Antibacterial potential and modes of action of the methanol extracts of Elephantopus mollis Kunth (Asteraceae) against multidrug-resistant Gram-negative bacteria overexpressing efflux pumps

Abstract

Background: Bacterial drug resistance still constitutes a major clinical issue. In the present study, the in vitro antibacterial potential, and modes of action of Elephantopus mollis were investigated.
Methods: The antibacterial activity of methanol extracts of the various parts of E. mollis, their association with an efflux pump inhibitor, phenylalanine-arginine β-naphthylamide (PAβN), and the potentiating effect of several standard antibiotics were determined using the broth microdilution method. The effects of E. mollis leaf extract on H+-proton pump/ATPase function and bacterial growth kinetics were determined using standard methods. Phytochemical screening of the extracts was carried out using standard qualitative methods.
Results: The crude extract (botanicals) from E. mollis leaf and flower had antibacterial activities with a 100% inhibition spectrum against bacterial strains and isolates, and the MIC values ranging from 16 to 256 µg/mL and 64 to 1024 µg/mL respectively. Botanical from the leaf showed excellent activity with a MIC of 16 µg/mL against K. pneumoniae KP55, a MIC of 32 µg/mL against K. pneumoniae (K2), and P. stuartti (NEA16). Botanicals from the leaf inhibited the exponential growth phase and H+-proton pump/ATPases of K. pneumoniae ATCC11296. In the presence of PAβN, the activity of E. mollis extracts was increased on 90% (leaves and flowers) and 63% (roots) of the multidrug-resistant (MDR) bacteria tested. The various extracts of E. mollis potentiated the activities of the antibiotics: doxycycline, levofloxacin, vancomycin, imipenem, ceftriaxone, and ciprofloxacin against at least 70% of bacterial strains and isolates, with factors of increase in activity ranging from 2 to 128. Extracts from all parts of E. mollis contained alkaloids, flavonoids, tannins, and phenols.
Conclusion: The results show that E. mollis is a source of antibacterial phytomedicine that can be used to treat bacterial infections caused by Gram-negative bacteria expressing MDR phenotypes.

Full text

Mapie Tiwa et al. Investigational Medicinal Chemistry and Pharmacology 2024 7(1):86 Page 1 of 10
Investigational
Medicinal Chemistry & Pharmacology
Research Article Open Access
Antibacterial potential and modes of action of the methanol
extracts of Elephantopus mollis Kunth (Asteraceae) against
multidrug-resistant Gram-negative bacteria overexpressing efflux
pumps
Stephanie Mapie Tiwa1, Valaire Y. Matieta1, Ramelle Ngakam1, Gaelle Kengne Fonkou1, Junior
F. Megaptche1, Paul Nayim1, Armelle T. Mbaveng1*, Victor Kuete1**
Abstract
Background: Bacterial drug resistance still constitutes a major clinical issue. In the present study, the in vitro antibacterial potential, and modes
of action of Elephantopus mollis were investigated.
Methods: The antibacterial activity of methanol extracts of the various parts of E. mollis, their association with an efflux pump inhibitor,
phenylalanine-arginine β-naphthylamide (PAβN), and the potentiating effect of several standard antibiotics were determined using the broth
microdilution method. The effects of E. mollis leaf extract on H+-proton pump/ATPase function and bacterial growth kinetics were determined
using standard methods. Phytochemical screening of the extracts was carried out using standard qualitative methods.
Results: The crude extract (botanicals) from E. mollis leaf and flower had antibacterial activities with a 100% inhibition spectrum against
bacterial strains and isolates, and the MIC values ranging from 16 to 256 µg/mL and 64 to 1024 µg/mL respectively. Botanical from the leaf
showed excellent activity with a MIC of 16 µg/mL against K. pneumoniae KP55, a MIC of 32 µg/mL against K. pneumoniae (K2), and P. stuartti
(NEA16). Botanicals from the leaf inhibited the exponential growth phase and H+-proton pump/ATPases of K. pneumoniae ATCC11296. In the
presence of PAβN, the activity of E. mollis extracts was increased on 90% (leaves and flowers) and 63% (roots) of the multidrug-resistant (MDR)
bacteria tested. The various extracts of E. mollis potentiated the activities of the antibiotics: doxycycline, levofloxacin, vancomycin, imipenem,
ceftriaxone, and ciprofloxacin against at least 70% of bacterial strains and isolates, with factors of increase in activity ranging from 2 to 128.
Extracts from all parts of E. mollis contained alkaloids, flavonoids, tannins, and phenols.
Conclusion: The results show that E. mollis is a source of antibacterial phytomedicine that can be used to treat bacterial infections caused by
Gram-negative bacteria expressing MDR phenotypes.
Keywords: Antibiotics; Asteraceae; bacteria; efflux pumps; Elephantopus mollis; multidrug resistance.
Correspondence: *Tel.: +237 676542386; E-mail: armbatsa@yahoo.fr; ORCID: https://orcid.org/0000-0003-4178-4967 (Armelle T. Mbaveng); ** Tel.: +237
677355927; E-mail: kuetevictor@yahoo.fr; ORCID: http://orcid.org/0000-0002-1070-1236 (Victor Kuete)
1Department of Biochemistry, Faculty of Science, University of Dschang, Dschang, Cameroon
Other authors:
E-mail: stetmapie@gmail.com (Stephanie Mapie Tiwa); E-mail: yvmatieta@yahoo.com (Valaire Y. Matieta); E-mail: ramellengakam@gmail.com (Ramelle Ngakam);
E-mail: gaellefonkou15@gmail.com (Gaelle Kengne Fonkou) ; E-mail: megapfabrice@gmail.com (Junior F. Megaptche); E-mail: nayimpaul@yahoo.fr (Paul Nayim).
Citation on this article: Mapie Tiwa S, Matieta VY, Ngakam R, Kengne Fonkou G, Megaptche JF, Nayim P, Mbaveng AT, Kuete V. Antibacterial potential and modes
of action of methanol extracts of Elephantopus mollis Kunth (Asteraceae) against multidrug-resistant Gram-negative bacteria overexpressing efflux pumps.
Investigational Medicinal Chemistry and Pharmacology (2024) 7(1):86; Doi: https://dx.doi.org/10.31183/imcp.2024.00086
Invest. Med. Chem. Pharmacol. (IMCP) ISSN: 2617-0019 (Print)/ 2617-0027 (Online); © The Author(s). 2024 Open Access This article is
available at https://investchempharma.com/

Mapie Tiwa et al. Investigational Medicinal Chemistry and Pharmacology 2024 7(1):86 Page 2 of 10
Background
Bacterial multidrug resistance is the ability of a pathogenic
bacterium to survive at least two antibiotics belonging to different
families, thus leading to an increasing mortality rate and a
considerable economic impact [1]. According to the World Health
Organization (WHO), of the 2.7 million neonatal deaths recorded
each year, 560,000 cases are caused by microbial infections.
However, half of this mortality rate is in developing countries,
particularly in South Asia and sub-Saharan Africa [2]. In 2019, the
death rate due to antimicrobial resistance was estimated at
approximately 4.19 million deaths worldwide while 1.27 million of
these deaths were attributed to infectious diseases due to
multidrug-resistant (MDR) pathogenic bacteria [3]. Several bacteria
have been increasingly implicated in infectious diseases in
humans, specifically, Enterococcus spp, Enterobacter spp,
Klebsiella pneumoniae, Staphylococcus aureus, Acinetobacter
baumanii, Pseudomonas aeruginosa, as well as Escherichia coli
[4]. The inappropriate and abusive use of antibiotics in humans and
animals is the main reason for the occurrence of antibiotic
resistance. The most predominant resistance mechanisms in
bacteria are, among others: enzymatic inactivation, modification of
the target, modification of membrane permeability, formation of
biofilm, and overexpression of efflux pumps. Indeed, efflux pump
systems can identify and expel from the bacterial cell a wide range
of chemically unrelated substances, including antibiotics. In Gram-
negative bacteria, efflux pumps of the resistance nodulation cell
division (RND) type are responsible for resistance to many families
of antibiotics: these are AcrAB-TolC pumps in Enterobacteriaceae
and MexAB-OprM in P. aeruginosa [5, 6].
Good strategies for effectively combating bacterial
resistance and multidrug resistance are based on the search and
development of novel antibacterial molecules from the plant
kingdom [7-11]. Several African medicinal plants and their
phytochemicals previously displayed good efficiency against MDR
Gram-negative bacteria [12-19]. To improve our library of the
antibacterial plant acting in MDR bacteria, the present study
focused on Elephantopus mollis Kunth (Asteraceae), a plant native
to South America [20]. The plant is commonly known as brown
tobacco, false tobacco, and elephant foot. The plant is traditionally
used for the treatment of pathologies such as cough, dysentery,
hepatitis, cancer, and liver infection [21]; it is also used against
fever, wounds, skin conditions, and intestinal disorders [22].
Herein, the antibacterial activity of botanicals from various parts of
the plant was determined in a panel of MDR Gram-negative
bacteria. The modes of action of the botanicals from the botanicals
were also determined.
