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Tiotsop et al.

Investigational Medicinal Chemistry and Pharmacology 2023 6(2):80 Page 1 of 10

Investigational
Medicinal Chemistry & Pharmacology

Research Article Open Access

Psidium guajava (Myrtaceae) re-sensitizes multidrug-resistant


Pseudomonas aeruginosa over-expressing MexAB-OprM efflux
pumps to commonly prescribed antibiotics

Romario S. Tiotsop1, Armelle T. Mbaveng1*, Armel Jackson Seukep2**, Valaire Y. Matieta 1,


Paul Nayim1, Brice E. N. Wamba1, Michel-Gael F. Guefack1, Victor Kuete1***

Abstract
Background: Pseudomonas aeruginosa is a critical-class priority pathogen showing high resistance to almost all classes of conventional
antibiotics. As a result, discovering new drugs capable of combating Pseudomonas infections becomes critical. The current study looked at the
antibacterial and antibiotic-resistance reversal properties of the leaf (PGL) and bark (PGB) methanol extracts of Psidium guajava (Myrtaceae), a
popular food plant, towards multidrug-resistant (MDR) P. aeruginosa over-expressing active efflux.
Methods: The activities were assessed using a 96-well plate microdilution technique, with iodonitrotetrazolium chloride (INT) to detect living
bacteria. The action of herbals was further carried out on Pseudomonas kinetic growth, cell membrane, and H+-ATPase mediated proton pumps,
using standards.
Results: PGL and PGB produced inhibitory effects on all the fourteen MDR strains and clinical isolates of P. aeruginosa, with the minimum
inhibitory concentrations (MIC) recorded ranging from 64 to 2048 μg/mL. PGB was shown to be more effective, having MICs ≤ 512 μg/mL on
100% of the MDR pathogens tested. It also exhibited the lowest MICs (best activity) of 64 μg/mL against three MDR clinical isolates P124, P57,
and P29, with activity higher than that of the reference medication (chloramphenicol). PGL and PGB were shown to have significant antibiotic-
resistance reversal action when combined with conventional antibiotics, with PGB enhancing the efficacy of all standard drugs employed. PGB
was shown to lengthen the latent phase of kinetic growth, also, it significantly inhibited the H+-ATPase-mediated proton pump and altered cell
membrane integrity, at MIC and 2×MIC.
Conclusion: The current investigation provides justification for considering P. guajava extracts, alone or in combination with antibiotics, as
potential treatments for MDR P. aeruginosa infections.

Keywords: Antibiotics; efflux pumps; food plants; multidrug-resistant bacteria; Pseudomonas aeruginosa; Psidium guajava.

*Correspondence: *Tel.: +237 676542386; E-mail address: armbatsa@yahoo.fr; ORCID: https://orcid.org/0000-0003-4178-4967 (Armelle T. Mbaveng); ** Tel.: +237
690606559; E-mail address: seukep.armel@ubuea.cm; ORCID: https://orcid.org/ 0000-0002-5501-3111 (Armel Jackson Seukep); *** Tel.: +237 677355927; E-mail
address: kuetevictor@yahoo.fr; ORCID: http://orcid.org/0000-0002-1070-1236 (Victor Kuete)

1
Department of Biochemistry, Faculty of Science, University of Dschang, P.O Box 67, Dschang, Cameroon; 2Department of Biomedical Sciences, Faculty of Health
Sciences, University of Buea, P.O Box 63, Buea, Cameroon

Other authors:
romariotiotsop@gmail.com (Romario S. Tiotsop); yvmatieta@yahoo.com (Valaire Y. Matieta); nayimpaul@yahoo.fr (Paul Nayim); wambaelvis@yahoo.fr (Brice E. N.
Wamba); michelfofack@gmail.com (Michel-Gael F. Guefack)

Citation on this article: Tiotsop RS, Mbaveng AT, Seukep AJ, Matieta VY, Nayim P, Wamba BEN, Guefack MF, Kuete V. Psidium guajava (Myrtaceae) re-sensitizes
multidrug-resistant Pseudomonas aeruginosa over-expressing MexAB-OprM efflux pumps to commonly prescribed antibiotics. Investigational Medicinal Chemistry
and Pharmacology (2023) 6(2):80; Doi: https://dx.doi.org/10.31183/imcp.2023.00080

Invest. Med. Chem. Pharmacol. (IMCP) ISSN: 2617-0019 (Print)/ 2617-0027 (Online); © The Author(s). 2023 Open Access This article is
available at https://investchempharma.com/
Tiotsop et al. Investigational Medicinal Chemistry and Pharmacology 2023 6(2):80 Page 2 of 10

