Hindawi Publishing Corporation
International Journal of Bacteriology
Volume 2015, Article ID 162028, 10 pages
http://dx.doi.org/10.1155/2015/162028
Research Article
Antibacterial Effects of Cissus welwitschii and
Triumfetta welwitschii Extracts against Escherichia coli and
Bacillus cereus
Batanai Moyo and Stanley Mukanganyama
Biomolecular Interactions Analyses Laboratory, Department of Biochemistry, University of Zimbabwe, P.O. Box MP167,
Mount Pleasant, Harare, Zimbabwe
Correspondence should be addressed to Stanley Mukanganyama; smukanganyama@medic.uz.ac.zw
Received 6 July 2015; Revised 22 September 2015; Accepted 19 October 2015
Academic Editor: Ramakrishna Nannapaneni
Copyright © 2015 B. Moyo and S. Mukanganyama. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Antibiotic resistance has increased sharply, while the pace for the development of new antimicrobials has slowed down. Plants
provide an alternative source for new drugs. This study aimed to screen extracts from Cissus welwitschii and Triumfetta welwitschii
for antibacterial activity against Escherichia coli and Bacillus cereus. The tests conducted included a susceptibility determination test,
analysis of the effect of T. welwitschii on cell wall integrity, and transport across the membrane. It was found that the T. welwitschii
methanol extracts were more effective than the water extracts and had the lowest minimum inhibitory concentration and minimum
bactericidal concentration at 0.125 mg/mL and 0.5 mg/mL, respectively, against E. coli and B. cereus. The C. welwitschii extract caused
the most drug accumulation in E. coli. In B. cereus, no significant drug accumulation was observed. Nucleic acid leakage in B. cereus
and E. coli and protein leakage in E. coli were observed after exposure to the T. welwitschii extract. The extracts from T. welwitschii
had greater antibacterial activity than the extracts from C. welwitschii. T. welwitschii may be a potential source of lead compounds
for that could be developed into antibacterial agents.
1. Introduction
Antibiotic resistance has become a major problem worldwide
in recent years. The unnecessary prescription of antibiotics
to treat viral infections, incorrect prescriptions due to a
lack of culturing to determine the exact cause of infection,
patients not completing their antibiotic treatments, and the
excessive use of antibiotics in stock feed are some of the
causes of the increased selective pressures placed on bacteria
by antibiotics, leading to resistance [1]. There has been
a decrease in the development of antimicrobial agents by
pharmaceutical companies [2] as their development is not as
profitable as developing drugs that treat chronic and lifestyle
diseases [3]. In the last four decades, only three new classes
of antibiotics have been discovered which are lipopeptides,
oxazolidinones, and streptogramins [4]. As most of the old
and cheap antibiotics are no longer effective, the use of second
or third line drugs has become necessary, and these may have
side effects [5]. When patients are infected with organisms
resistant to all available antimicrobials, surgery is required to
remove the nidus of infection, increasing the risk of death
[6]. Drug resistance is of particular concern among patients
with compromised immunity [7]. Infectious diseases account
for half of all deaths in tropical countries [8] and 90% of
infections are caused by bacteria [9].
Bacteria have the ability to acquire resistance mechanisms
such as genes encoding enzymes such as 𝛽-lactamases, genes
that alter the bacterial cell wall resulting in no binding
site for the antimicrobial agent and efflux pumps through
conjugation, translation, and transduction [10]. Efflux pumps
are transport proteins involved in the extrusion of toxic
substrates (including almost all classes of clinically relevant
antibiotics) [9]. Efflux pumps either can be selective or can be
multidrug efflux pumps, and the extrusion of antimicrobial
agents via these efflux pumps is a major component of resistance [11]. Cells can use proton driven antiporters and/or ATP
driven (ATP-binding cassette) transporters to expel drugs
[12]. Efflux pump inhibitors can be used to restrict the efflux
2
of antibiotics from bacterial cells, meaning that resistance to
antibiotics such as ciprofloxacin can be reversed and drug
resistance can be inhibited [13]. Examples of commonly used
efflux pump inhibitors are verapamil and MC-207,110 [12]. As
a result of antibiotic resistance, new antimicrobials that will
either replace or be used with antibiotics are required.
In African countries, traditional medicines are used
by approximately 80% of the population, while usage of
alternative medicines in developed countries is increasing
[14]. Traditional medicines are extensively incorporated into
the public health system of some countries [15]. To date,
studies have confirmed that some plant extracts can be used
to treat infectious diseases caused by bacteria as they have
antibacterial properties [16–18]. Studies have been conducted
to determine the effects of plant extracts on the activity of
antibiotics against resistant bacteria [7, 9]. Plant extracts have
been used to inhibit microbial growth in food and drinks [19].
Interest in herbal medicines has increased in recent years
due to the fact that they are cheap, readily available, and
effective, as well as the high cost of industrialized medicines,
lack of access to healthcare, and the side effects caused
by taking synthetic medicines [20]. Natural products offer
a large diversity of chemical structures which often serve
as lead molecules whose activities can be enhanced by
manipulation through combinations with chemicals and by
synthetic chemistry [21, 22]. Novel compounds from plants
may not have the problem of antimicrobial resistance [21].
