Effect of Vancomycin, tetracycline, Persia americana leaf extract and combinations thereof on antibacterial activity against pathogenic organisms

Abstract

Many pathogenic bacteria have emerged resistance to drugs leading to high cost of treatment. Alternatively, people simultaneously use herbal and conventional medicine to combat infections without scientific evidence on their effectiveness. In an attempt to find out effectiveness of vancomycin, tetracycline, P. americana leaf extract and their combinations against S. aureus, S. pyogenes and P. aeruginosa a laboratory experiment was conducted. MIC and antibacterial activity were determined by macrodilution and disc diffusion methods respectively. Lowest values of MIC and MBC of extract were observed against S. aureus. Zone of inhibition (ZI) ranged from 0 to 31.00mm for antibiotics. Extract was more effective against S. aureus with ZI of 13.00 mm. Combination of vancomycin with extract showed highest improvement of 36.12% against S. aureus. Potency reduction was observed for tetracycline against P. aeruginosa. Therefore, addition of plant extract to antibiotics is being recommended as a best way of improving their potency.

Full text

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Journal of Pharmacognosy and Phytochemistry 2019; 8(1): 2733-2737
E-ISSN: 2278-4136
P-ISSN: 2349-8234
JPP 2019; 8(1): 2733-2737
Received: 06-11-2018
Accepted: 10-12-2018
Martin Henderson Kalumbi
Department of Biomedical
Science, Malawi Adventist
University- Malamulo College of
Health Sciences Campus,
Makwasa, Thyolo, Malawi, East
Africa
Correspondence
Martin Henderson Kalumbi
Department of Biomedical
Science, Malawi Adventist
University- Malamulo College of
Health Sciences Campus,
Makwasa, Thyolo, Malawi, East
Africa
Effect of Vancomycin, tetracycline, Persia
americana leaf extract and combinations thereof
on antibacterial activity against pathogenic
organisms
Martin Henderson Kalumbi
Abstract
Many pathogenic bacteria have emerged resistance to drugs leading to high cost of treatment.
Alternatively, people simultaneously use herbal and conventional medicine to combat infections without
scientific evidence on their effectiveness. In an attempt to find out effectiveness of vancomycin,
tetracycline, P. americana leaf extract and their combinations against S. aureus, S. pyogenes and P.
aeruginosa a laboratory experiment was conducted. MIC and antibacterial activity were determined by
macrodilution and disc diffusion methods respectively. Lowest values of MIC and MBC of extract were
observed against S. aureus. Zone of inhibition (ZI) ranged from 0 to 31.00mm for antibiotics. Extract was
more effective against S. aureus with ZI of 13.00 mm. Combination of vancomycin with extract showed
highest improvement of 36.12% against S. aureus. Potency reduction was observed for tetracycline
against P. aeruginosa. Therefore, addition of plant extract to antibiotics is being recommended as a best
way of improving their potency.
Keywords: Effectiveness, Persia americana, leaf extract, antibacterial, potency, combination
1. Introduction
The impact of antibiotics in treatment of infectious diseases has dramatically declined in
various settings. Often it is pathogens that cause foodborne and nosocomial infections such as
Staphylococcus aureus, Streptococcus pyogenes and Pseudomonas aeruginosa which have
emerged resistance to several drugs due to improper use [1-5]. In Malawi, several attempts and
efforts have been made to contain antibacterial resistance such as surveillance, training on
proper prescription and drug use. Previously, there had been a call by WHO to integrate
traditional and biomedical health systems in order to prevent, control and treat infections in
Sub-Saharan Africa and no country has yet implemented this collaborative healthcare system
including Malawi [6-8]. Alternatively, people use herbal medicine to fight antibiotic resistance
either as a stand-alone or complementary therapy due to high cost of effective drugs and lack
of adequate health facilities [9, 10]. Complementing antibiotics with herbal remedies may either
improve or reduce their potency. However, studies on synergistic effect of local Persia
americanas leaf extract with Vancomycin and tetracycline in treatment of S. aureus, S.
pyogenes and P. aeruginosa have not been done on Malawian plants or elsewhere.
P. ameriacan leaf contains bioactive compounds such as tannins, flavonoids, saponins,
terpenoids, steroids, alkaloids and glycosides [11-15]. These compounds are responsible for their
antibiotic properties and are considered comparatively safe when used correctly [14, 11, 16-18]. But
others argued that the use of herbal medicine is not safe as people tend to sell them without
proper scientific validity, lack of suitable policies and the fact that they can be contaminated
by biological and chemical contaminants [19-23]. Until recently, there is no national policy,
registration system laws and regulation of herbal medicine in Malawi [24]. One of the most used
medicinal plants in Malawi is P. mericana which is used to treat diarrhoea, dysentery,
toothache, chronic gastritis and diabetes mellitus despite lack of recommended dosage.
Dissatisfaction with conventional medicine has prompted Malawians to take conventional
medicine (CM) and traditional medicine (TM) simultaneously in order to enhance recovery
rate and alleviate sufferings associated with various diseases. The prevalence of
simultaneously use of CM and TM is yet to be assessed, however, anecdotal evidence suggests
that utilization of medical pluralism is common in Malawi and other Sub-Saharan Africa.
Scientifically, it is safe to combine bioactive compounds from medicinal plants and
conventional medicine [25]. Growing evidence suggests that herbal medicine enhances the
effectiveness,

