Tiostop Et Al. 2023-InvestChemPharma
Tiostop Et Al. 2023-InvestChemPharma
Tiostop Et Al. 2023-InvestChemPharma
Investigational
Medicinal Chemistry & Pharmacology
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
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
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).
Table 2. Influence of PAβN (efflux pump inhibitor) on the efficacy of P. guajava leaf and bark methanol extracts against selected MDR P.
aeruginosa.
Table 3. Efficacy of antibiotics in combination with Psidium guajava extracts at MIC/2 and MIC/4 towards selected MDR P. aeruginosa.