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Antibacterial mechanism of Eugenia stipitata McVaugh essential oil and
synergistic effect against Staphylococcus aureus
W^
endeo Kennedy Costa*, Alisson Mac
ario de Oliveira, Izabelly Bianca da Silva Santos,
Valquíria Bruna Guimar~
aes Silva, Elys Karine Carvalho da Silva, Jo~
ao Victor de Oliveira Alves,
Ana Paula Sant’Anna da Silva, Vera L
ucia de Menezes Lima, Maria Tereza dos Santos Correia,
M
arcia Vanusa da Silva
Departamento de Bioquímica, Universidade Federal de Pernambuco, Recife - PE, 50670-901, Brazil
ARTICLE INFO
Article History:
Received 24 September 2021
Revised 13 February 2022
Accepted 6 March 2022
Available online xxx
Edited by: Dr. L. McGaw
ABSTRACT
The purpose of this study was to evaluate the antibacterial and antivirulence activities of an essential oil from
E. stipitata (EsEO) leaves against Staphylococcus aureus as well as its mechanism of action.The determination
of antibacterial activity was based on the MIC and MBC. The antibacterial mechanism was explored with
growth curve, synergistic effect, antivirulence and cell membrane permeability assays. In addition, the anti-
microbial activity in a model of S. aureus-infected Tenebrio mollitor larvae and the cytotoxicity on fibroblasts
and macrophages were evaluated. The MIC and the MBC of EsEO were 128512 mg/mL and 2561024 mg/
mL, respectively. The growth curve test showed that S. aureus was inhibited by EsEO. EsEO showed a syner-
gistic effect with all investigated antibiotics and showed a high capacity to reduce hemolysis (78.18-92.48%)
and staphyloxanthin (67.27-91.89%). The increase in the efflux of ions, proteins, and nucleic acids revealed
that the integrity of the membrane had been affected. In addition, EsEO promoted protection in T. mollitor
larvae infected with S. aureus. The essential oil from the leaves of E. stipitata did not present cytotoxic effects
in machophages and fibroblast human cells and demonstrated antibacterial activity against S. aureus strains
including multidrug resistant strains.
© 2022 Published by Elsevier B.V. on behalf of SAAB.
Keywords:
Eugenia stipitata
Antimicrobial activity
Synergistic antimicrobial effect
Myrtaceae
methicillin-resistant Staphylococcus aureus
1. Introduction
Staphylococcus aureus is a gram-positive bacterium found mainly
in the nasal cavity, skin, throat, armpits, vagina, and intestine that
can develop serious infections when immunity decreases (Rahimi;
Katouli; Karimi, 2016). According to the World Health Organization,
the wide variety of infections caused by S. aureus are alarming and
represent some of the main causes of nosocomial infections world-
wide, which generated the emergence of antibiotic resistant strains,
such as those resistant to methicillin, promoting an ineffectiveness to
conventional treatments (WHO, 2017;Oliveira Galv~
ao et al., 2020).
These facts raised the alert for infections by S. aureus for presenting
high pathogenicity and virulence promoting serious complications to
the host (Cong; Yang; Rao, 2020). Before this scenario, the search for
new drugs that have multiple targets that can be used individually or
combined with existing antibiotics is extremely relevant, a fact that
has been promising to escape bacterial resistance mechanisms
(Chan et al., 2011). Thus, natural antimicrobial agents with multiple
destination sites are of great interest, especially essential oils that
have been showing relevant antimicrobial properties (Wu et al.,
2019).
Eugenia is widely distributed in the Caatinga, an important bio-
geographic zone found exclusively in Brazil and has a hot climate,
experiences no rain for several months, and contains soil poor in
nutrients and minerals (Fernandes et al., 2020). Because of the envi-
ronmental conditions to which they are exposed, Caatinga plants
have developed interesting chemical characteristics, making them
useful medicinally (Magalh~
aes et al., 2019). Studies have indicated
the pharmacological potential of Eugenia against antibiotic-resistant
pathogenic microorganisms, a worldwide health problem
(Pereira et al., 2017;Lazarini et al., 2018). Thus, the search for new
antimicrobial and therapeutic agents of natural origin with safe use
has been of great interest in recent decades (Saleem et al., 2020).