Background biopharmaceutical potential, we tested the antibacterial and


antibiotic-resistance reversal activities of its leaf and bark extracts
against a critical class pathogen, P. aeruginosa, overexpressing
Antimicrobial resistance (AMR) is a serious hazard to public health,
active efflux pumps. The potential modes of action of extracts,
driving the ongoing search for novel and effective medicines. The
tested alone, have also been investigated.
World Health Organization (WHO) has identified Pseudomonas
aeruginosa as a critical priority pathogen to guide and support
research and development of novel antibacterial drugs [1, 2]. P. Methods
aeruginosa infections are most common in hospitalized patients or
those with compromised immune systems. It is especially harmful Plant material and extraction procedure
to those suffering from chronic pulmonary disorders. Some types of
multidrug-resistant (MDR) P. aeruginosa are resistant to nearly all The leaves (PGL) and barks (PGB) of Psidium guajava were
antibiotics (pan-drug resistant), including carbapenems [3, 4]. P. harvested in Dschang (West Region, Cameroon, 5.4459° N,
aeruginosa has evolved many resistance tactics, including active 10.0472° E) in October 2020. The plant was identified and
efflux, which appears to be one of the key processes contributing authenticated at the Cameroon National Herbarium (HNC) by
to MDR [5, 6]. Several measures are being made to combat MDR comparing it to a sample registered under the reference number
in bacteria. Recent decades have seen a surge in interest in 2884/SRFCam. The plant parts harvested were cleaned and dried
medicinal plants and their derived phytoconstituents, including food in the shade before being ground. The resulting powders were
plants, where research has revealed immense potential in the macerated in methanol 95° (1:3 w/v) for 48 hours. The mixture was
battle against resistant pathogens, either alone or in combination then agitated 3 to 4 times each day to increase the yield, followed
with conventional treatments [7-18]. The scholarly literature on the by filtration through Whatman paper N°1. The filtrate was
utilization of food plants for their many health advantages and evaporated under a vacuum using a rotary evaporator (BÜCHI R-
possible therapeutic uses has lately bloomed in pharmaceutical 200) at 40 °C. The crude extracts were then recovered in dry and
and nutritional sciences. Health practitioners increasingly sterile vials and dried in an oven at 40 °C for complete solvent
acknowledge that combining drugs and diet may yield the best evaporation. The extraction yield of each extract was determined
results in the battle against infectious diseases. Because of the relative to the mass of dry plant powder, which was 13.51% and
existence of pharmacologically active chemicals, the preventive 14.66% for PGL and PGB, respectively. The extracts were then
advantages of food plants are being researched for possible placed in a freezer at 4°C for future experiments.
application as innovative therapeutic therapies [19-22].
Furthermore, various studies have shown that food plants have a Chemicals
remarkable ability to combat MDR in bacteria [23-31]. Although
various food plant extracts have been investigated for their Dimethylsulfoxide (DMSO) was used to solubilize plant extracts
antibacterial effects against MDR microbes, few explicitly indicate and antibiotics, and para-iodonitrotetrazolium chloride (INT) was
antipseudomonal activity. employed to detect living bacteria during susceptibility testing.
The plant of interest in this study is Psidium guajava Phenylalanine arginine beta naphthylamide was used as an efflux
(Myrtaceae), a common food plant. The plant is a well-known tropic pump inhibitor. Antibiotics belonging to different classes were
tree that is often planted for its fruit. This herb is used to treat a included, comprising beta-lactams (ampicillin, penicillin), cyclins
variety of disorders including dysentery, gastroenteritis, diarrhea, (tetracycline and doxycycline), phenicols (chloramphenicol),
malaria, diabetes, hypertension, cavities, rheumatism, and pain. P. cephalosporins (ceftriaxone, cefixime), fluoroquinolones
guajava leaf extract is used to treat coughs, diarrhea, oral ulcers, (levofloxacin, ciprofloxacin), carbapenem (imipenem),
and wounds with swollen gums [32, 33]. P. guajava has a high aminopenicillin (augmentin), and polypeptide (polymixin B). All
concentration of organic and inorganic substances, such as chemicals were provided by Sigma-Aldrich (Germany).
secondary metabolites, especially polyphenols and terpenes [32].
The bioactive constituents extracted from P. guajava include Microorganisms and culture media
phytochemicals and essential oils. The main phytochemicals are
gallic acid, casuariin, catechin, chlorogenic acid, rutin, vanillic acid, A multidrug-resistant (MDR) clinical strain PA124 overexpressing
quercetin, syringic acid, kaempferol, apigenin, cinnamic acid, MexAB-OprM and a reference strain PA01, as well as 12 clinical
luteolin, quercetin-3-O-α-L-arabinopyranoside, morin, ellagic acid, isolates of P. aeruginosa expressing MDR characteristics, were
guaijaverin, pedunculoside, asiastic acid, ursolic acid, oleanolic employed. In a recent study, Tiotsop et al. [34] provided the
acid, methyl gallate and epicatechin, whereas the major essential bacterial characteristics of the examined P. aeruginosa strains and
oils include limonene, trans-caryophyllene, α-humulene, γ- laboratory collection. Cetrimide agar (Titan Biotech Ltd, Rajasthan,
muurolene, selinene, caryophyllene oxide, bisabolol, India) was used to first confirm the P. aeruginosa strains and
isocaryophyllene, δ-cadinene, α-copaene, α-cedrene, β-eudesmol, isolates. Microorganisms were then grown in Mueller Hinton Agar
α-pinene, β-pinene, β-myrcene, linalool, α-terpineol and eucalyptol (MHA, Liofilchem, Italy) before any susceptibility testing. The
[33]. These chemicals are responsible for P. guajava's diverse Mueller Hinton broth (MHB, Titan Biotech LTD, India) served in the
bioactivity. The most active antioxidant discovered in P. guajava is microdilution test for the determination of minimal inhibitory
quercetin. Furthermore, various in vitro and in vivo investigations concentration (MIC), minimal bactericidal concentration (MBC),
have shown that P. guajava has pharmacological activity such as antibiotic-resistance modulating factor (AMF), and the antibacterial
antidiarrheal, hepatoprotective, anticancer, anti-inflammatory, mechanisms of test samples. All culture media were prepared
antiestrogenic, and antibacterial properties, which support its according to the manufacturer's instructions.
traditional usage [32]. According to the findings, P. guajava is a
strong herbal product with remarkable pharmacological qualities Preparation of working solutions
that should be used in the development of pharmaceutical agents
and functional foods. To further demonstrate P. guajava's
Tiotsop et al. Investigational Medicinal Chemistry and Pharmacology 2023 6(2):80 Page 3 of 10

