microorganisms

Article
Antimicrobial Activity of Psidium guajava Aqueous Extract
against Sensitive and Resistant Bacterial Strains
Geraldo Augusto Pereira 1 , Douglas Siqueira de Almeida Chaves 2,† , Taynara Monsores e Silva 1 ,
Raissa Emidio de Araújo Motta 2 , Adriana Barbosa Rocha da Silva 2 , Thereza Cristina da Costa Patricio 1 ,
Anna Julia Bessa Fernandes 1 , Shana de Mattos de Oliveira Coelho 1,3 , Marcin Ożarowski 4 ,
Yara Peluso Cid 1,2, *,† and Tomasz M. Karpiński 5, *,†

1 Pos Graduation Program of Veterinary Science, Veterinary Institute, Federal Rural University of Rio de
Janeiro, BR 465, km 7, Seropédica 23897-000, RJ, Brazil
2 Pharmaceutical Science Department, Health and Biological Science Institute, Federal Rural University of Rio
de Janeiro, BR 465, km 7, Seropédica 23897-000, RJ, Brazil
3 Veterinary Microbiology and Immunology Department, Veterinary Institute, Federal Rural University of Rio
de Janeiro, BR 465, km 7, Seropédica 23897-000, RJ, Brazil
4 Department of Biotechnology, Institute of Natural Fibres and Medicinal Plants, Wojska Polskiego 71b,
60-630 Poznań, Poland
5 Chair and Department of Medical Microbiology, Poznań University of Medical Sciences, Rokietnicka 10,
60-806 Poznań, Poland
* Correspondence: [email protected] (Y.P.C.); [email protected] (T.M.K.)
† These authors contributed equally to this work.

Abstract: The inappropriate use of antimicrobials, along with environmental conditions, can lead to
the emergence of resistant microorganisms. The use of phytopharmaceuticals and herbal medicines
has a positive impact and represents a promising alternative. Psidium guajava extracts have been
widely reported to have antimicrobial potential; however, studies reporting their activity against
resistant bacterial strains are scarce. Because of the emerging resistance, the aim of this study was
Citation: Pereira, G.A.; Chaves,
to analyze the antimicrobial capacity of the aqueous extract of guava leaves against wild-type and
D.S.d.A.; Silva, T.M.e.; Motta,
R.E.d.A.; Silva, A.B.R.d.; Patricio,
resistant bacterial strains. The aqueous extract obtained from the leaves of P. guajava was evaluated by
T.C.d.C.; Fernandes, A.J.B.; Coelho, HPLC for the content of total phenolics and tannins, antioxidant activity, and chemical composition.
S.d.M.d.O.; Ożarowski, M.; Cid, Y.P.; The antimicrobial activity of the extracts was analyzed by the disk diffusion and broth microdilution
et al. Antimicrobial Activity of methods. The results of the chemical analysis of the extracts showed total phenolics content of
Psidium guajava Aqueous Extract 17.02 ± 6.87 mg/g of dry extract, total tannin content of 14.09 ± 1.20 mg of tannic acid equivalents/g
against Sensitive and Resistant of dry extract, and moderate antioxidant capacity with an EC50 value of 140 µg/mL. Flavonoids are
Bacterial Strains. Microorganisms 2023, the major compounds (rutin, hesperidin, and quercetin), followed by phenolic acids. Disk diffusion
11, 1784. https://doi.org/10.3390/ test results showed the presence of inhibition halos for Gram-positive bacteria (Staphylococcus aureus,
microorganisms11071784
sensitive and resistant; Staphylococcus pseudintermedius, sensitive and resistant; and Streptococcus
Academic Editor: Giuseppe Comi spp., beta-hemolytic), while for Gram-negative bacteria (Escherichia coli, sensitive and resistant),
there was no inhibition in the tested concentration range. The Minimal Inhibitory Concentration was
Received: 28 May 2023
6.8 mg/mL for all Gram-positive strains evaluated. The present study demonstrated the antimicrobial
Revised: 25 June 2023
activity of the aqueous extract of P. guajava against sensitive and resistant Gram-positive bacteria. The
Accepted: 6 July 2023
Published: 10 July 2023
better antimicrobial activity found in the present study compared with previously reported activity
should be highlighted and may be related to the higher concentration of total phenolics present in
the tested extract. Moreover, the content of tannins found suggests a species with high quality that
produces tannins. These new findings suggest an innovative profile regarding therapeutic resources
Copyright: © 2023 by the authors. that can be adopted to combat resistant microbial strains.
Licensee MDPI, Basel, Switzerland.
This article is an open access article Keywords: phytopharmaceuticals; Psidium guajava; antimicrobial resistance; phenolics; antioxidant
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Microorganisms 2023, 11, 1784. https://doi.org/10.3390/microorganisms11071784 https://www.mdpi.com/journal/microorganisms