Eugenia stipitata McVaugh (Myrtaceae), a shrub of approximately
3 m, with a light green globular-concave berry fruit that has a thick,
juicy, and aromatic pulp, is a medicinally important species
(Baldini et al., 2017;Saltos et al., 2018). It has been used in folk medi-
cine to treat problems in the gastro-intestinal tract, urinary tract, and
respiratory infections, however, these treatments with a rational sci-
entific basis have not been proven (Fern
andez-Tujillo et al., 2011).
* Corresponding author at: Avenida Prof. Moraes Rego, s/n - Cidade Universit
aria,
Recife - PE, 50670-420.
E-mail address: wendeocosta@gmail.com (W.K. Costa).
https://doi.org/10.1016/j.sajb.2022.03.012
0254-6299/© 2022 Published by Elsevier B.V. on behalf of SAAB.
South African Journal of Botany 147 (2022) 724730
Contents lists available at ScienceDirect
South African Journal of Botany
journal homepage: www.elsevier.com/locate/sajb
Studies have demonstrated the antioxidant, antiproliferative, anti-
mutagenic, antinociceptive, antipyretic, and anti-inflammatory
potential of this species (Neri-Numa et al., 2013;Baldini et al., 2017;
Costa et al., 2020b). We investigated the mechanism of action, syner-
gistic effect, and antivirulence of the antimicrobial activity as well as
the cytotoxic potential of the essential oil extracted from E. stipitata
(EsEO) leaves from the Brazilian Caatinga region.
2. Materials and methods
2.1. Herbal material and essential oil extraction
Leaves from E. stipitata McVaugh (Myrtaceae) were collected in
January 2017 (drought period) in the municipality of Exu (07° 30 '43
"S 39° 43027" W), Pernambuco, Brazil, the Caatinga region. The
botanical identification was made by Dra. Arlene Pessoa da Silva and
an exsicata was deposited in the Herbarium D
ardano de Andrade
Lima of the Universidade Regional do Cariri (URCA) voucher speci-
men 13,054. The plant material was registered in the Sistema Nacio-
nal de Gest~
ao do Patrim^
onio Gen
etico e do Conhecimento Tradicional
Associado (SisGen) at number A08E18B.
The collected leaves were washed, dried and pulverized to a fine
powder that was subsequently hydrodistilled for 4 h in a Clevenger-
type apparatus to obtain the essential oil. The EO used in the present
work was characterized by gas chromatography-mass spectrometry
(GC/MS). The EO contains a complex chemical profile, with the pres-
ence of 49 compounds, representing 97.04% of the EsEO composition
and the majority of the compounds were identified with guaiol
(13.77%), trans-caryophyllene (11.36%), b-eudesmol (8.13%), and
g-eudesmol (6.55%). (Costa et al., 2020b).
2.2. Determination of minimum inhibitory (MIC) and bactericidal (MBC)
concentrations
The minimum inhibitory concentration (MIC) was determined in a
96-well culture plate. The microorganisms tested were Staphylococ-
cus aureus (ATCC 29213) and clinical isolates of antibiotic-resistant S.
aureus (UFPEDA-659, UFPEDA-671, UFPEDA-691, UFPEDA-705,
UFPEDA-731, and UFPEDA-802) obtained from the Collection of
Microorganisms of the Universidade Federal de Pernambuco
(UFPEDA). The susceptibility of these isolates was previously evalu-
ated using the disk diffusion technique (Santos et al., 2019).
The stock cultures were maintained at 20 °C in sterile nonfat
milk powder with 10% (v/v) glycerol. Bacteria were cultured in Muel-
ler Hinton Agar (MHA) overnight at 37 °C. Final cell concentrations
were adjusted to 1 £10
7
CFU/mL using the McFarland scale. Mueller-
Hilton growth medium was added to each well along with previously
prepared microorganisms and different concentrations (2 1024
mg/mL) of EsEO.Ciprofloxacin (CIP) and gentamicin (GEN) was used
as reference antibiotics (0,125 1024 mg/mL).Wells containing
microorganisms in the absence of the EsEO or antibiotic corre-
sponded to 100% growth control. The plates were incubated at 37°C
for 24 h. Resazurin solution (0.01%) was used as an indicator of bacte-
rial growth. The lowest concentration at which the solution showed
no colour change (from purple to pink) was taken as the MIC.