Methods
Plant material and extraction
The leaves, flowers, and roots-stems of Elephantopus mollis Kunth
were collected in the locality of Fokoué, Menoua Department, West
Region of Cameroon in December 2022. The identification of the
plant was made at the Herbarium Cameroon National (HNC) in
Yaoundé under voucher number 35121/HNC. The different parts of
E. mollis were air-dried and powdered. The powder resulting from
the different parts was macerated in methanol at a ratio of 1/3 (m/v)
for 48 hours. Subsequently, the macerate obtained was filtered
using Whatman filter paper n°1. The filtrate obtained was
concentrated under a vacuum at 65°C. The crude extract obtained
was completely dried in an oven at 40°C to remove the residual
solvent and kept at 4°C until further use.
Chemicals and culture media
para-Iodonitrotetrazolium chloride ≥ 97% (INT) was used as the
bacterial growth indicator. Dimethyl sulfoxide (DMSO) served to
solubilize plant extracts. Eight antibiotics from four families, namely
ampicillin, ceftriaxone, imipenem, tetracycline, doxycycline,
vancomycin, levofloxacin, and ciprofloxacin were used. Five culture
media were used: Mueller Hinton Agar (MHA), for the activation of
bacterial strains and isolates; Mueller Hinton Broth (MHB), used
during microdilution as a nutrient medium for bacteria; Eosin
methylene blue (EMB), specific and differential culture medium to
confirm the purity of bacterial strains and isolates belonging to
species of the genus Escherichia coli and K. pneumoniae;
MacConkey, specific and differential culture medium to confirm the
purity of bacterial strains and isolates belonging to species of the
genus E. coli; and Cetrimide, specific and differential culture
medium to confirm the purity of P. aeruginosa. All chemicals were
purchased from Sigma-Aldrich (St. Quentin Fallavier, France).
Bacterial strains and isolates
Five Gram-negative bacterial species, each including three
bacterial strains or isolates were used in this work. They were
Escherischia coli (ATCC10536, AG102, and AG100), Klebsiella
pneumoniae (ATCC11296, KP55, and K2), Pseudomonas
aeruginosa (PA01 and PA124), Enterobacter aerogenes (EA3,
EA298, and EA27), and Providencia stuartii (ATCC29916, PS2636,
and NEA16). Their bacterial features are shown in Table 1.
Determination of minimal inhibitory (MIC) and bactericidal (MBC)
concentrations
The bacterial inoculum was prepared as previously described [23-
29] in comparison to the turbidity of a standard McFarland 0.5
(1.5x108 CFU/mL). The various plant extracts and the reference
drug (imipenem) were dissolved in DMSO-MHB. Plant extracts
were prepared at 8192 µg/mL, and antibiotics at 1024 µg/mL.
PAβN was prepared at 100 µg/mL. Botanicals were tested alone,
then in the presence of PAβN (EPI). The combination of plant
extracts with EPI was intended to evaluate the function of efflux
pumps in bacterial resistance to botanicals [28, 30-32]. The
minimal inhibitory (MIC) and bactericidal (MBC) concentrations of
botanicals alone were determined using a 96-well broth
microdilution method combined with the rapid INT colorimetric
method [32-34]. The reference drug used was imipenem for
positive control, whereas DMSO 2.5%+MHB and MHB alone were
used as negative controls. MIC was considered the lowest
concentration of plant extract which produced complete inhibition of
bacterial growth (the least concentration for which no color change
is observed) after 18 to 24 hours of incubation at 37°C, whereas
MBC was considered the lowest concentration of a sample that did
not induce a color change with the addition of INT upon 48 h of
additional incubation [35-37]. Each experiment was repeated three
times in triplicate.
Evaluation of the effect of the methanol extract of Elephantopus
mollis leaves on growth kinetics of K. pneumoniae ATCC11296.
To evaluate the effect of the crude extract from the leaf of
Elephantopus mollis on the kinetics of bacterial growth, the optical
densities (OD) were measured following the protocol previously

Mapie Tiwa et al. Investigational Medicinal Chemistry and Pharmacology 2024 7(1):86 Page 3 of 10
described [24]. The P. stuartii ATCC29916 strain was activated
onto MHA at 37°C for 18 h. Subsequently, a few colonies of this
bacterial culture were removed to prepare a suspension with
turbidity corresponding to McFarland 0.5 (1.5×108 CFU/mL). With
MHB, 20 mL of inoculum solution was prepared at a concentration
of 106 CFU/mL. These inocula were treated with the botanicals at
MIC/2, MIC, and 2×MIC, and the whole was incubated with stirring
at a speed of 130 rpm using a magnetic stirrer to allow good
dispersion of these. A positive control contained CIP at MIC while
the negative control was MHB + the bacterial suspension. After
incubation times of 0 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h,
16 h, 18 h, and 20 h, 200 μL of each solution were introduced into
the wells of flat-bottomed microplates and the OD were read at 600
nm. Each test was repeated 3 times.
Evaluation of the effect of E. mollis leaf extract on the H+-ATPases
pumps
The effects of leaf methanol extract were assessed on the kinetic
growth and H+-ATPase-mediated proton pumping of K.
pneumoniae ATCC11296, at 0.5×MIC, MIC, and 2×MIC as earlier
described [29]. The action on kinetic growth consisted of
measuring the absorbance (600 nm) of the bacterial solution
treated with extracts at various concentrations over 20 hours,
whereas the action on H+-ATPase-mediated proton pumping was
done by controlling the acidification of the bacterial growth medium
over 60 min. Elaborated procedures were previously described [38,
39].
Determination of the antibiotic-potentiating effects of the botanicals
The effects of the association of the botanicals with antibiotics were
determined against the MDR bacteria. Extracts were used at the
sub-inhibitory concentrations of MIC/2, MIC/4, MIC/8, and MIC/16
for a preliminary assay on P. aeruginosa PA01, which then allowed
the selection of appropriate sub-inhibitory concentrations of MIC/2
and MIC/4 for further combination testing (Data not shown).
Antibiotic-resistance modulating factor (AMF) was calculated as the
ratio of the MIC of the antibiotic alone versus MIC in combination
with the plant extract. The potentiation effect was considered for
AMF ≥ 2 [40].
Phytochemical screening of E. mollis extracts
Phytochemical screening was done following the standard methods
described for alkaloids, anthocyanins, flavonoids (Shinoda test),
phenols, saponins, tannins, and triterpenes (Liebermann-Burchard
test) [9, 41].
Interpretation of antibacterial data
Several cutoff points are available for the interpretation of the
antibacterial activity of plant products including extracts from edible
plants [7, 42]. According to Kuete [7], the following threshold values
are applied to botanicals: significant activity (MIC <100 µg/mL),
moderate (100 <MIC ≤ 625 µg/mL), and low or negligible (MIC>
625 µg/mL). According to Tamokou et al. [42], the cutoff point for
the antibacterial activity of botanicals from edible plants are as
follows: highly active (MIC below 100 µg/mL), significantly active
(100 ≤ MIC ≤ 512 µg/mL), moderately active (512 < MIC ≤ 2048
µg/mL), low activity (MIC > 2048 µg/mL), and considered not active
(MIC > 10 mg/mL). However, updated and rationally defined cutoff
points of the antibacterial botanicals have been defined,
considering the various bacterial species [43-46]. For
Enterobacteria: outstanding activity (MIC ≤8 µg/mL), excellent
activity (8 < MIC ≤64 µg/mL), very good activity (64 < MIC ≤128
µg/mL), good activity (128 < MIC ≤256 µg/mL), average activity
(256 < MIC ≤512 µg/mL), weak activity (512 < MIC ≤1024 µg/mL),
and not active (MIC values >1024 µg/mL) [43]. For P. aeruginosa:
outstanding activity (MIC ≤ 32 µg/mL), excellent activity (32 < MIC
≤ 128 µg/mL), very good activity (128 < MIC ≤ 256 µg/mL), good
activity (256 < MIC ≤ 512 µg/mL), average activity (512 < MIC ≤
1024 µg/mL), weak activity or not active (MIC values >1024 µg/mL)
[44]. The above appreciation criteria have been used to discuss the
antibacterial activities of samples reported in the present study.
Results
Antibacterial activity of the crude extracts
The antibacterial activity of the botanicals from leaves, flowers, and
roots of E. mollis was evaluated by determining the MICs and
MBCs on a panel of 15 strains and isolates belonging to 5 bacterial
species: P. aeruginosa, K. pneumoniae, E. coli, E. aerogenes, and
P. stuartti. To determine whether the extracts of E. mollis had
bactericidal or bacteriostatic effects, the MMC/MIC ratio was
calculated, and all the results are recorded in Table 2. The different
botanicals displayed MICs varying from 16 to 2048 μg/mL. The
botanical from the leaves had an inhibition spectrum of 100%
against the bacteria tested, with MICs ranging from 16 to 256
µg/mL. It showed excellent activity with a MIC of 16 µg/mL against
K. pneumoniae ATCC11295, a MIC of 32 µg/mL against K.
pneumoniae K2 and P. stuartti NEA16, a MIC of 64 µg/mL against
K. pneumoniae KP55, P. stuartti (ATCC29761 and PS2636), E.
aerogenes (EA27 and EA298) and E. coli AG100. However,
against the other tested enterobacteria, it had good activities.
Against P. aeruginosa PA124, the botanical from the leaf had
excellent activity with a MIC of 128 µg/mL and very good activity
against P. aeruginosa (PA01 and PA121) with a MIC of 256 µg/mL.