The stock solution for PGL and PGB was prepared at 8192 µg/mL,
whereas the antibiotics solution was made based on their Antibacterial activity of P. guajava extracts in the presence of
respective potency. Therefore, chloramphenicol was prepared at PAßN
1024 µg/mL, ampicillin and penicillin at 512 µg/mL, ceftriaxone,
cefixime, imipenem, augmentin at 256 µg/mL, levofloxacin and The procedure was carried out in the presence of an efflux pump
ciprofloxacin at 64 µg/mL, and the remaining antibiotics at 32 inhibitor (PAßN), as reported by Kuete et al. [41]. The MIC was
µg/mL. Plant extracts and antibiotics were dissolved in DMSO (For determined in the same way as described above except that 50 µL
innocuity, the final DMSO content was less than 2%), then the of the inhibitor solution was then introduced, followed by a 50 µL of
mixture was homogenized, and the final volume was obtained by bacterial inoculum (4 × 106 CFU/mL) for a final volume of 200
adding MHB. µL/well. A total of four controls were provided: two negative
controls, one formed by wells consisting of inoculum, MHB, and the
Inoculum preparation inhibitor, the other consisting of inoculum, MHB, and 10% DMSO,
the neutral control consisting of wells containing only MHB and the
Bacterial suspensions were prepared from 18-hour-old bacterial positive control consisting of a reference antibiotic
culture colonies. A bacterial colony was dissolved in 10 mL sterile (chloramphenicol). The work was done in triplicate and repeated
distilled water, comparing the turbidity of the bacterial suspension three times.
obtained after homogenization with that of the corresponding
McFarland 0.5 (1.5x108 CFU/mL). Then, a volume of prepared Action of P. guajava bark extract on kinetic of bacterial growth
bacterial suspension was collected and introduced into the MHB to
obtain a corresponding solution (2x106 CFU/mL). The effect of PGB on the kinetic of bacterial growth was examined
on P. aeruginosa PA124 using optical densities (OD) at 600 nm
Rapid colorimetric INT assay for MIC and MBC determination [42]. Briefly, the bacterial suspensions were produced at a
concentration of 108 CFU/mL in the corresponding flasks. They
The minimum inhibitory concentration (MIC) and bactericidal were subsequently treated with PGB at 0.5×MIC, MIC, and 2×MIC.
concentration (MBC) of PGL and PGB were determined by The vials were incubated at 37 °C under an orbital shaker (REMI)
microdilution using a rapid colorimetric INT test as reported by Eloff at 200 rpm. The positive and negative controls consisted of the
[35] and thoroughly described in our previous studies [34, 36-38]. vials containing ciprofloxacin at MIC as well as the vial containing
The negative control consisted of MHB and inoculum, whereas the MHB and the bacterial suspension, respectively. A volume of 500
positive control consisted of chloramphenicol, a reference μL was then collected at different time intervals (0 h, 1 h, 2 h, 4 h, 6
antibiotic, with a concentration range of 2 to 256 µg/mL. Bacterial h, 10 h, 12 h, 14 h, 16 h, 18 h, and 20 h) and the OD was read
growth was revealed using INT (0.2%) and the MICs were defined using a spectrophotometer (Thermo Scientific, Langenselbold,
as the minimum concentrations for which no bacterial growth Germany) at 600 nm. Each test was repeated 3 times. OD = f(t)
(absence of pink coloration in the wells) was observed. For the was plotted using the OD values in Microsoft Office Excel 2016.
MBCs, a volume of 50 µL of the content of the wells corresponding Action of P. guajava bark extract on H+-ATPase-mediated proton
to concentrations greater than or equal to the MIC was taken and pump
introduced into the wells of microplates containing 150 µL of MHB. The ability of PGB to inhibit the H+-ATPase-mediated proton pump
Afterward, the plates were incubated for 48 h at 37°C followed by of P. aeruginosa PA124 was performed by controlling the
the addition of INT. The concentrations of the wells in which no acidification of the bacterial growth medium as described
pink staining was present were taken as bactericidal and the lowest previously [43] with minimal modifications. Briefly, 100 mL of
of these was noted as MBC [39]. The work was done in triplicate bacterial suspension was cultured in MHB for 18 hours at 37 °C.
and repeated three times. The resulting culture was centrifuged (3000 rpm) for 10 minutes at
4 °C. Afterward, the pellet was washed twice in distilled water, then
Antibiotic-resistance reversal potential of P. guajava extracts in in 50 mM KCl, and re-suspended in 50 mL of KCl (50 mM). After
association with standard antibiotics that, the cell solution was incubated at 4 °C for 18 hours to induce
glucose deprivation. To 4 mL of the cell medium, 0.5 mL of the
P. guajava extracts at sub-inhibitory concentrations were combined PGB at 0.5×MIC, MIC, and 2×MIC, as well as an antibiotic
with antibiotics. The assay was performed in the same manner as (ciprofloxacin) were added. The pH was adjusted to 6.4 with 1 M
previously described for the MIC test, with the exception that HCl or 0.1 M NaOH. After 10 min of pre-incubation at 37 °C,
successive dilutions were performed in the presence of the acidification of the medium was started by adding 0.5 mL of 20%
antibiotics, followed by the addition of 50 µL of extract at sub- glucose. The pH was measured every 10 min for 1 h using a pH
inhibitory concentrations in each well. Afterward, the volume was meter (Thermo Scientific, USA). The extract was replaced with
completed to 200 µL by adding 50 µL of bacterial inoculum (4 × 106 DMSO to create the negative control. pH=f(t) was plotted using the
CFU/mL). The activity of PGL and PGB at sub-inhibitory reported pH values in Microsoft Office Excel 2016.
concentrations (MIC/2, MIC/4, MIC/8, MIC/16) was first evaluated
by a preliminary test performed on the most resistant strain Action of P. guajava bark extract on the bacterial cell membrane
(PA124) which allowed to select MIC/2 and MIC/4 for further
testing on extended MDR P. aeruginosa strains. The antibiotic- The intracellular content of P. aeruginosa PA124 after exposure to
resistance modulating factor (AMF) was calculated from the different concentrations of PGB (0.5×MIC, MIC, and 2×MIC) and
following formula: polymixin B was performed by measuring the absorbance of the
supernatant at 260 nm as previously reported [44, 45], with few
AMF = MICantibiotic alone/MICantibiotic in combination changes. Briefly, young colonies of bacteria were harvested from a
fresh culture on MHA medium and a suspension of 1 x 106 CFU
The biological significance of antibiotic-resistance modifying effects was prepared. Bacterial cells were then treated with PGB at
was set at AMF ≥ 2 [40]. 0.5×MIC, MIC, and 2×MIC, then incubated for 12 hours at 37°C.
Tiotsop et al. Investigational Medicinal Chemistry and Pharmacology 2023 6(2):80 Page 4 of 10