For MBC evaluation, an aliquot (10 mL) of the well suspension was
transferred to Mueller-Hinton Agar plates and incubated for 24 h at
37°C. MBC was determined as the lowest concentration of essential
oil capable of preventing bacterial growth.
2.3. Synergism assay
The combination of EsEO and ciprofloxacin or gentamicin was
studied using the microdilution assay. Dilutions of EsEO (40 mL) were
added separately to microwells containing antibiotic dilutions
(40 mL), then 110 mL of Mueller Hinton Broth and 10 mL of cell
suspension (1 £10
7
CFU/mL) were added. Then, the 96-microwell
plates were incubated at 37 °C for 24 h.
The fraction inhibitory concentration index (SFIC), as follows:
SFIC = (MIC of EsEO in combination/MIC of EsEO alone) + (MIC of
antibiotic in combination/MIC of antibiotic alone). The combinations
were classified as total synergism (FIC 0.5), partial synergism (0.5
<FIC 0.75), no effect (0.75 <FIC 2), or antagonism (FIC >2)
(Ferreira et al. 2018).
2.4. Growth curve
S. aureus ATCC 29213 (1 £10
7
CFU/mL) was treated with the
essential oil at 1 £MIC (128 mg/mL) and 2 £MIC (256 mg/mL), and
the control containing only DMSO (1%). The bacteria were then culti-
vated at 37°C with shaking 180 (r minutes
1
) for 0, 1, 2, 4, 8, 12, and
24 h, respectively. At selected time intervals, the OD
600
of the super-
natants was determined using a spectrophotometer. All measure-
ments were performed in triplicate (Zhou et al., 2015).
2.5. Antivirulence action
2.5.1. Antihaemolytic evaluation
S. aureus ATCC 29213 was used to investigate the effects of EsEO
on haemolysis inhibition in human blood. The hemolysis test was
performed as described by Almaaytah et al. (2014). In summary, the
log phase cultures were re-inoculated [1:100 (v/v)] in brain heart
infusion (BHI) with subinhibitory concentrations of the EsEO (128,
64, and 32 mg/mL, corresponding, respectively, to MIC, MIC/2, and
MIC/4). After 16 h, 500 mL of these cultures was added to 1 mL of
human erythrocyte solution (3%) and incubated at 37 §1°C for 1 h
with shaking (250 rpm). The supernatant was collected by centrifuga-
tion (3000 £g for 10 min) and the optical density was measured at
540 nm. The tests were performed in triplicates.
2.5.2. Staphyloxanthin (STX) inhibition assessment
S. aureus ATCC 29213 cultures were inoculated in brain heart infu-
sion (BHI) medium and incubated for 24 h at 37°C. Subsequently, the
inoculum was diluted (1:100) in BHI and the samples (1 mL) of this
suspension were incubated with subinhibitory concentrations of
EsEO (128, 64, and 32 mg/mL, corresponding to MIC, MIC/2, and MIC/
4) and incubated for 24 h at 37°C. The bacterial cells were then har-
vested by centrifugation (6700 £g for 10 min), washed twice with
saline, and re-centrifuged. For the extraction of carotenoid pigments,
the pellet was resuspended in 0.2 mL of methanol before heating at
55°C for 3 min. Extracted pigment was separated from cell debris by
centrifugation (6700 £g for 10 min). The pigment extraction proce-
dure was performed three times to maximize the extraction of staph-
yloxanthin. The optical densities of the collected extractions were
measured at 465 nm (Bezerra Filho et al., 2020).
2.6. Integrity of cell membrane
2.6.1. Efflux of potassium ions through the membrane
S. aureus ATCC 29213 suspensions were incubated overnight in
brain heart infusion broth (BHI) at 37°C for 12 h. Cells were washed
three times with deionized water and resuspended in deionized
water at a concentration of 1 £10
7
CFU. The suspensions were then
treated with EsEO at 1 £MIC (128 mg/mL) and 2 £MIC (256 mg/mL)
at 37 °C for 30, 60, 90, and 120 min. After centrifugation (10,000 £g
for 15 min), the quantities of K+ leakage in the suspensions were
determined using a selective ion analyzer 9180 Electrolyte Analyzer
(Roche). Control contained only DMSO (1%), tests were performed in
triplicate and the results were expressed in mEq/L (Cox et al. 2001a).