The extract from the leaves of E. mollis had a bactericidal effect
against K. pneumoniae ATCC11295, E. aerogenes EA298, and P.
aeruginosa (PA01, PA121, and PA124). The botanical from the
flowers exhibited an inhibition spectrum of 100% against the tested
bacteria with MIC values ranging from 64 to 1024 µg/mL. It showed
excellent activity with a MIC of 64 µg/mL against K. pneumoniae
ATCC11295 and very good activity with a MIC value of 128 µg/mL
against P. stuartti ATCC29761 and E. coli (AG100 and AG102).
However, it had good activities with other Enterobacteria. The
extract of E. mollis flowers showed good activity (256 µg/mL)
against P. aeruginosa PA124 and moderate activity against all
other strains and isolates of P. aeruginosa tested. The extract of E.
mollis flowers had a bacteriostatic effect against P. stuartti
ATCC29761 and E. coli (AG100 and AG102); it was bactericidal
against the other bacterial strains and isolates. The methanol
extract of the roots of E. mollis showed an antibacterial inhibition
spectrum of 86.66% with MIC values ranging from 128 to 2048
µg/mL. In general, the root extract had activities ranging from
moderate to low. Nevertheless, this extract displayed very good
activity against K. pneumoniae ATCC11295 with a MIC value of
128 µg/mL. The extract from the roots of E. mollis showed
bactericidal effects against K. pneumoniae ATCC11295 and P.
stuartti PS2636.

Mapie Tiwa et al. Investigational Medicinal Chemistry and Pharmacology 2024 7(1):86 Page 4 of 10
Effect of methanol extract of E. mollis leaves on the growth kinetics
of K. pneumoniae ATCC11296
The kinetics of the growth of K. pneumoniae ATCC11296 in the
presence of the leaf extract as well as the control drug,
ciprofloxacin was evaluated, and the results are depicted in Figure
1. It was found that the growth curve of K. pneumoniae
ATCC11296 in the absence of extract at MIC/2 presents all the
phases of bacterial growth except the last phase: a latency phase
(0 - 2 h), an exponential phase (2 - 10 h), and a stationary phase
(10 -20 h). The curve in the presence of the extract from the leaves
of E. mollis at the MIC shows a decrease in the exponential phase
ranging from (2 -8 h) and an extension of the stationary phase from
(8 -20 h). In the presence of the extract at 2MIC and ciprofloxacin
at MIC, inhibition of growth in the exponential phase ranges from 2
- 6 h, and a prolongation of the stationary phase lasted from 6 - 20
h.
Effect of E. mollis leaf extract on H+-ATPase pumps of K.
pneumoniae ATCC11296
The ability of E. mollis leaf extract to interfere with the functioning
of the H+-ATPase proton pumps of K. pneumoniae ATCC11296
was assessed by measuring at different times the pH of the
medium containing K. pneumoniae ATCC11296 in the presence of
the leaf extract (Figure 2). At MIC/2 there was a decrease in pH
values of the culture medium, indicating its acidification, from pH
6.4 to pH 4; i.e., a decrease of 2.4. At MIC and 2MIC, less
pronounced acidification of the medium (pH 4.65 and 5.15,
respectively) was observed. This is an indication that the extracts
exert a dose-dependent inhibition of the H+-ATPase proton pumps.
PAβN improves the activity of botanicals from E. mollis
The MICs botanicals alone and in the presence of PAβN are
shown in Table 3. PAβN enhanced the activity of the extracts of E.
mollis with an increase factor ranging from 2- to 64-fold. The
increase was recorded in 90.90% (10/11) of the bacteria tested in
the cases of leaves and flower extracts, and 63.63% (7/11) in the
case of the root extract. The combination of the root extract with
PAβN showed the highest increase in activity of up to 64-fold on P.
aeruginosa (PA01 and PA124). This is an indication that the
constituents of the botanicals are the substrates of bacterial efflux
pumps.
Antibiotic-potentiating effects of the botanicals
Botanicals at MIC/2 and MIC/4 were tested in combination with
antibiotics, and the results are shown in Tables 4 to 6. The
activities of the antibiotics were improved by the extracts on at
least one tested bacterium, with activity increase factors ranging
from 2- to 128-fold. Botanical from the roots of E. mollis potentiated
(at MIC/2 and MIC/4) the activity of doxycycline, vancomycin,
ciprofloxacin, imipenem, and levofloxacin on at least 80% of the
bacteria tested. it potentiated the effects of ceftriaxone on at least
70% of bacteria tested. The root extract potentiated the effects of
tetracycline and ampicillin on at least 60% and 40% of the bacteria
tested, respectively (Table 4). It was found that the methanol
extract of the leaf (at MIC/2 and MIC/4) enhanced the activity of
doxycycline, vancomycin, ciprofloxacin, imipenem, ceftriaxone, and
levofloxacin vis-a-vis at least 80% of bacteria tested. This extract
potentiated tetracycline and ampicillin against at least 60% and
40% of bacteria, respectively (Table 5). The botanical from the
flowers (at MIC/2 and MIC/4) potentiated the activity of
ciprofloxacin, imipenem, ceftriaxone doxycycline, vancomycin, and
levofloxacin vis-a-vis at least 80% of bacteria tested. It potentiated
the effects of tetracycline and ampicillin against at least 70% and
30% of bacteria, respectively (Table 6).
Phytochemical composition of the botanicals
The crude extract from the leaf of E. mollis contained all the
investigated classes of secondary metabolites, namely alkaloids,
anthocyanins, flavonoids, phenols, saponins, tannins, and
triterpenes (Table 7). They were selectively present in roots and
flower extracts.
Discussion
Medicinal plants are an undeniable source of effective and low-
toxic natural substances that can help fight against recalcitrant
human pathologies such as microbial, parasitic, viral infections,
and MDR cancer phenotypes [47-70]. This work constitutes a good
model for the discovery of substances to counteract bacterial
resistance, given the MDR features of many bacteria tested.
According to the established classification scales, the extract from
the leaves of E. mollis showed excellent activity [43] against K.
pneumoniae ATCC11295, K. pneumoniae K2 and KP55, P. stuartti
(NEA16, ATCC29761 and PS2636), E. aerogenes (EA27 and
EA298) and E. coli AG100. It also displayed excellent and very
good activities against aeruginosa PA124 and P. aeruginosa
(PA121 and PA01), respectively. These results are in agreement
with those obtained by Nguyen et al. [71] who highlighted the
significant antibacterial activity of the water decoction of the leaves
of E. mollis against the Enterobacteriaceae E. coli, S. Typhi, and S.
flexneri. Ohana et al. [22] also demonstrated that the hydro-
ethanolic extract of E. mollis leaves has exceptional [43, 44]
antibacterial activity against susceptible strains of E. coli, P.
aeruginosa and K. pneumoniae with a MIC value of 5 µg/mL, thus
confirming the interesting antibacterial activity of the leaves of E.
mollis. A significant shortening of the exponential growth phase of
this bacterium in the presence of the extract of the leaves of E.
mollis was observed. A decrease in the bacterial population at this
phase of bacterial growth could be because the extract from the
leaves of E. mollis denatures the enzymes and proteins, and
inhibits the transport systems of the bacteria, leading to the death
of certain bacteria.
H+-ATPase proton pumps are involved in the regulation
of bacterial cytoplasmic pH and the supply of energy in the form of
ATP to the bacterium. These two elements are necessary for the
growth of bacteria [72]. An increase in the environmental pH in the
presence of an antibacterial substance can lead to the inhibition by
this substance of the H+-ATPase-dependent proton pumps leading
to the death of the bacterium [73]. K. pneumoniae has an optimal
growth pH between 6-8 [74]. According to the results obtained,
there was a considerable decrease in pH at the level in the
negative control and the extract of the leaves of E. mollis at MIC/2;
in the presence of the extract of the leaves of E. mollis at MIC and
2MIC, there was a slowing down of the acidification of the medium
marked, indicating that at these concentrations, the botanical
inhibits the functioning of the proton pumps of K. pneumoniae
ATCC11296. The H+-ATPase proton pumps would be the target of
the action of the botanical from the leaves of E. mollis. MDR Gram-
negative bacteria including Enterobacteriaceae and P. aeruginosa
actively over-express efflux pumps, and consequently are resistant
to several antibiotics. The use of EPI in combination with the

Mapie Tiwa et al. Investigational Medicinal Chemistry and Pharmacology 2024 7(1):86 Page 5 of 10
botanicals tested in this study could be helpful for the antimicrobial
fight against MDR bacteria. The effect of the association of extracts
from the leaves, flowers, and roots of E. mollis and imipenem could
also be useful to fight bacterial drug resistance. These results are
similar to those of Kuete et al. [30] and Youmbi et al. [75] who
showed that the combination of plant extracts with PAβN improved
their activities.
Figure 1. Effect of the methanol extract of Elephantopus mollis leaves on growth kinetics of K. pneumoniae ATCC11296.
Figure 2. Effect of the methanol extract of Elephantopus mollis leaves on H+-proton pumps/ATPases of K. pneumoniae ATCC11296.
Table 1. Features of bacterial strains and isolates used.