The preparations were centrifuged, and the absorbance of the After treatment of P. aeruginosa PA124 with PGB at 0.5×MIC, MIC,
supernatant was measured at 260 nm for control and treated cells, and 2×MIC, a significant increase of OD at 260 nm was observed,
using a spectrophotometer (Thermo Scientific, Langenselbold, as compared with the control (Figure 2). The highest OD was
Germany). As a negative control, the tube containing the inoculum recorded at MIC and 2×MIC.
and DMSO was employed. Each assay was performed in triplicate.
H+-ATPase-mediated proton pumps

PGB induced a significant inhibition of PA124 H+-ATPase-mediated


Results proton pumps, from 20 min of exposure up to 60 min, as evidenced
by the reduction of acidity of the solution (Figure 3). Better effects
Antibacterial activity were obtained at MIC and 2×MIC.

P. guajava extracts (PGL and PGB) were examined for their


antibacterial activity on a panel of fourteen P. aeruginosa strains
Discussion
and clinical isolates expressing MDR phenotypes, through
determination of MIC and MBC. The bacteriostatic or bactericidal African medicinal plants have shown promising activity against
effect of test extracts was ascertained following the determination various types of multidrug-resistance phenotypes of bacterial and
of the MBC/MIC ratio. Table 1 summarizes the findings. PGL and parasitic infections as well as cancer [46-59]. Their role as source
PGB produced inhibitory effects against all tested bacteria, with of antibacterial phytomedicines and potential pharmaceuticals have
MIC recorded ranging from 64 to 1024 μg/mL. PGB displayed also been demonstrated [25, 60-64]. The antibacterial activity of
better efficacy than PGL, with MIC ≤ 512 μg/mL against all the the leaf (PGL) and bark (PGB) methanol extracts of P. guajava was
studied MDR P. aeruginosa. More so, PGB exhibited the lowest investigated towards MDR strains and clinical isolates of P.
MICs (best activity) of 64 μg/mL towards the clinical isolates P124, aeruginosa, overexpressing efflux pumps. The testing included
P57, and P29, with activity higher than that of the reference drug determining the activity of herbals alone, in the presence of an
(chloramphenicol). PGL showed MBC/MIC ≤ 4 in 13 of 14 studied efflux pump inhibitor (EPI), and association with standard
microorganisms, compared to 11 of 14 with PGB. MBC of the antibiotics. Also, the possible modes of action of the most active
reference antibiotic exceeded the initial test concentration of 256 extract were examined. P. aeruginosa has been identified as a
μg/mL in all cases (Table 1). critical priority class pathogen for guiding antibiotic research and
development [1]. This, therefore, emphasizes the relevance of the
Influence of PAβN on the efficacy of P. guajava leaf and bark present study. P. aeruginosa is highly resistant to conventional
extracts treatments leading to high prevalence of hard-to-treat infections.
Interestingly, PGL and PGB displayed remarkable
As presented in Table 2, the MICs of PGL and PGB, as well as the antipseudomonal potential, acting on all tested Pseudomonas
reference antibiotic (chloramphenicol) significantly improved in the strains with MIC recorded ranging from 64 to 1024 μg/mL (Table
presence of the efflux pump inhibitor, PAβN, with enhancement 1). The MIC range suggests the best to moderate activity of test
factor ranging from 2 to >128. herbals [65]. PGB produced better inhibitory potential than PGL.
Indeed, most of the MIC obtained with PGB were ≤ 512 μg/mL
Antibiotic-resistance modifying activity of P. guajava extracts against all the studied MDR P. aeruginosa. Furthermore, PGB
depicted the lowest MIC (best activity) of 64 μg/mL towards three
The ability of P. guajava extracts to re-sensitize MDR P. clinical MDR isolates (P124, P57, and P29), which activity was
aeruginosa strain and clinical isolates to standard antibiotics was found higher than that of the reference drug used
assessed. The results from the preliminary investigations showed a (chloramphenicol). This suggests that PGB may contain potent
better potentiation of antibiotics at MIC/2 and MIC/4, which were antipseudomonal phytoconstituents that work synergistically in the
then selected for testing on further MDR isolates (Table 3). The herbal complex. Further research using bio-guided separation of
results depicted outstanding antibiotic-resistance breaker potential these phytoconstituents will be beneficial in identifying the bioactive
of PGB, which significantly improved the activity of all standard components responsible for the observed actions. The genetic
antibiotics against all studied MDR Pseudomonas. Similar effects difference which influences the synthesis of secondary metabolites
were obtained with PGL in combination with ceftriaxone, imipenem, in plant parts could explain the variation in the potency of PGL and