W.K. Costa, A.M. de Oliveira, I.B.d.S. Santos et al. South African Journal of Botany 147 (2022) 724730
725
2.6.2. Leakage of DNA and RNA through the membrane
S. aureus ATCC 29213 suspensions were incubated overnight in
nutrient broth (NB) medium at 37°C for 12 h. Final cell concentrations
were adjusted to 1 £10
7
CFU/mL, and the suspensions were then
treated with EsEO at 1 £MIC (128 mg/mL) and 2 £MIC (256 mg/mL)
value, except for the control (DMSO 1%). Then, the samples were
incubated at 37°C for 1, 2, 4, 8, 12, and 24 h. To determine the
amounts of DNA and RNA released from the cytoplasm, the superna-
tant was immediately filtered through a 0.22 mm Millipore filter, and
the optical density was measured at 260 nm (Chen et al., 2002).
2.6.3. Leakage of proteins through the membrane
S. aureus ATCC 29213 suspensions were incubated overnight in
brain heart infusion broth (BHI) at 37°C for 12 h. Final cell concentra-
tions were adjusted to 1 £10
7
CFU/mL and the suspensions were
then treated with EsEO at 1 £MIC (128 mg/mL) and 2 £MIC (256
mg/mL) values, except for the control (DMSO 1%). Then, all samples
were incubated at 37°C for 1, 2, 4, 8, 12, and 24 h. Then, bacteria were
separated by centrifugation (10,000 £g for 5 min at 4°C). To deter-
mine the concentrations of proteins released from the cytoplasm, the
supernatant was used to measure the optical density at 595 nm
(Meng et al. 2016).
2.7. Infection model using Tenebrio molitor
Larvae of Tenebrio molitor (approximately 100 mg) were randomly
distributed into groups of 10 subjects. 10 microliters of S. aureus ATCC
29213 suspension (1 £10
7
CFU/mL) was injected into the last left
proleg, followed by incubation at 37°C for 2 h. The larvae were then
treated with 10 mL of EsEO 128 or 256 mg/mL (resulting in doses of
12.8 or 25.6 mg/kg). Infected-animals treated with PBS (vehicle)
were used as control; while uninfected-larvae treated with EsEO
were also used as controls. Larval viability was evaluated daily for
five days (Czarniewska et al., 2018).
2.8. Cytotoxic activity
The cytotoxicity assay was performed as described by
Politi et al. (2016). Macrophages (J774) and fibroblasts (NIH/3T3)
were cultured in Dulbecco's modified Eagle’s medium (DMEM) sup-
plemented with 10% heat-inactivated fetal bovine serum (FBS), peni-
cillin G (100 U/mL), and streptomycin (100 mg/mL). The cultures
were maintained at 37°C in a humid atmosphere with 5% CO
2
. Cells
were plated in 96-well microplates at a final concentration of 1 £10
4
cells/well. Thereafter, cells were treated with different concentra-
tions of EsEO (62.50 2,000 mg/mL) or untreated and re-cultured for
24 h. After incubation, the supernatant was aspirated and 50 mLof
MTT solution at 1 mg/mL in phosphate-buffered saline (PBS) was
added to each well. Plates were incubated for a further 4 h. Acidified
isopropanol (150 mL) was then added to solubilize the formazan crys-
tals produced by mitochondrial activity. Cell survival was quantified
by measuring absorbance at 570 nm in a Beckman Coulter Microplate
reader.
2.9. Statistical analysis
Experimental results are presented as the mean §standard devia-
tion (SD). Statistical analyses were performed using one-way ANOVA
followed by Tukey’s test. All in vitro tests were performed at least
three times in triplicate. Differences were considered statistically sig-
nificant at a probability of less than 5% (p <0.05).
3. Results and Discussion
Essential oils from leaves plants of the genus Eugenia demonstrate
antimicrobial potential against S. aureus, including E. brasiliensis
(156.2 mg/mL), E. beaurepaireana (1,110.0 mg/mL), E. umbeliflora
(119.2 mg/mL) (Magina et al., 2009), E. brejoensis (256 mg/mL)
(Bezerra-Filho et al., 2020), and E. uniflora (156 mg/mL)
(Ogunwande et al., 2005). Studies have shown that sesquiterpenes
guaiol (Choudhary et al., 2007) and trans-caryophyllene
(Selestino Neta et al., 2016), are potentially active against strains of S.
aureus. Costa et al. (2020b) reported that ingestion of E. stipitata
essential oil (EsEO) resulted in a decrease in pain, inflammation, and
fever without showing toxicity in mice. In the present study, we
examined this essential oil (previously characterized as mentioned in
section 2.1) for its antimicrobial and antivirulence activities against S.
aureus strains as well as its synergistic effect with several antibiotics.