Microorganisms 2023, 11, 1784 2 of 12

1. Introduction
Antimicrobial resistance can be described as the ability of a microorganism to resist the
action of antimicrobials, which regularly occurs through continuous exposure to them. The
level of resistance of a mutant strain can vary widely depending on the mechanism of resis-
tance resulting in its evolution, either by spreading between similar or dissimilar strains [1].
The emergence and spread of antimicrobial-resistant bacteria have been classified by the
World Health Organization (WHO), the United States Center for Disease Control and
Prevention (CDC), and the European Center for Disease Prevention and Control (ECDC)
not only as an emerging global disease but as one of the three most significant threats to
public health in the 21st century [2]. The Review on Antimicrobial Resistance (2016) [3]
report warned of the likelihood of a catastrophic increase in global rates of antimicrobial
resistance (AMR), raising the 700,000 annual deaths attributable to infections by resistant
pathogens to an alarming 10 million cases in 2050, with significant public spending in the
order of USD 100 trillion.
The improper use of antimicrobials stimulated the emergence of genetic modifica-
tions that contributed to circumventing the mechanism of action of drugs. Therefore, the
expansion of resistant strains results in damage to public health as it leads to infectious
conditions that require difficult treatment [1]. The use of phytopharmaceuticals and herbal
medicines has a positive impact on therapy, representing a promising alternative since
many microorganisms have developed resistance to synthetic drugs [4,5].
P. guajava, popularly known as guava [6], is widely cultivated in Brazil. In addition
to food, its various extracts have traditionally been used in Brazil for medicinal purposes.
The origin of the guava tree is in tropical and subtropical areas of the Americas, with a
prevalence in dry climates. Still, it expands naturally throughout Tropical America, between
southern Mexico and northern South America. It is considered one of the tropical and
subtropical fruits with high value and importance due to its natural sources of vitamins
and minerals.
The chemical composition of P. guajava includes tannins such as guavins A-D and
flavonoids [7,8], and its extracts have already been widely reported to have antimicro-
bial potential. Many studies have described the antimicrobial activity of different ex-
tracts from the leaves of P. guajava, such as methanolic [9–16], ethanolic [9,16–20], and
aqueous [9,11–13,16,19–23], including the activity of methanolic extracts against methicillin-
resistant Staphylococcus aureus (MRSA) strains [18]. The antimicrobial activity of the root [12]
and fruit extracts [24] of P. guajava has also been reported.
Because of the emerging resistance of microorganisms, the aim of this study was to
analyze the antimicrobial capacity of the aqueous extract of guava leaves against wild-type
and resistant strains.

2. Materials and Methods

2.1. Chemicals and Reagents
Solvents for extraction and preliminary analysis such as methanol, Folin-Ciocalteu,
DPPH (2,2-Diphenyl-1-picrylhydrazyl) (Sigma, Brazil), sodium carbonate, gallic acid, tannic
acid, and ferric chloride were obtained from Sigma-Aldrich (Brazil). Ultrapure water was
obtained from MilliQ equipment from Merck, São Paulo, Brazil. Culture medium (yeast
extract, glucose, peptone, and agar) were acquired from Difco, IL, USA, and hydrogen
peroxide and dimethylsulfoxide (DMSO) from Sigma-Aldrich, Brazil.

2.2. Plant Material, Extraction, and High Performance Liquid Chromatography

Leaves of P. guajava L. (Myrtaceae) were obtained from the Federal Rural University
of Rio de Janeiro in Brazil (UFRRJ)—GPS 22 460 300 S and 43 410 3600 W. A voucher specimen
classified by Dr. Marcelo de Souza (RBRv90000025) is deposited in the Botanical Garden
of the Botany Department of UFRRJ. The plant material was dried at 45 C for 72 h in an
oven with controlled air circulation. The dried guajava leaves were crushed using a knife
mill and then submitted to extraction by decoction in water at 80 C at 10% w/v for 15
Microorganisms 2023, 11, 1784 3 of 12

min. After the material was filtered, concentrated, frozen, and lyophilized to obtain the
final yield.
Identification of secondary metabolites present in the extracts of leaves from gua-
java was performed on a Shimadzu liquid chromatograph LC-20AT with a diode-array
wavelength SPD-M20A detector using a Merck reverse-phase column C-18 (5 µm, 150 mm,
4 mm). The mobile phase consisted of water adjusted to pH 3.0 with 0.1% formic acid
(eluent A) and methanol (eluent B). The samples were run for 45 min at 1 mL/min, and
absorbance was monitored between 200 and 600 nm. The gradient used in chromatography
analysis was 0 min = 10%B, 5 min = 10%B, 10 min = 30%B, 15 min = 30%B, 20 min = 70%B,
27 min = 90%B, 35 min = 90%B, 37 min = 10%B, and 45 min = 10%B. Psidium guajava extract
(2.5 mg) was dissolved in deionized water (2 mL), ultrasonicated (30 min), and filtered on a
Millipore filter. An amount of 20 µL was injected for analysis. Tannic acid (0.5 mg/mL),
gallic acid (0.5 mg/mL), protocatechuic acid (0.5 mg/mL), chlorogenic acid (0.5 mg/mL),
syringic acid (0.5 mg/mL), ellagic acid (0.5 mg/mL), rosmarinic acid (0.5 mg/mL), vitexin
(0.5 mg/mL), quercetin (0.5 mg/mL), and rutin (0.5 mg/mL) were used as standards.
Phenolics and flavonoids were quantified by the area of the extract.