and augmentin at sub-inhibitory concentrations of MIC/2 and PGB [66]. PGL and PGB showed MBC/MIC ratio ≤ 4 in most
MIC/4. cases, suggesting bactericidal effects [67]. The high antimicrobial
activity of various parts of P. guajava has been reported in the
Antibacterial mechanisms of P. guajava bark extract literature [32, 68]. However, a few research have been published
on its antipseudomonal activity involving MDR strains. As a result,
Kinetic growth the current study adds essential data to the field on the
antipseudomonal properties of P. guajava leaf and bark methanol
A significant and concentration-dependent reduction of bacterial extracts. Aqueous and ethanol extracts have been shown to have
growth was recorded over 20 hours of exposure of P. aeruginosa limited antimicrobial activity whereas methanol extracts show great
PA124 to PGB (Figure 1). More so, a prolonged latent phase was efficiency [69], as shown in the current investigation. As a result, it
observed, up to 2 hours for PGB and 4 hours for the reference drug is possible to infer that methanol should be considered the solvent
ciprofloxacin. of choice for P. guajava antimicrobial research. The
antipseudomonal activity of PGL and PGB was enhanced in the
Cell membrane presence of PAβN (Table 2). This validates efflux activity as the
primary mechanism in studied MDR P. aeruginosa. It was reported
that resistance to antibiotics in P. aeruginosa is mediated through
Tiotsop et al. Investigational Medicinal Chemistry and Pharmacology 2023 6(2):80 Page 5 of 10

various efflux pumps [70]. The strains tested in the present study PA124 over 20 hours of exposure at 0.5×MIC, MIC, and 2×MIC; an
are characterized as over-expressing MexAB-OprM efflux pumps. extended latent phase was also recorded (Figure 2). This
Indeed, MexAB-OprM pumps are responsible for resistance to demonstrates PGB's ability to suppress Pseudomonas growth. The
various classes of antibiotics such as β-lactams, β-lactam constituents of PGB may act by preventing the bacteria from
inhibitors, fluoroquinolones, tetracyclines, tigecycline, novobiocin, effectively using the nutrients of the milieu, probably by inhibiting
thiolactomycin, sulfonamides, macrolides, aminoglycosides, etc some key digestive or metabolic enzymes produced by bacteria. A
[70]. Thus, the addition of an EPI could be considered an significant increase of OD at 260 nm was observed when PA124
interesting approach to improve the antibacterial efficacy of PGL was exposed to PGB, better effects were recorded at MIC and
and PGB in the process of drug development. The ability of PGL 2×MIC. This suggests the disturbance of cell membrane
and PGB to re-sensitize MDR P. aeruginosa to standard antibiotics composition and alteration of cell membrane integrity induced by
(penicillin, ceftriaxone, cefixime, doxycycline, tetracycline, PGB, resulting in cell membrane leakage and release of
chloramphenicol, levofloxacin, ampicillin, imipenem, and intracellular constituents (DNA, RNA). Another noteworthy effect of
augmentin) was assessed; outstanding results were obtained. Both PGB was registered on H+-ATPase-mediated proton pumps of
extracts demonstrated a strong capacity to increase the efficiency PA124, with a significant reduction of the pH as compared with the
of chosen antibiotics against the most resistant P. aeruginosa control; marked effects were obtained at MIC and 2×MIC (Figure
strains or isolates tested (Table 3). PGB improved the activity of all 3). This shows that PGB may be an effective bacterial proton pump
standard antibiotics used against all studied MDR Pseudomonas, inhibitor. P. guajava's antibacterial activity is attributed to its rich
whereas PGL enhanced the activity of ceftriaxone, imipenem, and and diverse bioactive secondary metabolites. The reported
augmentin against all tested strains. The results are relevant and chemical composition includes compounds such as tannins,
could be exploited in rejuvenating the old antibiotics which have phenols, flavonoids, saponins, carbohydrates, alkaloids, sterols,
lost their efficacy due to resistance. P. guajava methanol and and terpenoids [71]. Some major P. guajava constituents
aqueous extracts synergized the efficacy of various common comprising oleanolic acid, guaijavarin, quercetin, and flavonoids
antibiotics against drug-resistant Salmonella bacteria, according to are well-established antibacterial phytochemicals [19, 32, 72]. The
recent research by Ngwanguong et al. [31]. This shows that the flavonoid compounds and their derivatives isolated from P. guajava
phytochemicals contained in P. guajava are potent antibiotic- have been reported to inhibit the growth of different bacteria in
resistance breakers that should be studied further. PGB produced different dilutions [20]. Terpinene and pinene found in the aqueous
a significant decrease in the bacterial count of P. aeruginosa extract of P. guajava’s leaves showed antimicrobial activity [32].

