with
1024 μg/mL for all strains. In the efflux pump inhibition effect, caffeic acid inhibited the MrsA pumps of the
strain RN-4220 and NorA of the strain 1199B. Caffeic acid showed greater efficacy in the docking model, in
agreement with the demonstrated experimental efficacy. Isolated compounds can be indicated as efficient op-
tions in the inhibition of resistance mechanisms.

1. Introduction generally contained in plasmids and transposons, and by mutations that

Bacterial infections have been increasing worldwide, being re-
sponsible for the increase in mortality rates and the increase in public
health costs [1,2]. Among the microorganisms that cause death, a group
of the most studied are bacteria from the genus Staphylococcus. Sta-
phylococcus aureus is one of the most difficult microorganisms to treat
because it has a high capacity for host colonization, as well as for the
manifestation of virulence factors and the development of resistance to
a great variety of drugs [3,4]. This is a Gram-positive bacterium asso-
ciated with skin, wound and soft tissue infections, in addition to being
pointed to as the cause of endocarditis and infections linked to medical
device implants, such as valves and catheters [5]. In addition, the
number of S. aureus strains present in clinical isolates resistant to
multiple drugs is increasing [6].
Bacteria become resistant to drugs by obtaining resistance genes
produce changes in the active site of antibiotics, leading to the emer-
gence of resistance mechanisms [7,8]. These mechanisms include
changes in membrane permeability, inactivation by enzymes,
target alterations and active efflux [9,10].
Active efflux is caused by transmembrane proteins capable of ac-
tively expelling or exchanging toxic compounds out of the bacterial cell,
enabling the survival of the micro-organism, denominated as efflux
pumps [11]. There are several types of pumps distributed among five
different families according to their characteristics. Among them, three
of the most studied are NorA, TetK and MrsA. The NorA pump is re-
sponsible for the efflux of various drugs such as fluoroquinolones,
quinolones, verapamil and omeprazole, as well as dyes such as acridine
and ethidium bromide, belonging to the Major Facilitator Superfamily
(MFS) [12–16]. TetK, a member of the MFS family, acts on reducing
tetracycline concentration from the intracellular medium [17]. The

⁎
Corresponding author at: Universidade Regional do Cariri – URCA, Centro de Ciências Biológicas e da Saúde – CCBS, Departamento de Química Biológica – DQB, Laboratório de
Microbiologia e Biologia Molecular – LMBM. Av. Cel. Antonio Luiz, 1161. Pimenta. Crato – CE CEP:63105-000, Brazil.
E-mail addresses: joycy.sampaio22@gmail.com (J.F.S. dos Santos), saulorelison@gmail.com (S.R. Tintino), thiagocrato@hotmail.com (T.S. de Freitas),
fabiacampina@gmail.com (F.F. Campina), irwinalencar@yahoo.com.br (I.R. de A. Menezes), jpsiq@uol.com.br (J.P. Siqueira-Júnior), hdmcoutinho@urca.br (H.D.M. Coutinho),
cunha.urca@gmail.com (F.A.B. Cunha).

https://doi.org/10.1016/j.cimid.2018.03.001
Received 25 November 2017; Accepted 5 March 2018
0147-9571/ © 2018 Elsevier Ltd. All rights reserved.
J.F.S. dos Santos et al.
MrsA pump is part of the RND family, is ATP-dependent and causes the
efflux of erythromycin [18].
Many studies are being carried out in order to find new substances
capable of reversing the bacterial resistance promoted by efflux pumps.
Several compounds isolated from plants, as well as extracts and es-
sential oils, have shown good results in relation to the inhibition of this
mechanism [19,20]. Among these substances, phenolic compounds
have been widely reported as promising alternatives in the search for
new treatment sources [9,21].
Phenolic compounds can contribute satisfactorily as a new com-
plementary treatment source, since they are able to modify bacterial
performance, impairing their locomotion, surface adhesion, biofilm
formation and formation of virulence determinants [22–30]. Phenolic
compounds also present a variety of properties such as anti-allergic,
antiviral, anticancer and anti-inflammatory properties, as well as acting
in the elimination of free radicals and metal chelators [31,32]. Con-
sidered as one of the groups with the greatest diversity of secondary
metabolites, phenolic compounds contain polyphenol categories such
as flavonoids, tannins, lignins, phenolic acids, quinones, flavones, fla-
vonols and coumarins [32,33].