Bacterial strains/isolates
Features
References
Escherichia coli
ATCC10536
Reference ATCC strains
[30, 31]
AG102
Wild-type strain of E. coli K-12 overexpressing AcrAB and Mar A pumps
[76, 77]
AG100
Wild-type E. coli K-12 expressing AcrAB efflux pumps
[78, 79]
Klebsiella pneumoniae
ATCC11296
Reference ATCC strains
[30, 31]
KP55
Clinical MDR isolate, Tetr, Ampr, Atmr, Cefr
[80, 81]
K2
Clinical over-expressing MDR AcrA-TolC pumps
Laboratory collection of UNR-MD1,
University of Marseille,France
Pseudomonas aeruginosa
PA01
Reference ATCC strains
[30, 31]
PA124
Clinical over-expressing MDR MexAB-OprM pumps
[78, 82]
P121
Clinical over-expressing MDR MexAB-OprM pumps
Laboratory collection of URMSA,
University of Dschang, Cameroon
Enterobacter aerogenes
EA3
Clinical MDR isolate Chlr, Norr,
[31, 83]
EA298
Clinical MDR isolate Moxr, Cftr, Atmr, Fepr
[5, 6]
EA27
Clinical MDR isolate, Kanr, Ampr, Nalr, Strr, Tetr; expressing the energy-
dependent efflux of norfloxacin and chloramphenicol
[6, 84]
Providencia stuartii
ATCC29916
Reference ATCC strains
[30, 31]
PS2636
Clinical MDR isolate of Providencia stuartii expressing AcrAB-TolC
pumps
[85]
NEA16
Clinical MDR isolate of Providencia stuartii expressing AcrAB-TolC
pumps
[86, 87]
ATCC: American Type Culture Collection; MDR: multidrug-resistant; Ofxar, Kanr, Tetr, Ermr, Ampr, Nalr, Strr, Atmr, Cefr, Cipr, Im/Csr, Chlr, Genr, Nisr, Flxr, Doxr, Cror, TOBr resistance respectively
to: Ofloxacin, kanamycin, tetracycline , erythromycin, ampicillin, nalidixic acid, streptomycin, aztreoname, cefepime, ciprofloxacin, imipenem/cilastatin sodium, chloramphenicol, gentamicin, nisin,
flomoxef, doxycycline, ceftriaxone and Tobramycin AcrAB-TolC, AcrAB and Mar A: efflux pumps.

Mapie Tiwa et al. Investigational Medicinal Chemistry and Pharmacology 2024 7(1):86 Page 6 of 10
Table 2. MICs and MBCs (µg/mL) of extracts from different parts of Elephantopus mollis.
Bacteria
Botanicals and ATB
Roots
Leaves
Flowers
ATB (imipenem)
MIC
MBC
R
MIC
MBC
R
MIC
MBC
R
MIC
MBC
R
K. pneumoniae
K2
512
-
nd
32
256
8
512
-
nd
<4
64
>16
KP55
2048
-
nd
64
2048
32
512
-
nd
<4
64
>16
ATCC11296
128
1024
4
16
64
4
64
256
4
<4
-
nd
P. stuartti
ATCC29761
512
-
nd
64
512
8
128
1024
8
<4
16
>4
NEA16
2048
-
nd
32
512
16
256
1024
4
8
64
8
PS2636
1024
2048
2
64
512
8
256
1024
4
8
64
8
E. aerogenes
EA3
1024
-
nd
128
-
nd
256
-
nd
8
64
8
EA27
1024
-
nd
64
512
8
256
1024
4
8
16
2
EA298
1024
-
nd
64
1024
4
512
-
nd
8
8
1
P. aeruginosa
PAO1
>2048
-
nd
256
1024
4
1024
2048
2
16
64
4
PA121
2048
-
nd
256
512
2
1024
-
nd
16
128
8
PA124
>2048
-
nd
128
256
2
512
1024
2
4
32
8
E. coli
AG100
512
-
nd
64
512
8
128
1024
8
8
64
8
AG102
1024
-
nd
128
1024
8
128
2048
8
16
64
4
ATCC10536
512
-
nd
128
1024
8
512
2048
4
8
64
8
R: MBC/MIC ratio; >2048: or inactive; nd: not determined; MIC: minimal inhibitory concentration; MBC: minimum bactericidal concentration, ATB: Antibiotic.
Table 3. Effects of the combination of Elephantopus mollis extracts with PAβN.
Bacteria
Botanicals and ATB
Roots
Leaves
Flowers
ATB (imipenem)
MIC
alone
MIC with
PAβN
R
MIC
alone
MIC with
PAβN
R
MIC
alone
MIC with
PAβN
R
MIC
alone
MIC with
PAβN
R
E. coli
ATCC10536
512
512
1
128
16
8
512
16
32
8
2
4
AG100
512
512
1
64
32
2
64
16
4
8
˂1/2
16
P. aeruginosa
PA01
>2048
32
64
256
256
1
256
128
2
16
8
2
PA121
2048
32
64
256
16
16
256
16
16
16
˂8
2
PA124
>2048
1024
2
128
˂16
8
512
˂16
32
4
˂1
4
K. pneumoniae
K2
512
64
8
32
16
2
512
128
2
˂4
2
2
KP55
2048
1024
2
64
˂16
4
512
256
2
˂ 4
1/4
16
E. aerogenes
EA3
1024
32
32
128
64
2
256
128
2
8
8
1
E298
1024
1024
1
64
32
2
512
16
32
8
˂1
8
P. stuartti
NEA16
2048
2048
1
32
16
2
256
128
2
8
8
1
PS2636
1024
64
16
64
32
2
256
256
1
8
2
4
R: MIC alone vs MIC with PAβN ratio; MIC alone: Minimal inhibitory Concentration; MIC with PAβN: Minimal inhibitory Concentration in the presence of PAβN; ATB: Antibiotic

Mapie Tiwa et al. Investigational Medicinal Chemistry and Pharmacology 2024 7(1):86 Page 7 of 10
Table 4. Activity of antibiotics combined with the root extract of Elephantopus mollis against bacterial strains and isolates.
ATB
Extract
concen-
tration
MIC of antibiotics in the presence of extract and Antibiotic-resistance modulating factor (AMF)
PSP
(%)
E. coli
E. aerogenes
K. pneumoniae
P. aeruginosa
P. stuartii
AG100
ATC10536
EA298
EA3
KP55
K2
PA124
PA121
PS2636
NEA16
TET
0
2
8
4
1/8
1
1/2
1
8
8
1/8
MIC/2
1/2
8
˂1/16(64)
1/16(2)
˂1/16(16)
˂1/16(8)
˂1/16(16)
8(1)
8(1)
˂1/16(2)
60%
MIC/4
2
8
˂1/16(64)
1/8(1)
˂1/16(16)
˂1/16(8)
˂1/16(16)
8(1)
8(1)
˂1/16(2)
50%
CIP
0
1/4
1/4
1/4
1/4
1/2
1/4
1
1
1/4
1/2
MIC/2
˂1/16(4)
˂1/16(4)
˂1/16(4)
1/16(4)
˂1/16(8)
˂1/16(4)
˂1/16(16)
1/16(16)
1/8(2)
˂1/16(8)
100%
MIC/4
1/8(2)
1/8(2)
˂1/16(4)
1/8(2)
˂1/16(8)
˂1/16(4)
˂1/16(16)
1/2(2)
1/8(2)
˂1/16(8)
100%
IMI
0
8
8
8
8
˂4
˂4
4
16
8
8
MIC/2
4(2)
8(1)
2(4)
˂1/4(32)
˂1/8(32)
1(4)
˂1/2(8)
˂1/2(32)
4(2)
1(8)
90%
MIC/4
4(2)
8(1)
8(1)
2(4)
˂1/8(32)
1(4)
˂1/2(8)
˂1/2(32)
4(2)
8(1)
70%
CEF
0
16
16
8
128
16
256
64
32
8
8
MIC/2
16(1)
8(2)
˂2(4)
˂2(64)
˂2(8)
256(1)
2(32)
2(16)
4(2)
8(1)
70%
MIC/4
32(0,5)
8(2)
4(2)
4(32)
˂2(8)
256(1)
4(16)
˂2(16)
8(1)
8(1)
60%
DOX
0
1
2
2
8
2
4
1/4
2
1
2
MIC/2
1/2(2)
1(2)
1(2)
8(1)
1/16(32)
1/16(64)
˂1/16(4)
˂1/16(32)
1(1)
1(2)
80%
MIC/4
1/2(2)
1/2(4)
1(2)
8(1)
1/16(32)
1/16(64)
˂1/16(4)
˂1/16(32)
1(1)
1(2)
80%
LEV
0
1/4
1/4
1/4
1/2
1/2
1/2
1/2
4
1
1/2
MIC/2
1/4(1)
1/16(4)
˂1/16(4)
1/16(8)
˂1/16(8)
1/2(1)
1/16(8)
1(4)
˂1/16(16)
˂1/16(8)
80%
MIC/4
1/4(1)
1/4(1)
˂1/16(4)
1/16(8)
˂1/16(8)
˂ 1/16(8)
1/16(8)
1(4)
˂1/16(16)
˂1/16(8)
80%
VAN
0
8
64
256
128
256
64
256
64
2
2
MIC/2
4(2)
1(64)
8(32)
64(2)
˂2(128)
8(8)
128(2)
4(16)
˂ 1/2(4)
˂ 1/2(4)
100%
MIC/4
4(2)
1(64)
8(32)
64(2)
˂2(128)
8(8)
128(2)
4(16)
1(2)
˂ 1/2(4)
100%
AMP
0
256
256
256
256
256
256
256
256
256
256
MIC/2
256(1)
256(1)
256(1)
8(32)
˂2(128)
256(1)
256(1)
˂2(128)
256(1)
˂2(128)
40%
MIC/4
256(1)
256(1)
256(1)
8(32)
˂2(128)
256(1)
256(1)
˂2(128)
256(1)
˂2(128)
40%
MIC: minimal inhibitory concentration; (): Antibiotic-resistance modulating factor (AMF); PSP (%): percentage of strain where potentiation effect was observed; ATB: Antibiotics; DOX:
Doxycycline, LEV: Levofloxacin; VAN: Vancomycin; AMP: Ampicillin; TET: Tetracycline; CIP: Ciprofloxacin; IMI: Imipenem; CEF: Ceftriaxone.