Natural plant extracts have been found to be an important
source of secondary metabolites such as tannins, terpenoids,
alkaloids, and flavonoids, which have been found in vitro
to have antimicrobial properties [22]. Some plant extracts
have also been classified as resistance modifiers because they
can enhance antibiotic activity or reverse antibiotic resistance
[22]. In Zimbabwe, plants have been used for centuries
to treat various ailments. Examples are Vernonia adoensis
leaves for the treatment of tuberculosis and Salons delagoense
leaves and fruits for the treatment of scabies in children
[23]. Aloe excelsa, Cymbopogon nardus, pepper, coconut milk,
and coconut oil are some plants used for the preparation of
ointments, lotions, and creams [24].
The aim of this study was to investigate the antibacterial
properties of the aqueous and methanol extracts of Cissus
welwitschii and Triumfetta welwitschii var. welwitschii. The
genus Cissus contains 350 species and is found in tropical and
subtropical locations [25]. Cissus is a member of the grape
family Vitaceae [25]. C. welwitschii is found in tropical Africa,
often on granite outcrops and termite mounds and in forests,
semievergreen bushland, and woodland [26]. The plant is a
vigorous grower, that is, 1.8–9 m long or shrubby, and has
aerial roots [26]. C. welwitschii has hairless, cylindrical stems
that are often spotted black and granular [26]. The plant has
simple leaves and tendril, and is closely related to C. fragilis
[27]. C. welwitschii is used traditionally in Zimbabwe for the
treatment of cancer.
Triumfetta welwitschii var. welwitschii is a perennial
herb that grows annual stems from a woody rootstock and
usually flowers before the leaves develop [28]. The plant
is a conspicuous species of burnt roadsides, grassland, and
woodland [28]. Three varieties of T. welwitschii exist, namely,
International Journal of Bacteriology
welwitschii, descampsii, and hirsuta [28]. In Zimbabwe, T.
welwitschii is used to treat diarrhoea [29], suggesting that it
has antibacterial effects. Microorganisms contribute to food
spoilage and in turn diarrhoea, so treatment of diarrhoea
using the T. welwitschii plant extract may indicate that the
plant has antibacterial activity. In South Africa, a decoction
of the tuber is mixed with milk and drunk as a fever remedy
[30] suggesting that it has antipyretic effects.
Escherichia coli and Bacillus cereus were the two bacteria
used in this study. Nonpathogenic strains of E. coli live in
the human colon. However, several strains are important
foodborne pathogens. Pathogenic strains include E. coli
O157:H7 which was first discovered in 1982 [1]. This strain
is found in meat products, unpasteurised fruit juices, fruits,
vegetables, and untreated water and is known to cause
haemolytic uremic syndrome [1]. E. coli O55, O111, and
O127 have been associated with infant diarrhoea, while other
strains have been associated with nosocomial infections in
the skin, urinary tract, and surgical wounds [1]. E. coli strains
resistant to drugs including penicillins and cephalosporins
have been discovered [1].
B. cereus causes food poisoning and is especially problematic in starchy foods [1]. The emetic toxin produced by B.
cereus has been associated with improperly stored boiled and
fried rice (causes nausea and vomiting) while the diarrheal
type is associated with a wider range of foods [1]. As B. cereus
and E. coli cause very serious illnesses, new drugs are needed
that can adequately manage the infections that they cause.
The plants used in traditional medicine are potential
sources of antibacterial agents. Many plants used traditionally
achieve good results, so studying these plants may give rise
to new potent antimicrobials with different mechanisms of
action. The results of this study can be used to either validate
or invalidate the use of T. welwitschii and C. welwitschii in
traditional medicine.
2. Materials and Methods
2.1. Chemicals. All chemicals and antibiotics used were purchased from Sigma-Aldrich, Steinheim, Germany, and were
of analytical grade. The major chemicals and antibiotics used
were methanol, for extractions, reserpine, an efflux pump
inhibitor, rhodamine 6G, a probe compound for the drug
efflux assay, propidium iodide dye, used to stain nucleic
acids, Bradford’s Reagent for protein determination using the
Bradford’s assay, and the three antibiotics used in the study:
ampicillin, kanamycin, and norfloxacin.
2.2. Plant Material. Cissus welwitschii and Triumfetta welwitschii were collected from Centenary (16.8∘ S, 31.1167∘ E, and
1 156 m above sea level), Mashonaland Central Province,
Zimbabwe. The plants were identified and authenticated
by Mr. Christopher Chapano, a botanist with the National
Botanical and Herbarium Garden (Harare, Zimbabwe).
2.3. Preparation of Extracts. The dried leaves and roots of
each plant were ground separately in a blender (Philips Co.,
Shanghai, China). To 10 g of plant material, either 100 mL
International Journal of Bacteriology
of methanol or 200 mL of distilled water was added. Two
hundred millilitres of distilled water was used as the mixture
became viscous during stirring. The mixtures were stirred
using a magnetic stirrer for 20 minutes. The methanol
extracts were filtered using No. 1 Whatman filter paper, while
the water extracts were filtered using mutton cloth. The
filtrate was evaporated to dryness, collected, and stored at
room temperature.