In the search for the reversion of bacterial resistance, recent studies
with phenolic compounds have shown satisfactory advances. Diniz-
Silva indicated in their work the antibiotic modifying activity of quer-
cetin in relation to norfloxacin in S. aureus carrying the NorA pump
[34]. Lima demonstrated the synergistic effect of the combination of
gentamicin and norfloxacin with pyrogallol against S. aureus strains
[35]. Tintino verified the inhibitory action of tannic acid on the NorA
efflux pump in the 1199B strain of the aforementioned bacterium [36].
Phenolic acids can often be identified in foods [37]. Caffeic acid
(3,4-dihydroxycinnamic acid) is a compound derived from hydro-
xycinnamic acid, considered to be one of the most commonly found
phenolic acids, and can be represented in the ester form [38,39]. It has
several biological activities such as antioxidant, antibacterial and fun-
gicide [40,41].
The tannin gallic acid (3,4,5-trihydroxybenzoic acid) is a water-
soluble polyphenol found in pomegranate peel, in processed beverages
such as red wine and in various types of vegetables [42–44]. Considered
the primordial initiator of ellagitannins and gallotannins [45,46]. It is
widely mentioned for its antiviral, antibacterial, anticancer and anti-
oxidant properties [47,35]. In this context, the objective of this work
was to evaluate the inhibitory action of caffeic acid and gallic acid on
efflux pumps from Staphylococcus aureus resistant strains and demon-
strate this mechanism through in silico studies.
2. Materials and methods
2.1. Substances
Caffeic acid, gallic acid and ethidium bromide (EtBr), as well as the
antibiotics tetracycline, norfloxacin and erythromycin, were obtained
from SIGMA Chemical Co., St. Louis, U.S.A.
2.2. Strains utilized
We used the following S. aureus strains in the tests: RN4220 carrying
the MrsA efflux pump; IS-58, which possesses the TetK pump; 1199B
with resistance to hydrophilic fluoroquinolones via the NorA efflux
protein and the 1199 strain which is considered wild-type. Bacteria
were kept on blood agar for testing of their type (Laboratories Difco
Ltda., Brazil) and then transferred and maintained in two stocks, one in
glycerol at −80 °C and the other in Heart Infusion Agar slants (HIA,
Difco) at 4 °C.
2.3. Culture medium
During the assays we used the following culture media: Heart
23
Comparative Immunology, Microbiology and Infectious Diseases 57 (2018) 22–28
Infusion Agar (HIA, Difco laboratorises Ltd.), Brain Heart Infusion (BHI,
difco Laboratories Ltda.), glycerol and blood agar.
2.4. Preparation of the substances
Each antibiotic was used for a particular bacterial efflux pump: er-
ythromycin for the MrsA pump; tetracycline for the TetK pump; nor-
floxacin for the NorA pump and the wild-type strain. All antibiotics,
caffeic acid, gallic acid and EtBr were prepared according to the CLSI
guidelines [48]. Substances were diluted in DMSO with 10 mg/mL and
then diluted in water, decreasing the concentration to 1024 μg/mL.
EtBr was diluted only in water.
2.5. Preparation and standardization of inocula
Inoculations from the stocks were prepared for all assays using the
standard method determined by CLSI [48]. Bacteria were cultured
again on solid Heart Infusion Agar slants and maintained at 37 °C for
24 h. Deriving from this solid medium, the inocula were made using test
tubes containing sterile saline solution which were standardized ac-
cording to the McFarland 0.5 scale corresponding to 106 CFU (Colony
Forming Unit).
2.6. Minimum inhibitory concentration (MIC) assays
All microbiological procedures as well as the readings were per-
formed following the CLSI guidelines with some adaptations [48]. In
the minimum inhibitory concentration assays with caffeic and gallic
acid, ethidium bromide and the antibiotics, the distribution media were
prepared in eppendorfs using 900 μL of BHI liquid culture medium and
100 μL of the inoculum. Subsequently, the solutions contained in the
eppendorfs were transferred to a 96-well microdilution plate, in a hor-
izontal fashion. 100 μL were placed in each well, making up 10 wells.