Table 5. Activity of antibiotics combined with antibiotics and the leaves extract of Elephantopus mollis against bacterial strains and isolates.
ATB
Extract
concen-
tration
MIC of antibiotics in the presence extract and Antibiotic-resistance modulating factor (AMF)
PSP
(%)
E. coli
E. aerogenes
K. pneumoniae
P. aeruginosa
P. stuartii
AG100
ATCC10536
EA298
EA3
KP55
K2
PA124
PA121
PS2636
NEA16
TET
0
2
8
4
1/8
1
1/2
1
8
8
1/8
MIC/2
2(1)
8(1)
1/8(32)
1/16(2)
˂1/16(16)
˂1/16(8)
˂1/16(16)
8(1)
8(1)
˂1/16(2)
60%
MIC/4
2(1)
8(1)
1/2(8)
1/16(2)
˂1/16(16)
˂1/16(8)
˂1/16(16)
8(1)
8(1)
1/8(1)
50%
CIP
0
1/4
1/4
1/4
1/4
1/2
1/4
1
1
1/4
1/2
MIC/2
1/8(2)
˂1/16(4)
1/4(1)
1/16(4)
˂1/16(8)
˂1/16(4)
˂1/16(16)
1/16(16)
1/8(2)
1/4(2)
90%
MIC/4
1/4(1)
˂1/16(4)
1/8(2)
1/16(4)
˂1/16(8)
˂1/16(4)
˂1/16(16)
1/2(2)
1/8(2)
1/4(2)
90%
IMI
0
8
8
8
8
˂4
˂4
4
16
8
8
MIC/2
8(1)
2(4)
˂1/2(16)
2(8)
2(2)
1(4)
˂1/2(8)
4(4)
1(8)
8(1)
80%
MIC/4
8(1)
2(4)
1(8)
2(4)
2(2)
1(4)
2(2)
4(4)
1(8)
8(1)
80%
CEF
0
16
16
8
128
16
256
64
32
8
8
MIC/2
8(2)
2(8)
4(2)
2(64)
˂2(8)
256(1)
2(32)
˂2(16)
4(2)
8(1)
80%
MIC/4
8(2)
8(2)
8(1)
16(8)
˂2(8)
256(1)
2(32)
8(4)
4(2)
16(0,5)
70%
DOX
0
1
2
2
8
2
4
1/4
2
1
2
MIC/2
1/2(2)
1/8(16)
1(2)
8(1)
1/16(32)
˂1/16(64)
˂1/16(4)
˂1/16(32)
˂1/16(16)
1/8(16)
90%
MIC/4
1(1)
1/4(8)
1(2)
8(1)
1/16(32)
˂1/16(64)
˂1/16(4)
˂1/16(32)
˂1/16(16)
1/8(16)
80%
LEV
0
1/4
1/4
1/4
1/2
1/2
1/2
1/2
4
1
1/2
MIC/2
1/4(1)
1/16(4)
1/8(2)
1/16(8)
˂1/16(8)
˂1/16(8)
1/16(8)
1/16(64)
1/2(2)
1/4(2)
90%
MIC/4
1/4(1)
1/8(2)
1/8(2)
1/4(2)
˂1/16(8)
˂1/16(8)
1/8(4)
1/16(64)
1/2(2)
1/2(1)
80%
VAN
0
8
64
256
128
256
64
256
64
2
2
MIC/2
4(2)
1/2(128)
8(32)
2(64)
˂2(128)
8(8)
128(2)
4(16)
1(2)
˂ 1/2(4)
100%
MIC/4
4(2)
1/2(128)
8(32)
32(4)
˂2(128)
8(8)
256(1)
4(16)
1(2)
1(2)
90%
AMP
0
256
256
256
256
256
256
256
256
256
256
MIC/2
256(1)
256(1)
256(1)
2(128)
˂2(128)
256(1)
256(1)
˂2(128)
256(1)
˂2(128)
40%
MIC/4
256(1)
256(1)
256(1)
256(1)
˂2(128)
256(1)
256(1)
˂2(128)
256(1)
128(2)
30%
MIC: minimal inhibitory concentration; (): Antibiotic-resistance modulating factor (AMF); PSP (%): percentage of strain where potentiation effect was observed; ATB: Antibiotics; DOX:
Doxycycline, LEV: Levofloxacin; VAN: Vancomycin; AMP: Ampicillin; TET: Tetracycline; CIP: Ciprofloxacin; IMI: Imipenem; CEF: Ceftriaxone.

Mapie Tiwa et al. Investigational Medicinal Chemistry and Pharmacology 2024 7(1):86 Page 8 of 10
Table 6. Activity of antibiotics combined with antibiotics and the flower’s extract of Elephantopus mollis against bacterial strains and isolates.
ATB
Extract
concen-
tration
MIC of antibiotics in the presence extract and Antibiotic-resistance modulating factor (AMF)
PSP
(%)
E. coli
E. aerogenes
K. pneumoniae
P. aeruginosa
P. stuartii
AG100
ATCC10536
EA298
EA3
KP55
K2
PA124
PA121
PS2636
NEA16
TET
0
2
8
4
1/8
1
1/2
1
8
8
1/8
MIC/2
1/2(4)
8(1)
˂1/16(64)
1/8(1)
1/2(2)
˂1/16(8)
˂1/16(16)
8(1)
8(1)
˂1/16(2)
60%
MIC/4
1(2)
8(1)
˂1/16(64)
1/16(2)
1/2(2)
˂1/16(8)
˂1/16(16)
8(1)
8(1)
1/16(2)
70%
CIP
0
1/4
1/4
1/4
1/4
1/2
1/4
1
1
1/4
1/2
MIC/2
˂1/16(4)
˂1/16(4)
˂1/16(4)
1/8(2)
˂1/16(8)
˂1/16(4)
˂1/16(16)
1/16(16)
˂1/16(4)
1/4(2)
100%
MIC/4
1/8(2)
˂1/16(4)
˂1/16(4)
1/4(1)
˂1/16(8)
1/8(2)
˂1/16(16)
1/16(16)
1/8(2)
1/4(2)
90%
IMI
0
8
8
8
8
˂4
˂4
4
16
8
8
MIC/2
4(2)
˂ 1(8)
1/2(16)
2(4)
˂1/8(32)
˂ 1(4)
˂1/2(8)
16(1)
2(4)
8(1)
80%
MIC/4
8(1)
1(8)
4(2)
4(2)
2(2)
1/2(8)
2(2)
16(1)
2(4)
8(1)
70%
CEF
0
16
16
8
128
16
256
64
32
8
8
MIC/2
˂2(8)
2(8)
˂2(4)
8(16)
˂2(8)
256(1)
8(8)
8(4)
2(4)
8(1)
80%
MIC/4
16(1)
4(4)
˂2(4)
8(16)
˂2(8)
256(1)
4(16)
8(4)
4(2)
8(1)
70%
DOX
0
1
2
2
8
2
4
1/4
2
1
2
MIC/2
˂1/16(16)
1/16(32)
1(2)
1/4(32)
˂1/16(32)
˂1/16(64)
˂1/16(4)
1/16(32)
˂1/16(16)
1/2(4)
100%
MIC/4
1/8(8)
1/16(32)
1(2)
8(1)
˂1/16(32)
˂1/16(64)
˂1/16(4)
1/16(32)
˂1/16(16)
1/2(4)
90%
LEV
0
1/4
1/4
1/4
1/2
1/2
1/2
1/2
4
1
1/2
MIC/2
1/8(2)
1/16(4)
˂1/16(4)
1/8(4)
˂1/16(8)
1/16(8)
1/16(8)
1/2(8)
˂1/16(16)
1/4(2)
100%
MIC/4
1/4(1)
1/16(4)
˂1/16(4)
1/2(1)
˂1/16(8)
1/16(8)
1/16(8)
1/2(8)
1/8(8)
1/2(1)
70%
VAN
0
8
64
256
128
256
64
256
64
2
2
MIC/2
4(2)
4(16)
8(32)
128(1)
˂2(128)
8(8)
64(4)
4(16)
˂ 1/2(4)
1/2(4)
90%
MIC/4
4(2)
8(8)
8(32)
128(1)
˂2(128)
8(8)
64(4)
4(16)
1(2)
1(2)
90%
AMP
0
256
256
256
256
256
256
256
256
256
256
MIC/2
256(1)
256(1)
256(1)
256(1)
˂2(128)
256(1)
256(1)
˂2(128)
256(1)
˂2(128)
30%
MIC/4
256(1
256(1)
256(1)
256(1)
˂2(128)
256(1
256(1
˂2(128)
256(1)
64(4)
30%
MIC: minimal inhibitory concentration; (): Antibiotic-resistance modulating factor (AMF); PSP (%): percentage of strain where potentiation effect was observed; ATB: Antibiotics; DOX:
Doxycycline, LEV: Levofloxacin; VAN: Vancomycin; AMP: Ampicillin; TET: Tetracycline; CIP: Ciprofloxacin; IMI: Imipenem; CEF: Ceftriaxone.