2.4. Bacteria. Escherichia coli ATCC 11229 and Bacillus cereus
ATCC 11778 were used in this study. The bacteria were grown
from glycerol stocks stored at −33∘ C. On nutrient agar plates,
25 𝜇L of bacteria was plated and grown overnight at 37∘ C
in an incubator (Lab Design Engineering (Pty) Ltd., Maraisburg, South Africa). From the plates, 1 colony was inoculated
into nutrient broth and grown overnight at 37∘ C with shaking
at 160 revolutions per minutes (rpm) (Lab Companion, Jeio
Tech, South Korea). The plates and bacterial culture tubes
were stored at 4∘ C. Fresh stocks were prepared for each assay.
2.5. Determination of Antibacterial Activity Using the Agar
Disk Diffusion Method. Molten nutrient agar was inoculated
with bacteria to a final concentration of 1 × 106 cfu/mL. The
agar was poured into petri dish plates and allowed to solidify.
Stock solutions of C. welwitschii, T. welwitschii, and the antibiotic ampicillin were prepared to a concentration of 25 mg/mL
by dissolving the plant extracts in the extracting solvent and
ampicillin in distilled water. Filter paper disks measuring
6 mm in diameter were prepared. To each disk, 20 𝜇L of
extract or antibiotic was added. The samples were prepared
in quadruplicate. The disks were allowed to dry before being
placed onto the plates impregnated with bacteria. The plates
were stored at 4∘ C for two hours to allow the extracts and
antibiotics to diffuse into the agar. The plates were incubated
overnight at 37∘ C. The diameters of the zones of inhibition
were measured in millimetres.
2.6. Minimum Inhibitory Concentration (MIC) and Minimum
Bactericidal Concentration (MBC). The MICs of the plant
extracts and antibiotics against B. cereus and E. coli were
determined using the broth dilution method. The assay was
based on the method described by Eloff [31]. One in two serial
dilutions of the extracts and antibiotics were prepared. For the
plant extracts and ampicillin, the concentrations used ranged
from 0.008 mg/mL to 4 mg/mL, while, for kanamycin and
norfloxacin, the concentrations used ranged from 1 𝜇g/mL
to 1 mg/mL. One hundred microliters of the extracts and
antibiotics was added to wells in microwell plates. To each
well, 100 𝜇L of bacteria was added to achieve a final concentration of 1 × 106 cfu/mL. Wells containing 200 𝜇L of broth
only, 100 𝜇L of the extracts or antibiotics and 100 𝜇L of broth,
and 100 𝜇L of broth and 100 𝜇L of bacteria were used as the
controls. The plates were incubated at 37∘ C with shaking at
30 rpm overnight.
After incubation, the absorbance of the wells was
measured at 600 nm using a microplate spectrophotometer (SpectraMaxPlus, Molecular Devices, Sunnyvale, USA).
3
To each well, 25 𝜇L of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) was then added. MTT
turns dark blue (from yellow) in the presence of living cells
[32]. The plates were incubated for 1 hour at 37∘ C at 30 rpm.
The absorbance was measured at 570 nm.
To determine the MBC, one loopful of the bacteria in
the wells that contained the MIC was streaked, in duplicate,
onto nutrient agar plates without antibiotics. The wells that
contained extracts at one and two concentrations higher and
one concentration lower than the MIC were also plated. The
plates were incubated for 24 hours at 37∘ C.
2.7. Effects of Plant Extracts on Drug Accumulation. The drug
accumulation assay was used to determine the effects of the
plant extracts on drug accumulation in E. coli and B. cereus.
The method described by Chitemerere and Mukanganyama
[23] was used with some modifications. The bacteria were
grown overnight at 37∘ C in two separate flasks containing 400 mL nutrient broth (120 rpm). The bacteria were
centrifuged at 4000 rpm for 10 minutes (MSE Minor 35,
England). The supernatant was discarded. The pellet was
washed twice in phosphate buffered saline (PBS) (pH 7.2).
The cells were centrifuged at 4000 rpm for 5 minutes in a
preweighed tube. The supernatant was discarded and the
pellet was weighed using a Kern EG balance (Kern & Sohn,
Germany). PBS containing 10 mM sodium azide (NaN3 ) was
added to the tube to achieve a concentration of cells of
40 mg/mL. The tube was gently inverted to disperse the cells
within the PBS. Rhodamine 6G (R6G) to a final concentration
of 10 𝜇M was immediately added and cells were incubated at
90 rpm for 1 hour.
