Hindawi

BioMed Research International

Volume 2023, Article ID 5584412, 9 pages
https://doi.org/10.1155/2023/5584412

Research Article
Antibacterial Effect of Ethanolic Extracts of Dodonaea viscosa L.
Jacq. and Mammea americana L. against Staphylococci
Isolated from Skin Lesions

Oscar Alberto Pérez-Narváez,1 Sandra Loruhama Castillo Hernández S,2

Catalina Leos-Rivas,1 Raymundo Alejandro Pérez-Hernández,1 Abelardo Chávez-Montes,1
Jorge Armando Verduzco-Martínez,3 and Eduardo Sánchez-García 1
1
Department of Chemistry, Laboratory of Analytical Chemistry, Universidad Autónoma de Nuevo León, Faculty of
Biological Sciences, San Nicolás de los Garza, Nuevo León C.P. 66455, Mexico
2
Food Department, Laboratory of Functional Foods, Universidad Autónoma de Nuevo León, San Nicolás de los Garza,
Nuevo León C.P. 66455, Mexico
3
Department of Cell Biology and Genetics, Faculty of Biological Sciences, Universidad Autónoma de Nuevo León, San Nicolás de
los Garza, Nuevo León C.P. 66455, Mexico

Correspondence should be addressed to Eduardo Sánchez-García; eduardo.sanchezgrc@uanl.edu.mx

Received 18 January 2023; Revised 30 June 2023; Accepted 22 August 2023; Published 4 September 2023

Academic Editor: Rahul Shivahare

Copyright © 2023 Oscar Alberto Pérez-Narváez et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work
is properly cited.

Background. The resistance to antibiotics shown by some dermatological pathogenic microorganisms has increased the interest of
pharmaceutical and cosmetic industries in developing natural products that possess different biological activities, including
antimicrobial effects. Methods. In the present investigation, the antibacterial activity of ethanolic extracts of Dodonaea viscosa
aerial part and Mammea americana leaves and seed was evaluated against resistant strains of Staphylococcus isolated from skin
lesions and against S. aureus ATCC 25923 (reference strain). Column chromatography (CC) and preparative thin-layer
chromatography (PTLC) were used to obtain separate fractions of the seed extract of M. americana. We also determined the
antimicrobial resistance of the strains against antibiotics using the agar disc diffusion assay. In addition, phytochemical
screening was performed by colorimetric standard techniques. Results. M. americana seed extract showed the highest
antibacterial activity with MBC from 2.3 μg/mL to 19.5 μg/mL without differences with gentamicin (p = 0:998). The isolated
strain S. epidermidis I showed the highest antimicrobial resistance against the tested antibiotics. PTLC-fractions of M.
americana seed extract showed MBC from 3.2 μg/mL to 40.7 μg/mL against S. epidermidis I and S. aureus 25923 (reference),
respectively, which suggests a synergistic effect of the secondary metabolites present in the crude ethanolic extract compared to
its active PTLC-fractions, where only coumarins and compounds with lactone groups were detected in the phytochemical
screening. Conclusion. M. americana seed extract has promising effects that should be considered in further studies as an
alternative or adjuvant in treating skin infections caused by staphylococci.

1. Introduction their mortality rate is relatively low, they are often persistent
and challenging to treat [1, 3]. According to the Global Bur-
Human skin is the body’s most extensive organ; it has an den of Disease (GBD), an estimated 66,500 deaths yearly are
average surface area of 2 m2 in adults and a variable chemical due to bacterial infections in this organ [2]. The inappropri-
constitution. Its functions include protection, permeability, ate use of antibiotics has favored the appearance of
and stabilization of body temperature [1]. Skin diseases antibiotic-resistant microorganisms, making it difficult to
affect about 900 million people of all ages [2], and although combat the diseases they cause [4]. Due to this and the
2 BioMed Research International