2.3. Total Phenolic and Tannin Content

The total phenolic content was determined by the Folin-Ciocalteu (FC) method using a
standard curve of an alcoholic solution of gallic acid at five different concentrations (0, 1, 3,
5, 7, and 9 µg/mL). To these dilutions, 5.0 mL of water and 2.5 mL of diluted Folin-Ciocalteu
reagent (1:10 in distilled water) were added. From a methanolic solution of the extract
(1 mg/mL), a 0.5 mL aliquot was placed in an amber flask with a cap, and 5.0 mL of water
and 2.5 mL of FC solution (10% v/v) were added, stirred quickly, and left to stand for
5 min. A 2.0 mL aliquot of a 4% sodium carbonate solution was added, and the mixture was
allowed to stand for 2 h, after which the optical density was measured at 765 nm against
a blank. The total phenolic contents were calculated on the basis of the calibration curve
of gallic acid and expressed as gallic acid equivalents (GAE) in milligrams per g of dry
extract [25].
The total tannin content in the aqueous guava leaf extract was determined using the
aluminum chloride colorimetric method, adapted from Quettier-Deleu et al. [26]. For this,
aliquots of 0.5 mL, in triplicate, of each sample of the hydroalcoholic extract were added to
an equal volume of a methanolic solution of 5% aluminum chloride (AlCl3 ). After standing
for 15 min, the absorbance was read at 420 nm. Total tannin content was determined using
a standard tannic acid curve at concentrations of 0, 5, 10, 20, 30, 40, and 50 µg/mL. Samples
were independently analyzed in triplicate, and the total tannin content was expressed as
mg tannic acid (TAE) equivalents per g of dry extract.

2.4. Antioxidant Activity

The antioxidant activity of the extract was evaluated by the DPPH free radical assay,
which involves the measurement of the decrease in absorbance of the extract (using a UV
spectrophotometer). The stabilization of free radicals is seen by the change in color from
dark violet to light violet [27]. Aqueous solutions were composed of 50% methanol and
70% acetone, 0.06 mM methanolic solution, and methanolic dilutions of the extract (sample)
(1, 2, 5, 7, and 10 µg/mL). The reaction mixture was produced by adding 0.1 mL of sample
to 3.9 mL of 0.06 mM DPPH solution. The absorbance reading was measured at 515 nm
in pentaplicate. The DPPH calibration curve (1, 2, 3, 4, and 5 µM) used methanol as a
blank to create the first linear equation. At the same wavelength, another reading was
taken after 55 min of reaction, and the EC50 curve (sample and DPPH), in triplicate, was
plotted to create the second linear equation. The elimination activity value, EC50 , expresses
the amount of extract needed to decrease the absorbance of DPPH by 50%, which was
determined graphically by plotting the linear regression of the absorbance against the
extract concentration. This experiment followed the method described by Rufino et al. [27]
and De Menezes Epifanio et al. [25].
Microorganisms 2023, 11, 1784 4 of 12

2.5. Antimicrobial Activity of the Extract

The antimicrobial activity of the extract was analyzed by the disk diffusion and
broth microdilution methods to determine the initial concentration of extract capable of
enlarging the inhibition halo and to determine the minimum inhibitory concentration
(MIC) and minimum bactericidal concentration (MBC), respectively. For the antimicrobial
evaluation, dilutions of the extract (in water) were tested against the strains Escherichia coli
ESBL (CMY-2), Escherichia coli (ATCC 25922), Staphylococcus aureus MRSA (ATCC 43300),
Staphylococcus aureus (ATCC 23923), Staphylococcus pseudintermedius (B19), Staphylococcus
pseudintermedius (B20), and Streptococcus spp. beta-hemolytic, described in Table 1.

Table 1. Description of the strains tested against the guava leaf extract.

Strains Code Resistance Pattern

Betalactamic resistance (Penicillins and cephalosporins
E. coli (ESBL) CMY-2
up to third generation)
E.coli ATCC 25922 Wild-type E. coli
S. aureus (MRSA) ATCC 43300 Methicillin resistant
S.aureus ATCC 23923 Wild-Type S. aureus
S.pseudintermedius B19 Resistant to Sulfametoxazole + trimetoprim
S.pseudintermedius B20 No expressed resistance to any tested antimicrobials
Streptococcus spp. beta-hemolytic - No expressed resistance to any tested antimicrobials
Legend: ESBL—Extended Spectrum Betalactamase; MRSA—Methicillin resistant Staphylococcus aureus.