The cells were divided into two tubes, A and B, in the ratio
of 1 : 2 but both with the same concentration of cells. Both
tubes were centrifuged at 4000 rpm for 5 min. For tube A,
the supernatant was discarded, and PBS alone was added to
achieve a final concentration of cells of 40 mg/mL. For tube B,
the supernatant was discarded and the pellet was resuspended
in PBS containing 1 M glucose. The contents of tube B were
then divided into five tubes, B1 to B5 . Reserpine was added to
tube B1 to a final concentration of 60 ng/mL. Tube B2 served
as a control with glucose alone. Plant extracts were added to
tubes B3 to B5 to achieve final concentrations of 60 ng/mL
of plant extract in each tube. Equal concentrations of plant
extracts and reserpine were used to allow comparisons to
be conducted. All of the tubes were mixed on a vortex
mixer (Barnstead/Thermolyne, USA) before being incubated
for 30 minutes (90 rpm, 37∘ C) in a Lab Companion SI-300
incubator.
After incubation, the tubes were centrifuged at 4000 rpm
for 10 minutes and the supernatant was discarded. The pellets
were resuspended in 0.1 M glycine HCl, pH 3. The glycine HCl
was used to lyse the cells. The cells were mixed on a vortex
mixer before being incubated at 37∘ C, 90 rpm overnight.
The tubes were centrifuged at 4000 rpm for 10 minutes and
the supernatant was collected. The absorbance of R6G was
measured at 527 nm. The standards were prepared by diluting
R6G in glycine HCl, and the concentrations used ranged from
0 𝜇M to 5 𝜇M.
4
International Journal of Bacteriology
Table 1: Zones of inhibition produced after exposure of bacteria to plant extracts and antibiotics. The zone of inhibition refers to the diameter
of the circle in which no growth of cells was observed, minus the diameter of the filter paper disk (6 mm). The assay was conducted twice.
Plant extract/antibiotic
C. welwitschii leaf methanol
C. welwitschii leaf water
C. welwitschii root methanol
T. welwitschii leaf methanol
T. welwitschii leaf water
T. welwitschii root methanol
T. welwitschii root water
Ampicillin
Methanol
Distilled water
B. cereus zone of inhibition (mm)
2.5 ± 0.6
0
0.8 ± 0.29
3.3 ± 0.5
0
6±0
1.8 ± 0.5
9 ± 0.0
0
0
2.8. Determination of the Effect of T. welwitschii Extracts on
Nucleic Acids Leakage. Propidium iodide is a dye that is
capable of binding to nucleic acids. The dye is unable to
enter viable cells, making it useful for determining the effects
of plant extracts on bacterial membranes. B. cereus and E.
coli cells were suspended in 0.9% saline solution (OD600 =
1.5). The cell suspensions were exposed to plant extracts at
concentrations of the MIC and double the MIC in duplicate
for 10 minutes. The bacteria (1 mL) were centrifuged for 1
minute at 11000 ×g (Centrifuge 5415C, Eppendorf, Berlin,
Germany). The pellet was washed with 1 mL 0.9% saline
solution. Three microliters of propidium iodide was added to
each sample and the solution was mixed. The samples were
kept in the dark for 10 min. Fluorescence was measured at
excitation and emission wavelengths of 544 nm and 612 nm,
respectively, using an 𝑓max microplate spectrofluorometer
(Molecular Devices, Sunnyvale, USA). The controls used
were nontreated cells, 3% DMSO, 0.1% SDS, and kanamycin.
2.9. Determination of the Effect of T. welwitschii Extracts on
Protein Leakage. B. cereus and E. coli cells were suspended
in 0.9% saline solution (OD600 = 1.5). The cell suspensions
were exposed to plant extracts at concentrations of the MIC
and double the MIC. The samples were incubated at 37∘ C
with shaking (120 rpm) for 120 min. Five hundred microlitres
of cell suspension was centrifuged at 7000 rpm for 2 min.
To 50 𝜇L of the supernatant, 950 𝜇L of Coomassie brilliant
blue G-250 was added to measure the protein content by
Bradford’s method. The colour was allowed to develop for
10 min before the absorbance was measured at 595 nm using
a spectrophotometer. The controls used were kanamycin,
3% DMSO, 0.1% SDS, and untreated cells. Bovine serum
albumin (BSA) was used as a standard to determine protein
concentration.
2.10. Statistical Analysis. The one-way analysis of variance
test (ANOVA) with Dunnett’s Multiple Comparison Test was
used to analyse the results. The values with a 𝑝 value of 0.05
or less were considered statistically significant. GraphPad
Prism 5 for Windows (GraphPad Software Inc., San Diego,
California, USA) version 5.03 was used.
E. coli zone of inhibition (mm)
1.3 ± 0.5
0
0.5 ± 0.0
2.3 ± 0.5
0
4.5 ± 1.2
2 ± 0.6
12 ± 0.8
0
0
3. Results
3.1. Determination of Antibacterial Activity Using the Disk
Diffusion Method. The results of the disk diffusion assay are
shown in Table 1. The zone of inhibition refers to the diameter
of the zone in which no growth of bacteria was observed
minus the diameter of the filter paper disk (6 mm). The root
methanol extract from T. welwitschii had the largest zones
of inhibition against both B. cereus and E. coli at 6 mm and
4.5 mm, respectively. T. welwitschii leaf and root methanol
extracts produced larger zones of inhibition against the
bacteria than C. welwitschii leaf and root methanol extracts.