possible side effects of conventional treatments [5], in recent Finally, the dried extracts were stored in light-protected vials
years, there has been an increased interest in natural prod- at 4°C until use [16].
ucts by both the pharmaceutical and cosmetic industries in
the search for new, safe, and more effective treatments [6], 2.3. Bacterial Strains and Culture Conditions. Bacterial
among which are the antimicrobial and antioxidant proper- strains used in this investigation (five strains of S. epidermi-
ties of plants [7]. The secondary metabolites, synthesized by dis, one strain of S. saprophyticus, and one strain of S.
plants [8], could provide beneficial effects on skin health [9]; aureus) were obtained from the culture collection of the
such products have been accepted for generations due to Analytical Chemistry Laboratory of the Faculty of Biological
their efficacy, biodegradability, low environmental impact, Sciences. These strains were previously isolated from skin
and the fact that, when used in appropriate doses, they do lesions during an independent investigation, carried out
not generate harmful effects [6]. For these reasons, develop- from April to June 2018. S. aureus ATCC 25923 was used
ing new antimicrobial plant products as auxiliaries in treat- as a reference. All strains were maintained in tubes with
ing skin infections is a growing trend [4]. Antimicrobial Mueller-Hinton Agar slants (DIFCO) at 4°C until use.
properties of D. viscosa and M. americana have been previ- Before the antimicrobial assay, active cultures were obtained
ously reported. Regarding this, a significant number of by inoculating a loopful of each strain into 5 mL of Mueller-
reports highlight the biological activities of M. americana, Hinton Broth (DIFCO) and incubated overnight at 35°C
such as potential source of natural antioxidants, antimicro- ±2°C [15].
bial and even for the treatment of skin diseases [10, 11]).
2.4. Antibiotic Susceptibility Assay. Bacterial susceptibility
Furthermore, a recent study by Pajaro-Gonzalez et al. [12]
was evaluated by the agar disc diffusion assay, according to
reported antibacterial activity at low concentrations of the
the Kirby-Bauer technique and following the standards
ethanol extract of the seeds of M. americana against S.
established by the Clinical and Laboratory Standards Insti-
aureus (ATCC and clinical) strains that are sensitive and
tute (CLSI). For these, activated cultures were seeded by
resistant to methicillin. Regarding D. viscosa, several reports
extension in MH agar plates; subsequently, discs containing
of its antibacterial activity can be found in the literature.
antibiotics (MultiBac I.D.) were placed on the agar plate and
Getie et al. [13] report that D. viscosa, possess antibacterial
incubated at 35°C ±2°C for 16 h to 18 h. Finally, the measure-
activity against S. aureus, suggesting the potential of this
ment of inhibition zones (mm) was determined and
plant to treat bacterial infections of the skin. Similarly,
recorded [17]. Antibiotics used were ampicillin (AM)
Mothana et al. [14] mention that D. viscosa, could be a
10 μg, cephalothin (CF) 30 μg, cefoxitin (CFX) 30 μg, cipro-
source for antibacterial drugs against gram-positive bacteria,
floxacin (CPF) 5 μg, clindamycin (CLM) 30 μg, dicloxacillin
especially against multiresistant microorganisms. In the
(DC) 1 μg, erythromycin (E) 15 μg, gentamicin (GE) 10 μg,
present study, we evaluated the antibacterial activity of etha-
penicillin (P) 10 U, sulfamethoxazole+trimethoprim (SXT)
nolic extracts of D. viscosa aerial part, and M. americana
25 μg, tetracycline (T) 30 μg, and vancomycin (V) 30 μg
leaves and seed against Staphylococcus strains isolated from
(Multibac Investigación Diagnóstica I.D. Gram positives).
skin lesions and S. aureus ATCC 25923. Besides, the antibac-
terial activity of fractions obtained by column chromatogra- 2.5. Agar Well Diffusion Assay. The antimicrobial activity of
phy and PTLC of M. americana seed extract was also extracts was determined by the agar well diffusion technique.
evaluated. Each strain was spread using a Digralsky loop onto MH agar
plates. Then, wells were cut on the agar using an inverted
2. Materials and Methods sterile tube (Ø =8 mm in diameter). Subsequently, 100 μL
of the extracts were deposited in each well; ethanol was eval-
2.1. Plant Material. D. viscosa was collected in Durango, uated alone as a control. Plates were incubated overnight at
México, and M. americana in Quintana Roo, México. The 35°C ±2°C, and after the incubation period, inhibition zones
plant material was dried at room temperature in the shade; were recorded. This assay was performed three times in trip-
afterward, the leaves of D. viscosa and the aerial part and licate [18].
seeds of M. americana were separated. Each plant sample
was crushed and pulverized in a manual grain mill. The 2.6. Minimum Inhibitory Concentration (MIC) and
dried and ground plants were stored in manila paper enve- Minimum Bactericidal Concentration (MBC). The micro-
lopes and in a fresh and dry place [15]. plate dilution technique was used for MIC determination.
For this, different concentrations of the extracts were evalu-
2.2. Preparation of Extracts. For extract preparation, 100 g of ated in MH broth inoculated with 1% of the bacterial strain
dry plant material was weighed on an analytical balance [19]. Plates were incubated for 18 h at 35°C ±2°C. The MIC
(Nimbus® NBL-214e). Plant material was placed in value was determined as the concentration of the extract
1,000 mL Erlenmeyer flasks, to which 500 mL of absolute resulting in complete inhibition of visible growth of the
ethanol (CTR Scientific) was added. Maceration was per- microorganisms evaluated. For MBC, an aliquot of 25 μL
formed by constant agitation in an orbital shaker (Luzeren® was taken from each well without visible growth. Subse-
THZ-100) at room temperature for 24 h. Subsequently, the quently, drops were dispensed onto an MH agar plate [20].
extracts were filtered through filter paper (WHATMAN Plates were incubated under previously established condi-
No.1); the solvent recovered was evaporated using a rotary tions. MBC was defined as the concentration of extracts that
evaporator (Yamato BM 100) under reduced pressure. eliminated 99.9% of the microorganisms evaluated [21].
BioMed Research International 3