2.5.1. Disk Diffusion Test

The strains were incubated for 24 h on brain heart infusion (BHI) agar at 35 C. Filter
paper discs (0.38 cm2 ) impregnated with different concentrations of the extract (2.3, 4.6, 6.8,
9.1, and 11.4 mg/mL) were used, placed on Mueller-Hinton (MH) agar plates inoculated
with the strains previously adjusted to 0.5 on the McFarland scale, and incubated for
24 h [28]. The tests were carried out in triplicate, and the impregnation volume was 6 µL,
using water and 10% methanol as blanks.

2.5.2. Broth Microdilution Test

The broth microdilution test was carried out using a 96-well microtiter plate to evaluate
the minimum inhibitory concentration (MIC). The strains were previously inoculated in
MH broth at 35 C and adjusted to 0.5 on the McFarland scale. Concentrations of P. guajava
aqueous extract were set based on the results of the disk diffusion test. Imipenem was used
as a negative control. For positive control, only strains without extract were used, and the
blank was broth with extract. UV spectroscopy was used at 655 nm [29] to measure the
turbidity, and the MIC was determined by the concentration of the extract of the sample
that did not show turbidity when compared to the blank.

2.5.3. Minimum Bactericidal Concentration Determination

The minimum bactericidal concentration (MBC) was evaluated with aliquots from
the sample wells (strain and extract), where no turbidity was observed, inoculated in
BHI culture medium at 36 C, and read after 24 h. The MBC was defined as the lowest
concentration of the extract capable of preventing the growth (meaning death) of the
inoculum [28].

2.6. Statistical Analysis

In the statistical analysis, the averages of the inhibition halos and turbidity were
compared. Initially, the data were assessed for normal distribution using the Shapiro-
Wilk test. The data with a normal distribution (parametric) were submitted to analysis of
variance (ANOVA) and the Tukey test. The data that did not present a normal distribution
(nonparametric) were evaluated using the Kruskal–Wallis test. The level of significance
 GraphPad Prism 7 statistical program (GraphPad Software Inc., San Diego, CA, USA).

3. Results and Discussion

3.1. Chemical Analysis of Plant Extract
Microorganisms 2023, 11, 1784 5 of 12
HPLC analysis led to the identification of 12 peaks with different λmax. benzoic acids,
phenolic acids, and flavonoids. The chemical analyses are shown in Figure 1 and Table 2.
The compoundconsidered
1 (Rt = 9.711 min) was identified as tannic acid, followed by gallic acid
in all tests was 95% (p  0.05). Statistical analyses were performed using the
(2, Rt = 15.076 min).GraphPad
The compound 3 (Rt = 17.838
Prism 7 statistical program min) showsSoftware
(GraphPad λmax 242Inc.,
and 294
San nm,CA,
Diego, corre-
USA).
sponding to protocatechuic acid. Rutin, quercetin, hesperidin, and cinnamic derivatives
3. Results and Discussion
were identified as major compounds (4–7), and compound 8 was not identified. Other
3.1. Chemical Analysis of Plant Extract
compounds such as syringic, ellagic, and rosmarinic acids can be found in the guava ex-
HPLC analysis led to the identification of 12 peaks with different max . benzoic acids,
tract. phenolic acids, and flavonoids. The chemical analyses are shown in Figure 1 and Table 2.

Figure 1. HPLC analyzes of Psidium guajava crude extract using co-injection of standards.
Figure 1. HPLC analyzes of Psidium
(1—tannic acid, guajava
2—galliccrude extract using co-injection
acid, 3—protocatechuic of standards.
acid, 4—rutin, (1—tannic
5—hesperidin, 6—quercetin,
acid, 2—gallic acid, 3—protocatechuic acid, 4—rutin, 5—hesperidin, 6—quercetin, 7—cinnamic de- acid,
7—cinnamic derivative, 8—not identified, 9—syringic acid, 10—cinnamic derivative, 11—ellagic
12—rosmarinic
rivative, 8—not identified, acid).acid, 10—cinnamic derivative, 11—ellagic acid, 12—rosma-
9—syringic
rinic acid).
Table 2. Chemical composition identified from Psidium guajava extract.

Retention Time Concentration

Peak max (nm) Name
(Rt ) (%)
1 9.711 1.079 278 Tannic acid
2 15.068 3.699 270 Gallic acid
3 17.838 1.316 242, 294 Protocatechuic acid
4 18.865 16.549 257, 358 Rutin
5 19.579 14.131 270, 323 Hesperidin
6 23.807 19.305 258, 378 Quercetin
7 24.243 13.965 240, 280 Cinnamic derivative
8 24.794 0.444 - n.i.
9 25.785 0.836 267 Syringic acid
10 27.481 10.071 235, 322 Cinnamic derivative
11 28.071 5.553 252, 352, 369 Ellagic acid
12 28.517 7.221 220, 329 Rosmarinic acid
n.i.—not identified.