The leaf water extracts of both C. welwitschii and T. welwitschii
produced no zones of inhibition, making them the least
effective extracts. Generally the zones of inhibition are larger
against B. cereus than against E. coli. The ampicillin was more
effective at inhibiting E. coli than B. cereus (12 mm and 9 mm,
resp.).
3.2. Minimum Inhibitory Concentration and Minimum Bactericidal Concentration. The results of the MIC assay, shown
in Table 2, show that C. welwitschii leaf and root methanol
extracts were more effective against E. coli than B. cereus.
The T. welwitschii root water and methanol extracts were
more effective against B. cereus than E. coli. The T. welwitschii leaf methanol showed significant growth inhibitory
effect against both E. coli and B. cereus with an MIC of
0.125 mg/mL. All 3 antibiotics, particularly kanamycin and
norfloxacin, inhibited B. cereus and E. coli at very low concentrations. B. cereus and E. coli were inhibited at 0.5 𝜇g/mL
and 1 𝜇g/mL, respectively, by kanamycin and 0.25 𝜇g/mL
by norfloxacin. Of the plant extracts, T. welwitschii leaf
methanol extract had the lowest MBC of >0.5 mg/mL against
B. cereus and E. coli. C. welwitschii leaf and root methanol
extracts and T. welwitschii root water extract were the highest
at >4 mg/mL. The antibiotics had lower MICs and MBCs
against B. cereus than E. coli, except norfloxacin which
had the same MIC for both bacteria. Of note was that at
4 mg/mL of C. welwitschii root methanol extract, E. coli
grew fairly well, but at 2 mg/mL the extract killed the
bacteria.
International Journal of Bacteriology
5
Table 2: MICs and MBCs of plant extracts and antibiotics against B. cereus and E. coli. The assay was conducted twice.
Bacterium
Extract/antibiotic
C. welwitschii leaf methanol
C. welwitschii root methanol
T. welwitschii leaf methanol
T. welwitschii root methanol
T. welwitschii root water
Ampicillin
Kanamycin
Norfloxacin
C. welwitschii leaf methanol
C. welwitschii root methanol
T. welwitschii leaf methanol
T. welwitschii root methanol
T. welwitschii root water
Ampicillin
Kanamycin
Norfloxacin
B. cereus
E. coli
MIC (mg/mL)
2
2
0.125
0.25
0.5
0.004
0.0005
0.00025
0.5
1
0.125
0.5
>4
0.008
0.001
0.00025
MBC (mg/mL)
>4
>4
>0.5
>1
>2
0.008
0.004
0.002
2
a
>0.5
2
>4
0.016
0.008
0.004
The > indicates that the MIC and MBC were higher than the tested concentrations.
At 4 mg/mL of the C. welwitschii root methanol extract, E. coli cells were able to grow, but they were unable to grow at 2 mg/mL.
a
∗∗∗
∗∗∗
10
∗∗∗
8
R6G concentration (𝜇M)
6
4
2
∗∗∗ ∗∗∗ ∗∗∗
8
6
4
2
Glucose
Glucose + root
methanol extract
Glucose + leaf
methanol extract
Glucose + leaf
water extract
Glucose + reserpine
No glucose
Glucose + leaf
water extract
Glucose + leaf
methanol extract
Glucose
Sample
Sample
B. cereus
E. coli
Glucose + root
methanol extract
Glucose + root
methanol extract
Glucose + leaf
methanol extract
Glucose
Glucose + reserpine
No glucose
Glucose + root
methanol extract
Glucose
Glucose + leaf
methanol extract
Glucose + reserpine
No glucose
No glucose
0
0
Glucose + reserpine
R6G concentration (𝜇M)
10
B. cereus
E. coli
(a)
(b)
Figure 1: Drug accumulation in B. cereus and E. coli cells. The graphs show the accumulation of R6G in B. cereus and E. coli cells after exposure
to T. welwitschii (a) and C. welwitschii (b) extracts. The amount of R6G accumulated is measured in 𝜇M. The mean and standard deviation are
shown. 𝑛 = 3. The test was conducted three times. The test for significance was carried out by comparing glucose + reserpine/plant extracts
to glucose only. ∗∗∗ 𝑃 < 0.001.
3.3. Effects of Plant Extracts on Drug Accumulation. The
results of the R6G accumulation assay using T. welwitschii
extracts are shown in Figure 1(a). Generally more drug
accumulation was observed in E. coli (>4 𝜇M) than in B.
cereus (<4 𝜇M). In the presence of the leaf methanol and root
methanol extracts 5.10 𝜇M and 5.39 𝜇M R6G, respectively,
accumulated in E. coli, while in the presence of reserpine
4.89 𝜇M R6G was accumulated. For E. coli none of these
results were significantly higher than the control: cells
exposed to glucose only (4.38 𝜇M). When the B. cereus cells
were exposed to reserpine, 5.01 𝜇M R6G was accumulated in
the cells. There was a significant decrease in R6G accumulation when B. cereus cells were exposed to the T. welwitschii
root methanol extract.