Table 7. Phytochemical composition of extracts from different parts of Elephantopus mollis.
Phytochemical classes
Botanicals
Roots
Leaves
Flowers
Alkaloids
+
+
+
Polyphenols
+
+
+
Flavonoids
+
+
+
Tannins
+
+
+
Triterpenes
-
+
+
Saponins
-
+
-
Anthocyanins
-
+
-
(+): present; (-): absent
Conclusion
In the present study, the antibacterial potential, and modes of
action of botanicals from Elephantopus mollis against MDR Gram-
negative bacteria were evaluated. It was shown that botanicals
from E. mollis leaf and flower are potent sources of antibacterial
agents against MDR bacteria. The botanical from the leaf of E.
mollis exerts its antibacterial activity at the exponential phase of
bacterial growth, probably through the inhibition of the H+-ATPase
proton pumps. The constituents from the methanol extracts are
potential substrates for bacterial efflux pumps. Botanicals from this
plant have potentiating effects with doxycycline, ciprofloxacin,
levofloxacin, imipenem, ceftriaxone, and vancomycin. Finally, the
methanol extracts of the leaf, flower, and root of E. mollis are
potential sources of effective antibacterial molecules that could be
used alone and in combination with antibiotics or efflux pump
inhibitors to overcome MDR pathogenic bacteria.
Abbreviations
AMF, antibiotic-resistance modulating factor; DMSO,
dimethylsulfoxide, HNC, Cameroon national herbarium; INT, para-
Iodonitrotetrazolium chloride; MDR, multidrug-resistant; MBC,
minimal bactericidal concentrations; MHA, Mueller Hinton Agar;
MHB, Mueller Hinton Broth; MIC, minimal inhibitory concentrations.
Authors’ Contribution
SMT, VYM, RN, GKF, JFM, and PN carried out the study; ATM and
VK supervised the study; All authors read and approved the final
version of the manuscript.
Acknowledgments
The authors are grateful to the Cameroon National Herbarium for
identifying the plant.
Conflict of interest
The authors declare no conflict of interest.
Article history:
Received: 5 June 2023
Received in revised form: 26 July 2023
Accepted: 01 August 2023
Available online: 01 August 2023

Mapie Tiwa et al. Investigational Medicinal Chemistry and Pharmacology 2024 7(1):86 Page 9 of 10
References
1. Chee E, Brown AC. 2020. Biomimetic antimicrobial material strategies for combating
antibiotic resistant bacteria. Biomater Sci. 8(4):1089-1100.
2. WHO.: WHO/UNICEF joint statement. Management of a potentially serious bacterial
infection in young infants aged 0 to 59 days when transfer to a hospital structure is
impossible. Geneva: World Health Organization. License: CC BY-NC-SA 3.0 IGO. .
https://appswhoint/iris/handle/10665/254502 2017, Accessed on May 23, 2023. .
3. Collaborators AR. 2022. Global burden of bacterial antimicrobial resistance in 2019:
a systematic analysis. Lancet. 399(10325):629-655.
4. Fongang H, Mbaveng AT, Kuete V. 2023. Chapter One - Global burden of bacterial
infections and drug resistance. Advances in Botanical Research 106: 1-20.
https://doi.org/10.1016/bs.abr.2022.08.001.
5. Pradel E, Pages JM. 2002. The AcrAB-TolC efflux pump contributes to multidrug
resistance in the nosocomial pathogen Enterobacter aerogenes. Antimicrob Agents
Chemother. 46(8):2640-2643.
6. Ghisalberti D, Masi M, Pages JM, Chevalier J. 2005. Chloramphenicol and
expression of multidrug efflux pump in Enterobacter aerogenes. Biochem Biophys
Res Commun. 328(4):1113-1118.
7. Kuete V. 2010. Potential of Cameroonian plants and derived products against
microbial infections: a review. Planta Med. 76(14):1479-1491.
8. Kuete V, Efferth T. 2010. Cameroonian medicinal plants: pharmacology and derived
natural products. Front Pharmacol. 1:123.
9. Kuete V. 2013. Medicinal Plant Research in Africa In: Pharmacology and Chemistry.
Edited by Kuete V, 1 edn. Oxford: Elsevier.
10. Kuete JRN, Kuete V. 2023. Chapter Three - Harvesting and processing medicinal
plants for antibacterial testing. Advances in Botanical Research 106: 47-60.
https://doi.org/10.1016/bs.abr.2022.08.003.
11. Hashim I, Omosa LK, Nchiozem-Ngnitedem VA, Onyari JM, Maru SM, Guefack
MGF, Mbaveng AT, Kuete V. 2021. Antibacterial activities and phytochemical
screening of crude extracts from Kenyan Macaranga species towards MDR
phenotypes expressing efflux pumps. Pharmacogn Commun. 11(2):119-126.
12. Mbaveng AT, Omosa LK, Bitchagno GTM, Kuete JRN, Nchiozem-Ngnitedem V-A,
Kuete V. 2023. Chapter Eleven - Potential antibacterial pharmaceuticals from the
flora of Africa. Advances in Botanical Research 107: 307-352.
https://doi.org/10.1016/bs.abr.2022.08.021.
13. Ngameni B, Kuete V, Simo IK, Mbaveng AT, Awoussong PK, Patnam R, Roy R,
Ngadjui BT. 2009. Antibacterial and antifungal activities of the crude extract and
compounds from Dorstenia turbinata (Moraceae). S Afr J Bot. 75(2):256-261.
14. Kuete V, Tangmouo JG, Penlap Beng V, Ngounou FN, Lontsi D. 2006. Antimicrobial
activity of the methanolic extract from the stem bark of Tridesmostemon
omphalocarpoides (Sapotaceae). J Ethnopharmacol. 104(1-2):5-11.
15. Tchinda CF, Voukeng KI, Beng VP, Kuete V. 2016. Antibacterial activities of the
methanol extracts of Albizia adianthifolia, Alchornea laxiflora, Laportea ovalifolia and
three other Cameroonian plants against multi-drug resistant Gram-negative bacteria
Saudi J Biol Sci. 24:950-955.
16. Voukeng IK, Beng VP, Kuete V. 2016. Antibacterial activity of six medicinal
Cameroonian plants against Gram-positive and Gram-negative multidrug resistant
phenotypes. BMC Complement Altern Med. 16(1):388.
17. Omosa LK, Midiwo JO, Mbaveng AT, Tankeo SB, Seukep JA, Voukeng IK, Dzotam
JK, Isemeki J, Derese S, Omolle RA, Efferth T, Kuete V. 2016. Antibacterial activity
and structure-activity relationships of a panel of 48 compounds from kenyan plants
against multidrug resistant phenotypes. SpringerPlus. 5:901.
18. Kuete V, Betrandteponno R, Mbaveng AT, Tapondjou LA, Meyer JJ, Barboni L, Lall
N. 2012. Antibacterial activities of the extracts, fractions and compounds from
Dioscorea bulbifera. BMC Complement Altern Med. 12:228.
19. Mbaveng AT, Sandjo LP, Tankeo SB, Ndifor AR, Pantaleon A, Nagdjui BT, Kuete V.
2015. Antibacterial activity of nineteen selected natural products against multi-drug
resistant Gram-negative phenotypes. Springerplus. 4:823.
20. Kabiru A, Por L, Y. 2013. Elephantopus species: traditional uses, pharmacological
actions and chemical composition. Adv Life Sci Technol. 15(0):6-13.
21. Ooi KL, Muhammad TS, Tan ML, Sulaiman SF. 2011. Cytotoxic, apoptotic and anti-
α-glucosidase activities of 3,4-di-O-caffeoyl quinic acid, an antioxidant isolated from
the polyphenolic-rich extract of Elephantopus mollis Kunth. J Ethnopharmacol.
135(3):685-695.
22. Ohana AAJ, Eutrophe K, Doux L, Martin OA, Lazare SS, Nadia AH, Aren A, Mirlene
N, Nga N, Joseph N et al. 2020. Phytochemical screening and in-vitro evaluation of
antimicrobial and antioxidant activities of ethanolic extracts of Elephantopus mollis
Kunth.(Asteraceae). J Pharmacogn Phytochem. 9(1):1711-1715.
23. Nguemeving JR, Azebaze AG, Kuete V, Eric Carly NN, Beng VP, Meyer M, Blond A,
Bodo B, Nkengfack AE. 2006. Laurentixanthones A and B, antimicrobial xanthones
from Vismia laurentii. Phytochemistry. 67(13):1341-1346.
24. Guefack M-GF, Messina NDM, Mbaveng AT, Nayim P, Kuete JRN, Matieta VY, Chi
GF, Ngadjui BT, Kuete V. 2022. Antibacterial and antibiotic-potentiation activities of
the hydro-ethanolic extract and protoberberine alkaloids from the stem bark of
Enantia chlorantha against multidrug-resistant bacteria expressing active efflux
pumps. J Ethnopharmacol. 296:115518.
25. Matieta VY, Kuete V, Mbaveng AT. 2023. Anti-Klebsiella and antibiotic-potentiation
activities of the methanol extracts of seven Cameroonian dietary plants against
multidrug-resistant phenotypes over-expressing AcrAB-TolC efflux pumps. Invest
Med Chem Pharmacol. 6(1):73.