Subsequently, serial microdilution of the substances was performed,
where 100 μL of each substance was microdiluted up to the penultimate
well (1:1), where the last well is intended for growth control. The
concentrations are varied, from 512 μg/mL to 1.0 μg/mL. The reading
was performed after 24 h by visualization of the color change of the
medium, as indicated by the addition of 20 μL of Resazurin (7-hydroxy-
3H-phenoxazine-3-one 10-oxide). The color change of the medium from
blue to red points to the presence of bacterial growth, while the pre-
sence of the blue color indicates the absence of growth. All experiments
were prepared in triplicates.
2.7. Efflux pump inhibition assays by the reduction of the MIC of ethidium
bromide
The analysis of the effect of decreasing the MIC of EtBr was per-
formed in triplicates. Firstly, the distribution media of the test as well as
that of the control were prepared in eppendorfs. In the test 150 μL of the
inoculum and the substances at the sub-inhibitory concentration (MIC/
8) were added and the volume of the eppendorfs were supplemented
with liquid medium up to 1.5 mL. The control contains the same
amount of inoculum as the test and 1350 μL of the medium.
Subsequently, the prepared solution was transferred to 96-well micro-
dilution plates, the distribution was performed vertically by the addi-
tion of 100 μL of the eppendorfs content in each well. Then, the micro-
dilution of EtBr was performed with 100 μL added to this medium until
the penultimate cavity (1:1). The last cavity did not receive any of the
substances under evaluation, because it is considered as the growth
control. Concentration ranges start from 1024 μg/mL to 0.5 μg/mL.
After 24 h, the plates were read by observing the color change of the
medium, determined by the addition of 20 μL Resazurin. The reading
has as an indicative the color change of the medium from blue to red
pointing to the presence of bacterial growth and the persistence in blue,
the nonexistence of growth. The decreased MIC of ethidium bromide or
J.F.S. dos Santos et al.
specific antibiotics in strains carrying an efflux pump is a characteristic
of efflux pump inhibition.
2.8. Efflux pump inhibition assays by reduction of antibiotic MIC
To verify the effect of reducing the MIC of the antibiotic, the media
for the tests as well as the control were prepared in eppendorfs. In the
procedure, 150 μL of the inoculum, the substances at a sub-inhibitory
concentration (MIC/8) were added to eppendorfs and these were filled
up to the volume of 1.5 mL. In the preparation of the control, the same
inoculum volume of the test was added and the eppendorf volume was
completed up to 1.5 mL. The solutions were then transferred to 96-well
microdilution plates, where 100 μL of the eppendorf content was placed
in each well. Subsequently, the microdilution of 100 μL of the antibiotic
was performed in this medium until the penultimate cavity (1:1). The
last cavity was reserved as the control. Concentrations ranged from
1024 μg/mL to 0.5 μg/mL. After 24 h the plates were read by visuali-
zation of the color modification of the medium characterized by the
addition of 20 μL of Resazurin, as previously cited in the assay with
EtBr.
2.9. Statistical analysis of microbiological results
The results were processed in triplicate and shown as the geometric
mean. The results were subjected to statistical analysis using the sta-
tistical program GraphPad Prism 6.0. A Two-Way ANOVA test was
performed using the geometric mean obtained through the triplicates as
central data ± standard deviation of the mean. Continuing, a
Bonferroni post hoc test was performed (where p < 0.05 and
p < 0.0001 were considered significant and p > 0.05 not significant).
2.10. In Silico tests with the NorA efflux pump
Prediction of the 3D structure and identification of potential efflux
binding pockets of NorA.
In the present work, monitoring of NorA protein and all equivalent
proteins (PDB ID: 1PV4, 1PV7, 3WDO, 2GFP) were acquired in the
FASTA format through the NCBI resources. Identification of the se-
quence of the other proteins and the NorA sequence was 6.89%, 5.87%,
14.80% and 3.47% identity, respectively. The transmembrane segment
prediction of the NorA equence was obtained using MODELLER 9.17v
[49] found on the ModWeb server (https://modbase.compbio.ucsf.edu/
modweb/) using the crystallographic structure of YajR.