2.7. Phytochemical Screening. Standard phytochemical tests which showed inhibition zones of 15 mm and 18 mm,
(Supplementary materials (available here)) to detect metab- respectively, against S. aureus ATCC 6538 [29].
olites such as carbohydrates, sterols, triterpenes, sesquiter- Regarding the D. viscosa leaf extract against Staphylococ-
penes, lactones, tannins, alkaloids, flavonoids, coumarins, cus strains, the inhibition zones showed ranges from
quinones, and saponins were performed following standard 12.5 mm (S. saprophyticus) to 18.8 mm (S. aureus), and sig-
procedures [22]. nificant differences were found with the extracts of M. amer-
icana as well as with the positive control (Table 1). However,
2.8. Chromatographic Separation. The extract with the high- these results showed superior activity to those reported by
est antibacterial activity was fractionated by column chro- Getie et al. [30]. They reported that the methanolic extract
matography (CC) using silica gel 60 Ä (Merck®). The of D. viscosa showed inhibition zones of 8 mm to 9 mm
extract was eluted with 1 L of the solvent system CHCl3- against S. aureus ATCC 29213. Al-Haj et al. [31] reported
MeOH in different proportions. Fifty-mL portions were col- inhibition zones of 16.3 mm against S. aureus, similar to
lected in 250 mL beakers, and the obtained fractions were those obtained in this investigation. Mothana et al. [32]
concentrated by reduced pressure and resuspended in etha- reported the effect of aqueous and methanolic extracts of
nol (EtOH). The purity of fractions was evaluated using D. viscosa against S. aureus ATCC 6538, showing inhibition
thin-layer chromatography (TLC) [23]. zones of 14 mm and 15 mm, respectively. Furthermore,
methanolic extract was effective against multidrug-resistant
2.9. Detection and Semipurification of Antimicrobial
S. aureus from northern Germany (IZ = 12 mm) and against
Compounds. The bioautography technique was used to
multidrug-resistant S. epidermidis 847 (IZ = 12 mm). Like-
detect the active compounds of the fractions obtained by
wise, Monreal [33] reported that the 80% ethanolic extract
CC. Developed TLC plates were placed face down inside
of D. viscosa presented inhibition zones of 10-20 mm against
the MH agar plates with the microorganism of interest pre-
S. aureus INDRE-LIBM-01001.
viously seeded by extension. Plates were incubated at 35°C
±2°C for 24 h to enable the compounds’ diffusion. Subse-
3.2. Minimum Inhibitory Concentration (MIC) and
quently, fractions with antimicrobial activity were evidenced
Minimum Bactericidal Concentration (MBC). The MIC
by the presence of inhibition zones on the agar surface, cor-
values obtained in this work range from 1.7 μg/mL to
responding to the antimicrobial compound. Retention fac-
1,800 μg/mL, depending on the extract and the strain tested
tors (Rf) were recorded for further analysis. Finally, active
(Table 2). M. americana seed extract showed the lowest MIC
compounds were obtained using preparative thin-layer
against the strains of S. epidermidis and S. saprophyticus with
chromatography (PTLC) by scarping plates eluted as bioau-
values from 1.7 μg/mL to 2.9 μg/mL. Moreover, the MIC
tography and according to the RF obtained in the bioauto-
obtained for S. aureus (isolated from skin) was 8.8 μg/mL
graphy technique [24, 25].
and 17.0 μg/mL against S. aureus ATCC 25923. Similarly,
2.10. Statistical Analysis. Data were reported as mean ± SD low MICs were reported by Pajaro-Gonzalez et al. [12], with
and were analyzed by ANOVA with Tukey and Dunnett’s MIC90 values between 2 μg/mL and 4 μg/mL of the ethanol
test. It has statistical significance p ≤ 0:05. extract of the seeds of M. americana against S. aureus
strains. However, all the results mentioned above are lower
3. Results and Discussion than those obtained by Yasunaka et al. [34] who reported
64 μg/mL of methanolic extracts of the seed of M. americana
3.1. Preliminary Inhibitory Effect of Ethanolic Extracts. The against methicillin-resistant S. aureus strains.
antibacterial properties of plant extracts have been demon- D. viscosa extract showed higher MIC values from
strated in a wide variety of studies. [7, 26, 27]. The present 200 μg/mL to 960 μg/mL, according to the tested strain. Sim-
investigation observed such activity in the ethanolic extracts ilarly, Verotta et al. [35] reported ranges from 125 μg/mL to
of M. americana and D. viscosa. Both extracts showed inhi- 1,000 μg/mL against S. aureus ATCC 25923. However, our
bition zones (IZ) from 12.5 mm to 30.5 mm against the results are lower than those obtained by Al-Haj et al. [31],
Staphylococcus strains. The activity of the M. americana seed who obtained a MIC of 20,000 μg/mL with the methanolic
extract is outstanding since it produced the highest inhibi- extract of D. viscosa against S. aureus.
tion effect, ranging from 21.3 mm (S. aureus ATCC 25923) The results of MBC (Table 2) show that M. americana
to 30.5 mm (S. epidermidis I) and because no significant dif- seed extract presented the lowest values, ranging from
ferences were observed with the positive control (gentamy- 2.3 μg/mL (S. epidermidis IV strain) to 19.5 μg/mL (S. aureus
cin). M. americana aerial part extract was less active ATCC 25923). The MBC reported in this research are lower
showing inhibition zones from 15.2 mm (S, saprophyticus) than those found in the literature. Manjulatha [36] men-
to 22.5 mm (S. aureus) (Table 1). Similar results were tioned an MBC ranging from 125 μg/mL to 500 μg/mL using
reported by Poojary et al. [28] using different extracts of the ethanolic seed extracts of M. americana against Strepto-
M. suriga root bark against S. aureus ATCC 25923 (IZ = 28 coccus mutans ATCC 25175 (gram-positive bacteria). Simi-
mm-33 mm), attributing this effect to the alkaloids and fla- larly, Pajaro-Gonzalez et al. [12] report MBC higher than
vonoids found in the extracts. Likewise, the inhibition zones 64 μg/mL (>64 μg/mL) establishing in their investigation a
generated by our M. americana seed extract were similar to bacteriostatic effect of the extract. Interestingly, the ethanolic
the inhibition zones of two hydroxycoumarin isolated from extract of the aerial part of M. americana presented greater
M. africana named Mammea B/BB, and Mammea B/BA activity against S. epidermidis strains (91 μg/mL to 540 μg/
4 BioMed Research International