Figure 1. Effect of Psidium guajava (bark) extract on the kinetic growth of P. aeruginosa PA124
Tiotsop et al. Investigational Medicinal Chemistry and Pharmacology 2023 6(2):80 Page 6 of 10

Figure 2. Effect of Psidium guajava (bark) extract on the cell membrane of P. aeruginosa PA124

Figure 3. Effect of Psidium guajava (bark) extract on H+-ATPase-mediated proton pumps of P. aeruginosa PA124

Table 1. Antipseudomonal activity (MIC and MBC) of P. guajava leaf and bark methanol extracts (in µg/mL).

Psidium guajava Chloramphenicol


Bacterial strains Leaves Bark
MIC MBC R MIC MBC R MIC MBC R
PA124 256 512 2 128 256 2 256 >256 nd
PA01 1024 1024 1 256 512 2 256 >256 nd
P081 512 2048 4 512 1024 2 128 >256 nd
P61 256 1024 4 256 512 2 256 >256 nd
P120 256 1024 4 256 512 2 128 >256 nd
P124 256 512 2 64 512 8 128 >256 nd
P57 128 256 2 64 512 8 128 >256 nd
P29 256 2048 4 64 512 8 128 >256 nd
P2 512 1024 2 256 512 2 128 >256 nd
P121 1024 1024 1 256 512 2 128 >256 nd
P97 128 2048 16 256 1024 4 128 >256 nd
P060 256 1024 4 256 1024 4 128 >256 nd
P21 512 1024 2 256 512 2 128 >256 nd
P1 256 1024 4 256 1024 4 128 >256 nd
MIC: minimal inhibitory concentration (in µg/mL); MBC: minimal bactericidal concentration (in µg/mL); R: MBC/MIC ratio; nd: not determined
Tiotsop et al. Investigational Medicinal Chemistry and Pharmacology 2023 6(2):80 Page 7 of 10

Table 2. Influence of PAβN (efflux pump inhibitor) on the efficacy of P. guajava leaf and bark methanol extracts against selected MDR P.
aeruginosa.

Bacterial P. guajava Chloramphenicol


strains Leaves Bark
MIC alone +PAβN R MIC alone +PAβN R MIC alone +PAβN R
PA124 256 <8 >32 128 <8 >16 256 <8 >32
PA01 1024 <8 >128 256 <8 >32 256 128 2
P97 128 >8 >16 256 <8 >32 128 16 8
P21 512 <8 >64 256 <8 >32 128 <8 >16
P081 512 <8 >64 512 <8 >64 128 128 1
P121 1024 <8 >128 256 <8 >32 128 <8 >16
P124 256 128 2 64 <8 >8 128 64 2
P1 256 128 2 256 <8 >32 128 <8 >16
R: Ratio of MIC (+PAβN)/MIC (alone); MIC: minimal inhibitory concentration (in µg/mL); Values in bold: improved activity.

Table 3. Efficacy of antibiotics in combination with Psidium guajava extracts at MIC/2 and MIC/4 towards selected MDR P. aeruginosa.