In addition, its mechanism of action and cytotoxic potential were
investigated.
The antimicrobial activity of EsEO or selected antibiotics against S.
aureus, including isolates with multidrug resistance (MDR) profile,
was determined by MIC and MBC. EsEO showed activity in all bacteria
tested, with MIC and MBC ranges of 128512 mg/mL and 2561024
mg/mL, respectively. Isolates UFPEDA 671, 691, 731, and 802 showed
resistance to all antibiotics tested in the present study (Table 1).
Antibiotic resistance has become a global health problem, and
most commercial antibiotics have side effects and are potentially
toxic to the body (Ili
c et al., 2014). Therefore, the identification of
new agents that can be used alone or in combination with the exist-
ing therapies is required to inhibit the growth of antibiotic-resistant
microorganisms. The action of essential oils on bacterial cells involves
multiple targets, which could prove helpful against antibiotic-resis-
tant strains and resistance development (Salem, 2018). These oils
may exert their action on the external bacterial membrane, promot-
ing changes such as protein and enzyme denaturation, and K
+
and H
+
ion concentration imbalance, leading to the modification of the entire
cell morphology and the death of the microorganism (Hu et al., 2017;
Zhang et al., 2017). Thus, the combined effect of essential oils and
antibiotics may be a promising strategy to increase treatment efficacy
and decrease the minimum effective dose of antimicrobial drugs
(Ferreira et al., 2018).
Therefore, we evaluated the synergistic effect of EsEO plus antibi-
otics, and the results are summarized in Table 2. EsEO enhanced the
antistaphylococcal activity of all investigated antimicrobial agents.
The combination of EsEO-gentamicin showed total synergism against
all S. aureus strains.Ciprofloxacin demonstrated total synergism
against S. aureus ATCC 29213, UFPEDA 659, UFPEDA 691, and UFPEDA
705. A partial synergistic effect was observed for S. aureus UFPEDA
671, UFPEDA 731, and UFPEDA 802.
The synergistic interaction of antibiotics and essential oils from
the genus Eugenia, which is rich in sesquiterpenes, has been reported
in E. uniflora in combination with gentamicin (Coutinho et al., 2010),
E. pyriformis with vancomycin (Souza et al., 2014), and E. jambolona
with erythromycin (Pereira et al., 2017). This synergistic effect may
be due to the interaction of sesquiterpenes with the bacterial
Table 1
Antimicrobial activity in vitro of E. stipitata essential oil and antibiotics (mg/mL) against
S. aureus.
S. aureus EsEO CIP GEN
MIC MBC MIC MBC MIC MBC
ATCC 29213 128 256 0.5 4 0.5 4
UFPEDA 659 128 256 1 4 1 4
UFPEDA 671 512 1024 32 64 8 16
UFPEDA 691 128 512 8 16 8 32
UFPEDA 705 512 1024 8 16 1 8
UFPEDA 731 512 1024 32 64 16 32
UFPEDA 802 512 1024 64 128 16 64
EsEO: Eugenia stipitata essential oil; CIP: ciprofloxacin; GEN: gentamicin. MIC: mini-
mum inhibitory concentration (mg/mL); MBC: minimum bactericidal concentration
(mg/mL). The experiments were performed in triplicate.
W.K. Costa, A.M. de Oliveira, I.B.d.S. Santos et al. South African Journal of Botany 147 (2022) 724730
726
membrane, causing loss of integrity, making bacteria more perme-
able, and facilitating the action of antibiotic (Elhidar et al., 2019).
Although the MIC results are important for assessing the antimi-
crobial activity of essential oils, this analysis is static. Thus, studies
that assess the activity over time are necessary to provide informa-
tion on the time of action of the essential oil (Meng et al., 2016).