Table 1: Inhibition zones (mm) of the ethanolic extracts.

Ethanolic extract Antibiotic

Strains
M. americana seed extractc M. americana aerial part extractb D. viscosa leaf extracta Positive control, Gentamicinc
S. saprophyticus 28:9 ± 0:4∗ 15:2 ± 0:0 12:5 ± 8:2 30:0 ± 0:0
S. epidermidis I 30:5 ± 0:3 16:8 ± 8:9 14:1 ± 1:29 30:0 ± 1:93
S. epidermidis II 30:4 ± 1:0 16:3 ± 6:3 13:6 ± 2:88 31:3 ± 0:02
S. epidermidis III 28:9 ± 0:3 16:2 ± 1:9 14:7 ± 8:9 28:3 ± 0:02
S. epidermidis IV 29:4 ± 0:3 16:2 ± 3:5 14:4 ± 5:7 27:7 ± 00
S. epidermidis V 26:9 ± 0:3 16:5 ± 2:2 14:6 ± 1:10 29:7 ± 0:02
S. aureus 28:8 ± 1:0 22:5 ± 1:16 18:8 ± 1:34 21:0 ± 0:03
25923
S. aureus 21:3 ± 1:1 16:7 ± 7:6 16:0 ± 4:11 24:7 ± 0:04
∗
Mean ± SD (3×) of inhibition zone by well diffusion on MH agar. Diameter (Ø = 8 mm) included. a,b,c,d
ANOVA-Tukey and Dunnet, p ≤ 0:05.

Table 2: MIC and MBC (μg/mL) of the ethanolic extracts against strains of staphylococci.

Ethanolic extract Antibiotic

M. americana seed M. americana aerial part Positive control,
Strains D. viscosa leaf extracta
extractc extractb Gentamicinc
MIC MBC MIC MBC MIC MBC MIC MBC
∗
Ss 2:9 ± 0:4 3:9 ± 0:4 1000 ± 0:0 1750 ± 267 356 ± 82 450 ± 93 <0:05 ± 0 <0:05 ± 0
Se I 1:8 ± 0:3 2:5 ± 0:5 440 ± 89 540 ± 89 417 ± 129 833 ± 258 21:5 ± 1:93 22:75 ± 1:75
Se II 8:8 ± 1:0 10:0 ± 0:0 300 ± 63 350 ± 63 822 ± 288 1478 ± 632 0:09 ± 0:02 0:14 ± 0:03
Se III 1:8 ± 0:3 2:6 ± 0:6 243 ± 19 293 ± 19 960 ± 89 1800 ± 447 0:06 ± 0:02 0:10 ± 0:03
Se IV 1:7 ± 0:3 2:3 ± 0:6 81 ± 35 91 ± 39 200 ± 57 345 ± 166 0:05 ± 00 0:1 ± 0:00
Se V 1:7 ± 0:3 2:4 ± 0:5 150 ± 22 194 ± 30 920 ± 110 1120 ± 110 0:11 ± 0:02 0:16 ± 0:02
Sa 8:8 ± 1:0 10:0 ± 0:0 313 ± 116 625 ± 231 375 ± 134 750 ± 267 0:23 ± 0:03 0:35 ± 0:05
25923
Sa 17:0 ± 1:1 19:5 ± 0:9 550 ± 76 650 ± 76 700 ± 411 1100 ± 548 0:1 ± 0:04 0:15 ± 0:06
∗
Media ± SD of 3 experiments. MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration. a,b,cANOVA-Tukey and Dunnet, p ≤
0:05. Mean initial and final inoculation: 2:3 × 108 and 5:8 × 1010 CFU/mL, respectively. Ss: S. saprophyticus; Se: S. epidermidis; Sa: S. aureus; Sa25923: S.
aureus ATCC 25923.