The compound 1 (Rt = 9.711 min) was identified as tannic acid, followed by gallic
acid (2, Rt = 15.076 min). The compound 3 (Rt = 17.838 min) shows max 242 and 294 nm,
corresponding to protocatechuic acid. Rutin, quercetin, hesperidin, and cinnamic deriva-
tives were identified as major compounds (4–7), and compound 8 was not identified. Other
compounds such as syringic, ellagic, and rosmarinic acids can be found in the guava extract.
P. guajava is a species rich in phenolic chemical compounds in its leaves, such as
flavonoids (+)-psiflavanone A, ( )-psiflavanone A, (+)-psiflavanone B, ( )-psiflavanone
Microorganisms 2023, 11, 1784 6 of 12

B [8], The yield of the extract was 4.3% (w/w), similar to reports in the literature. The total phe-
nolic content of the aqueous extract, quantified from the standard curve (y = 0.1401x + 0.0047;
R2 = 0.9971), was 17.02 ± 6.87 mg/g of dry extract. The total tannin content of the aqueous
extract was quantified using a standard curve equation: y = 0.0018x + 0.0328; R2 = 0.9741,
affording 14.09 ± 1.20 mg of tannic acid equivalents/g of dry extract. A comparison of our
results with those reported in the literature indicated significant differences in the guava leaf
extracts’ phenolic compounds. However, the content of tannins found in our study suggests a
species with high quality that produces tannins.
Nantitanon et al. [30] investigated the influence of certain factors on the yield, antioxi-
dant activity (AA), and total phenolic content (TPC) of guava leaf extract, as well as the
effects of pretreatment of leaf samples prior to extraction, the extraction method, and the
leaf age. Folin–Ciocalteu was used to determine the TPC, and the values reported ranged
from 80.28 mg ± 1.58 to 136.02 mg ± 5.55 EAG/g of extract from the different extrac-
tion methods (maceration with/without stirring, ultrasonication, and Soxhlet extraction).
Haida et al. [31] found phenolic contents ranging from 158.29 to 165.07 EAG mg/g dry
extract of white guava and from 160.61 to 175.10 EAG mg/g for red guava. Camarena-Tello
et al. [32] reported the total phenolic compounds of P. guajava in different solvents. The ace-
tone fraction had the greatest quantity of phenolic compounds of both varieties, followed by
the aqueous fraction, and finally the chloroform fraction (71.69 ± 3.69–374.63 ± 29.92 mg
GAE/g extract).
Some authors have reported that the pretreatment process of the guava leaves be-
fore extraction and the extraction method are important factors that affect the amount
of active principles and antioxidant activity of the extracts. The maturity stage of the
guava leaves and the extraction solvent are other important factors that result in different
phenolic contents.
The genus Psidium belongs to the Myrtaceae family and comprises important botanical
species, especially the guava tree (Psidium guajava L.). The health benefits of the phenolic
composition of guava fruits and leaves have been studied due to their chemical composition
and pharmacological properties, such as antifungal and antimicrobial activities.
From the absorbances obtained from the different dilutions of P. guajava extract, it was
possible to calculate the total antioxidant activity (EC50 ) as the absorbance equivalent to
50% of the DPPH concentration by the standard curves of DPPH (y = 0.1415x 0.005). So,
for the aqueous extract from the leaves of P. guajava, we obtained an EC50 of 140.0 µg/mL
(y = 0.044x + 0.7214), considered to be the moderate antioxidant capacity of the extract,
corroborating the results described by Iha et al. [24] (EC50 = 150.0 µg/mL) and Camarena-
Tello et al. [32] (EC50 = 269.78 µg/mL).

3.2. Antimicrobial Extract Evaluation

In the disk diffusion tests, inhibition halos were found for Gram-positive bacteria,
while for Gram-negative bacteria (Escherichia coli, sensitive and resistant), there was no
inhibition at the tested concentrations (Table 3 and Figure 2), corroborating the results
reported by Araújo et al. [33] and Kidaha et al. [12], where no inhibition was found for
E. coli. Gram-negative bacteria are generally more resistant to antimicrobials than Gram-
positive bacteria because they have an additional outer membrane that can protect them
from antimicrobial compounds [34].
Microorganisms 2023, 11, 1784 7 of 12

Table 3. Inhibition halos (mm) obtained in the disk diffusion test (mean ± sd) of different con-
centrations of Psidium guajava aqueous extract against the strains Staphylococcus aureus (resistant),
Staphylococcus aureus (wild-type), Staphylococcus pseudintermedius (resistant), Staphylococcus pseudinter-
medius (wild-type), Streptococcus beta-hemolytic (wild-type) (n = 3).