6
International Journal of Bacteriology
E. coli
∗∗∗
6
Fluorescence (F/units)
4
2
∗∗∗
∗∗∗
∗∗∗
4
2
Cells only
0.1% SDS
3% DMSO
Kan. 0.125 mg·mL−1
Cells only
0.1% SDS
3% DMSO
Kan. 0.125 mg·mL−1
RM 0.5 mg·mL−1
RM 0.25 mg·mL−1
LM 0.25 mg·mL−1
LM 0.125 mg·mL−1
Sample
LM 0.25 mg·mL−1
0
0
LM 0.125 mg·mL−1
Fluorescence (F/units)
∗
RM 1 mg·mL−1
∗∗
6
RM 0.5 mg·mL−1
B. cereus
Sample
Figure 2: Fluorescence of nucleic acid binding propidium iodide after exposure of B. cereus and E. coli to T. welwitschii plant extracts. The
T. welwitschii extracts were tested at the MIC and 2-fold the MIC, while kanamycin, 3% DMSO, 0.1% SDS, and untreated cells were used as
controls. 𝑛 = 2. The test was conducted twice. The test for significance was carried out by comparing all samples to cells only. ∗ 𝑃 < 0.05.
∗∗
∗∗∗
𝑃 < 0.01.
𝑃 < 0.001. LM: leaf methanol; RM: root methanol; Kan.: kanamycin; DMSO: dimethyl sulfoxide; SDS: sodium dodecyl
sulphate.
Figure 1(b) shows the accumulation in B. cereus and E.
coli after exposure to C. welwitschii extracts. In E. coli, the leaf
methanol extract caused the accumulation of large amounts
of R6G (9.25 𝜇M). The remaining C. welwitschii extracts,
the root methanol and leaf water extracts, caused much
less accumulation at 3.06 𝜇M and 2.93 𝜇M, respectively, than
the control cells that were exposed to glucose. In E. coli,
more R6G accumulated inside the cells treated with glucose
than in those treated without glucose (4.65 𝜇M and 1.98 𝜇M,
resp.). In B. cereus, C. welwitschii extracts caused more drug
accumulation than that observed in cells treated with reserpine, but the accumulation was not significantly different
to cells exposed to glucose only. The root methanol, leaf
methanol,and leaf water extracts caused the accumulation of
4.98 𝜇M, 4.95 𝜇M, and 4.92 𝜇M R6G, while reserpine caused
the accumulation of 4.03 𝜇M. The cells exposed to glucose
had more accumulation of R6G than those without glucose.
3.4. Determination of the Effect of T. welwitschii Extracts
on Nucleic Acids Leakage. The fluorescence of propidium
iodide in B. cereus and E. coli cells after exposure to the
T. welwitschii leaf methanol and root methanol extracts is
shown in Figure 2. Against B. cereus, the fluorescence of
propidium iodide increased as the concentration of the root
methanol extract increased. The root methanol extract caused
the most nucleic acids leakage. At the MIC (0.25 mg/mL) and
double the MIC (0.5 mg/mL), the fluorescence of propidium
iodide was 1.94 fluorescence units (F/units) and 2.53 F/units,
respectively. The fluorescence at 0.5 mg/mL was significantly
higher than the fluorescence of propidium iodide in the
untreated B. cereus cells, which was 1.72 F/units.
In E. coli, following exposure to the leaf methanol extract,
the fluorescence of propidium iodide was 1.741 F/units
and 1.743 F/units after exposure to the MIC (0.125 mg/mL)
and double the MIC (0.25 mg/mL), respectively. The root
methanol extract at 0.5 mg/mL and 1 mg/mL (MIC and
double the MIC, resp.) caused nucleic acid leakage in E.
coli cells. However, as the concentration of plant extracts
increased, the fluorescence of propidium iodide decreased:
3.386 F/units at the MIC and 2.871 F/units at 2-fold the MIC.
Untreated E. coli cells had a propidium iodide fluorescence of
1.61 F/units. SDS was able to cause nucleic acid leakage in B.
cereus and E. coli. In B. cereus the fluorescence of propidium
iodide in cells exposed to DMSO was 1.692 F/units which was
similar to that in untreated cells. The same was true for E.
coli cells, where the fluorescence of propidium iodide was
1.403 F/units for cells treated with DMSO.
3.5. Determination of the Effect of T. welwitschii Extracts on
Protein Leakage. In B. cereus cells the T. welwitschii extracts
do not cause protein leakage (Figure 3). In the presence of the
plant extracts, less protein is lost compared to untreated cells.
The root methanol extract at 0.25 mg/mL and 0.5 mg/mL (the
MIC and double the MIC, resp.) caused significantly less
protein leakage than the untreated cells. Ampicillin caused
the leakage of large amounts of protein (0.073 mg/mL). In
the cells treated with SDS, 0.46 mg/mL of protein was leaked,
while in those treated with DMSO 0.057 mg/mL of protein
was leaked out. These amounts were similar to the untreated
cells: 0.052 mg/mL of protein was leaked from the cells.
In E. coli (Figure 3), the root methanol extract caused
protein leakage at 1 mg/mL (double the MIC): 0.048 mg/mL.