26. Matieta VY, Seukep AJ, Kuete JRN, Megaptche JF, Guefack MGF, Nayim P,
Mbaveng AT, Kuete V. 2023. Unveiling the antibacterial potential and antibiotic-
resistance breaker activity of Syzygium jambos (Myrtaceae) towards critical-class
priority pathogen Klebsiella isolates. Invest Med Chem Pharmacol. 6(2):82.
27. Tiotsop RS, Mbaveng AT, Seukep AJ, Matieta VY, Nayim P, Wamba BEN, Guefack
MF, Kuete V. 2023. Psidium guajava (Myrtaceae) re-sensitizes multidrug-resistant
Pseudomonas aeruginosa over-expressing MexAB-OprM efflux pumps to commonly
prescribed antibiotics. Invest Med Chem Pharmcol. 6(2):80.
28. Manekeng HT, Mbaveng AT, Nguenang GS, Seukep JA, Wamba BEN, Nayim P,
Yinkfu NR, Fankam AG, Kuete V. 2018. Anti-staphylococcal and antibiotic-
potentiating activities of seven Cameroonian edible plants against resistant
phenotypes. Investig Med Chem Pharmacol. 1:7.
29. Ekamgue B, Mbaveng AT, Seukep AJ, Matieta VY, Kuete JRN, Megaptche JF,
Guefack MGF, Nayim P, Kuete V. 2023. Exploring Mangifera indica
(Anacardiaceae) leaf and bark methanol extracts as potential adjuvant therapy in the
management of multidrug-resistant Staphylococcus aureus. Invest Med Chem
Pharmcol. 6(2):84.
30. Kuete V, Alibert-Franco S, Eyong KO, Ngameni B, Folefoc GN, Nguemeving JR,
Tangmouo JG, Fotso GW, Komguem J, Ouahouo BMW et al: Antibacterial activity of
some natural products against bacteria expressing a multidrug -resistant phenotype.
International Journal of Antimicrobial Agents 2011, 37(2):156-161.
31. Kuete V, Ngameni B, Tangmouo JG, Bolla JM, Alibert-Franco S, Ngadjui BT, Pages
JM. 2010. Efflux pumps are involved in the defense of Gram-negative bacteria
against the natural products isobavachalcone and diospyrone. Antimicrob Agents
Chemother. 54(5):1749-1752.
32. Ekamgue B, Mbaveng AT, Kuete V. 2023. Anti-staphylococcal and antibiotic-
potentiating activities of botanicals from nine Cameroonian food plants towards
multidrug-resistant phenotypes. Invest Med Chem Pharmacol. 6(1):75.
33. Eloff JN. 1998. A sensitive and quick microplate method to determine the minimal
inhibitory concentration of plant extracts for bacteria. Planta Med. 64(8):711-713.
34. Moungoue Ngwaneu LS, Mbaveng AT, Nayim P, Wamba BEN, Youmbi LM, Bonsou
IN, Ashu F, Kuete V. 2022. Antibacterial and antibiotic potentiation activity of Coffea
arabica and six other Cameroonian edible plants against multidrug-resistant
phenotypes. Invest Med Chem Pharmacol. 5(2):68.
35. Djeussi DE, Sandjo LP, Noumedem JA, Omosa LK, B TN, Kuete V. 2015.
Antibacterial activities of the methanol extracts and compounds from Erythrina
sigmoidea against Gram-negative multi-drug resistant phenotypes. BMC
Complement Altern Med. 15(1):453.
36. Nayim P, Mbaveng AT, Wamba BEN, Fankam AG, Dzotam JK, Kuete V. 2018.
Antibacterial and antibiotic-potentiating activities of thirteen Cameroonian edible
plants against gram-negative resistant phenotypes. ScientificWorldJournal.
2018:4020294.
37. Tchana ME, Fankam AG, Mbaveng AT, Nkwengoua ET, Seukep JA, Tchouani FK,
Nyassé B, Kuete V. 2014. Activities of selected medicinal plants against multi-drug
resistant Gram-negative bacteria in Cameroon. Afr Health Sci. 14(1):167-172.
38. Seukep AJ, Fan M, Sarker SD, Kuete V, Guo MQ. 2020. Plukenetia
huayllabambana fruits: analysis of bioactive compounds, antibacterial activity and
relative action mechanisms. Plants (Basel). 9(9):doi: 10.3390/plants9091111.
39. Demgne OMF, Mbougnia JFT, Seukep AJ, Mbaveng AT, Tene M, Nayim P, Wamba
BEN, Guefack MGF, Beng VP, Tane P, Kuete V. 2021. Antibacterial
phytocomplexes and compounds from Psychotria sycophylla (Rubiaceae) against
drug-resistant bacteria. Adv Trad Med.10.1007/s13596-13021-00608-13590.
40. Kovač J, Gavarić N, Bucar F, Smole Možina S. 2014. Antimicrobial and resistance
modulatory activity of Alpinia katsumadai seed phenolic extract, essential oil and
post-distillation extract. Food Technol Biotechnol. 52(2):248-254.
41. Harborne J. 1973. Phytochemical methods, London, Chapman Hall Ltd.
42. Tamokou JDD, Mbaveng AT, Kuete V. 2017. Chapter 8 - Antimicrobial Activities of
African Medicinal Spices and Vegetables. In: Medicinal Spices and Vegetables from
Africa. edn.: Academic Press: 207-237.
43. Kuete V. 2023. Chapter Six - Potential of African medicinal plants against
Enterobacteria: Classification of plants antibacterial agents. Advances in Botanical
Research 106: 151-335. https://doi.org/10.1016/bs.abr.2022.1008.1006.
44. Tankeo SB, Kuete V. 2023. Chapter Seven - African plants acting on Pseudomonas
aeruginosa: Cut-off points for the antipseudomonal agents from plants. Advances in
Botanical Research 106: 337-412. https://doi.org/10.1016/bs.abr.2022.08.007.
45. Tchinda CF, Kuete V. 2023. Chapter Nine - Potential of African flora to combat
tuberculosis and drug resistance of Mycobacteria: Rationale classification of
antimycobacterial agents from a natural source. Advances in Botanical Research
106: 523-598. https://doi.org/10.1016/bs.abr.2022.08.009.
46. Wamba BEN, Mbaveng AT, Kuete V. 2023. Chapter Eight - Fighting Gram-positive
bacteria with African medicinal plants: Cut-off values for the classification of the
activity of natural products. In: Advances in Botanical Research 106: 413-522.
https://doi.org/10.1016/bs.abr.2022.08.008.
47. Kuete V, Sandjo LP, Djeussi DE, Zeino M, Kwamou GM, Ngadjui B, Efferth T. 2014.
Cytotoxic flavonoids and isoflavonoids from Erythrina sigmoidea towards multi-
factorial drug resistant cancer cells. Invest New Drugs. 32:1053–1062.
48. Tekwu EM, Askun T, Kuete V, Nkengfack AE, Nyasse B, Etoa FX, Beng VP. 2012.
Antibacterial activity of selected Cameroonian dietary spices ethno-medically used
against strains of Mycobacterium tuberculosis. J Ethnopharmacol. 142(2):374-382.
49. Kuete V, Ngameni B, Mbaveng AT, Ngadjui B, Meyer JJ, Lall N. 2010. Evaluation of
flavonoids from Dorstenia barteri for their antimycobacterial, antigonorrheal and anti-
reverse transcriptase activities. Acta Trop. 116(1):100-104.
50. Kuete V, Tangmouo JG, Marion Meyer JJ, Lall N. 2009. Diospyrone, crassiflorone
and plumbagin: three antimycobacterial and antigonorrhoeal naphthoquinones from
two Diospyros spp. Int J Antimicrob Ag. 34(4):322-325.
51. Dzoyem JP, Nkuete AH, Kuete V, Tala MF, Wabo HK, Guru SK, Rajput VS, Sharma
A, Tane P, Khan IA et al. 2012. Cytotoxicity and antimicrobial activity of the

Mapie Tiwa et al. Investigational Medicinal Chemistry and Pharmacology 2024 7(1):86 Page 10 of 10
methanol extract and compounds from Polygonum limbatum. Planta Med.
78(8):787-792.
52. Kuete V, Sandjo LP. 2012. Isobavachalcone: an overview. Chin J Integr Med.
18(7):543-547.
53. Kuete V, Ngameni B, Wiench B, Krusche B, Horwedel C, Ngadjui BT, Efferth T.
2011. Cytotoxicity and mode of action of four naturally occuring flavonoids from the
genus Dorstenia: gancaonin Q, 4-hydroxylonchocarpin, 6-prenylapigenin, and 6,8-
diprenyleriodictyol. Planta Med. 77(18):1984-1989.
54. Mbaveng AT, Kuete V, Efferth T. 2017. Potential of Central, Eastern and Western
Africa medicinal plants for cancer therapy: spotlight on resistant cells and molecular
targets. Front Pharmacol 8:343.
55. Kuete V, Efferth T. 2015. African flora has the potential to fight multidrug resistance
of cancer. BioMed Res Int. 2015:914813.
56. Kuete V, Nkuete AHL, Mbaveng AT, Wiench B, Wabo HK, Tane P, Efferth T. 2014.
Cytotoxicity and modes of action of 4′-hydroxy-2′,6′-dimethoxychalcone and other
flavonoids toward drug-sensitive and multidrug-resistant cancer cell lines.