Based on the susceptibility testing results, S. aureus ATCC 29213 was
selected for growth curve tests (Figure 1). EsEO showed bactericidal
activity against S. aureus ATCC 29213 at a concentration of 2 £MIC
(256 mg/mL) and was capable of strongly reducing growth at a con-
centration of 1 £MIC (128 mg/mL) in the first 12 h (p <0.05).
EsEO significantly decreased the growth kinetics, indicating that
different incubation and concentration times had great impacts on
this activity. Similar results with other essential oils have previously
been reported, E. brejoensis after 3 h decreased 50% in the number of
viable colonies of S. aureus when compared to control (Bezerra Filho
et al., 2020). Essential oil of O. compactum proved the reduction in the
bacterial growth of B. subtilis, a gram-positive bacterium
(Bouyahya et al., 2019).
S. aureus produces several virulence factors that contribute to the
spread and evasion of the immune response. For example, a-hemoly-
sin is a cytotoxin that promotes damage in several cells, especially
erythrocytes, which allows the growth and spread of the bacteria and
aggravates the infection process (Costa et al., 2020a). Another viru-
lence factor secreted by S. aureus is staphyloxanthin (STX), which is
responsible for resistance to attack by the reactive oxygen species
(ROS) produced by phagocytic and neutrophil cells (Bezerra Filho
et al., 2020). Therefore, we evaluated the influence of EsEO on the
hemolytic activity and STX production of S. aureus ATCC 29213. EsEO
showed a high capacity to inhibit the hemolysis caused by S. aureus
at MIC and MIC/2 concentrations (p <0.05), with 92.48% and 78.18%
inhibition, respectively (Figure 2A). At the MIC/4 concentration, the
inhibition was only 2.43%. In the STX assay, 91.89%, 89.04%, and
67.27% reductions in STX levels were observed when S. aureus was
treated with EsEO MIC, MIC/2, and MIC/4 concentrations, respectively
(Figure 2B).
These results indicated that EsEO can act as a therapeutic agent
with multiple targets (Silva et al., 2017). Other essential oils from
plants of the Myrtaceae family have been explored and shown to
inhibit S. aureus virulence factors. Subinhibitory concentrations (64,
32 and 16 mg/mL) of E. brejoensis decreased hemolytic activity and
the levels of staphyloxanthin, and increased susceptibility of S. aureus
to hydrogen peroxide (Bezerra Filho et al., 2020). Myrciaria pilosa (2.5
mg/mL) showed reduction of 92.0% in haemolytic action and 47.2%
production of staphyloxanthin (Costa et al., 2020a).
Further, we investigated the possible mechanisms behind the
antimicrobial potential of EsEO. To that aim, we evaluated K
+
efflux
and leakage of nucleic acids or proteins through the bacterial mem-
brane. The bacterial membrane is an essential barrier that maintains
an adequate intracellular environment for vital processes. Damage to
this structure, even if relatively small, can seriously affect cell metab-
olism, leading to the death of the bacterium (Bouhdid et al., 2009).
Data summarizing K
+
efflux from S. aureus cells treated with EsEO
are shown in Figure 3A. K
+
efflux peaked at 90 min of incubation
when the S. aureus cells were treated with a concentration of 1 £MIC
(128 mg/mL), with values of 1.20 §0.05 mEq/L. When the EsEO con-
centration was 2 £MIC (256 mg/mL), K
+
efflux reached 165 §0.09
mEq/L at 60 min of incubation. The K
+
release values were kept con-
stant in the control group during all the time intervals of the experi-
ment. According to Cox et al. (2001b), the extravasation of potassium
ions (K
+
) is considered the first indication of cell membrane damage
in microorganisms. This occurs when the bacterial membranes are
compromised by the interaction with bacterial inhibiting agents,
which ends in the release of low molecular weight molecules, such as
K
+
and PO
43
(Chen et al., 2002).
EsEO at concentrations of 1 £CIM (128 mg/mL) and 2 £CIM (256
mg/mL) induced the release of important genetic materials from S.
aureus ATCC 29213, with OD
260
values of the supernatant of 0.50§
0.02 and 0.75§0.03, respectively. The results indicated that EsEO
induces the release of cellular constituents (DNA and RNA) in a dose-
dependent manner (Figure 3B).