mL) followed by the MBC of isolated and ATCC strains of S. 36.1 μg/mL (PTLC fraction I), it was determined that the
aureus (625 μg/mL and 650 μg/mL, respectively) and finally fraction with the highest antimicrobial activity is fraction II
by S. saprophyticus (1750 μg/mL). On the other hand, we (Table 3). S. epidermidis I strain and S. aureus ATCC
consider that the D. viscosa extract was the least active 25923 were selected for antimicrobial determination of
against staph strains, since four strains presented CMB PTLC fractions because they are the lowest and highest anti-
greater than 1000 μg/mL and two strains are above 700 μg/ bacterial sensitivity strains (Table 4). Interestingly, the
mL. However, Herrera et al. [37] report MBCs ranging from PTLC-fraction II eluted using a mixture of CHCl3:MeOH
20,000 μg/mL to 40,000 μg/mL against S. aureus strains, (9.5 : 0.5) presented a similar MIC to the M. americana etha-
using 80% ethanol and diethyl ether extracts of D. viscosa nolic seed extract against the same strain of S. epidermidis I,
leaves, results that are higher than those reported in this suggesting this effect is due to the coumarins since these
investigation. compounds were only detected with the Baljet test for lac-
tones in the phytochemical screening (Table 5). More than
3.3. Antimicrobial Determination of Isolated PTLC Fractions. 1,800 natural coumarins are known, and many have been
Phytochemicals can be antimicrobial when MIC values evidenced to possess high biological activity [39]. In this
range from 100 μg/mL to 1,000 μg/mL [38]. Our results sense, our MIC results for the PTLC-fractions of the M.
show that secondary metabolites are present in the ethanolic americana seed extract may be comparable with MICs
seed extract and the isolated PTLC-fractions of M. ameri- reported by Verotta et al. [40], with Mesua ferrea extract,
cana are promising. Two isolated PTLC-fractions showed mesuol compound, and coumarin mixtures against
antimicrobial activity and presented MIC values of 1.8 μg/ multidrug-resistant S. epidermidis 3112. Liu et al. [40]
mL (PTLC fraction II) and 5.6 μg/mL (PTLC fraction I) reported MICs of 2 μg/mL against one strain of S. epidermi-
against S. epidermidis I strain. MIC values for S. aureus dis and 4 μg/mL against two strains of S. aureus, using the
ATCC 25923 were 7.7 μg/mL (PTLC fraction II) and extract of M. ferrea. It should be mentioned that the MICs
BioMed Research International 5