Concentration (mg/mL) 2.3 4.6 6.8 9.1 11.4

Staphylococcus aureus (resistant) 14.0 ± 2.00 a 15.3 ± 2.31 a 16.7 ± 1.15 a 17.3 ± 2.31 a 18.0 ± 3.46 a
Staphylococcus aureus (wild-type) 9.3 ± 1.15 a 11.3 ± 1.15 a 14.0 ± 0.00 b 14.7 ± 1.15 b 16.0 ± 0.00 b
Staphylococcus pseudintermedius (resistant) 13.3 ± 1.15 a 15.3 ± 1.15 b 15.3 ± 1.15 b 16.0 ± 0.00 b 18.0 ± 0.00 b
Staphylococcus pseudintermedius (wild-type) 13.3 ± 1.15 a 15.3 ± 1.15 b 15.3 ± 1.15 b 16.0 ± 0.00 b 18.0 ± 0.00 c
Streptococcus beta-hemolytic (wild-type) 14.7 ± 1.15 a 18.7 ± 1.53 a 18.0 ± 4.0 a 19.3 ± 3.06 a 21.0 ± 2.0 a
Microorganisms 2023, 10, x FOR PEER REVIEW 8 of 13
Equal letters do not differ significantly between the concentration (p > 0.05). Different letters differ significantly
between the concentrations (p < 0.05).

Figure2.2.Inhibition
Figure Inhibitionhalos
halospresented
presentedby byPsidium guajavaaqueous
Psidiumguajava aqueousextract
extractininthe
theconcentration
concentrationrange
rangeof
2.3
of to
2.311.4 mg/mL
to 11.4 mg/mL(1–5) and and
(1–5) blank (B) against
blank the different
(B) against strains:
the different (A) Staphylococcus
strains: (A) Staphylococcus (resis-
aureusaureus
tant); (B) Staphylococcus
(resistant); (B) Staphylococcus (wild-type);
aureusaureus (C) Staphylococcus
(wild-type); pseudintermedius
(C) Staphylococcus (resistant);
pseudintermedius (D) Staphy-
(resistant); (D)
lococcus pseudintermedius
Staphylococcus (wild-type);
pseudintermedius (E) Escherichia
(wild-type); coli (wild-type);
(E) Escherichia (F) Escherichia
coli (wild-type); coli (resistant);
(F) Escherichia coli
(resistant);
(G) (G) Streptococcus
Streptococcus beta-hemolytic
beta-hemolytic (wild-type).(wild-type).

At
At the
the highest
highest concentration
concentration evaluated
evaluated (11.4 mg/mL),
mg/mL), we weobserved
observedinhibition
inhibitionhalo halo
values
values of 18.0
18.0±± 3.46
3.46 mm mmfor for resistant
resistant 16.0 ±16.0
S. aureus,
S. aureus, 0 mm±for 0 mm for sensitive
sensitive S. aureus,
S. aureus, 17.3 mm
17.3 mm
± 1.15 1.15 for resistant
for±resistant S. pseudintermedius, 18 mm ±18
S. pseudintermedius, mmsensitive
0 for ± 0 for sensitive S. pseudintermedius
S. pseudintermedius and 21
and
mm21 mmfor
± 2.00 ± 2.00 for sensitive
sensitive Streptococcus
Streptococcus beta-hemolytic.
beta-hemolytic. Inhibition
Inhibition halos were halos were dose-
dose-dependent
dependent for the gram-positive
for the gram-positive bacteria
bacteria tested. tested.
The same hadThe same been
already had already
reportedbeen reported
by Bolzan by
et al.
Bolzan et al. [17], who observed dose-dependence in the inhibition halos obtained against
[17], who observed dose-dependence in the inhibition halos obtained against the ethanolic
the ethanolic
extract of P. extract
guajava of P. guajava
leaves leaves in
in a clinical a clinical
trial carriedtrial
outcarried out with microorganisms
with microorganisms from the
from
caninetheoral
canine oral microbiota.
microbiota.
The
Theactivity
activityof ofthe
theaqueous
aqueousextract
extractofofP.P. guajava
guajavaagainst
against S. S.
aureus
aureus through
through thethe
disk dif-
disk
fusion
diffusiontesttest
hashas
already been
already reported
been reported in previous
in previous studies,
studies,butbut
thetheconcentrations
concentrations necessary
neces-
to obtain
sary the inhibition
to obtain halo were
the inhibition higher,
halo were 50.0 mg/mL
higher, [16] and
50.0 mg/mL [16] 62.5
and mg/mL
62.5 mg/mL [33],[33],
compared
com-
to our results
pared (11.40 mg/mL).
to our results The better
(11.40 mg/mL). antimicrobial
The better activity
antimicrobial foundfound
activity in theinpresent study
the present
may be related to the higher concentration of total phenolics present in the tested extract
study may be related to the higher concentration of total phenolics present in the tested
than thethan
extract values
the reported by Raj et
values reported byal.Raj[16] and
et al. Araújo
[16] et al. [33],
and Araújo et al.since
[33],these
since compounds
these com-
generally exhibit antibacterial
pounds generally activity. activity.
exhibit antibacterial
Based
Based on the theresults
resultsobtained
obtained in in
thethediskdisk diffusion
diffusion test, test, concentrations
concentrations of 6.8 ofand6.8 and
11.40
11.40
mg/mL,mg/mL, respectively,
respectively, were selected
were selected for MIC forand
MIC MBCandevaluations.
MBC evaluations. The broth The broth mi-
microdilu-
crodilution assay demonstrated
tion assay demonstrated that microbial
that microbial growth growth
inhibitioninhibition
occurredoccurred in allatstrains
in all strains both
at both concentrations
concentrations tested (6.8tested (6.8 mg/mL)
and 11.40 and 11.40 mg/mL)
(Figure (Figure 3).
3). Therefore, Therefore,
the MIC was 6.8the mg/mLMIC
was 6.8 mg/mL for all strains, a value similar to that reported by Metwally et al. [35]
for all strains, a value similar to that reported by Metwally et al. [35] (5.25 mg/mL) for S.
aureus (sensitive), higher than that reported by Sanches et al. [20] (500 µg/mL) for S. aureus
(sensitive), and lower than the values reported by Araújo et al. [33] (62.5 mg/mL) for S.
aureus (sensitive and resistant) and Ratnakara et al. [13] (30–40 mg/mL) for S. aureus (sen-
sitive).
 Microorganisms 2023, 11, 1784 8 of 12