International Journal of Bacteriology
7
B. cereus
∗∗
∗∗∗
0.10
∗
Protein concentration (mg/mL)
0.08
0.06
0.04
0.02
∗∗∗
0.08
0.06
0.04
0.02
Cells only
0.1% SDS
3% DMSO
Amp. 1 mg·mL−1
RM 1 mg·mL−1
RM 0.5 mg·mL−1
Cells only
0.1% SDS
3% DMSO
Amp. 1 mg·mL−1
RM 0.5 mg·mL−1
RM 0.25 mg·mL−1
LM 0.25 mg·mL−1
LM 0.125 mg·mL−1
Sample
LM 0.25 mg·mL−1
0.00
0.00
LM 0.125 mg·mL−1
Protein concentration (mg/mL)
0.10
E. coli
Sample
Figure 3: Graph of protein leakage in B. cereus and E. coli after exposure to the T. welwitschii leaf and root methanol extracts. The protein
concentration is shown in mg/mL. 𝑛 = 2. The test was conducted three times. The test for significance was carried out by comparing all
samples to cells only. ∗ 𝑃 < 0.05. ∗∗ 𝑃 < 0.01. ∗∗∗ 𝑃 < 0.001. LM: leaf methanol; RM: root methanol; Amp.: ampicillin; DMSO: dimethyl
sulfoxide; SDS: sodium dodecyl sulphate.
The protein leakage observed in the untreated cells was
0.0098 mg/mL. DMSO did not change the amount of protein
leakage in B. cereus cells.
4. Discussion
The search for new antimicrobial agents has been necessitated
by the increase in antimicrobial resistance in recent years.
Fifty thousand people die every day worldwide due to infectious diseases [33]. Natural plant products are a good source
of new antimicrobials as they generally have low toxicity,
cause minimal environmental pollution, have a low risk of
development of resistance by pathogens [34], are cheap, and
are generally safer than synthetic medicines [35]. The use of
known medicinal plants is advantageous as they have been
prescreened over thousands of years, resulting in a higher
probability of isolating useful and safe compounds from them
than from plants not in use by humans already [36]. Plant
materials are either present in or have provided models for
approximately 50% of drugs [35]. Screening T. welwitschii
and C. welwitschii for antibacterial activity was more likely
to be successful as these plants are already used for medicinal
purposes.
The extracts from the two plants were generally more
effective against B. cereus than E. coli. B. cereus is a Grampositive bacterium while E. coli is Gram-negative, suggesting
that the plant extracts are more effective against Grampositive than Gram-negative bacteria. According to the literature, plant extracts are generally more active against Grampositive than Gram-negative bacteria [37]. Gram-negative
bacteria are more resistant to antibacterial agents than Grampositive bacteria [38, 39] because of the presence of the outer
membrane that acts as a permeability barrier [38]. Seasotiya
and Dalal [40] and Narayan [38] found that Gram-positive
bacteria were more susceptible to the tested plant extracts
than Gram-negative bacteria.
The MBC test results showed that when exposed to
4 mg/mL of C. welwitschii root methanol extract, E. coli grew
fairly well, but at 2 mg/mL the extract killed the bacteria.
The anomalous result observed may have been caused by
high levels of nutrients present in the tuber that allowed the
bacteria to overcome any bactericidal substances in the root
extract. Nutrients would be more concentrated at 4 mg/mL
compared to lower concentrations.
All of the plant extracts except the T. welwitschii root
water extract against E. coli were able to inhibit the growth
of the bacteria. Many studies have shown that plant extracts
have antibacterial activity. Plants found to have antibacterial
activity include Leonotis nepetifolia [38], Melia azedarach
[21], Avicenna marina [35], L. erythrorhizon [19], Adansonia
digitata, Tamarindus indica, Aframomum alboviolaceum, and
Ocimum gratissimum [41].
The plant extracts had much higher MICs and MBCs
than those produced by the antibiotics against the bacteria,
showing that they are not as effective as the antibiotics. The
use of whole extracts as opposed to purified compounds may
be responsible. There may have been a lack of specificity as
there were many compounds working together at reduced
concentrations. Interactions between these compounds may
also reduce the effectiveness of the plant extract. Studies have
shown that plant extracts, though effective against bacteria,
are in most cases not as effective as antibiotics [9, 36, 42–44].
The presence of efflux pumps on the bacterial membrane
causes resistance in bacteria [45]. Bacteria use these efflux
8
pumps to expel antibiotics from the cell until their concentration is too low to be effective against the bacteria [46].
The extracts from T. welwitschii caused accumulation of more
R6G in E. coli cells than in B. cereus cells. T. welwitschii was
more capable of binding to the efflux pumps on E. coli than
those on B. cereus. However, T. welwitschii failed to cause
significant drug accumulation in both bacteria.
C. welwitschii in B. cereus was ineffective as an efflux
pump inhibitor. The C. welwitschii leaf methanol extract was a
more effective efflux pump inhibitor in E. coli than reserpine.