Table 3: MIC and MBC of subfractions from the M. americana detected by standard phytochemical screening (Table 5) and
seed extract. could be the cause of the effective antibacterial activity in the
M. americana and D. viscosa extracts. The inhibitory effect
Bioactive PTLC fractions of these compounds has been related to the perturbation of
Strain PTLC fraction I PTLC fraction II
membrane permeability and the inhibition of enzymes such
MIC CMB MIC CMB
as ATPase and phospholipase A2 [42]. Other secondary
S. epidermidis I 5:6 ± 1:7 12:8 ± 1:1 1:8 ± 0:4 3:2 ± 0:8 metabolites found in the extracts were tannins, which are
25923
S. aureus 36:1 ± 0:9 40:7 ± 1:9 7:7 ± 0:8 10:7 ± 1:6 abundant in plants, and their antimicrobial effect against
∗
Mean ± SD (μg/mL) of 3 experiments (3×). MIC: minimum inhibitory bacteria and fungi has been demonstrated [43]. On the other
concentration; MBC: minimum bactericidal concentration. Mean initial hand, alkaloids were not detected with Dragendorff’s
and final inoculation: 4 × 107 and 3 × 1010 CFU m-1, respectively. reagent, which could indicate antimicrobial activities are
mainly by phenolic compounds [19]. Saponins were detect-
able in all the extracts except in M. americana seed; these
reported for the mesuol compound were the same than those metabolites can cause membrane rupture, and their hemo-
obtained with the crude extract (2 μg/mL and 4 μg/mL), lytic activity is known [44]. Likewise, coumarins were also
which is comparable with the results obtained in this inves- revealed; these phenolic substances are benzene fused to α-
tigation with PTLC fraction II and the ethanolic extract of pyrone rings, many of which possess antimicrobial proper-
M. americana. Finally, the extract of M. ferrea and the ties. In the present investigation, we believe coumarins are
mesuol compound did not show activity against S. saprophy- the principal cause of antibacterial activity in M. americana
ticus strains. extracts because >120 coumarins have been identified in
In addition, MIC for PTLC fraction II (7.7 μg/mL) Mammea species with insecticidal, antioxidant, anti-HIV,
against S. aureus ATCC 25923 are comparable with the anticancer, antifungal, antibacterial, antimicrobial, and anti-
results reported by Yasunaka et al. [34]; they reported MICs biotic effects [34, 40, 45, 46].
values from 1 μg/mL to 8 μg/mL using two flavonoids iso-
lated from the leaf of Calophyllum brasiliense and the fruit 3.5. Antimicrobial-Resistant Profile. Antibiotic resistance has
skin of M. americana named Mammea A/BA and Mammea increased in recent years due to the inappropriate use of
A/AA, against methicillin-resistant (MRSA) and methicillin- these drugs and is a severe health problem worldwide [17].
sensitive (MSSA) strains of S. aureus. Besides this, El-Seedi In our study, we observed penicillin and ampicillin insensi-
[41] determined the MIC of the isolated coumarins asphode- tivity in isolated strains. It is worth mentioning that the S.
lin A 4′-O-β-D-glucoside and its aglycone asphodelin A iso- epidermidis I strain showed the lowest sensitivity against
lated from Asphodelus microcarpus and reported 128 μg/mL the gram-positive antimicrobials (Table 4). Likewise, S. epi-
and 16 μg/mL, respectively, against S. aureus IAM1011. It is dermidis I, S. epidermidis IV, and S. epidermidis V isolated
essential to mention that synthetic coumarin compounds strains showed multiresistance by presenting insensitivity