The E. coli transporter [PDB ID: 3WDO] was used as a standard. The
final shaped structure was identified using the PROCHECK [50] pro-
gram in Ramachandran Plot. The NorA protein structure modelled by
homology was reduced with the solvent implied by the Chimera pro-
gram using AMBER 8 [51] as the force field. The topographic structure
of the pockets and cavities can be intermediated by the CASTP server
(http://cast.engr.uic.edu) with a standard value of 1.4 Å of the radius of
Fig. 1. Inhibition of the efflux pump by Caffeic (A) and Gallic acid (B) on effluxing strains: CEPA 1 (IS-58/TetK efflux pump, using tetracycline), CEPA 2 (RN-4220/MRS efflux pump,
using erythromycin), CEPA 3 (SA–1199B/NorA efflux pump, using norfloxacin) and CEPA 4 (wild type). (****) P < 0,0001 compared with the control.
24
Comparative Immunology, Microbiology and Infectious Diseases 57 (2018) 22–28
the probe. To achieve a more satisfactory understanding of the mole-
cular basis of the binding compatibilities of NorA, potential NorA in-
hibitors were, through the identification of key residues, in charge of
the binding interactions [52]. Compounds struck by the NorA in-
hibitory activity are considered compounds with good docking scores.
2.11. Docking studies of NorA receptor in its predicted 3D structure
For the performance of docking calculations, DockingServer [53]
was used. Partial Gasteiger charges were placed at ligand atoms. Non-
polar hydrogen atoms were mixed and the rotational bonds were de-
termined. Calculations were performed on the protein content model of
caffeic acid and gallic acid. With the aid of AutoDock tools, the essential
hydrogen atoms, Kollman bound atom type charges and solvation
parameters were added [54]. The grid point affinity (grid) maps of
0.5 Å and spacing of 0.375 Å were generated using the Autogrid 54
program (Morris et al., 1998). In the Van der Waals calculation and the
electrostatic terms, AutoDock set-parameters and distance-dependent
dielectric functions were used, respectively. Coupling demonstrations
were performed using the Lamarckian (LGA) genetic algorithm and the
local localization method Solis & Wets [55]. The establishment of the
initial position, orientation and torsions of the ligand molecules were
random. During the docking, all the rotating torsions were released.
The docking experiments were derived from 10 different races that
were determined to finish after a maximum of 250,000 energy eva-
luations. The population size was adjusted to 150. During the research,
a 0.2 Â translation step and quaternion and torsion steps of 5 were
applied.
3. Results
3.1. Microbiological assays
The phenolic compounds showed results considered clinically irre-
levant, by obtaining MIC in the value of ≥1024 for all strains. In re-
lation to the inhibition of bacterial resistance promoted by efflux
pumps, the caffeic acid in strain 1 obtained a non-relevant result. In
strains 2, 3 and 4 there was a decrease in antibiotic MIC, characterizing
a synergistic action (Fig. 1A). In the ethidium bromide assay, the MIC
was reduced in all strains, however inhibition only occurred in the
MrsA and NorA pumps of strains 2 and 3, respectively (Fig. 2A).
With respect to gallic acid, in the efflux inhibition assay by anti-
biotic MIC reduction, strain 1 was found to be statistically irrelevant
(Fig. 1B). In strain 2 an antagonistic action occurred. In strains 3 and 4
the reduction of the MIC in the antibiotic association with gallic acid
was verified. With ethidium bromide, a reduction in the MIC in strains
1, 2 and 3 was observed. However, only in strain 3 which possess the
NorA pump was the reversal of bacterial resistance by efflux pump
inhibition observed (Fig. 2B).
In this work we used a docking assay to evaluate stabilizations of the
J.F.S. dos Santos et al.