In addition, protein leakage was evaluated. In the first hour, the
protein leakage of S. aureus ATCC 29213 treated with EsEO was 2.34
mg/mL (1 £MIC) and 2.82 mg/mL (2 £MIC), while that of the control
was 0.22 mg/mL (Figure 3C). Extravasation of proteins of S. aureus
ATCC 29213 treated with EsEO peaked in the first 8 h and remained
similar until 24 h, for both concentrations. Protein leakage indicates
the extent of EsEO involvement on bacterial membrane integrity, and
demonstrates structural damage of the membrane. These findings
imply that EsEO acts on the bacterial membrane, causing leakage of
cellular constituents such as nucleic acids and proteins, and resulting
in bacterial death (Bajpai et al., 2013).
Table 2
Synergistic interaction between E. stipitata essential oil and antibiotics against S. aureus.
S. aureus FIC SFIC Effect FIC SFIC Effect
EsEO CIP EsEO GEN
ATCC 29213 0,12 0,25 0,37 Total synergism 0,12 0,25 0,37 Total synergism
UFPEDA 659 0,12 0,25 0,37 Total synergism 0,12 0,12 0,25 Total synergism
UFPEDA 671 0,06 0,5 0,56 Partial synergism 0,06 0,06 0,12 Total synergism
UFPEDA 691 0,12 0,12 0,25 Total synergism 0,12 0,06 0,18 Total synergism
UFPEDA 705 0,12 0,12 0,25 Total synergism 0,06 0,25 0,31 Total synergism
UFPEDA 731 0,12 0,5 0,62 Partial synergism 0,03 0,25 0,28 Total synergism
UFPEDA 802 0,25 0,5 0,75 Partial synergism 0,06 0,25 0,31 Total synergism
EsEO: Eugenia stipitata essential oil; CIP: ciprofloxacin; GEN: gentamicin. FIC of EsEO= MIC of EsEO in combina-
tion with antibiotic/MIC of EsEO alone; FIC of Antibiotic = MIC of antibiotic in combination with EsEO/MIC of
antibiotic alone; SFIC= FIC of EsEO + FIC of antibiotic. The experiments were performed in triplicate.
Figure 1. Growth curves of S. aureus ATCC 29213 affected by the essential oil from the
leaves of E. stipitata (EsEO). S. aureus ATCC 29213 treated with EsEO at 1x MIC (128 mg/
mL) or 2x MIC (256 mg/mL). Control containing only DMSO (1%) Statistical analysis
was performed using ANOVA, with Tukey’s post-test (p <0.05). Results are expressed
as mean §standard error of three independent tests.
W.K. Costa, A.M. de Oliveira, I.B.d.S. Santos et al. South African Journal of Botany 147 (2022) 724730
727
Based on the results of in vitro antimicrobial activity, EsEO was
evaluated for its anti-infectious capacity in an infection model using
T. molitor. The larvae were infected with S. aureus ATCC 29213 and
treated with EsEO at a concentration of 12.8 and 25.6 mg/kg
(Figure 4). Infected untreated larvae showed 100% mortality on the
3rd test day, while infected treated larvae showed 30% and 90% sur-
vival at the end of the 5th day when the EsEO treating concentration
was 12.8 mg/kg and 25.6 mg/kg, respectively. Uninfected larvae
treated with 25.6 mg/kg EsEO did not show mortality during the trial.
T. molitor is a model for the analysis of antimicrobial activity in
vivo, and the results of the present study demonstrated that EsEO
can protect larvae from S. aureus infection Colasso et al. (2019). used
Euphorbia tirucalli latex to inhibit S. aureus infection, while
Silva et al., (2020) demonstrated that extracts and products derived
from Buchenavia tetraphylla confer protection against E. coli in
infected T. molitor. These data suggest that products of plant origin
can efficiently inhibit T. molitor larvae infection.
Sesquiterpenes represent one of the most prevalent classes of
metabolites present in natural products with diverse structural char-
acteristics and biologically significant for the discovery of new thera-
peutic sources, including in folk medicine. Essential oils demonstrate
strong antibacterial activity and have been linked to the presence of
sesquiterpenes in their composition (Miladinovi
c et al., 2021). How-
ever, its action is clearly described by the synergistic effect or the
mixture of secondary metabolites, being able to attribute the
Figure 2. Effects of essential oil from the leaves of E. stipitata (EsEO) on inhibition of hemolysis (A) and sthaphyloxantin production (B) caused by S. aureus ATCC 29213. S. aureus
ATCC 29213 treated with EsEO on MIC (128 mg/mL), MIC/2 (64 mg/mL) and MIC/4 (32 mg/mL). Control containing only DMSO (1%). Statistical analysis performed was ANOVA, with
post-test of Tukey (p <0.05). Results expressed as mean §standard error of three independent tests. a: significant difference when compared to the control group.