Bacterial strains MIC(µg/mL) Psidium guajava extracts


Bark Leaves
0 MIC/2 MIC/4 MIC/2 MIC/4
Penicillin
PA01 16 4(4) 4(4) 16(1) 16(1)
P081 64 4(16) 4(16) 8(8) 8(8)
P124 32 4(8) 4(8) 16(2) 16(2)
P21 64 4(16) 4(16) 4(16) 4(16)
P1 64 2(32) 2(32) 8(8) 8(8)
P121 32 8(4) 8(4) 8(4) 8(4)
P97 32 4(8) 4(8) 4(8) 8(4)
Ceftriaxone
PA01 32 2(16) 2(16) 16(2) 16(2)
P081 16 4(4) 4(4) 8(2) 8(2)
P124 64 16(4) 16(4) 32(2) 32(2)
P21 32 4(8) 4(8) 4(8) 4(8)
P1 32 4(4) 16(2) 16(2) 16(2)
P121 32 4(4) 4(4) 4(8) 4(8)
P97 64 8(4) 8(4) 32(2) 32(2)
Cefixime
PA01 256 128(2) 128(2) 128(2) 256(1)
P081 128 4(32) 4(32) 4(32) 4(32)
P124 256 128(2) 128(2) 256(1) 256(1)
P21 64 8(8) 16(4) 8(8) 8(8)
P1 128 32(4) 32(4) 128(1) 128(1)
P121 128 32(4) 32(4) 64(2) 64(2)
P 97 128 32(4) 32(4) 64(2) 64(2)
Doxycycline
PA01 8 4(2) 8(2) 16(0.5) 8(1)
P081 32 2(16) 2(16) 4(8) 2(16)
P124 16 4(4) 4(4) 16(1) 4(4)
P21 32 8(4) 8(4) 8(4) 8(4)
P1 8 0.5(16) 0.5(16) 1(8) 0.5(16)
P121 8 0.5(16) 0.5(16) 0.5(16) 0.5(16)
P97 32 4(8) 4(8) 8(4) 4(8)
Tetracycline
PA01 32 8(4) 8(4) 32(1) 32(1)
P081 8 0.25(32) 0.25(32) 0.5(16) 0.5(16)
P124 32 4(8) 4(8) 16(2) 16(2)
P21 32 16(2) 16(2) 32(2) 32(1)
P1 8 2(4) 4(8) 8(2) 8(2)
P121 8 1(8) 1(8) 2(4) 2(4)
P97 16 4(4) 8(2) 8(2) 8(2)
Tiotsop et al. Investigational Medicinal Chemistry and Pharmacology 2023 6(2):80 Page 8 of 10

Table 3. continued and end.