(5.25 mg/mL) for S. aureus (sensitive), higher than that reported by Sanches et al. [20]
(500
Microorganisms 2023, 10, x FOR PEER µg/mL) for S. aureus (sensitive), and lower than the values reported by Araújo et al. [33]
REVIEW
(62.5 mg/mL) for S. aureus (sensitive and resistant) and Ratnakara et al. [13] (30–40 mg/mL)
for S. aureus (sensitive).

Figure 3. Minimum Inhibition Concentration determination: broth microdilution test of Psidium

Figure 3. Minimum Inhibition Concentration determination: broth microdilution test o
guajava aqueous extract in the concentrations of 6.8 mg/mL (1–3 and 9) and 11.4 mg/mL (4–6 and
guajava aqueous extract in the concentrations of 6.8 mg/mL (1–3 and 9) and 11.4 mg/mL (4–
10) against the different strains: (A) Staphylococcus aureus (resistant); (B) Staphylococcus aureus (wild-
against
type); the different
(C) Staphylococcus strains: (A) (resistant);
pseudintermedius Staphylococcus aureus (resistant);
(D) Staphylococcus (B) Staphylococcus
pseudintermedius (wild-type); aure
type);
(E) (C) Staphylococcus
Streptococcus beta-hemolytic pseudintermedius (resistant);
(wild-type). Negative (D) Staphylococcus
control imipenem (8). F, G andpseudintermedius
H were not (w
(E) Streptococcus beta-hemolytic
utilized in the experiment. (wild-type). Negative control imipenem (8). F, G and H
utilized in the experiment.
The results of MBC determination indicated that for S. pseudintermedius (resistant), the
interaction between extract and microorganism prevented growth at a concentration of
The results of MBC determination indicated that for S. pseudintermedius (r
6.8 mg/mL. However, for the other strains, the MBC value was greater than 11.4 mg/mL
the interaction
(Figure 4). between extract and microorganism prevented growth at a conce
Most
of 6.8 previous
mg/mL. works highlight
However, for thethe antimicrobial
other activity
strains, the MBC of value
the aqueous extract of
was greater than 11.
guajava only
P.(Figure 4). against sensitive strains of Gram-positive bacteria, finding little or no effec-
tiveness against resistant strains. Likewise, the large variation in inhibitory concentration
between the studies suggests that it may be related to the proportion of total phenolic
and tannins present in each extract, which in turn may be a consequence of the extraction
method or the exposure of plant material to different environmental conditions, as reported
by Raj et al. [16] and Biswas et al. [9]. Moorthy et al. [36] suggest that tannins would be
primarily responsible for antimicrobial activity.
The influence of the extraction method, the part of the plant, and the extracting solvent
on the antimicrobial activity was demonstrated in a study carried out by Sanches et al. [20].
The aqueous extract of leaves, roots, and stem bark of P. guajava showed different potencies
against S. aureus (MICs = 500, 125, and 250 µg/mL, respectively) and practically inactiv-
ity against Gram-negative bacteria such as E. coli and P. aeruginosa (MICs > 1000 µg/mL).
Ethanol: water extracts showed greater antimicrobial activity when compared to aque-
ous extracts.
 (E) Streptococcus beta-hemolytic (wild-type). Negative control imipenem (8). F, G and H were not
utilized in the experiment.

The results of MBC determination indicated that for S. pseudintermedius (resistant),

Microorganisms 2023, 11, 1784
the interaction between extract and microorganism prevented growth at a concentration
9 of 12
of 6.8 mg/mL. However, for the other strains, the MBC value was greater than 11.4 mg/mL
(Figure 4).