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Journal of Pharmacognosy and Phytochemistry
reduces undesirable effects, increases stability and
bioavailability of conventional drugs when used correctly [26-
30].
This study will therefore help to curb antibiotic resistance,
reduce severity of infections and duration of illness by
improving effectiveness and bioavailability of aforementioned
conventional drugs which are already on the market. The
main aim of the study was to determine combined
antibacterial activity of methanolic P. americana leaf extract
with vancomycin and tetracycline against S. aureus, S.
pyogenes and P. aeruginosa.
2. Materials and Methods
2.1. Collection of plant materials and microorganisms
Fresh mature leaves of P. americana were collected from
Malawi Adventist University, Malamulo Campus in Thyolo
district, Malawi. The leaves were collected during rainy
season of February to March 2018. The plant’s identity was
confirmed by a botanist. The organisms that were used in this
study were gram positive Staphylococcus aureus (ATCC
29213), Streptococcus pyogenes (ATCC 19615) and
Pseudomonas aeruginosa (PSA). These organisms were
obtained from Malawi Liverpool Welcome Trust.
2.2. Culture media, chemicals and antibiotics
Culture media, antibiotics and chemicals used for the present
work were purchased from Lapken Suppliers in Malawi. All
the laboratory materials used for extraction were suitable for
laboratory use and were used as received without any further
purification or treatment. Muller Hinton agar culture media
was used for S. aureus and P. aeruginosa and blood agar for
S. pyogenes. Methanol was used for extraction and antibiotics
that were used are vancomycin and tetracycline.
2.3. Preparation of P. American leaf extract
The leaves were washed with running distilled water to
remove contaminants and then shed dried for 7 days. The
dried leaves were ground into fine powder using a laboratory
mill. About 20g of powder were weighed and soaked in
100ml of 80% methanol for a period of 48 hours. The mixture
was agitated to ensure proper mixing after addition of the
solvent. They were then filtered using Whatman filter paper
No.1 to obtain 100% concentration of the extract. The extract
was stored in a refrigerator at 4 oC until ready for experiment.
2.4. Standardization of test isolates
The 24 hour old five colonies of test organisms from pure
culture were transferred into 4 ml sterile saline solution which
was adjusted to obtain a turbidity visually comparable to 0.5
McFarland standard at a well illuminated place with a white
background [31]. The prepared inocula was used within 15
minutes from preparation time.
2.5. Determination of antibacterial activity of the
antibiotics, leaf extract and their combinations
Antibacterial activity was done using disc diffusion method
[32]. Filter paper discs (6 mm in diameter) were impregnated
with leaf extract of 100% concentration and ready-made
antibiotic discs were placed on the media that were previously
inoculated with the standard inocula. For synergistic
(combined) effect 10 µl of the extract was added to the
antibiotic discs and then placed on the surface of inoculated
media with S. aureus, S. pyogenes and P. aeruginosa
separately. They were incubated overnight at 37 oC together
with the controls. Antibacterial activity was assessed by
measuring the diameter of the zone of inhibition (clear area
around the disc) in mm.
2.6. Determination of Minimum Inhibitory Concentration
(MIC) and Minimum Bactericidal Concentration (MBC)
of the extract
Two-fold serial broth macro dilution method was used to
determine MIC [32]. The 100% concentration of the extract
was serially diluted to achieve 1.5 mg/ml, 3.13 mg/ml, 6.25
mg/ml, 12.5 mg/ml, 25 mg/ml and 50 mg/ml and then
standardized inocula of 0.1 ml of the test organism and extract
were poured into 2 ml peptone broth tubes. Broth plus
inoculum and uninoculated tubes containing broth plus extract
were included as controls to check viability of inoculum and
sterility respectively. Then the tubes were incubated at 37 oC
for 24 hours. MIC was determined as the lowest concentration
that completely inhibited visual growth of organisms. The
tubes that did not show growth were streaked on Muller
Hinton agar plates. The plates were incubated for 24hours at
37 oC. The lowest concentration of the extract that did not
show any growth of organisms was considered to be MBC.
2.7. Data analysis
Laboratory analysis was performed in triplicates. The data
was analyzed using Statistical Package for Social Science
version 20.0.
3. Results
3.1. MIC and MBC of methanolic P. americana leaf extract
against S. aureus and S. pyogenes
Data on the MIC and MBC of methanolic leaf extract of P.
americana against S. aureus and S. pyogenes are provided in
Table 1. The preliminary tests showed that the extract had
highest MIC of 12.5 mg/ml and MBC of 25 mg/ml against S.
aureus while lowest MIC and MBC were both observed for S.
pyogenes. The observable growth was recorded for the leaf
extract concentrations below 6.25 mg/ml while concentrations
above 12.5 mg/ml showed no observable growth for both
aforementioned bacteria. Nearly all the concentrations of the
extract did not inhibit growth of P. aeruginosa.
Table 1: MIC and MBC of methanolic leaf extracts of P. americana
against test organisms
Organism
MIC/MB
C
Concentration of P. americana leaf
extract (mg/ml)
1.5
3.125
6.25
12.5
25
50
S. aureus
MIC
+
+
+
–
–
–
MBC
+
+
+
+
–
–
S. pyogenes
MIC
+
+
–
–
–
–
MBC
+
+
+
–
–
–
P. aeruginosa
MIC
+
+
+
+
+
+
Key: + Observable growth (Turbidity/ colonies), - No observable
growth (No Turbidity/ No colonies).
3.2. Antibacterial activity of the antibiotics, leaf extract
and combinations thereof
The results of antibacterial activity of Vancomycin,
tetracycline, methanolic P. americana leaf extract and their
combinations against test organisms are summarized in Table
2. Vancomycin showed greatest potency against S. pyogenes
but not against P. aeruginosa. Similarly, S. pyogenes was the
most susceptible organism against tetracycline while P.
aeruginosa was the least susceptible to tetracycline.
Antibacterial activity test showed that extract alone was more
effective against S. aureus but did not show inhibiting effect