The C. welwitschii leaf methanol extract has potential for use
as an efflux pump inhibitor. R6G was expected to accumulate
in the bacterial cells in the absence of glucose while efflux
of R6G would occur in the cells exposed to glucose. R6G
may not have accumulated without glucose as cells may use
proton driven antiporters and/or ATP driven (ATP-binding
cassette (ABC)) transporters to expel drugs [12]. The R6G
may have been expelled from the cells through the proton
driven antiporters as opposed to the ABC transporters. E.
coli has 19 antiporters belonging to the major facilitator
superfamily, small multidrug resistance family and the resistance/nodulation/cell division family [12], and 80 ABC transporter proteins [47]. Proton driven antiporters expel drugs
by coupling drug efflux to the influx of a proton, H+ , while
ABC transporters couple drug efflux to the hydrolysis of ATP
[12]. The AcrAB-TolC pump is a member of the resistancenodulation-cell division (RND) family of tripartite multidrug
efflux pumps ubiquitous throughout Gram-negative bacteria
[48]. In E. coli, the multidrug efflux pump has been shown to
expel a wide range of antibacterial agents [48].
Many plant extracts with the ability to promote drug
accumulation in bacterial cells have been studied. Artemisia
absinthium has been found to contain efflux pump inhibitory
compounds [11]. Hydrastis canadensis, Curcuma longa, Capsicum annum, and Elettaria cardamomum have been found to
inhibit efflux pump activity [40, 49].
Some compounds are capable of disrupting the integrity
of the bacterial cell wall/membrane; for example, polymyxins
increase bacterial membrane permeability and lipopeptides
like daptomycin and crystallomycin bind to bacteria and
cause rapid depolarisation of the bacterial membrane and
eventual cell death [50]. The T. welwitschii root methanol
extract was capable of causing damage to the B. cereus and E.
coli membranes resulting in nucleic acid leakage (Figure 2).
DMSO did not cause damage to the bacterial membranes as
the protein and nucleic acid leakage from the bacterial cells
did not change significantly when the cells were exposed to
DMSO. Although the root methanol extract caused leakage of
nucleic acids in E. coli, the leakage decreased with increasing
concentration of plant extracts. Further studies are needed
to determine the cause of this phenomenon. Cocos nucifera
husk extracts caused leakage of nucleic acids in bacteria [51].
Plumbago zeylonica, Leucas aspera, and Hemidesmus indicus
were all found to be capable of causing nucleic acid leakage in
bacteria [52]. The plant extracts from these plants were found
to contain phenols and flavonoids, resulting in the authors
concluding that the membrane disrupting activities of the
extracts may be due to the activities of these phytochemicals
[52].
International Journal of Bacteriology
Ampicillin was able to cause significant protein leakage
in B. cereus but not in E. coli. Ampicillin inhibits bacterial
cell wall synthesis [1]. Ampicillin is thus expected to be more
effective against Gram-positive bacteria than Gram-negative
bacteria due to the differences in their structures. Grampositive bacteria have thick cell walls (20–80 nm) composed
mainly of peptidoglycan while Gram-negative bacteria have
thin, inner peptidoglycan layers (2–7 nm) and an outer
membrane (7-8 nm) of lipid, protein, and lipopolysaccharide
[1].
Protein leakage was only observed in E. coli after treatment with 1 mg/mL T. welwitschii root methanol extract. The
root methanol extract was able to bind to and disrupt the E.
coli membrane and not the B. cereus cell wall. Some authors
have found that plant extracts are capable of causing membrane disruption [53, 54]. Henie et al. [53] found that Psidium
guajava was capable of causing protein leakage in various
bacteria, while Akinpelu et al. [55] found that extracts from
Garcinia kola caused protein leakage in bacteria, including E.
coli.
5. Conclusions
In conclusion, the leaf and root extracts of C. welwitschii
and T. welwitschii were shown to have antibacterial activity
against B. cereus and E. coli. T. welwitschii extracts were
more potent growth inhibitory activity against the bacteria
than the C. welwitschii extracts. Further analysis is needed to
investigate the exact mode of activity of the plant extracts and
the in vivo toxicity of the plant extracts and to determine the
phytoconstituents present in the plant extracts. T. welwitschii
may be a potential source of antibacterial agents.
Disclosure
Batanai Moyo is co-author.
Conflict of Interests
The authors declare that there is no conflict of interests
regarding the publication of this paper.
Authors’ Contribution
Batanai Moyo performed the experiments, analysed and
interpreted the results obtained, and wrote the document.
Stanley Mukanganyama directed the study, obtained the plant
material used, analysed and interpreted the results, and edited
the paper. All authors have read and approved of the paper.
Acknowledgments
The authors would like to acknowledge Mr. Christopher
Chapano from the National Botanical and Herbarium Garden, Harare, who identified C. welwitschii and T. welwitschii var. welwitschii. They would like to thank Mr. Noel
Mukanganyama, a herbalist from Centenary, Zimbabwe, who
International Journal of Bacteriology
showed them the C. welwitschii plant and provided information on ethnomedicinal uses. This work was supported by
the IPICS-ZIM01 project from the International Program in
the Chemical Sciences (IPICS, Uppsala University, Sweden),
and the International Foundation for Science (IFS F/3413-03),
Stockholm, Sweden.
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