have shown relevant results, such as those observed by to four antibiotics [47]. Velásquez et al. [48] determined
Simões et al. [39], with seventy coumarin compounds from the antimicrobial profile of 101 isolates of the Staphylococcus
antibiotic novobiocin linked to a pyrazole ring against S. genus and observed strains that were resistant to 9 antibi-
aureus ATCC 12600; the MIC obtained using these synthetic otics. Furthermore, coagulase-negative staphylococci
compounds ranged from 0.5 μg/mL to 128 μg/mL. Those showed resistance to penicillin, ampicillin, erythromycin,
results can be compared with the obtained with PTLC- tetracycline, kanamycin, and clindamycin. In our work, iso-
fraction I (MIC = 36:1 μg/mL) and PTLC-fraction II lated S. aureus showed intermediate insensitivity to dicloxa-
(MIC = 7:7 μg/mL) against S. aureus ATCC 25923 cillin, whereas the S. epidermidis from our isolated
(Table 3). MICs and MBCs of the PTLC-fractions suggest coagulase-negative staphylococci strain was insensitive to
a synergistic effect from the M. americana seed extract; the gentamicin. Velásquez et al. [48] observed
PTLC-fraction II, on the other hand, required almost half sulfamethoxazole-trimethoprim insensitivity in one strain
the concentration to inhibit S. aureus ATCC 25923, suggest- of S. aureus, whereas coagulase-negative staphylococci did
ing these metabolites possess a more significant antibacterial not show resistance; in contrast, we observed resistance in
effect. S. epidermidis I, similarly with the work of Liu et al. [40],
who reported insensitivity in a strain of S. epidermidis, as
3.4. Phytochemical Screening. There is a wide variety of poly- well as to erythromycin and gentamicin. Regarding cipro-
phenols with antimicrobial properties [27]. The antibacterial floxacin, the strain S. epidermidis I showed intermediate
effect of our extracts can be due to the presence of different resistance, comparable to the one reported by Silva et al.
phenolic compounds whose biological activities are related [47]; they observed high susceptibility in coagulase-
to their molecular structures containing hydroxyl groups, negative staphylococci isolated from infected chronic
and phenolic rings bind to proteins and bacterial mem- wounds. Likewise, we observed similarities in sensitivity to
branes to form complexes that inhibit bacterial growth tetracycline except in S. epidermidis I. This resistance is
[19]. Flavonoids, flavones, and flavonols are phenolic com- probably a consequence of inappropriate antibiotic use, such
pounds synthesized by plants in response to microbial infec- as erythromycin, which is widely used to treat staphylococ-
tions and are generally effective in vitro against a wide range cal infection [49]. We observed resistance to clindamycin
of microorganisms [18]. In our study, these metabolites were in strains that also showed insensitivity to erythromycin, so
6 BioMed Research International

Table 4: Antimicrobial sensitivity profile of Staphylococcus strains.

Strains
Antibiotic S. epidermidis
S. saprophyticus S. aureus S. aureus 25923
I II III IV V
Ampicillin R R R R R R R S
Cephalothin S S S S S S S S
Cefotaxime S S S S S S S S
Ciprofloxacin S I S S S S S S
Clindamycin S S S I R R S S
Dicloxacillin S R S S S S I S
Erythromycin S R S S R R S S
Gentamicin S R S S S S S S
Penicillin R R R R R R R R
Sulfamethoxazole/Trimethoprim S R S S S S S S
Tetracycline S R S S S S S S
Vancomycin S S S S S S S S
∗
Antimicrobial sensitivity values represented by R: resistance; I: intermediate; S: sensitive.