Phytomedicine. 21(12):1651-1657.
57. Komguem J, Meli AL, Manfouo RN, Lontsi D, Ngounou FN, Kuete V , Kamdem HW,
Tane P, Ngadjui BT, Sondengam BL et al. 2005. Xanthones from Garcinia
smeathmannii (Oliver) and their antimicrobial activity. Phytochemistry. 66(14):1713-
1717.
58. Kuete V, Wiench B, Alsaid MS, Alyahya MA, Fankam AG, Shahat AA, Efferth T.
2013. Cytotoxicity, mode of action and antibacterial activities of selected Saudi
Arabian medicinal plants. BMC Complement Altern Med. 13:354.
59. Mbaveng AT, Bitchagno GTM, Kuete V, Tane P, Efferth T . 2019. Cytotoxicity of
ungeremine towards multi-factorial drug resistant cancer cells and induction of
apoptosis, ferroptosis, necroptosis and autophagy. Phytomedicine. 60:152832.
60. 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.
61. Mbaveng AT, Fotso GW, Ngnintedo D, Kuete V, Ngadjui BT, Keumedjio F, Andrae-
Marobela K, Efferth T. 2018. Cytotoxicity of epunctanone and four other
phytochemicals isolated from the medicinal plants Garcinia epunctata and
Ptycholobium contortum towards multi-factorial drug resistant cancer cells.
Phytomedicine 48:112-119.
62. Kuete V, Dzotam JK, Voukeng IK, Fankam AG, Efferth T. 2016. Cytotoxicity of
methanol extracts of Annona muricata, Passiflora edulis and nine other
Cameroonian medicinal plants towards multi-factorial drug-resistant cancer cell
lines. Springerplus. 5(1):1666.
63. Kuete V, Mbaveng AT, Sandjo LP, Zeino M, Efferth T. 2017. Cytotoxicity and mode
of action of a naturally occurring naphthoquinone, 2-acetyl-7-methoxynaphtho[2,3-
b]furan-4,9-quinone towards multi-factorial drug-resistant cancer cells.
Phytomedicine. 33:62-68.
64. Kuete V, Sandjo LP, Kwamou GM, Wiench B, Nkengfack AE, Efferth T. 2014.
Activity of three cytotoxic isoflavonoids from Erythrina excelsa and Erythrina
senegalensis (neobavaisoflavone, sigmoidin H and isoneorautenol) toward multi-
factorial drug resistant cancer cells. Phytomedicine. 21(5):682-688.
65. Kuete V. 2014. 21 - Health Effects of Alkaloids from African Medicinal Plants. In:
Toxicological Survey of African Medicinal Plants. edn. Edited by Kuete V: Elsevier:
611-633.
66. Kuete V, Sandjo LP, Mbaveng AT, Seukep JA, Ngadjui BT, Efferth T. 2015.
Cytotoxicity of selected Cameroonian medicinal plants and Nauclea pobeguinii
towards multi-factorial drug-resistant cancer cells. BMC Complement Altern Med.
15:309.
67. Poumale HMP, Hamm R, Zang Y, Shiono Y, Kuete V. 2013. 8 - Coumarins and
Related Compounds from the Medicinal Plants of Africa. In: Medicinal Plant
Research in Africa. edn. Edited by Kuete V. Oxford: Elsevier: 261-300.
68. Ngounou FN, Manfouo RN, Tapondjou LA, Lontsi D, Kuete V, Penlap V, Etoa FX,
Dubois MAL, Sondengam BL. 2005. Antimicrobial diterpenoid alkaloids from
Erythrophleum suaveolens (guill. & perr.) brenan. Bull Chem Soc Ethiop. 19(2):221-
226.
69. Omosa LK, Midiwo JO, Kuete V. 2017. Chapter 19 - Curcuma longa. In: Medicinal
Spices and Vegetables from Africa. edn. Edited by Kuete V: Academic Press: 425-
435.
70. Dzoyem JP, Tchuenguem RT, Kuiate JR, Teke GN, Kechia FA, Kuete V. 2014. In
vitro and in vivo antifungal activities of selected Cameroonian dietary spices. BMC
Complement Altern Med. 14:58.
71. Nguyen THP, Do TK, Nguyen TTN, Phan TD, Phung TH. 2020. Acute toxicity,
antibacterial and antioxidant abilities of Elephantopus mollis HBK and Elephantopus
scaber L. Can Tho Univ J Sci. 12(2):9-14.
72. Mambe FT, Tchinda CF, Wamba BEN, Nayim P, Ashu F, Manekeng T, Veronique
P, Kuete V. 2022. Modes of action of the methanol extract and 3-O-[β-
galactopyranosyl-(1→ 4)-β-D-galactopyranosyl]-oleanolic acid from Acacia
polyacantha against multidrug-resistant Gram-negative bacteria. Invest Med Chem
Pharmacol. 5:60.
73. Bavishi C, DuPont HL. 2011. Systematic review: The use of proton pump inhibitors
and increased susceptibility to enteric infection. Aliment Pharmacol Ther. 34(11–
12):1269–1281.
74. Mitrea L, Vodnar DC. 2019. Klebsiella pneumoniae-A useful pathogenic strain for
biotechnological purposes: Diols biosynthesis under controlled and uncontrolled ph
levels. Pathogens. 8(4):293.
75. Youmbi LM, Atontsa BCK, Tankeo SB, Wamba NEB, Nayim P, Nganou KB,
Bitchagno GTM, Simo KI, Mpetga JDS, Penlap VB. 2020. Antibacterial potential and
mechanism of action of botanicals and phytochemicals from Stachytarpheta
cayennensis (Verbenaceae) against Gram-negative multidrug-resistant phenotypes
expressing efflux pumps. Invest Med Chem Pharmacol. 3(1):35.
76. Chollet R, Chevalier J, Bryskier A, Pagès J-M. 2004. The AcrAB-TolC pump is
involved in macrolide resistance but not in telithromycin efflux in Enterobacter
aerogenes and Escherichia coli. Antimicrob Agents Chemother. 48(9):3621-3624.
77. Dzotam JK, Touani FK, Kuete V. 2016. Antibacterial activities of the methanol
extracts of Canarium schweinfurthii and four other Cameroonian dietary plants
against multi-drug resistant Gram-negative bacteria. Saudi J Biol Sci. 23:565-570.
78. Lorenzi V, Muselli A, Bernardini AF, Berti L, Pages JM, Amaral L, Bolla JM. 2009.
Geraniol restores antibiotic activities against multidrug-resistant isolates from Gram-
negative species. Antimicrob Agents Chemother. 53(5):2209-2211.
79. Dzotam JK, Simo IK, Bitchagno G, Celik I, Sandjo LP, Tane P, Kuete V. 2018. In
vitro antibacterial and antibiotic modifying activity of crude extract, fractions and
3',4',7-trihydroxyflavone from Myristica fragrans Houtt against MDR Gram-negative
enteric bacteria. BMC Complement Altern Med. 18(1):15.
80. Seukep JA, Sandjo LP, Ngadjui BT, Kuete V. 2016. Antibacterial and antibiotic-
resistance modifying activity of the extracts and compounds from Nauclea
pobeguinii against Gram-negative multi-drug resistant phenotypes. BMC
Complement Altern Med. 16:193.
81. Dzotam JK, Kuete V. 2017. Antibacterial and antibiotic-modifying activity of
methanol extracts from six cameroonian food plants against multidrug-resistant
enteric bacteria. BioMed Res Int. 2017:1583510.
82. Voukeng IK, Kuete V, Dzoyem JP, Fankam AG, Noumedem JA, Kuiate JR, Pages
JM. 2012. Antibacterial and antibiotic-potentiation activities of the methanol extract
of some Cameroonian spices against Gram-negative multi-drug resistant
phenotypes. BMC Res Notes. 5:299.
83. Fankam AG, Kuiate JR, Kuete V. 2015. Antibacterial and antibiotic resistance
modifying activity of the extracts from allanblackia gabonensis, combretum molle
and gladiolus quartinianus against Gram-negative bacteria including multi-drug
resistant phenotypes. BMC Complement Altern Med. 15:206.
84. Mallea M, Chevalier J, Bornet C, Eyraud A, Davin-Regli A, Bollet C, Pages JM.
1998. Porin alteration and active efflux: two in vivo drug resistance strategies used
by Enterobacter aerogenes. Microbiology. 144 ( Pt 11):3003-3009.
85. Seukep JA, Fankam AG, Djeussi DE, Voukeng IK, Tankeo SB, Noumdem JA, Kuete
AH, Kuete V: Antibacterial activities of the methanol extracts of seven Cameroonian
dietary plants against bacteria expressing MDR phenotypes. Springerplus 2013,
2:363.
86. Tran QT, Mahendran KR, Hajjar E, Ceccarelli M, Davin-Regli A, Winterhalter M,
Weingart H, Pages JM. 2010. Implication of porins in beta-lactam resistance of
Providencia stuartii. J Biol Chem. 285(42):32273-32281.
87. Touani FK, Seukep AJ, Djeussi DE, Fankam AG, Noumedem JA, Kuete V. 2014.
Antibiotic-potentiation activities of four Cameroonian dietary plants against
multidrug-resistant Gram-negative bacteria expressing efflux pumps. BMC
Complement Altern Med. 14:258.