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Journal of Pharmacognosy and Phytochemistry
on P. aeruginosa. Individually, it was observed that only
tetracycline had antibacterial activity against P. aeruginosa.
Although vancomycin and extract were combined, their effect
was not enough to inhibit growth of P. aeruginosa. However,
combined effect of vancomycin and extract showed greater
potency against S. pyogenes just like in tetracycline plus
extract.
Table 2: Antibacterial activity of antibiotics, extract and their combinations against test organisms
Organisms
Zone of inhibition (mm)
Vancomycin
Tetracycline
Extract
Treatment Combination
Vancomycin + extract
Tetracycline + extract
S. aureus
15.67 ± 1.53
28.00 ± 2.00
13.00 ± 0.00
21.33 ± 2.31
31.00 ± 2.00
S. pyogenes
19.33 ± 0.58
31.00 ± 1.41
10.00 ± 0.00
23.33 ± 1.15
35.33 ± 0.57
P. aeruginosa
0.00 ± 0.00
12.33 ± 1.15
0.00 ± 0.00
0.00 ± 0.00
8.00 ± 0.00
Values are means ± standard deviations of triplicate determinations.
Of all the two combinations, addition of the extract to
vancomycin showed the greatest improvement against S.
aureus while the least improvement was recorded for
tetracycline against aforementioned organism (Figure 1). It
was noted that when the extract was added to tetracycline a
reduction effect of -35.12% was observed while treating
vancomycin with the same concentration of the extract no
observable effect against P. aeruginosa was displayed. The
percent improvement of the antibiotics ranged from 10.71%
to 36.12%. Nearly all the combined formulations did not
show any improvement against P. aeruginosa.
Fig 1: Percent improvement of antibiotics following addition of the
extract.
4. Discussion
Medicinal plants produce various secondary metabolites in
expression of self-defense against several stimulants. P.
americana leaf extract contains bioactive compounds with
antibacterial activity against selected organisms [11, 16]. The
data from this study indicates that the extract was able to
inhibit growth of S. aureus and S. pyogenes but not P.
aeruginosa as evidenced by MIC. Low MIC and MBC
observed for P. pyogenes means that it would be cost
effective to use the extract alone against S. pyogenes because
management and treatment of S. pyogenes infection would
require little amount of plant materials hence conservation of
plants and money unlike S. aureus. It is likely that these
differences resulted from the variation in genetic makeup of
these organisms that determines susceptibility of an organism
to particular antibiotics. For instance, gram negative P.
aeruginosa as expected showed resistance to the extract due
to the nature of their cell wall [33, 34]. The MIC and MBC of
the extract against S. aureus and S. pyogenes reported herein
are comparatively lower than those that had been previously
published by others [11, 14]. Interestingly, others reported that
P. americana leaf extract inhibited growth of P. aeruginosa
and MIC had ranged from 30 mg/ml to 50 mg/ml [11, 14]. These
differences could be attributed to the variation in solvents and
their preparations, the strain of test organism and variety of P.
americana. It is also well known that metabolism and
accumulation of bioactive compounds in plants are highly
dependent on environmental factors such as altitude,
temperature, sunshine duration, light, soil, precipitation,
humidity and moisture, hence variation in these findings [35-
40]. An anecdotal evidence shows that level of maturity of the
leaf also influences antibacterial activity although we could
not find scientific evidence to support this claim.
There was clear indication that species type contributes to
susceptibility to bioactive components, thus different species
respond differently to the same drug due to genetic variation
[41]. Data from this study clearly indicate that vancomycin,
tetracycline and the extract were effective in inhibiting the
growth of S. aureus and P. aeruginosa separately while P.
aeruginosa was resistant to both vancomycin and the extract.
It is worth mentioning that in the preliminary determination of
MIC and MBC, the extract showed greatest potency against S.
pyogenes but surprisingly, disc diffusion method showed that
the extract greatly inhibited growth of S. aureus more than S.
pyogenes. These results may suggest that the potency of the
extract against S. pyogenes became compromised following
incorporation of the extract into the disc. Perhaps change of
physical nature of the extract from a solution state to solid
form of the disc favored activity against S. aureus. The zone
of inhibition for the extract against S. aureus was within the
range of 6.0 mm to 15 mm reported by others [11, 14]. The
contrast in these findings were due to various factors as
indicated above. Resistance of P. aeruginosa to vancomycin
was expected due to nature of the bacterial cell wall,
endogenous inactivation of the drug, efflux pump action and
possibility of acquired resistance therein [42-44]. Unlike in the
present study, others reported presence of zone of inhibition
for the extract against P. aeruginosa [11, 14]. The data on
antibacterial activity of the extract constitute a rational
evidence and scientific basis to justify and support the use of
P. americana leaf extract for the treatment of S. aureus and S.
pyogenes. This implies that the potency of these commercial
drugs can be improved by combining them with the leaf
extract.
In this study, the synergistic and additive interactions between
bioactive compounds of the extract and the antibiotics were
revealed. The presence of synergistic interaction implies that
combination of vancomycin and tetracycline with the extract
can be translated into useful clinical application in treatment
and control of S. aureus and S. pyogenes infections. These