Table 5: Phytochemical screening of extracts and active subfractions.

Extract PTLC fractions

Phytochemical test
M. americana seeds M. americana aerial parts D. viscosa leaf Rf 0.33 Rf 0.86
Liebermann-Burchard Triterpene Triterpene Sterol — —
Sesquiterpene Lactones + + — + +
Carbohydrates + + + — —
Tannins + + + — —
Alkaloids — — — — —
Flavonoids + ++ ++ — —
Coumarins + + + + +
Quinones + + + — —
Saponins — + + — —
+: present; -: not present.

cross-resistance should be considered. Isolated strains in this ATCC 25923, suggest a synergistic effect of the extract from
work were sensitive to vancomycin, which agrees with Peix- which they were obtained. Currently, the use of medicinal
oto et al. [50], who mention that vancomycin has become plants for the care and treatment of skin infections is of great
the first-line therapy for most infections caused by interest, and therefore, the search for alternatives to face
methicillin-resistant staphylococci. It is well known that such resistance is relevant.
resistance to antibiotics by pathogenic microorganisms hin-
ders the treatment of diseases [4]. In the case of infected skin Data Availability
wounds, healing is difficult [47], which may result in other
inconveniences, such as increased treatment costs, hospital- The data used to support the findings of this study are available
ization, and side effects [5]. from the corresponding author upon request.

Conflicts of Interest
4. Conclusion
The authors declare no conflict of interest.
Ethanolic extracts of D. viscosa leaf and M. americana aerial
part and seed showed good antibacterial activity against Authors’ Contributions
staphylococci isolated from skin lesions. The M. americana
seed extract was the most outstanding due to its low MIC/ Oscar Alberto Pérez Narváez did the writing, which
MBC, comparable with the gentamicin effect. The bioactive includes the original draft, and investigation. Sandra Loru-
PTLC-fractions from the seed extract against the most hama Castillo Hernández did the strain isolation and gen-
antimicrobial-resistant strain, S. epidermidis I and S. aureus eral review. Catalina Leos Rivas carried out the review and
BioMed Research International 7

methodology. Abelardo Chávez-Montes was responsible de microorganismos intervinientes en el biodeterioro del patri-
for the general review and methodology. Jorge Armando monio cultural,” in VII Congreso de Medio Ambiente Mayo 22-
Verduzco Martínez worked on the review and validation. 24, pp. 1–24, La Plata, Arg, 2012.
Eduardo Sánchez García was in charge of the supervision, [9] A. Saikia, V. Ryakala, P. Sharma, P. Goswami, and U. Bora,
review, editing, and correspondence. “Ethnobotany of medicinal plants used by Assamese people
for various skin ailments and cosmetics,” Journal of Ethno-
pharmacology, vol. 106, no. 2, pp. 149–157, 2006.
Acknowledgments
[10] C. Lemus, J. Smith-Ravin, and O. Marcelin, “Mammea ameri-
The authors acknowledge Programa de Apoyo a la Inves- cana: a review of traditional uses, phytochemistry and biolog-
tigación Científica y Tecnológica (PAICYT, UANL ical activities,” Journal of Herbal Medicine, vol. 29, article
100466, 2021.
SA834-19) for funding our research and Consejo Nacional
de Ciencia y Tecnología (CONACYT), for the Postgradu- [11] L. G. B. Lima, J. Montenegro, J. P. D. Abreu et al., “Metabolite
ate Scholarship (No. 722125). M.C. Sergio García Gonzá- profiling by UPLC-MSE, NMR, and antioxidant properties of
amazonian fruits: mamey apple (mammea americana),
lez, Facultad de Ciencias Biológicas, is acknowledged for
camapu (physalis angulata), and uxi (endopleura uchi),” Mol-
his experience and teaching. Dr. David Gilberto García is ecules, vol. 25, no. 2, p. 342, 2020.
acknowledged for his support with the Multidiscs for anti-
[12] Y. Pájaro-González, A. F. Oliveros-Díaz, J. Cabrera-Barraza
microbial susceptibility.
et al., “Mammea B/BA isolated from the seeds of Mammea
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