Figure
Figure4. Minimumbactericidal
4. Minimum bactericidalconcentration
concentration determination:
determination: aliquot-seeded
aliquot-seeded BHI BHI plates
plates after after
broth
broth microdilution
microdilution test. test. (A) Staphylococcus
(A) Staphylococcus aureus
aureus (resistant);
(resistant); (B) Staphylococcus
(B) Staphylococcus aureus
aureus (wild-type);
(wild-type); (C)
(C) Staphylococcus
Staphylococcus pseudintermedius
pseudintermedius (resistant);
(resistant); (D) (D) Staphylococcus
Staphylococcus pseudintermedius
pseudintermedius (wild-type);
(wild-type); (E)
Streptococcus
(E) beta-hemolytic
Streptococcus beta-hemolytic (wild-type).
(wild-type).

In comparative
Most previous studies of aqueous,
works highlight theethanolic, and methanolic
antimicrobial extracts,
activity of the aqueousthe extract
methanolic
of P.
extract
guajavahadonlygreater
againstactivity against
sensitive strainsgram-positive and bacteria,
of Gram-positive gram-negative
findingbacteria
little orthan the
no effec-
others,
tivenessasagainst
reported by Biswas
resistant [9], Dhiman
strains. Likewise, [10],
theRatnakaran [13],inand
large variation Nair [14].
inhibitory concentration
Furthermore, for biological purposes, it is important to analyze the association between
extracts from different plant species. The combination of extracts of P. guajava and Cannabis
sativa, or Trema orientalis, was able to promote inhibition against MRSA. The same activity
was observed when analyzing clinical samples of the same strain [18]. Another study
demonstrated the synergy between aqueous extracts of Psidium sp., Mongifera sp., and
Mentha sp. against Streptococcus sp. [23].
Likewise, it is also important to evaluate the potential activity of the different major
chemical components as well as search for additive or synergistic activity from the combi-
nation of major components in the same plant extract [37]. The flavonoids luteolin, morin,
naringin, rutin, and quercetin were effective in inhibiting the growth of gram-positive and
gram-negative bacteria of different genera, including S. aureus and E. coli [38]. Previously,
Amin et al. [39] indicated combined inhibitory activity against MRSA by the flavonoids
morin, rutin, and quercetin. In addition, it demonstrated additive efficacy when these
isolates were associated with existing commercial antibiotics.
It is important to highlight the significant evidence of the activity of the aqueous
extract of P. guajava against resistant strains of zoonotic importance, such as S. aureus and
S. pseudintermedius, observed by us, since there is a lack of studies reporting the activity of
P. guajava extracts against resistant strains.
As previously described by Gelatti [40] and Hughes [1], certain strains have become
less sensitive to different antimicrobials, thus indicating the importance of searching for
new forms of treatment to overcome resistance mechanisms. More specifically, Donkor [41]
and Jaradat [42] highlighted the problem of MRSA, with a high incidence of transmission
associated with the oral mucosa, as also previously reported by Turner [43], who described
the threat this scenario poses to public health. In addition to its extensive resistance to
antibiotics, MRSA is a cause of serious concern due to the high prevalence of its infections
and association with persistent outbreaks, which have serious economic implications [41].
Due to the problematic bacterial resistance in Brazil and around the world, a deeper
study of the potential of plants and their metabolites as therapeutic alternatives capable
of combating multiresistant microorganisms becomes significant and necessary. Mainly
Microorganisms 2023, 11, 1784 10 of 12

because it is a more inexpensive and accessible treatment, especially considering that Brazil
is a country of outstanding biodiversity.

4. Conclusions
The present study demonstrates the antimicrobial activity of the aqueous extract
of P. guajava leaves against sensitive and resistant Gram-positive bacteria. New findings
suggest an innovative profile regarding therapeutic resources that can be adopted to combat
resistant microbial strains. According to the estimate of the World Health Organization for
2050, it is important to continue the studies of this species and its compounds in the search
for a new innovative antibacterial drug.

Author Contributions: Conceptualization—Y.P.C. and D.S.d.A.C. Methodology—G.A.P., T.M.e.S.

and R.E.d.A.M. Writing—original draft preparation, G.A.P., A.J.B.F., S.d.M.d.O.C., A.B.R.d.S. and
T.C.d.C.P. Writing—review and editing, Y.P.C., D.S.d.A.C., M.O. and T.M.K.; Project administration,
Y.P.C., S.d.M.d.O.C. and D.S.d.A.C. All authors have read and agreed to the published version of
the manuscript.
Funding: This study was supported by Fundação de Apoio à Pesquisa Tecnológica da Universidade
Federal Rural do Rio de Janeiro (FAPUR-01), Fundação de Apoio a Pesquisa do Estado do Rio de
Janeiro (FAPERJ—E-26/201.277/2022), and Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior—Brasil (CAPES)—Finance Code 001.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.

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