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Journal of Pharmacognosy and Phytochemistry
findings significantly contribute to development of effective
antibiotics. On the other hand, the findings are also beneficial
to those who practice medical pluralism by taking
simultaneously herbal and conventional medicine.
Considering that in Malawi and Sub-Saharan Africa, people
simultaneously use herbal medicine and conventional
medicine, the present study has added evidence that taking
vancomycin or tetracycline together with P. americana leaf
extract would be beneficial. This will help to curb antibiotic
resistance, reduce severity of an infection and duration of
illness. It is noteworthy that addition of the extract to
tetracycline displayed antagonism (Figure 1). It is likely that
the reduction effect resulted from inability of the extract to
penetrate the outer membrane present in gram negative
organisms, lipopolysaccharide layer that hinders access of
most compounds. It is also assumed that some constituents of
the extract interfered with active components of tetracycline
resulting into inactive complexes [45].
Based on our results, public awareness is needed to cost
effectively use herbal remedies together with antibiotics.
However, it should be stressed that it is not ideal to combine
tetracycline with P. americana leaf extract against P.
aeruginosa as it reduced the activity of the antibiotic with -
35.12% reduction rate. However, combination of vancomycin
and the extract would give the best results due to high
improvement rate especially against S. aureus and S.
pyogenes. Further investigations on bioavailability of
combined treatments and their effects in the human body
should be high priority as these were in vitro analyses.
5. Conclusion and Recommendations
The present study has provided evidence that the potency of
the antibiotics can be improved by combining them with P.
americana leaf extract. The results obtained have shown that
the formulation of vancomycin with the extract greatly
inhibited the growth of S. aureus and P. aeruginosa. These
findings have potential implications in preventing antibiotic
resistance, accelerating recovery rate and reducing cost of
treatment. Based on our results, public awareness is needed to
cost effectively use herbal remedies together with antibiotics.
There is also a need to raise public’s knowledge about health
risks associated with combining tetracycline and the extract.
6. Acknowledgement
The author acknowledges Malamulo Mission Hospital and
Malawi Adventist University- Malamulo College of Health
Sciences Campus for the facility support. Many thanks to
Andrew Mbwembwe, George Selemani, Malawi Liverpool
Welcome Trust for your immerse support during material
collection and analysis.
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