Fig. 2. Inhibition of the efflux pump by Caffeic (A) and Gallic acid (B) on effluxing strains against the biocide activity of ethidium bromide: CEPA 1 (IS-58/TetK efflux pump), CEPA 2
(RN-4220/MRS efflux pump), CEPA 3 (SA–1199B/NorA efflux pump) and CEPA 4 (wild type). (****) P < 0,0001 compared with the control.
Table 1
Energy of interactio betwee caffeic and gallic acid from docking with the NorA structure obtained by molecular modelling.
substance Est. Free Energy of Binding Est. Inhibition Constant, Ki
Caffeic acid −4.93 kcal/mol 245.08 μM
Gallic acid −4.34 kcal/mol 653.95 μM
protein/caffeic acid or gallic acid complex to corroborate with the re-
sults in vitro. Table 1 shows the interaction energies and the inhibition
constant calculated by Autodock. In these results, gallic acid had a free
binding energy (−4.34 kcal/mol) and a better inhibition constant with
a theoretical value calculated at 654 μM, whereas caffeic acid presented
lower free binding energy (−4.93 kcal/mol) and better inhibition
constant with a theoretical value calculated at 245 μM. These results
corroborate with the modulation assays observed in Fig. 2 for strain 3
which presents the NorA efflux system, in view of the fact that caffeic
acid presented potency approximately three times higher than that of
gallic acid.
In Fig. 3 all interaction positions with the amino acids present at the
docking site for caffeic acid (Fig. 1A) and gallic acid (Fig. 1B) are
shown. In this sense, it has revealed the orientation of the ligands
within the binding site (as shown in Fig. 1), which allows their aliphatic
chain, from both the caffeic acid and gallic acid, to extend into the
hydrophobic cleft. Both the hydroxyl groups of caffeic acid and gallic
acid were able to form a hydrogen bond with side chain amino acids
ALA261 (D), ALA339 (A), LEU335 (D) and GLY265 (D) for gallic acid
and ALA261 (D), LEU335 (D), GLY342 (A) for caffeic acid, thereby
contributing additional stability to the ligand/NorA complex. These
results suggest that the hydrophobic and π-π interactions also con-
tribute to the stabilization of the ligand/NorA complexes, therefore, an
interaction of −3.72 kcal/mol was observed for caffeic acid, whereas
for gallic acid an interaction of −2.41 kcal/mol was observed.
4. Discussion
Isolated plant compounds have been consistently reported as pos-
sible modifiers of antibiotic activity, and may act to reverse bacterial
resistance through various mechanisms, such as in the inhibition of
active efflux, resulting in a change in susceptibility to antimicrobial
drugs [56]. Several studies have shown the inhibitory capacity of sec-
ondary metabolites on efflux proteins by reducing the MIC of antibiotics
and other toxic molecules such as ethidium bromide. According to the
study by Ponnusamy, it was verified that the phenolic compound in-
dirrubin presented synergistic interaction with ciprofloxacin, an effect
caused by the inhibition of S. aureus efflux pumps [57].
Several other studies have demonstrated the form of interaction of
phenolic compounds in relation to the inhibition of resistance caused by
efflux pumps. Among them, the blockade of action by iron complex
formation, interference in the production of ATP, membrane depolar-
ization, among others [58–60]. In Chan's work, the flavonoid diosmetin
in combination with erythromycin inhibited the resistance caused by
Comparative Immunology, Microbiology and Infectious Diseases 57 (2018) 22–28
vdW + Hbond + desolv Energy Electrostatic Energy Total Intermolec. Energy
−5.46 kcal/mol −0.12 kcal/mol −5.58 kcal/mol
−4.49 kcal/mol −0.07 kcal/mol −4.56 kcal/mol
the MrsA pump in the RN-4220 strain through the lack of ATP [59].
Although the results of the MICs of caffeic acid and gallic acid did not
demonstrate clinically relevant effects, which made their use as an
antibiotic unfeasible, these compounds presented a potential efflux
pump inhibitory effect. According to Bhardwaj, ideal efflux pump in-
hibitors should be free of any antibacterial activity [61,62].