Figure 3. Effect of E. stipitata essential oil treatment on the K+ leakage (A), leakage of nucleic acid (B), and leakage of protein (C) of S. aureus ATCC 29213. S. aureus ATCC 29213
treated with EsEO at 1xMIC MIC (128 mg/mL) or 2x MIC (256 mg/mL). Control containing only DMSO (1%) Statistical analysis was performed using ANOVA, with Tukey’s post-test
(p <0.05). Results are expressed as mean §standard error of three independent tests. a: significant difference when compared to the control group. b: significant difference when
compared to the group treated with 1xMIC.
Figure 4. In vivo antimicrobial activity of E. stipitata essential oil in Tenebrio molitor lar-
vae infected with S. aureus ATCC 29213. S. aureus ATCC 29213 treated with EsEO at
1xMIC MIC (128 mg/mL = 12,8 mg/kg) or 2x MIC (256 mg/mL = 25,6 mg/kg). Control
containing only S. aureus ATCC 29213 and DMSO (1%). Uninfected larvae treated with
EsEO at a concentration of 2x MIC (256 mg/mL = 25.6 mg/mg/kg) were used as con-
trols.
728
W.K. Costa, A.M. de Oliveira, I.B.d.S. Santos et al. South African Journal of Botany 147 (2022) 724730
biological effect to the presence of all components (Moumni et al.,
2020)Veras et al (2020). describe that sesquiterpenes promote dam-
age to the cell membrane, leading to the death of microorganisms.
Thus, the investigated EO showed a high content of sesquiterpenes
(85,33%), thus being able to relate that the strong antibacterial activ-
ity is related to this class of metabolites.
The application of natural products in complementary treatments
can be limited if their composition is toxic to the organism. Consider-
ing the potential use of EsEO in antimicrobial formulations, its cyto-
toxicity needed to be determined. A cytotoxicity assay using
macrophage (J774) and fibroblast (NIH/3T3) cell lines was performed.
EsEO had a greater cytotoxic effect on macrophages (IC
50
= 1,134 mg/
mL) than on fibroblasts (IC
50
= 2,460 mg/mL). The results indicated
that EsEO has low cytotoxicity for macrophages (J774) and fibroblasts
(NIH/3T3) and concur with those of Neri-Numa et al. (2013), in which
E. stipitata fruit extract did not show any cytotoxic effect against sev-
eral cell lines. Previous studies by our group have also shown that
EsEO has low toxicity, does not cause damage to the erythrocyte
membranes of mice, and does not show signs of toxicity when
administered orally at a single dose in mice (Costa et al., 2020b).
Conclusion
Eugenia stipitata essential oil inhibited the growth of resistant S.
aureus isolates and potentiated the effects of ciprofloxacin and genta-
micin, demonstrating total or partial synergism. In addition, EsEO
was able to reduce the production of STX and decrease the ability of
S. aureus to cause hemolysis, two important mechanisms of virulence.
Moreover, EsEO caused destabilization in the membrane of S. aureus,
promoting the extravasation of potassium ions, genetic material, and
proteins. The in vivo study demonstrated that EsEO increases the sur-
vival of T. molitor larvae infected with S. aureus. Moreover, EsEO did
not show cytotoxicity against human cells. Based on these results,
EsEO is a promising complementary alternative that could be used as
an adjuvant against antibiotic-resistant microorganisms of medical
importance.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgements
The authors express their gratitude to the Alexandre Gomes da
Silva (in memorian) for their support and Comunidade Serra dos Paus
D
oias for popular medicinal knowledge. This work was supported by
the Conselho Nacional de Desenvolvimento Científico e Tecnol
ogico
(CNPq); Coordena¸c~
ao de Aperfei¸coamento de Pessoal de Nível Superior
(CAPES) (Finance Code 001), and the Funda¸c~
ao de Amparo
aCi
^
encia e
Tecnologia do Estado de Pernambuco (FACEPE) [BFP-0122-4.03/19].
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