Bacterial strains Psidium guajava


MIC(µg/mL)
Bark Leaves
0 MIC/2 MIC/4 MIC/2 MIC/4
Chloramphenicol
PA01 128 64(4) 64(4) 128(1) 128(1)
P081 256 32(8) 32(8) 32(8) 32(8)
P124 256 64(4) 64(4) 128(2) 128(2)
P21 256 128(2) 128(2) 256(1) 256(1)
P1 128 16(8) 16(8) 128(1) 128(1)
P121 128 16(8) 16(8) 16(8) 32(8)
P97 256 64(4) 64(4) 128(2) 128(2)
Levofloxacin
PA01 8 2(4) 2(4) 8(1) 8(1)
P081 4 0.5(8) 0.5(8) 0.5(8) 0.5(8)
P124 4 1(4) 1(4) 4(1) 4(1)
P21 4 0.5(8) 0.5(8) 1(4) 1(4)
P1 8 1(8) 1(8) 2(4) 2(4)
P121 8 2(4) 2(4) 2(4) 2(4)
P97 4 1(4) 1(4) 1(4) 1(4)
Ampicillin
PA01 32 8(4) 8(4) 16(2) 16(2)
P081 32 2(16) 2(16) 4(8) 16(2)
P124 128 8(16) 8(16) 32(4) 32(4)
P21 32 4(8) 4(8) 8(4) 8(4)
P1 64 32(2) 32(2) 64(1) 64(1)
P121 128 8(32) 8(32) 16(8) 16(8)
P97 64 8(8) 8(8) 16(4) 16(4)
Imipenem
PA01 64 8(4) 8(4) 8(8) 16(4)
P081 32 2(16) 2(16) 2(16) 2(16)
P124 16 2(8) 2(8) 8(2) 8(2)
P21 16 4(4) 4(4) 8(2) 8(2)
P1 32 4(8) 4(8) 32(4) 32(4)
P121 32 2(16) 2(16) 4(8) 8(4)
P97 32 1(32) 1(32) 4(8) 8(4)
Augmentin
PA01 32 2(16) 2(16) 2(4) 2(16)
P081 32 2(16) 2(16) 2(16) 2(16)
P124 16 1(16) 1(16) 4(4) 4(4)
P21 8 2(4) 2(4) 2(4) 2(4)
P1 32 2(16) 2(16) 8(4) 16(2)
P121 32 8(4) 8(4) 16(2) 16(2)
P97 32 0.5(64) 0.5(64) 1(32) 1(32)
MIC: minimal inhibitory concentration (in µg/mL); (): antibiotic-resistance modulating factors (AMF); Values in bold: improved efficacy of antibiotics.
Tiotsop et al. Investigational Medicinal Chemistry and Pharmacology 2023 6(2):80 Page 9 of 10
6. Seukep AJ, Mbuntcha HG, Kuete V, Chu Y, Fan E, Guo M-Q. 2022. What
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7. Kuete V, Tangmouo JG, Penlap Beng V, Ngounou FN, Lontsi D. 2006.
The current study established the noteworthy antipseudomonal Antimicrobial activity of the methanolic extract from the stem bark of
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8. Nguemeving JR, Azebaze AG, Kuete V, Eric Carly NN, Beng VP, Meyer M,
expressing efflux pumps. The bark extract (PGB) was determined Blond A, Bodo B, Nkengfack AE. 2006. Laurentixanthones A and B,
to be the most active. Interestingly, both plant parts displayed antimicrobial xanthones from Vismia laurentii. Phytochemistry. 67(13):1341-
outstanding antibiotic-resistance reversal potential towards MDR P. 1346.
9. Ngameni B, Kuete V, Simo IK, Mbaveng AT, Awoussong PK, Patnam R,
aeruginosa tested, as evidenced by improved efficacy of standard Roy R, Ngadjui BT. 2009. Antibacterial and antifungal activities of the crude
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10. Tekwu EM, Askun T, Kuete V, Nkengfack AE, Nyasse B, Etoa F-X, Beng
pumps. More research is needed to uncover specific VP. 2012. Antibacterial activity of selected Cameroonian dietary spices
antipseudomonal phytochemicals from P. guajava, as well as the ethno-medically used against strains of Mycobacterium tuberculosis. J
combination's mechanisms of action, in addition to in vivo testing Ethnopharmacol. 142(2):374-382.
11. Kuete V, Wiench B, Hegazy ME, Mohamed TA, Fankam AG, Shahat AA,
and toxicity. Efferth T. 2012. Antibacterial activity and cytotoxicity of selected Egyptian
medicinal plants. Planta Med. 78(2):193-199.
12. Kuete V, Tangmouo JG, Marion Meyer JJ, Lall N. 2009. Diospyrone,
Abbreviations crassiflorone and plumbagin: three antimycobacterial and antigonorrhoeal
naphthoquinones from two Diospyros spp. Int J Antimicrob Ag. 34(4):322-
AMF, antibiotic-resistance modulating factor; DMSO, 325.
. Kuete V, Wansi JD, Mbaveng AT, Kana Sop MM, Tadjong AT, Beng VP,
dimethylsulfoxide, HNC, Cameroon national herbarium; INT, para- Etoa FX, Wandji J, Meyer JJM, Lall N. 2008. Antimicrobial activity of the
Iodonitrotetrazolium chloride; MDR, multidrug-resistant; PAβN, methanolic extract and compounds from Teclea afzelii (Rutaceae). S Afr J
phenylalanine arginine beta naphthylamide; MBC, minimal Bot. 74(4):572-576.
14. Komguem J, Meli AL, Manfouo RN, Lontsi D, Ngounou FN, Kuete V,
bactericidal concentrations; MHA, Mueller Hinton Agar; MHB, Kamdem HW, Tane P, Ngadjui BT, Sondengam BL et al. 2005. Xanthones
Mueller Hinton Broth; MIC, minimum inhibitory concentrations. from Garcinia smeathmannii (Oliver) and their antimicrobial activity.
Phytochemistry. 66(14):1713-1717.
15. Voukeng IK, Kuete V, Dzoyem JP, Fankam AG, Noumedem JA, Kuiate JR,
Authors’ Contribution Pages JM. 2012. Antibacterial and antibiotic-potentiation activities of the
methanol extract of some Cameroonian spices against Gram-negative multi-
RST, MYV, PN, BENW, and MGF carried out the study; AJS drug resistant phenotypes. BMC Res Notes. 5:299.
16. Omosa LK, Midiwo JO, Mbaveng AT, Tankeo SB, Seukep JA, Voukeng IK,
analyzed data and wrote the manuscript; ATM and VK supervised Dzotam JK, Isemeki J, Derese S, Omolle RA et al. 2016. Antibacterial
the study; All authors read and approved the final version of the activity and structure-activity relationships of a panel of 48 compounds from
manuscript. kenyan plants against multidrug resistant phenotypes. SpringerPlus. 5:901.
17. Tchinda CF, Voukeng KI, Beng VP, Kuete V. 2016. Antibacterial activities of
the methanol extracts of Albizia adianthifolia, Alchornea laxiflora, Laportea
Acknowledgments ovalifolia and three other Cameroonian plants against multi-drug resistant
Gram-negative bacteria Saudi J Biol Sci. 24:950-955.
18. Voukeng IK, Beng VP, Kuete V. 2016. Antibacterial activity of six medicinal
The authors are grateful to the Cameroon National Herbarium for Cameroonian plants against Gram-positive and Gram-negative multidrug
identifying the plant material. resistant phenotypes. BMC Complement Altern Med. 16(1):388.
19. Mbaveng AT, Zhao Q, Kuete V. 2014. 20 - Harmful and protective effects of
phenolic compounds from african medicinal plants. In: Toxicological Survey
of African Medicinal Plants. edn. Edited by Kuete V: Elsevier: 577-609.
Conflict of interest 20. Rattanachaikunsopon P, Phumkhachorn P: Contents and antibacterial
activity of flavonoids extracted from leaves of Psidium guajava. J Med
Plants Rese 2010, 4(5):393–396.
The authors declare no conflict of interest. 21. Nayim P, Mbaveng AT, Sudhir K, Wamba BEN, Sanjukta M, Kuete V. 2023.
Phytochemistry and pharmacology of Imperata cylindrica: A comprehensive
Article history: review. Invest Med Chem Pharmacol. 6(1):00076.
22. Kuete V. 2023. Ethnopharmacology, phytochemistry and pharmacology of
Received: 27 April 2023 potent antibacterial medicinal plants from Africa. Adv Bot Res. 107: 353-
Received in revised form: 8 June 2023 660.
Accepted: 14 June 2023 23. Djeussi DE, Noumedem JA, Seukep JA, Fankam AG, Voukeng IK, Tankeo
SB, Nkuete AH, Kuete V. 2013. Antibacterial activities of selected edible
Available online: 14 June 2023 plants extracts against multidrug-resistant Gram-negative bacteria. BMC
Complement Altern Med. 13(1):164.
24. Touani FK, Seukep AJ, Djeussi DE, Fankam AG, Noumedem JA, Kuete V.

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