In the results obtained by caffeic acid, the MIC of the antibiotic was
reduced in the RN-4220, SA–1199 B and wild-type strains character-
izing a synergistic action. In spite of not using the same strain, Lima
pointed out in their study the same synergistic effect of caffeic acid in
the association with antibiotics against S. aureus, Escherichia coli and
Pseudomonas aeruginosa [35]. In the verification of the reversal of
bacterial resistance by ethidium bromide (EtBr), synergism was seen in
all strains, however only the efflux pump inhibition in the RN-4220
strains carrying the MrsA pump and the SA-1199B, which possessed the
NorA pump, was verified. In the case of the IS-58 strain which possesses
the TetK pump, this action was due to the greater affinity with EtBr
than with the antibiotic, making it easier to extrude EtBr. In the re-
search carried out by Diniz-Silva [63], tannic acid, which, like caffeic
acid, is classified as a phenolic acid, was able to inhibit the NorA pump
in its sub-inhibitory concentration, affecting membrane-bound proteins,
preventing the performance of its function.
Gallic acid, on the other hand, demonstrated on the antibiotic
modifying action of the RN-4220 strain an antagonistic action requiring
a greater amount of the antibiotic to obtain the effect. The synergistic
action was identified in the SA–1199B and wild-type strains. In the
study developed by Lima, a synergistic action also occurred in the as-
sociation of gallic acid with gentamicin and norfloxacin against S.
aureus [35]. In the case of the wild-type strain, the synergism observed
with the two compounds may be associated with another factor since it
has no resistance mechanism. In the EtBr data, synergism was observed
in all strains. This may be justified by lower drug affinity and non-
chelation of EtBr. Chelation is characterized as the binding to the active
site of the drug by a particular substance preventing its action [64].
Studies have reported that the interaction of phenolic acid com-
pounds with efflux pumps may be linked not only to their permeability
to the cell membrane due to lipophilic [65] interactions, however these
can also interfere with the mechanism of the efflux pump. Chlorogenic
acid inhibited the active efflux of S. aureus by disturbing the cell
membrane [66]. Tannins, however, can act in a variety of ways, among
them, by the complexation of enzymes with substrates and metal ions as
well as membrane-associated toxicity microorganisms [67,68].
The results of the docking assay present in this study corroborate
with other studies present in the literature, thus, explaining the
25
J.F.S. dos Santos et al. Comparative Immunology, Microbiology and Infectious Diseases 57 (2018) 22–28

Fig. 3. Docking demonstrating all possible positions to the interaction between the NorA efflux pump and Caffeic acid (A) and Gallic acid (B), highliting the H+ bounds.

mechanism of molecular interaction between caffeic acid and gallic acid as gallic acid may be interfering with the NorA pump mechanism
acid and NorA. In studies by Zhang et al. [69] and Kalia et al. [70] it and thus reducing the ability to eliminate ethidium bromide.
was demonstrated that the π-π, hydrophobic and hydrogen bonding
interactions for the stabilization of the ginsenoside20 (S) −Rh2 ligand
5. Conclusion
complexes and capsaicin with NorA protein obtained by molecular
modelling described the mechanism of molecular interaction and in this
The results of this study demonstrated that both gallic acid and
way are able to reduce the activity observed in in vitro assays. The study
caffeic acid cause a reversal of the resistance phenotype, moreover it
by Sharma et al. [71] revealed that piperine is involved in the inter-
was observed that caffeic acid presented the best results, effectively
action of H (2.06A°) binding with Arg141 to form a protein-ligand
inhibiting the MrsA and NorA pumps belonging to the RN-4220 and
complex and the heterocyclic ring of His343 is seen to be favorably
SA–1199B strains, respectively, also demonstrating greater efficacy in
oriented to interact with a π-π interaction. In this way, similar inter-
the in silico model. In view thereof, caffeic acid would be a candidate
actions have also been observed that in part can be inferred as caffeic
drug as an adjuvant for a possible novel formulation using antibiotics

26
J.F.S. dos Santos et al.
such as quinolones against bacteria resistant to these drugs. However,
further studies on the toxicity of this possible formulation should be
performed in order to demonstrate the safety of this formulation. It is
evident from this work that isolated plant compounds may be efficient
alternatives in the search for new forms of treatment against MDR
bacteria.
Conflict of interest
The authors declare that there are no conflict of interest.
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